Hepatitis GB virus recombinant proteins and uses thereof

BACKGROUND OF THE INVENTION
This invention relates generally to a group of infectious viral agents now termed the "GB viruses" which cause hepatitis in man, and more particularly, relates to materials such as polynucleotides derived from this group of viruses, polypeptides encoded therein, antibodies which specifically bind to these polypeptides, and diagnostics and vaccines that employ these materials.
Hepatitis is one of the most important diseases transmitted from a donor to a recipient by transfusion of blood products, organ transplantation and hemodialysis; it also can be transmitted via ingestion of contaminated food stuffs and water, and by person to person contact. Viral hepatitis is known to include a group of viral agents with distinctive viral genes and modes of replication, causing hepatitis with differing degrees of severity of hepatic damage through different routes of transmission. In some cases, acute viral hepatitis is clinically diagnosed by well-defined patient symptoms including jaundice, hepatic tenderness and an elevated level of liver transaminases such as aspartate transaminase (AST), alanine transaminase (ALT) and isocitrate dehydrogenase (ISD). In other cases, acute viral hepatitis may be clinically inapparent. The viral agents of hepatitis include hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), hepatitis E virus (HEV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV).
Although specific serologic assays available by the late 1960's to screen blood donations for the presence of HBV surface antigen (HBsAg) were successful in reducing the incidence of post-transfusion hepatitis (PTH) in blood recipients, PTH continued to occur at a significant rate. H. J. Alter et al., Ann. Int. Med. 77:691-699 (1972); H. J. Alter et al., Lancet ii:838-841 (1975). Investigators began to search for a new agent, termed "non-A, non-B hepatitis" (NANBH), that caused viral hepatitis not associated with exposure to viruses previously known to cause hepatitis in man (HAV, HBV, CMV and EBV). See, for example, S. M. Feinstone et al., New Engl. J. Med. 292:767-770 (1975); Anonymous editorial, Lancet ii:64-65 (1975); F. B. Hollinger in B. N. Fields and D. M. Knipe et al., Virology, Raven Press, New York, pp. 2239-2273 (1990).
Several lines of epidemiological and laboratory evidence have suggested the existence of more than one parenterally transmitted NANB agent, including multiple attacks of acute NANBH in intravenous drug users; distinct incubation periods of patients acquiring NANBH post-transfusion; the outcome of cross-challenge chimpanzee experiments; the ultrastructural liver pathology of infected chimpanzees; and the differential resistance of the putative agents to chloroform. J. L. Dienstag, Gastroenterology 85:439-462 (1983); J. L. Dienstag, Gastroenterology 85:743-768 (1983); F. B. Hollinger et al., J. Infect. Dis. 142:400-407 (1980); D. W. Bradley in F. Chisari, ed., Advances in Hepatitis Research, Masson, N.Y., pp. 268-280 (1984); and D. W. Bradley et al., J. Infect. Dis. 148:254-265 (1983).
A serum sample obtained from a surgeon who had developed acute hepatitis was shown to induce hepatitis when inoculated into tamarins (Saguinus species). Four of four tamarins developed elevated liver enzymes within a few weeks following their inoculation, suggesting that an agent in the surgeon's serum could produce hepatitis in tamarins. Serial passage in various non-human primates demonstrated that this hepatitis was caused by a transmissible agent; filtration studies suggested the agent to be viral in nature. The transmissible agent responsible for these cases of hepatitis in the surgeon and tamarins was termed the "GB agent." F. Deinhardt et al., J. Exper. Med. 125:673-688 (1967). F. Deinhardt et al., J. Exper. Med., supra; E. Tabor et al., J. Med. Virol. 5:103-108 (1980); R. O. Whittington et al., Viral and Immunological Diseases in Nonhuman Primates, Alan R. Liss, Inc., New York, pp. 221-224 (1983)
Although it was suggested that the GB agent may be an agent causing NANBH in humans and that the GB agent was not related to the known NANBH agents studied in various laboratories, no definitive or conclusive studies on the GB agent are known, and no viral agent has been discovered or molecularly characterized. F. Deinhardt et al., Am. J. Med. Sci. 270:73-80 (1975); and J. L. Dienstag et al., Nature 264:260-261 (1976). See also E. Tabor et al., J. Med. Virol., supra, E. Tabor et al., J. Infect. Dis. 140:794-797 (1979); R. O. Whittington et al., supra; and P. Karayiannis et al., Hepatology 9:186-192 (1989).
Early studies indicated that the GB agent was unrelated to any known human hepatitis virus. S. M. Feinstone et al., Science 182:1026-1028 (1973); P. J. Provost et al., Proc. Soc. Exp. Biol. Med. 148:532-539 (1975); J. L. Melnick, Intervirology 18:105-106 (1982); A. W. Holmes et al., Nature 243:419-420 (1973); and F. Deinhardt et al., Am. J. Med. Sci., supra. However, questions were raised regarding whether the GB agent was a virus which induced hepatitis infection in humans, or a latent tamarin virus activated by the GB serum and once activated, easily passaged to other tamarins, inducing hepatitis in them. Also, a small percentage of marmosets inoculated with GB-positive serum did not develop clinical hepatitis (4 of 52, or 7.6%), suggesting that these animals may have been naturally immune and thus, that the GB agent may be a marmoset virus. W. P. Parks et al., J. Infect. Dis. 120:539-547 (1969); W. P. Parks et al., J. Infect. Dis. 120:548-559 (1969). Morphological studies have been equivocal, with immune electron microscopy studies in one report indicating that the GB agent formed immune complexes with a size distribution of 20-22 nm and resembling the spherical structure of a parvovirus, while another study reported that immune electron microscopy data obtained from liver homogenates of GB-positive tamarins indicated that aggregates of 34-36 nm with icosahedral symmetry were detected, suggesting that the GB agent was a calici-like virus. See, for example, J. D. Almeida et al., Nature 261:608-609 (1976); J. L. Dienstag et al., Nature, supra.
Two hepatitis-causing viruses recently have been discovered and reported: HCV, which occurs primarily through parenteral transmission, and HEV, which is transmitted enterically. See, for example, Q. L. Choo et al., Science 244:359-362 (1989), G. Kuo et al., Science 244:362-364 (1989), E. P. Publication No. 0 318 216 (published May 31, 1989), G. R. Reyes et al., Science 247:1335-1339 (1990). HCV is responsible for a majority of PTH ascribed to the NANBH agent(s) and many cases of acute NANBH not acquired by transfusion. Anonymous editorial, Lancet 335:1431-1432 (1990); J. L. Dienstag, Gastroenterology 99:1177-1180 (1990); and M. J. Alter et al., JAMA 264:2231-2235 (1990).
While the detection of HCV antibody in donor samples eliminates 70 to 80% of NANBH infected blood in the blood supply system, the discovery and detection of HCV has not totally prevented the transmission of hepatitis. H. Alter et al., New Eng. J. Med. 321:1494-1500 (1989). Recent publications have questioned whether additional hepatitis agents may be responsible for PTH and for community acquired acute and/or chronic hepatitis that is not associated with PTH. For example, of 181 patients monitored in a prospective clinical survey conducted in France from 1988 to 1990, investigators noted a total of 18 cases of PTH. Thirteen of these 18 patients tested negative for anti-HCV antibodies, HBsAg, HBV and HCV nucleic acids. The authors speculated as to the potential importance of a non-A, non-B, non-C agent causing PTH. V. Thiers et al., J. Hepatology 18:34-39 (1993). Also, of 1,476 patients monitored in another study conducted in Germany from 1985 to 1988, 22 cases of documented cases of PTH were not related to infection with HBV or HCV. T. Peters et al., J. Med. Virol. 39:139-145 (1993).
It would be advantageous to identify and provide materials such as the recombinant antigens disclosed herein derived from and encoding a group of novel and unique viruses causing hepatitis and diagnostics and vaccines that employ these materials. Such materials could greatly enhance the ability of the medical community to more accurately diagnose acute and/or chronic viral hepatitis and could provide a safer blood and organ supply by detecting non-A, non-B and non-C hepatitis in these blood and organ donations.
SUMMARY OF THE INVENTION
The present invention provides a recombinant polypeptide comprising an amino acid sequence or fragment thereof wherein said sequence is characterized by a positive stranded RNA genome wherein said genome comprises an open reading frame (ORF) encoding a polyprotein wherein said polyprotein comprises an amino acid sequence having at least 35% identity to an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C, wherein said recombinant polypeptide is produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3. pCHO/E2-315 and pAcGP67-E2C. Also provided is an antibody directed against at least one hepatitis GB virus (HGBV) epitope, wherein said antibody is produced as a result of the immunization of an individual with a recombinant polypeptide, wherein said recombinant polypeptide is produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3, pCHO/E2-315 and pAcGP67-E2C. The antibody is polyclonal or monoclonal.
The present invention also provides a fusion polypeptide comprising at least one hepatitis GB virus (HGBV) polypeptide or fragment thereof wherein said fusion polypeptide is produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3, pCHO/E2-315 and pAcGP67-E2C.
In addition, an assay kit for determining the presence of hepatitis GB virus (HGBV) antigen or antibody in a test sample comprising a container containing a recombinant polypeptide possessing at least one HGBV epitope present in an HGBV antigen, wherein said polypeptide is characterized by a positive stranded RNA genome wherein said genome comprises an open reading frame (ORF) encoding a polyprotein wherein said polyprotein comprises an amino acid sequence having at least 35% identity to an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C and is produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3, pCHO/E2-315 and pAcGP67-E2C, is provided. The polypeptide of the assay kit can be attached to a solid phase. Further, the polypeptide can be attached to a signal generating compound which generates a measurable signal. The signal generating compound is selected from the group consisting of an enzyme, a fluorescent compound or a chemiluminescent compound. Another assay kit for determining the presence of hepatitis GB virus (HGBV) antigen or antibody in a test sample is provided, which comprises a container containing an antibody which specifically binds to an HGBV antigen, wherein said antigen comprises an HGBV epitope encoded by a sequence having at least about 60% sequence similarity to a sequence of HGBV and wherein said antibody is produced as a result of the immunization of an individual with a recombinant polypeptide produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3/pCHO/E2-315 and pAcGP67-E2C. The antibody can be attached to a solid phase. Additionally, the antibody can be attached to an indicator reagents comprising a signal generating compound which generates a measurable signal. The signal generating compound is selected from the group consisting of an enzyme, a fluorescent compound or a chemiluminescent compound.
A method for producing a polypeptide containing at least one hepatitis GB virus (HGBV) epitope comprising incubating host cells transformed with an expression vector comprising a sequence encoding a polypeptide characterized by a positive stranded RNA genome wherein said genome comprises an open reading frame (ORF) encoding a polyprotein wherein said polyprotein comprises an amino acid sequence having at least 35% identity to an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C wherein said polypeptide is produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3, pCHO/E2-315 and pAcGP67-E2C also is provided by the present invention.
The present invention also provides a method for detecting hepatitis GB virus (HGBV) antigen in a test sample suspected of containing HGBV comprising contacting the test sample with an antibody or fragment thereof which specifically binds to at least one HGBV antigen, for a time and under conditions sufficient to allow the formation of antibody/antigen complexes and detecting said complex containing the antibody wherein said antibody is produced as a result of the immunization of an individual with a recombinant polypeptide produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3, pCHO/E2-315 and pAcGP67-E2C. The antibody, either monoclonal or polyclonal, can be attached to a solid phase. The complex can be attached to an indicator reagent which comprises a signal generating compound which generates a measurable signal. The signal generating compound is selected from the group consisting of an enzyme, a fluorescent compound or a chemiluminescent compound.
A method for detecting hepatitis GB virus (HGBV) antibodies in a test sample suspected of containing said antibodies is also provided, which comprises contacting the test sample with a recombinant polypeptide wherein said polypeptide contains at least one HGBV epitope comprising an amino acid sequence or fragment thereof is characterized by a positive stranded RNA genome wherein said genome comprises an open reading frame (ORF) encoding a polyprotein wherein said polyprotein comprises an amino acid sequence having at least 35% identity to an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C, for a time and under conditions sufficient to allow antigen/antibody complexes to form; and detecting said complexes which contain the probe polypeptide, wherein said polypeptide is produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3, pCHO/E2-315 and pAcGP67-E2C. The probe polypeptide can be attached to a solid phase. The solid phase is selected from the group consisting of beads, microtiter wells, walls of test tube, nitrocellulose strips, magnetic beads and non-magnetic beads. The complexes can be attached to an indicator reagent which comprises a signal generating compound which generates a measurable signal. The signal generating compound is selected from the group consisting of an enzyme, a fluorescent compound or a chemiluminescent compound.
The present invention further provides a vaccine for treatment of hepatitis GB virus (HGBV) infection comprising a pharmacologically effective dose of an immunogenic HGBV recombinant polypeptide or fragment thereof which polypeptide is characterized by a positive stranded RNA genome wherein said genome comprises an open reading frame (ORF) encoding a polyprotein wherein said polyprotein comprises an amino acid sequence having at least 35% identity to an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C, in a pharmaceutically acceptable excipient, wherein said polypeptide is produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3, pCHO/E2-315 and pAcGP67-E2C.
In addition, a method for producing antibodies to hepatitis GB virus (HGBV) comprising administering to an individual an isolated immunogenic recombinant polypeptide or fragment thereof comprising at least one HGBV epitope in an amount sufficient to produce an immune response and wherein said polypeptide is produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3, pCHO/E2-315 and pAcGP67-E2C is provided.
Further, a diagnostic reagent comprising a recombinant polypeptide or fragment thereof derived from hepatitis GB virus (HGBV), wherein said polypeptide or fragment thereof encodes at least one epitope of HGBV and is characterized by a positive stranded RNA genome wherein said genome comprises an open reading frame (ORF) encoding a polyprotein wherein said polyprotein comprises an amino acid sequence having at least 35% identity to an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C, wherein said polypeptide is produced from a plasmid selected from the group consisting of pSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, pSFV-ss/NS3, pCHO/E2-315 and pAcGP67-E2C is provided.
The present invention moreover provides a method for determining the clearance of hepatitis GB virus (HGBV) from a patient infected HGBV, which comprises contacting a test sample obtained from said patient with a recombinant polypeptide wherein said polypeptide contains at least one HGBV epitope comprising an amino acid sequence or fragment thereof characterized by a positive stranded RNA genome wherein said genome comprises an open reading frame (ORF) encoding a polyprotein wherein said polyprotein comprises an amino acid sequence of HGBV E2 having at least 35% identity to an amino acid sequence selected from the group consisting of HGBV-A, HGBV-B and HGBV-C, for a time and under conditions sufficient to allow antigen/antibody complexes to form, wherein said polypeptide is produced from a plasmid selected from the group consisting of sSinRep5/NS5A, pCHO/E2-336, pSFV-ss/E2-336, sSFV-ss/NS3, pCHOE2-315 and pAcGP67-E2C and detecting said complexes, wherein the presence of said complexes is an indication of the clearance of said HGBV virus from said patient. The recombinant polypeptide can be attached to a solid phase, and the solid phase can be selected from the group consisting of beads, microtiter wells, walls of test tube, nitrocellulose strips, magnetic beads and non-magnetic beads. The complexes can be attached to an indicator reagent comprising a signal generating compound which generates a measurable signal. The signal generating compound is selected from the group consisting of an enzyme, a fluorescent compound or a chemiluminescent compound.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 presents a schematic drawing of the HGBV-C large open reading frame (SEQUENCE I.D. NO. 33) and the expressed PCR products.
FIG. 2 is a graphic representation of plasmid 577.
FIG. 3 presents a graphic of anti-GBV-C E2 reactivity of 100 volunteer blood donors demonstrating the distribution for determining the preliminary cutoff.
FIG. 4 is a graph of a serological profile of an individual acutely infected with GBV-C wherein the GBV-CE2 ELISA's S/N is plotted against days post exposure, and the GBV-C PCR and GBV-CE2 RIPA reactivities corresponding to the days post exposure are presented.
FIG. 5 is a graph of a serological profile of an individual acutely infected with GBV-C wherein the GBV-CE2 ELISA's S/N is plotted against days post exposure, and the GBV-C PCR and RIPA GBV-CE2 reactivities corresponding to the days post exposure are presented.
FIG. 6 is a photograph depicting the GBV-CE2 RIPA results of another individual infected with GBV-C.
FIG. 7 shows a Coomassie Brilliant Blue R-250 stained SDS polyacrylamide gel of the GBV-C E2 315 protein, wherein M designates molecular weight markers, SM designates starting material (concentrated, clarified VAS medium from GBV-C E2 315-transfected cells, wherein non-binding fractions refer to protein which did not bind to anti-FLAG antibody affinity column, wherein GBV-C E2 315 refers to the protein eluted from anti-FLAG M2 antibody affinity column by competition with FLAG peptide, and wherein SDS-PAGE and staining with Coomassie Brilliant Blue R-250 was performed using standard methods and reagents known in the art.

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides characterization of a newly ascertained etiological agents of non-A, non-B, non-C, non-D and non-E hepatitis-causing agents, collectively so-termed "Hepatitis GB Virus," or "HGBV" The present invention provides a method for determining the presence of the HGBV etiological agents, methods for obtaining the nucleic acid of this etiological agents created from infected serum, plasma or liver homogenates from individuals, either humans or tamarins, with HGBV to detect newly synthesized antigens derived from the genome of heretofore unisolated viral agents, and of selecting clones which produced products which are only found in infectious individuals as compared to non-infected individuals.
Polypeptide sequences of HGBV antigens encoded within the HGBV genome(s) permit the production of polypeptides which are useful as standards or reagents in diagnostic tests and/or as components of vaccines. Monoclonal and polyclonal antibodies which have been produced as a result of inoculation of an animal with at least one recombinant antigen disclosed herein directed against at least one epitope contained within these polypeptide sequences, also are useful for diagnostic tests as well as therapeutic agents, for screening of antiviral agents, and for the isolation of the HGBV agent.
According to one aspect of the invention, there will be provided a recombinant polypeptide encoding an epitope of HGBV; a recombinant vector containing any of the above described recombinant polypeptides, and a host cell transformed with these vectors. These recombinant polypeptides may be used alone or in combination, or in conjunction with other substances representing epitopes of HGBV.
In yet another aspect of the invention there will be provided a recombinant expression system comprising an open reading frame (ORF) of DNA derived from an HGBV genome or from HGBV cDNA, wherein the ORF is operably linked to a control sequence compatible with a desired host, a cell transformed with the recombinant expression system, and a polypeptide produced by the transformed cell.
Additional aspects of the present invention include at least one recombinant HGBV polypeptide, at least one recombinant polypeptide comprised of a sequence derived from an HGBV genome or from HGBV cDNA; at least one recombinant polypeptide comprised of an HGBV epitope and at least one fusion polypeptide comprised of an HGBV polypeptide.
The present invention also provides methods for producing a monoclonal antibody which specifically binds to at least one epitope of HGBV; a purified preparation of polyclonal antibodies which specifically bind to at least one HGBV epitope; and methods for using these antibodies, which include diagnostic, prognostic and therapeutic uses.
In still another aspect of the invention there will be provided a particle which immunizes against HGBV infection comprising a non-HGBV polypeptide having an amino acid sequence capable of forming a particle when said sequence is produced in an eukaryotic host, and an HGBV epitope.
A polynucleotide probe for HGBV also will be provided.
The present invention provides kits containing reagents which can be used for the detection of the presence and/or amount of antigen or antibody derived from HGBV, such reagents comprising a recombinant antigen containing an amino acid sequence from HGBV of about 3 to 5 or more amino acids in a suitable container; a reagent for detecting the presence and/or amount of an HGBV antigen comprising an antibody directed against the HGBV antigen to be detected in a suitable container. Other kits for various assay formats also are provided by the present invention as described herein.
Other aspects of the present invention include a polypeptide comprising at least one HGBV epitope attached to a solid phase and an antibody to an HGBV epitope attached to a solid phase. Also included are methods for producing a polypeptide containing an HGBV epitope comprising incubating host cells transformed with an expression vector containing a sequence encoding a polypeptide containing an HGBV epitope under conditions which allow expression of the polypeptide, and a polypeptide containing an HGBV epitope produced by this method.
The present invention also provides assays which utilize the recombinant polypeptides provided by the invention, as well as the antibodies described herein in various formats, any of which may employ a signal generating compound in the assay. Assays which do not utilize signal generating compounds to provide a means of detection also are provided. All of the assays described generally detect either antigen or antibody, or both, and include contacting a test sample with at least one reagent provided herein to form at least one antigen/antibody complex and detecting the presence of the complex. These assays are described in detail herein.
Vaccines for treatment of HGBV infection comprising an immunogenic peptide containing an HGBV epitope, or an inactivated preparation of HGBV, or an attenuated preparation of HGBV, or the use of recombinant vaccines that express HGBV epitope(s) and/or the use of synthetic peptides, also are included in the present invention. An effective vaccine may make use of combinations of these immunogenic peptides (such as, a cocktail of recombinant antigens wherein at least one recombinant antigen is selected from those disclosed herein, synthetic peptides and native viral antigens administered simultaneously or at different times); some of these may be utilized alone and be supplemented with other representations of immunogenic epitopes at later times. Also included in the present invention is a method for producing antibodies to HGBV comprising administering to an individual an isolated immunogenic polypeptide containing an HGBV epitope in an amount sufficient to produce an immune response in the inoculated individual.
The term "Hepatitis GB Virus" or "HGBV", as used herein, collectively denotes a viral species which causes non-A, non-B, non-C, non-D, non-E hepatitis in man, and attenuated strains or defective interfering particles derived therefrom. This may include acute viral hepatitis transmitted by contaminated foodstuffs, drinking water, and the like; hepatitis due to HGBV transmitted via person to person contact (including sexual transmission, respiratory and parenteral routes) or via intraveneous drug use. The methods as described herein will allow the identification of individuals who have acquired HGBV. Individually, the HGBV isolates are specifically referred to as "HGBV-A", "HGBV-B" and "HGBV-C." As described herein, the HGBV genome is comprised of RNA. Analysis of the nucleotide sequence and deduced amino acid sequence of the HGBV reveals that viruses of this group have a genome organization similar to that of the Flaviridae family. Based primarily, but not exclusively, upon similarities in genome organization, the International Committee on the Taxonomy of Viruses has recommended that this family be composed of three genera: Flavivirus, Pestivirus, and the hepatitis C group. Similarity searches at the amino acid level reveal that the hepatitis GB virus subclones have some, albeit low, sequence resemblance to hepatitis C virus. The information provided herein is sufficient to allow classification of other strains of HGBV.
Several lines of evidence demonstrate that HGBV-C is not a genotype of HCV. First, sera containing HGB-C sequences were tested for the presence of HCV antibody. Routine detection of individuals exposed to or infected with HCV relies upon antibody tests which utilize antigens derived from three or more regions from HCV-1. These tests allow detection of antibodies to the known genotypes of HCV (See, for example, Sakamoto et al., J. Gen. Virol. 75:1761-1768 (1994) and Stuyver et al., J. Gen. Virol. 74:1093-1102 (1993). HCV-specific ELISAs failed to detect sera containing GB-C sequences in six of eight cases. Second, several human sera that were seronegative for HCV antibodies have been shown to be positive for HCV genomic RNA by a highly sensitive RT-PCR assay (Sugitani, Lancet 339:1018-1019 (1992). This assay failed to detect HCV RNA in seven of eight sera containing HGB-C sequences (TABLE A). Thus, HGBV-C is not a genotype of HCV based on both serologic and molecular assays.
The alignment of a portion of the predicted translation product of HGB-C within the helicase region with the homologous region of HGBV-A, HGBV-B, HCV-1 and additional members of the Flaviviridae, followed by phylogenetic analysis of the aligned sequences suggests that HGBV-C is more closely related to HGBV-A than to any member of the HCV group. The sequences of HGBV-C and HGBV-A, while exhibiting an evolutionary distance of 0.42, are not as divergent as HGBV-C is from HGBV-B, which shows an evolutionary distance of 0.92. Thus, HGBV-A and HGBV-C may be considered to be members of one subgroup of the GB viruses and GBV-B a member of its own subgroup. The phylogenetic analysis of the helicase sequences from various HCV isolates show that they form a much less diverged group, exhibiting a maximum evolutionary distance of 0.20. A comparison of the HCV group and the HGBV group shows a minimum evolutionary distance between any two sequences from each group of 0.69. The distance values reported hereinabove were used to generate a phylogenic tree. The relatively high degree of divergence among these viruses suggests that the GB viruses are not merely types or subtypes within the hepatitis C group; rather, they constitute their own phylogenetic group (or groups). Phylogenetic analysis using sequence information derived from a small portion of HCV viral genomes has been shown to be an acceptable method for the assignment of new isolates into genotypic groups (Simmonds et al., Hepatology 19:1321-1324 (1994). In the current analysis, the use of a 110 amino acid sequence within the helicase gene from representative HCV isolates has properly grouped them into their respective genotypes (Simmonds et al., J. Gen. Virol. 75:1053-1061 (1994). Therefore, the evolutionary distances shown, in all likelihood, accurately reflect the high degree of divergence between the GB viruses and the hepatitis C virus.
The techniques for determining amino acid sequence "similarity" and/or "identity" are well-known in the art and include, for example, directly determining the amino acid sequence and comparing it to the sequences provided herein; determining the nucleotide sequence of the genomic material of the putative HGBV (usually via a cDNA intermediate), and determining the amino acid sequence encoded therein, and comparing the corresponding regions. In general, by "identity" is meant the exact match-up of either the nucleotide sequence of HGBV and that of another strain(s) or the amino acid sequence of HGBV and that of another strain(s) at the appropriate place on each genome. Also, in general, by "similarity" is meant the exact match-up of amino acid sequence of HGBV and that of another strain(s) at the appropriate place, where the amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. The programs available in the Wisconsin Sequence Analysis Package, Version 8 (available from the Genetics Computer Group, Madison, Wis., 53711), for example, the GAP program, are capable of calculating both the identity and similarity between two polynucleotide or two polypeptide sequences. Other programs for calculating identity and similarity between two sequences are known in the art.
Additionally, the following parameters are applicable, either alone or in combination, in identifying a strain of HGBV-A, HGBV-B or HGBV-C. It is expected that the overall nucleotide sequence identity of the genomes between HGBV-A, HGBV-B or HGBV-C and a strain of one of these hepatitis GB viruses will be about 45% or greater, since it is now believed that the HGBV strains may be genetically related, preferably about 60% or greater, and more preferably, about 80% or greater.
Also, it is expected that the overall sequence identity of the genomes between HGBV-A and a strain of HGBV-A at the amino acid level will be about 35% or greater since it is now believed that the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater. In addition, there will be corresponding contiguous sequences of at least about 13 nucleotides, which may be provided in combination of more than one contiguous sequence. Also, it is expected that the overall sequence identity of the genomes between HGBV-B and a strain of HGBV-B at the amino acid level will be about 35% or greater since it is now believed that the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater. In addition, there will be corresponding contiguous sequences of at least about 13 nucleotides, which may be provided in combination of more than one contiguous sequence. Also, it is expected that the overall sequence identity of the genomes between HGBV-C and a strain of HGBV-C at the amino acid level will be about 35% or greater since it is now believed that the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater. In addition, there will be corresponding contiguous sequences of at least about 13 nucleotides, which may be provided in combination of more than one contiguous sequence.
The compositions and methods described herein will enable the propagation, identification, detection and isolation of HGBV and its possible strains. Moreover, they also will allow the preparation of diagnostics and vaccines for the possible different strains of HGBV, and will have utility in screening procedures for anti-viral agents. The information will be sufficient to allow a viral taxonomist to identify other strains which fall within the species. We believe that HGBV encodes the sequences that are included herein. Methods for assaying for the presence of these sequences are known in the art and include, for example, amplification methods such as ligase chain reaction (LCR), polymerase chain reaction (PCR) and hybridization. In addition, these sequences contain open reading frames from which an immunogenic viral epitope may be found. This epitope is unique to HGBV when compared to other known hepatitis-causing viruses. The uniqueness of the epitope may be determined by its immunological reactivity with HGBV and lack of immunological reactivity with Hepatitis A, B, C, D and E viruses. Methods for determining immunological reactivity are known in the art and include, for example, radioimmunoassay (RIA), enzyme-linked immunosorbant assay (ELISA), hemagglutination (HA), fluorescence polarization immunoassay (FPIA) and several examples of suitable techniques are described herein.
A polynucleotide "derived from" a designated sequence for example, the HGBV cDNA, or from the HGBV genome, refers to a polynucleotide sequence which is comprised of a sequence of approximately at least about 6 nucleotides, is preferably at least about 8 nucleotides, is more preferably at least about 10-12 nucleotides, and even more preferably is at least about 15-20 nucleotides corresponding, i.e., similar to or complementary to, a region of the designated nucleotide sequence. Preferably, the sequence of the region from which the polynucleotide is derived is similar to or complementary to a sequence which is unique to the HGBV genome. Whether or not a sequence is complementary to or similar to a sequence which is unique to an HGBV genome can be determined by techniques known to those skilled in the art. Comparisons to sequences in databanks, for example, can be used as a method to determine the uniqueness of a designated sequence. Regions from which sequences may be derived include but are not limited to regions encoding specific epitopes, as well as non-translated and/or non-transcribed regions.
The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of HGBV, but may be generated in any manner, including but not limited to chemical synthesis, replication or reverse transcription or transcription, which are based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. In addition, combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with an intended use.
A "polypeptide" or "amino acid" sequence derived from a designated nucleic acid sequence or from the HGBV genome refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence or a portion thereof wherein the portion consists of at least 3 to 5 amino acids, and more preferably at least 8 to 10 amino acids, and even more preferably 15 to 20 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence.
A "recombinant polypeptide" as used herein means at least a polypeptide of genomic, semisynthetic or synthetic origin which by virtue of its origin or manipulation is not associated with all or a portion of the polypeptide with which it is associated in nature or in the form of a library and/or is linked to a polynucleotide other than that to which it is linked in nature. A recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence of HGBV or from an HGBV genome. It also may be generated in any manner, including chemical synthesis or expression of a recombinant expression system, or isolation from mutated HGBV.
The term "synthetic peptide" as used herein means a polymeric form of amino acids of any length, which may be chemically synthesized by methods well-known to the routineer. These synthetic peptides are useful in various applications.
The term "polynucleotide" as used herein means a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modifications, either by methylation and/or by capping, and unmodified forms of the polynucleotide.
"HGBV containing a sequence corresponding to a cDNA" means that the HGBV contains a polynucleotide sequence which is similar to or complementary to a sequence in the designated DNA. The degree of similarity or complementarity to the cDNA will be approximately 50% or greater, will preferably be at least about 70%, and even more preferably will be at least about 90%. The sequence which corresponds will be at least about 70 nucleotides, preferably at least about 80 nucleotides, and even more preferably at least about 90 nucleotides in length. The correspondence between the HGBV and the cDNA can be determined by methods known in the art, and include, for example, a direct comparison of the sequenced material with the cDNAs described, or hybridization and digestion with single strand nucleases, followed by size determination of the digested fragments.
"Purified viral polynucleotide" refers to an HGBV genome or fragment thereof which is essentially free, i.e., contains less than about 50%, preferably less than about 70%, and even more preferably, less than about 90% of polypeptides with which the viral polynucleotide is naturally associated. Techniques for purifying viral polynucleotides are well known in the art and include, for example, disruption of the particle with a chaotropic agent, and separation of the polynucleotide(s) and polypeptides by ion-exchange chromatography, affinity chromatography, and sedimentation according to density. Thus, "purified viral polypeptide" means an HGBV polypeptide or fragment thereof which is essentially free, that is, contains less than about 50%, preferably less than about 70%, and even more preferably, less than about 90% of cellular components with which the viral polypeptide is naturally associated. Methods for purifying are known to the routineer.
"Polypeptide" as used herein indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term, however, is not intended to refer to post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
"Recombinant host cells," "host cells," "cells," "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the original progeny of the original cell which has been transfected.
As used herein "replicon" means any genetic element, such as a plasmid, a chromosome or a virus, that behaves as an autonomous unit of polynucleotide replication within a cell. That is, it is capable of replication under its own control.
A "vector" is a replicon in which another polynucleotide segment is attached, such as to bring about the replication and/or expression of the attached segment.
The term "control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, such control sequences generally include promoter, ribosomal binding site and terminators; in eukaryotes, such control sequences generally include promoters, terminators and, in some instances, enhancers. The term "control sequence thus is intended to include at a minimum all components whose presence is necessary for expression, and also may include additional components whose presence is advantageous, for example, leader sequences.
"Operably linked" refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner. Thus, for example, a control sequence "operably linked" to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences.
The term "open reading frame" or "ORF" refers to a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence.
A "coding sequence" is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.
The term "immunologically identifiable with/as" refers to the presence of epitope(s) and polypeptide(s) which also are present in and are unique to the designated polypeptide(s), usually HGBV proteins. Immunological identity may be determined by antibody binding and/or competition in binding. These techniques are known to the routineer and also are described herein. The uniqueness of an epitope also can be determined by computer searches of known data banks, such as GenBank, for the polynucleotide sequences which encode the epitope, and by amino acid sequence comparisons with other known proteins.
As used herein, "epitope" means an antigenic determinant of a polypeptide. Conceivably, an epitope can comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually, it consists of at least eight to ten amino acids. Methods of examining spatial conformation are known in the art and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
A polypeptide is "immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope contained within the polypeptide. Immunological reactivity may be determined by antibody binding, more particularly by the kinetics of antibody binding, and/or by competition in binding using as competitor(s) a known polypeptide(s) containing an epitope against which the antibody is directed. The methods for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.
As used herein, the term "immunogenic polypeptide containing an HGBV epitope" means naturally occurring HGBV polypeptides or fragments thereof, as well as polypeptides prepared by other means, for example, chemical synthesis or the expression of the polypeptide in a recombinant organism.
The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction, or f-mating are included. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
"Treatment" refers to prophylaxis and/or therapy.
The term "individual" as used herein refers to vertebrates, particularly members of the mammalian species and includes but is not limited to domestic animals, sports animals, primates and humans; more particularly the term refers to tamarins and humans.
The term "plus strand" (or "+") as used herein denotes a nucleic acid that contains the sequence that encodes the polypeptide. The term "minus strand" (or "-") denotes a nucleic acid that contains a sequence that is complementary to that of the "plus" strand.
"Positive stranded genome" of a virus denotes that the genome, whether RNA or DNA, is single-stranded and which encodes a viral polypeptide(s).
The term "test sample" refers to a component of an individual's body which is the source of the analyte (such as, antibodies of interest or antigens of interest). These components are well known in the art. These test samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens; and fixed cell specimens.
After preparing the recombinant antigen as described, the recombinant antigen can be used to develop unique assays as described herein to detect either the presence of antigen or antibody to HGBV. These compositions also can be used to develop monoclonal and/or polyclonal antibodies with a specific recombinant antigen which specifically bind to the immunological epitope of HGBV which is desired by the routineer. Also, it is contemplated that at least one recombinant antigen of the invention can be used to develop vaccines by following methods known in the art.
It is contemplated that the reagent employed for the assay can be provided in the form of a test kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a monoclonal antibody, or a cocktail of monoclonal antibodies, or a recombinant antigen employed in the assay. Other components such as buffers, controls, and the like, known to those of ordinary skill in art, may be included in such test kits.
"Analyte," as used herein, is the substance to be detected which may be present in the test sample. The analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared. Thus, an analyte is a substance that can bind to one or more specific binding members in an assay. "Analyte" also includes any antigenic substances, haptens, antibodies, and combinations thereof. As a member of a specific binding pair, the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B12, the use of folate-binding protein to determine folic acid, or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate. The analyte can include a protein, a peptide, an amino acid, a nucleotide target, and the like.
The present invention provides assays which utilize specific binding members. A "specific binding member," as used herein, is a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal, and complexes thereof, including those formed by recombinant DNA molecules. The term "hapten", as used herein, refers to a partial antigen or non-protein binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unless coupled to a carrier protein.
A "capture reagent", as used herein, refers to an unlabeled specific binding member which is specific either for the analyte as in a sandwich assay, for the indicator reagent or analyte as in a competitive assay, or for an ancillary specific binding member, which itself is specific for the analyte, as in an indirect assay. The capture reagent can be directly or indirectly bound to a solid phase material before the performance of the assay or during the performance of the assay, thereby enabling the separation of immobilized complexes from the test sample.
The "indicator reagent" comprises a "signal generating compound" (label) which is capable of generating and generates a measurable signal detectable by external means conjugated (attached) to a specific binding member for HGBV. "Specific binding member" as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. In addition to being an antibody member of a specific binding pair for HGBV, the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like. An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to HGBV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
The various "signal generating compounds" (labels) contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums and luminol, radioactive elements, and direct visual labels. Examples of enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
"Solid phases" ("solid supports") are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, Duracytes.RTM. (derivatized red blood cells, available from Abbott Laboratories, Abbott Park, Ill.), and others. The "solid phase" is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and Duracytes.RTM. are all suitable examples. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like. A "solid phase", as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, Duracyte.RTM. and other configurations known to those of ordinary skill in the art.
It is contemplated and within the scope of the invention that the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens. Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well. All of these materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics.
Other embodiments which utilize various other solid phases also are contemplated and are within the scope of this invention. For example, ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer, described in co-pending U.S. patent application Ser. No. 150,278 corresponding to EP publication 0326100 and U.S. patent application Ser. No. 375,029 (EP publication no. 0406473), can be employed according to the present invention to effect a fast solution-phase immunochemical reaction. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-anion/immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described, including those described in chemiluminescent signal measurements as described in co-pending U.S. patent application Ser. No. 921,979 corresponding to EPO Publication No. 0 273,115.
Also, the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in automated and semi-automated systems wherein the solid phase comprises a microparticle (magnetic or non-magnetic). Such systems include those described in pending U.S. patent applications Ser. Nos. 425,651 and 425,643, which correspond to published EPO applications Nos. EP 0 425 633 and EP 0 424 634, respectively.
The use of scanning probe microscopy (SPM) for immunoassays also is a technology to which the monoclonal antibodies of the present invention are easily adaptable. In scanning probe microscopy, in particular in atomic force microscopy, the capture phase, for example, at least one of the monoclonal antibodies of the invention, is adhered to a solid phase and a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase. The use of scanning tunneling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes. The use of SPM to monitor specific binding reactions can occur in many ways. In one embodiment, one member of a specific binding partner (analyte specific substance which is the monoclonal antibody of the invention) is attached to a surface suitable for scanning. The attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art. Or, covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized. Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece. If the test piece is silicon or glass, the surface must be activated prior to attaching the specific binding partner. Also, polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a test piece by using techniques and chemistries described by pending U.S. patent applications Ser. No. 150,278, filed Jan. 29, 1988, and Ser. No. 375,029, filed Jul. 7, 1989. The preferred method of attachment is by covalent means. Following attachment of a specific binding member, the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding. The surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.
Various other assay formats may be used, including "sandwich" immunoassays and probe assays. For example, the monoclonal antibodies of the present invention can be employed in various assay systems to determine the presence, if any, of HGBV proteins in a test sample. Fragments of these monoclonal antibodies provided also may be used. For example, in a first assay format, a polyclonal or monoclonal anti-HGBV antibody or fragment thereof, or a combination of these antibodies, which has been coated on a solid phase, is contacted with a test sample which may contain HGBV proteins, to form a mixture. This mixture is incubated for a time and under conditions sufficient to form antigen/antibody complexes. Then, an indicator reagent comprising a monoclonal or a polyclonal antibody or a fragment thereof, which specifically binds to an HGBV region, or a combination of these antibodies, to which a signal generating compound has been attached, is contacted with the antigen/antibody complexes to form a second mixture. This second mixture then is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes. The presence of HGBV antigen present in the test sample and captured on the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating compound. The amount of HGBV antigen present in the test sample is proportional to the signal generated.
Alternatively, a polyclonal or monoclonal anti-HGBV antibody or fragment thereof, or a combination of these antibodies which is bound to a solid support, the test sample and an indicator reagent comprising a monoclonal or polyclonal antibody or fragments thereof, which specifically binds to HGBV antigen, or a combination of these antibodies to which a signal generating compound is attached, are contacted to form a mixture. This mixture is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes. The presence, if any, of HGBV proteins present in the test sample and captured on the solid phase is determined by detecting the measurable signal generated by the signal generating compound. The amount of HGBV proteins present in the test sample is proportional to the signal generated.
In another alternate assay format, one or a combination of at least two monoclonal antibodies of the invention can be employed as a competitive probe for the detection of antibodies to HGBV protein. For example, HGBV proteins such as the recombinant antigens disclosed herein, either alone or in combination, can be coated on a solid phase. A test sample suspected of containing antibody to HGBV antigen then is incubated with an indicator reagent comprising a signal generating compound and at least one monoclonal antibody of the invention for a time and under conditions sufficient to form antigen/antibody complexes of either the test sample and indicator reagent to the solid phase or the indicator reagent to the solid phase. The reduction in binding of the monoclonal antibody to the solid phase can be quantitatively measured. A measurable reduction in the signal compared to the signal generated from a confirmed negative NANB, non-C, non-D, non-E hepatitis test sample indicates the presence of anti-HGBV antibody in the test sample.
In yet another detection method, each of the monoclonal or polyclonal antibodies of the present invention can be employed in the detection of HGBV antigens in fixed tissue sections, as well as fixed cells by immunohistochemical analysis. Cytochemical analysis wherein these antibodies are labeled directly (fluorescein, colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) or are labeled by using secondary labeled anti-species antibodies (with various labels as exemplified herein) to track the histopathology of disease also are within the scope of the present invention.
In addition, these monoclonal antibodies can be bound to matrices similar to CNBr-activated Sepharose and used for the affinity purification of specific HGBV proteins from cell cultures, or biological tissues such as blood and liver such as to purify recombinant and native viral HGBV antigens and proteins.
The monoclonal antibodies of the invention can also be used for the generation of chimeric antibodies for therapeutic use, or other similar applications.
The monoclonal antibodies or fragments thereof can be provided individually to detect HGBV antigens. Combinations of the monoclonal antibodies (and fragments thereof) provided herein also may be used together as components in a mixture or "cocktail" of at least one anti-HGBV antibody of the invention with antibodies to other HGBV regions, each having different binding specificities. Thus, this cocktail can include the monoclonal antibodies of the invention which are directed to HGBV proteins and other monoclonal antibodies to other antigenic determinants of the HGBV genome.
The polyclonal antibody or fragment thereof which can be used in the assay formats should specifically bind to a specific HGBV region or other HGBV proteins used in the assay. The polyclonal antibody used preferably is of mammalian origin; human, goat, rabbit or sheep anti-HGBV polyclonal antibody can be used. Most preferably, the polyclonal antibody is rabbit polyclonal anti-HGBV antibody. The polyclonal antibodies used in the assays can be used either alone or as a cocktail of polyclonal antibodies. Since the cocktails used in the assay formats are comprised of either monoclonal antibodies or polyclonal antibodies having different HGBV specificity, they would be useful for diagnosis, evaluation and prognosis of HGBV infection, as well as for studying HGBV protein differentiation and specificity.
It is contemplated and within the scope of the present invention that the HGBV group of viruses may be detectable in assays by use of a recombinant antigen as well as by use of a synthetic peptide or native peptide, which contain an amino acid sequence that is common to all HGBV viruses. It also is within the scope of the present invention that different synthetic, recombinant or native peptides identifying different epitopes from HGBV-A, HGBV-B, HGBV-C, or yet other HGBV viruses, can be used in assay formats. In the later case, these can be coated onto one solid phase, or each separate peptide may be coated on separate solid phases, such as microparticles, and then combined to form a mixture of peptides which can be later used in assays. Such variations of assay formats are known to those of ordinary skill in the art and are discussed hereinbelow.
In another assay format, the presence of antibody and/or antigen to HGBV can be detected in a simultaneous assay, as follows. A test sample is simultaneously contacted with a capture reagent of a first analyte, wherein said capture reagent comprises a first binding member specific for a first analyte attached to a solid phase and a capture reagent for a second analyte, wherein said capture reagent comprises a first binding member for a second analyte attached to a second solid phase, to thereby form a mixture. This mixture is incubated for a time and under conditions sufficient to form capture reagent/first analyte and capture reagent/second analyte complexes. These so-formed complexes then are contacted with an indicator reagent comprising a member of a binding pair specific for the first analyte labeled with a signal generating compound and an indicator reagent comprising a member of a binding pair specific for the second analyte labeled with a signal generating compound to form a second mixture. This second mixture is incubated for a time and under conditions sufficient to form capture reagent/first analyte/indicator reagent complexes and capture reagent/second analyte/indicator reagent complexes. The presence of one or more analytes is determined by detecting a signal generated in connection with the complexes formed on either or both solid phases as an indication of the presence of one or more analytes in the test sample. In this assay format, recombinant antigens derived from human expression systems may be utilized as well as monoclonal antibodies produced from the proteins derived from the mammalian expression systems as disclosed herein. Such assay systems are described in greater detail in pending U.S. patent application Ser. No. 07/574,821 entitled Simultaneous Assay for Detecting One Or More Analytes, which corresponds to EP Publication No. 0473065.
In yet other assay formats, the recombinant antigens disclosed herein may be utilized to detect the presence of anti-HGBV in test samples. For example, a test sample is incubated with a solid phase to which at least one recombinant protein has been attached. These are reacted for a time and under conditions sufficient to form antigen/antibody complexes. Following incubation, the antigen/antibody complex is detected. Indicator reagents may be used to facilitate detection, depending upon the assay system chosen. In another assay format, a test sample is contacted with a solid phase to which a recombinant protein produced as described herein is attached and also is contacted with a monoclonal or polyclonal antibody specific for the protein, which preferably has been labeled with an indicator reagent. After incubation for a time and under conditions sufficient for antibody/antigen complexes to form, the solid phase is separated from the free phase, and the label is detected in either the solid or free phase as an indication of the presence of HGBV antibody. Other assay formats utilizing the recombinant antigens disclosed herein are contemplated. These include contacting a test sample with a solid phase to which at least one antigen from a first source has been attached, incubating the solid phase and test sample for a time and under conditions sufficient to form antigen/antibody complexes, and then contacting the solid phase with a labeled antigen, which antigen is derived from a second source different from the first source. For example, a recombinant protein derived from a first source such as E. coli is used as a capture antigen on a solid phase, a test sample is added to the so-prepared solid phase, and a recombinant protein derived from a different source (i.e., non-E. coli) is utilized as a part of an indicator reagent. Likewise, combinations of a recombinant antigen on a solid phase and synthetic peptide in the indicator phase also are possible. Any assay format which utilizes an antigen specific for HGBV from a first source as the capture antigen and an antigen specific for HGBV from a different second source are contemplated. Thus, various combinations of recombinant antigens, as well as the use of synthetic peptides, purified viral proteins, and the like, are within the scope of this invention. Assays such as this and others are described in U.S. Pat. No. 5,254,458, which enjoys common ownership and is incorporated herein by reference.
Other assay systems which utilize an antibody (polyclonal, monoclonal or naturally-occurring) which specifically binds HGBV viral particles or sub-viral particles housing the viral genome (or fragments thereof) by virtue of a contact between the specific antibody and the viral protein (peptide, etc.). This captured particle then can be analyzed by methods such as LCR or PCR to determine whether the viral genome is present in the test sample. Test samples which can be assayed according to this method include blood, liver, sputum, urine, fecal material, saliva, and the like. The advantage of utilizing such an antigen capture amplification method is that it can separate the viral genome from other molecules in the test specimen by use of a specific antibody. Such a method has been described in pending U.S. patent application Ser. No. 08/141,429.
While the present invention discloses the preference for the use of solid phases, it is contemplated that the reagents such as antibodies, proteins and peptides of the present invention can be utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the present invention.
MATERIALS AND METHODS
General Techniques
Conventional and well-known techniques and methods in the fields of molecular biology, microbiology, recombinant DNA and immunology are employed in the practice of the invention unless otherwise noted. Such techniques are explained and detailed in the literature. See, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); D. N. Glover, ed., DNA Cloning, Volumes I and II (1985); M. J. Gait ed., Oligonucleotide Synthesis, (1984); B. D. Hames et al., eds., Nucleic Acid Hybridization, (1984); B. D. Hames et al., eds., Transcription and Translation, (1984); R. L Freshney ed., Animal Cell Culture, (1986); Immobilized Cells and Enzymes, IRL Press (1986); B. Perbal, A Practical Guide to Molecular Cloning, (1984); the series, Methods in Enzymology, Academic Press, Inc., Orlando, Fla.; J. H. Miller et al., eds., Gene Transfer Vectors For Mammalian Cells, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1987); Wu et al., eds., Methods in Enzymology, Vol. 154 and 155; Mayer et al., eds., Immunological Methods In Cell and Molecular Biology, Academic Press, London (1987); Scopes, Protein Purification: Principles and Practice, 2nd ed., Springer-Verlag, N.Y.; and D. Weir et al., eds., Handbook Of Experimental Immunology, Volumes I-IV (1986); N. Lisitisyn et al., Science 259:946-951 (1993).
The reagents and methods of the present invention are made possible by the provision of a family of closely related nucleotide sequences, isolated by representational difference analysis modified as described herein, present in the plasma, serum or liver homogenate of an HGBV infected individual, either tamarin or human. This family of nucleotide sequences is not of human or tamarin origin, since it hybridizes to neither human nor tamarin genomic DNA from uninfected individuals, since nucleotides of this family of sequences are present only in liver (or liver homogenates), plasma or serum of individuals infected with HGBV, and since the sequence is not present in GenBank.RTM.. In addition, the family of sequences shows no significant identity at the nucleic acid level to sequences contained within the HAV, HBV, HCV, HDV and HEV genome, and low level identity, considered not significant, as translation products. Infectious sera, plasma or liver homogenates from HGBV infected humans contain these polynucleotide sequences, whereas sera, plasma or liver homogenates from non-infected humans do not contain these sequences. Northern blot analysis of infected liver with some of these polynucleotide sequences demonstrate that they are derived from a large RNA transcript similar in size to a viral genome. Sera, plasma or liver homogenates from HGBV-infected humans contain antibodies which bind to this polypeptide, whereas sera, plasma or liver homogenates from non-infected humans do not contain antibodies to this polypeptide; these antibodies are induced in individuals following acute non-A, non-B, non-C, non-D and non-E infection. By these criteria, it is believed that the sequence is a viral sequence, wherein the virus causes or is associated with non-A, non-B, non-C, non-D and non-E hepatitis.
The recombinant antigens disclosed herein are useful in diagnosing non-A, non-B, non-C, non-D, non-E hepatitis due to HGBV infections, and in screening blood donors, donated blood, blood products and individuals for infection. The family of polypeptides derived from these sequences, as well as antibodies directed against these polypeptides, also are useful in the isolation and identification of the HGBV etiological agent(s). For example, antibodies directed against HGBV epitopes contained in polypeptides derived from the nucleic acid sequences may be used in methods based upon affinity chromatography to isolate the virus. Alternatively, the antibodies can be used to identify viral particles isolated by other techniques. The viral antigens and the genomic material within the isolated viral particles then may be further characterized.
The information obtained from further sequencing of the HGBV genome(s), as well as from further characterization of the HGBV antigens and characterization of the genome enables the design and synthesis of additional probes and polypeptides and antibodies which may be used for diagnosis, prevention and therapy of HGBV induced non-A, non-B, non-C non-D, non-E hepatitis, and for screening of infected blood and blood-related products.
Deposit of Strains
Strains replicated (clones 2, 4, 10, 16, 18, 23 and 50) from the HGBV nucleic acid sequence library have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, as of Feb. 10, 1994, under the terms of the Budapest Treaty and will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, or for the enforceable period of the U.S. patent, whichever is longer. The deposits and any other deposited material described herein are provided for convenience only, and are not required to practice the present invention in view of the teachings provided herein. The HGBV cDNA sequences in all of the deposited materials are incorporated herein by reference. The plasmids were accorded the following A.T.C.C. deposit numbers: Clone 2 was accorded A.T.C.C. Deposit No. 69556; Clone 4 was accorded A.T.C.C. Deposit No. 69557; Clone 10 was accorded A.T.C.C. Deposit No. 69558; Clone 16 was accorded A.T.C.C. Deposit No. 69559; Clone 18 was accorded A.T.C.C. Deposit No. 69560; Clone 23 was accorded A.T.C.C. Deposit No. 69561; and Clone 50 was accorded A.T.C.C. Deposit No. 69562.
Strains replicated (clones 11, 13, 48 and 119) from the HGBV nucleic acid sequence library have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, as of Apr. 29, 1994, under the terms of the Budapest Treaty and will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, or for the enforceable period of the U.S. patent, whichever is longer. The deposits and any other deposited material described herein are provided for convenience only, and are not required to practice the present invention in view of the teachings provided herein. The HGBV cDNA sequences in all of the deposited materials are incorporated herein by reference. The plasmids were accorded the following A.T.C.C. deposit numbers: Clone 11 was accorded A.T.C.C. Deposit No. No. 69613; Clone 13 was accorded A.T.C.C. Deposit No. 69611; Clone 48 was accorded A.T.C.C. Deposit No. 69610; and Clone 119 was accorded A.T.C.C. Deposit No. 69612.
Additional strains (clones 4B1.1, 66-3A1.49, 70-3A1.37 and 78-1C1.17) from the HGBV nucleic acid sequence library have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, as of Jul. 28, 1994, under the terms of the Budapest Treaty and will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, or for the enforceable period of the U.S. patent, whichever is longer. The deposits and any other deposited material described herein are provided for convenience only, and are not required to practice the present invention in view of the teachings provided herein. The HGBV cDNA sequences in all of the deposited materials are incorporated herein by reference. The plasmids were accorded the following A.T.C.C. deposit numbers: Clone 4-B1.1 was accorded A.T.C.C. Deposit No. No. 69666; Clone 66-3A1.49 was accorded A.T.C.C. Deposit No. 69665; Clone 70-3A1.37 was accorded A.T.C.C. Deposit No. 69664; and Clone 78-1C1.17 was accorded A.T.C.C. Deposit No. 69663.
Clone pHGBV-C clone #1 was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 as of Nov. 8, 1994, under the terms of the Budapest Treaty and will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, or for the enforceable period of the U.S. patent, whichever is longer. The deposits and any other deposited material described herein are provided for convenience only, and are not required to practice the present invention in view of the teachings provided herein. pHGBV-C clone #1 was accorded A.T.C.C. Deposit No. 69711. The HGBV cDNA sequences in all of the deposited materials are incorporated herein by reference.
Preparation of Viral Polypeptides and Fragments
The availability of nucleic acid sequences permits the construction of expression vectors encoding antigenically active regions of the polypeptide encoded in either strand. These antigenically active regions may be derived from structural regions of the virus, including, for example, envelope (coat) or core antigens, in addition to nonstructural regions of the virus, including, for example, polynucleotide binding proteins, polynucleotide polymerase(s), and other viral proteins necessary for replication and/or assembly of the viral particle. Fragments encoding the desired polypeptides are derived from the genomic or cDNA clones using conventional restriction digestion or by synthetic methods, and are ligated into vectors which may, for example, contain portions of fusion sequences such as beta-galactosidase (b-gal) or superoxide dismutase (SOD) or CMP-KDO synthetase (CKS). Methods and vectors which are useful for the production of polypeptides which contain fusion sequences of SOD are described in EPO 0196056, published Oct. 1, 1986, and those of CKS are described in EPO Publication No. 0331961, published Sep. 13, 1989. Any desired portion of the nucleic acid sequence containing an open reading frame, in either sense strand, can be obtained as a recombinant protein, such as a mature or fusion protein; alternatively, a polypeptide encoded in the HGBV genome or cDNA can be provided by chemical synthesis.
An additional expression system useful for producing the recombinant antigens disclosed herein utilizes the lambda pL vector system. This expression system has the following features: (1) a strong lambda pL promoter, (2) a strong three-frame translation terminator rrnBt1, and (3) translation starts at an ATG codon, eight base pairs from the ribosome binding site located within an accessible Nco1 restriction site.
The nucleic acid sequence encoding the desired polypeptide, whether in fused or mature form, and whether or not containing a signal sequence to permit secretion, may be ligated into expression vectors suitable for any convenient host. Both eukaryotic and prokaryotic host systems are used in the art to form recombinant proteins, and some of these are listed herein. The polypeptide then is isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification can be performed by techniques known in the art, and include salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, among others. Such polypeptides may be used as diagnostic reagents, or for passive immunotherapy. In addition, antibodies to these polypeptides are useful for isolating and identifying HGBV particles. The HGBV antigens also may be isolated from HGBV virions. These virions can be grown in HGBV infected cells in tissue culture, or in an infected individual.
Preparation of Antigenic Polypeptides and Conjugation With Solid Phase
An antigenic region or fragment of a polypeptide generally is relatively small, usually about 8 to 10 amino acids or less in length. Fragments of as few as 5 amino acids may characterize an antigenic region. These segments may correspond to regions of HGBV antigen. By using the HGBV genomic or cDNA sequences as a basis, nucleic acid sequences encoding short segments of HGBV polypeptides can be expressed recombinantly either as fusion proteins or as isolated polypeptides. These short amino acid sequences also can be obtained by chemical synthesis. The small chemically synthesized polypeptides may be linked to a suitable carrier molecule when the synthesized polypeptide provided is correctly configured to provide the correct epitope but too small to be antigenic. Linking methods are known in the art and include but are not limited to using N-succinimidyl-3-(2-pyrdylthio)propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Polypeptides lacking sulfhydryl groups can be modified by adding a cysteine residue. These reagents create a disulfide linkage between themselves and peptide cysteine residues on one protein and an amide linkage through the epsilon-amino on a lysine, or other free amino group in the other. A variety of such disulfide/amide-forming agents are known. Other bifunctional coupling agents form a thioester rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and are known to those of ordinary skill in the art. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Any carrier which does not itself induce the production of antibodies harmful to the host can be used. Suitable carriers include proteins, polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose beads, polymeric amino acids such as polyglutamic acid, polylysine, amino acid copolymers and inactive virus particles, among others. Examples of protein substrates include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and yet other proteins known to those skilled in the art.
Preparation of Hybrid Particle Immunogens Containing HGBV Epitopes
The immunogenicity of HGBV epitopes also may be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteins such as those associated with HBV surface antigen. Constructs wherein the HGBV epitope is linked directly to the particle-forming protein coding sequences produce hybrids which are immunogenic with respect to the HGBV epitope. In addition, all of the vectors prepared include epitopes specific for HGBV, having varying degrees of immunogenicity. Particles constructed from particle forming protein which include HGBV sequences are immunogenic with respect to HGBV and HBV.
Hepatitis B surface antigen has been determined to be formed and assembled into particles in S. cerevisiae and mammalian cells; the formation of these particles has been reported to enhance the immunogenicity of the monomer subunit. P. Valenzuela et al., Nature 298:334 (1982); P. Valenzuela et al., in I. Millman et al., eds., Hepatitis B, Plenum Press, pp. 225-236 (1984). The constructs may include immunodominant epitopes of HBsAg. Such constructs have been reported expressible in yeast, and hybrids including heterologous viral sequences for yeast expression have been disclosed. See, for example, EPO 174, 444 and EPO 174,261. These constructs also have been reported capable of being expressed in mammalian cells such as Chinese hamster ovary (CHO) cells. Michelle et al., International Symposium on Viral Hepatitis, 1984. In HGBV, portions of the particle-forming protein coding sequence may be replaced with codons encoding an HGBV epitope. In this replacement, regions that are not required to mediate the aggregation of the units to form immunogenic particles in yeast or mammals can be deleted, thus eliminating additional HGBV antigenic sites from competition with the HGBV epitope.
Vaccine Preparation
Vaccines may be prepared from one or more immunogenic polypeptides or nucleic acids derived from HGBV nucleic acid sequences or from the HGBV genome to which they correspond. Vaccines may comprise recombinant polypeptides containing epitope(s) of HGBV. These polypeptides may be expressed in bacteria, yeast or mammalian cells, or alternatively may be isolated from viral preparations. It also is anticipated that various structural proteins may contain epitopes of HGBV which give rise to protective anti-HGBV antibodies. Synthetic peptides therefore also can be utilized when preparing these vaccines. Thus, polypeptides containing at least one epitope of HGBV may be used, either singly or in combinations, in HGBV vaccines. It also is contemplated that nonstructural proteins as well as structural proteins may provide protection against viral pathogenicity, even if they do not cause the production of neutralizing antibodies.
Considering the above, multivalent vaccines against HGBV may comprise one or more structural proteins, and/or one or more nonstructural proteins. These vaccines may be comprised of, for example, recombinant HGBV polypeptides and/or polypeptides isolated from the virions and/or synthetic peptides. These immunogenic epitopes can be used in combinations, i.e., as a mixture of recombinant proteins, synthetic peptides and/or polypeptides isolated from the virion; these may be administered at the same or different time. Additionally, it may be possible to use inactivated HGBV in vaccines. Such inactivation may be preparation of viral lysates, or by other means known in the art to cause inactivation of hepatitis-like viruses, for example, treatment with organic solvents or detergents, or treatment with formalin. Attenuated HGBV strain preparation also is disclosed in the present invention. It is contemplated that some of the proteins in HGBV may cross-react with other known viruses, and thus that shared epitopes may exist between HGBV and other viruses which would then give rise to protective antibodies against one or more of the disorders caused by these pathogenic agents. It is contemplated that it may be possible to design multiple purpose vaccines based upon this belief.
The preparation of vaccines which contain at least one immunogenic peptide as an active ingredient is known to one skilled in the art. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection also may be prepared. The preparation may be emulsified or the protein may be encapsulated in liposomes. The active immunogenic ingredients often are mixed with pharmacologically acceptable excipients which are compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol and the like; combinations of these excipients in various amounts also may be used. The vaccine also may contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. For example, such adjuvants can include aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), and RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80.RTM. emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing an HGBV antigenic sequence resulting from administration of this polypeptide in vaccines which also are comprised of the various adjuvants.
The vaccines usually are administered by intraveneous or intramuscular injection. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include but are not limited to polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably, about 1% to about 2%. Oral formulation include such normally employed excipients as, for example pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
The proteins used in the vaccine may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts such as acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and others known to those skilled in the art. Salts formed with the free carboxyl groups also may be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxides and the like, and such organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine procaine, and others known to those skilled in the art.
Vaccines are administered in a way compatible with the dosage formulation, and in such amounts as will be prophylactically and/or therapeutically effective. The quantity to be administered generally is in the range of about 5 micrograms to about 250 micrograms of antigen per dose, and depends upon the subject to be dosed, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection sought. Precise amounts of active ingredient required to be administered also may depend upon the judgment of the practitioner and may be unique to each subject. The vaccine may be given in a single or multiple dose schedule. A multiple dose is one in which a primary course of vaccination may be with one to ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reinforce the immune response, for example, at one to four months for a second dose, and if required by the individual, a subsequent dose(s) after several months. The dosage regimen also will be determined, at least in part, by the need of the individual, and be dependent upon the practitioner's judgment. It is contemplated that the vaccine containing the immunogenic HGBV antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, with immune globulins.
Preparation of Antibodies Against HGBV Epitopes
The immunogenic peptides prepared as described herein are used to produce antibodies, either polyclonal or monoclonal. When preparing polyclonal antibodies, a selected mammal (for example, a mouse, rabbit, goat, horse or the like) is immunized with an immunogenic polypeptide bearing at least one HGBV epitope. Serum from the immunized animal is collected after an appropriate incubation period and treated according to known procedures. If serum containing polyclonal antibodies to an HGBV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by, for example, immunoaffinity chromatography. Techniques for producing and processing polyclonal antibodies are known in the art and are described in, among others, Mayer and Walker, eds., Immunochemical Methods In Cell and Molecular Biology, Academic Press, London (1987). Polyclonal antibodies also may be obtained from a mammal previously infected with HGBV. An example of a method for purifying antibodies to HGBV epitopes from serum of an individual infected with HGBV using affinity chromatography is provided herein.
Monoclonal antibodies directed against HGBV epitopes also can be produced by one skilled in the art. The general methodology for producing such antibodies is well-known and has been described in, for example, Kohler and Milstein, Nature 256:494 (1975) and reviewed in J. G. R. Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press Inc., Boco Raton, Fla. (1982), as well as that taught by L. T. Mimms et al., Virology 176:604-619 (1990). Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See also, M. Schreier et al., Hybridoma Techniques, Scopes (1980) Protein Purification, Principles and Practice, 2nd Edition, Springer-Verlag, New York (1984); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas (1981); Kennet et al., Monoclonal Antibodies (1980). Examples of uses and techniques of monoclonal antibodies are disclosed in U.S. patent applications Ser. Nos. 748,292; 748,563; 610,175, 648,473; 648,477; and 648,475.
Monoclonal and polyclonal antibodies thus developed, directed against HGBV epitopes, are useful in diagnostic and prognostic applications, and also, those which are neutralizing are useful in passive immunotherapy. Monoclonal antibodies especially can be used to produce anti-idiotype antibodies. These anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired. See, for example, A. Nisonoff et al., Clin. Immunol. Immunopath. 21:397-406 (1981), and Dreesman et al., J. Infect. Dis. 151:761 (1985). Techniques for raising such idiotype antibodies are known in the art and exemplified, for example, in Grych et al., Nature 316:74 (1985); MacNamara et al., Science 226:1325 (1984); and Uytdehaag et al., J. Immunol. 134:1225 (1985). These anti-idiotypic antibodies also may be useful for treatment of HGBV infection, as well as for elucidation of the immunogenic regions of HGBV antigens.
Immunoassay and Diagnostic Kits
Both the polypeptides which react immunologically with serum containing HGBV antibodies and composites thereof, and the antibodies raised against the HGBV specific epitopes in these polypeptides are useful in immunoassays to detect the presence of HGBV antibodies, or the presence of the virus and/or viral antigens in biological test samples. The design of these immunoassays is subject to variation, and a variety of these are known in the art; a variety of these have been described herein. The immunoassay may utilize one viral antigen, such as a polypeptide derived from any clone-containing HGBV nucleic acid sequence, or from the composite nucleic acid sequences derived from the HGBV nucleic acid sequences in these clones, or from the HGBV genome from which the nucleic acid sequences in these clones is derived. Or, the immunoassay may use a combination of viral antigens derived from these sources. It may use, for example, a monoclonal antibody directed against the same viral antigen, or polyclonal antibodies directed against different viral antigens. Assays can include but are not limited to those based on competition, direct reaction or sandwich-type assays. Assays may use solid phases or may be performed by immunoprecipitation or any other methods which do not utilize solid phases. Examples of assays which utilize labels as the signal generating compound and those labels are described herein. Signals also may be amplified by using biotin and avidin, enzyme labels or biotin anti-biotin systems, such as that described in pending U.S. patent application Ser. Nos. 608,849; 070,647; 418,981; and 687,785 and as known in the art. Recombinant polypeptides which include epitopes from immunodominant regions of HGBV may be useful for the detection of viral antibodies in biological test samples of infected individuals. It also is contemplated that GBV antibodies may be useful in discriminating acute from non-acute infections. Kits suitable for immunodiagnosis and containing the appropriate reagents are constructed by packaging the appropriate materials, including the polypeptides of the invention containing HGBV epitopes or antibodies directed against HGBV epitopes in suitable containers, along with the remaining reagents and materials required for the conduct of the assay, as well as suitable assay instructions.
Assay formats can be designed which utilize the recombinant proteins detailed herein, and although we describe and detail CKS-fused proteins, it also is contemplated and within the scope of the present invention that other expression systems, such as pL, superoxide dismutase (SOD), and others, can be used in the present invention to generate fusion proteins capable of use in a variety of ways, including as antigens in immunoassays, immunogens for antibody production, and the like. In an assay format to detect the presence of antibody against a specific analyte (for example, an infectious agent such as a virus) in a human test sample, the human test sample is contacted and incubated with a solid phase coated with at least one recombinant protein (polypeptide). If antibodies are present in the test sample, they will form a complex with the antigenic polypeptide and become affixed to the solid phase. After the complex has formed, unbound materials and reagents are removed by washing the solid phase. The complex is reacted with an indicator reagent and allowed to incubate for a time and under conditions for second complexes to form. The presence of antibody in the test sample to the CKS recombinant polypeptide(s) is determined by detecting the signal generated. Signal generated above a cut-off value is indicative of antibody to the analyte present in the test sample. With many indicator reagents, such as enzymes, the amount of antibody present is proportional to the signal generated. Depending upon the type of test sample, it may be diluted with a suitable buffer reagent, concentrated, or contacted with the solid phase without any manipulation ("neat"). For example, it usually is preferred to test serum or plasma samples which previously have been diluted, or concentrate specimens such as urine, in order to determine the presence and/or amount of antibody present.
In addition, more than one recombinant protein can be used in the assay format just described to test for the presence of antibody against a specific infectious agent by utilizing CKS fusion proteins against various antigenic epitopes of the viral genome of the infectious agent under study. Thus, it may be preferred to use recombinant polypeptides which contain epitopes within a specific viral antigenic region as well as epitopes from other antigenic regions from the viral genome to provide assays which have increased sensitivity and perhaps greater specificity than using a polypeptide from one epitope. Such an assay can be utilized as a confirmatory assay. In this particular assay format, a known amount of test sample is contacted with (a) known amount(s) of at least one solid support coated with at least one recombinant protein for a time and under conditions sufficient to form recombinant protein/antibody complexes. The complexes are contacted with known amount(s) of appropriate indicator reagent(s)s for a time and under suitable conditions for a reaction to occur, wherein the resultant signal generated is compared to a negative test sample in order to determine the presence of antibody to the analyte in the test sample. It further is contemplated that, when using certain solid phases such as microparticles, each recombinant protein utilized in the assay can be attached to a separate microparticle, and a mixture of these microparticles made by combining the various coated microparticles, which can be optimized for each assay.
Variations to the above-described assay formats include the incorporation of CKS- recombinant proteins of different analytes attached to the same or to different solid phases for the detection of the presence of antibody to either analyte (for example, CKS- recombinant proteins specific for certain antigenic regions of one infective agent coated on the same or different solid phase with CKS- recombinant proteins specific for certain antigenic region(s) of a different infective agent, to detect the presence of either (or both) infective agents.
In yet another assay format, CKS recombinant proteins containing antigenic epitopes are useful in competitive assays such as neutralization assays. To perform a neutralization assay, a recombinant polypeptide representing epitopes of an antigenic region of an infectious agent such as a virus, is solubilized and mixed with a sample diluent to a final concentration of between 0.5 to 50.0 .mu.g/ml. A known amount of test sample (preferably 10 .mu.l), either diluted or non-diluted, is added to a reaction well, followed by 400 .mu.l of the sample diluent containing the recombinant polypeptide. If desired, the mixture may be preincubated for approximately 15 minutes to two hours. A solid phase coated with the CKS recombinant protein described herein then is added to the reaction well, and incubated for one hour at approximately 40.degree. C. After washing, a known amount of an indicator reagent, for example, 200 .mu.l of a peroxidase labeled goat anti-human IgG in a conjugate diluent is added and incubated for one hour at 40.degree. C. After washing and when using an enzyme conjugate such as described, an enzyme substrate, for example, OPD substrate, is added and incubated at room temperature for thirty minutes. The reaction is terminated by adding a stopping reagent such as 1N sulfuric acid to the reaction well. Absorbance is read at 492 nm. Test samples which contain antibody to the specific polypeptide generate a reduced signal caused by the competitive binding of the peptides to these antibodies in solution. The percentage of competitive binding may be calculated by comparing absorbance value of the sample in the presence of recombinant polypeptide to the absorbance value of the sample assayed in the absence of a recombinant polypeptide at the same dilution. Thus, the difference in the signals generated between the sample in the presence of recombinant protein and the sample in the absence of recombinant protein is the measurement used to determine the presence or absence of antibody.
In another assay format, the recombinant proteins can be used in immunodot blot assay systems. The immunodot blot assay system uses a panel of purified recombinant polypeptides placed in an array on a nitrocellulose solid support. The prepared solid support is contacted with a sample and captures specific antibodies (specific binding member) to the recombinant protein (other specific binding member) to form specific binding member pairs. The captured antibodies are detected by reaction with an indicator reagent. Preferably, the conjugate specific reaction is quantified using a reflectance optics assembly within an instrument which has been described in U.S. patent application Ser. No. 07/227,408 filed Aug. 2, 1988. The related U.S. patent application Ser. Nos. 07/227,586 and 07/227,590 (both of which were filed on Aug. 2, 1988) further described specific methods and apparatus useful to perform an immunodot assay, as well as U.S. Pat. No. 5,075,077 (U.S. Ser. No. 07/227,272 filed Aug. 2, 1988), which enjoys common ownership and is incorporated herein by reference. Briefly, a nitrocellulose-base test cartridge is treated with multiple antigenic polypeptides. Each polypeptide is contained within a specific reaction zone on the test cartridge. After all the antigenic polypeptides have been placed on the nitrocellulose, excess binding sites on the nitrocellulose are blocked. The test cartridge then is contacted with a test sample such that each antigenic polypeptide in each reaction zone will react if the test sample contains the appropriate antibody. After reaction, the test cartridge is washed and any antigen-antibody reactions are identified using suitable well-known reagents. As described in the patents and patent applications listed herein, the entire process is amenable to automation. The specifications of these applications related to the method and apparatus for performing an immunodot blot assay are incorporated herein by reference.
CKS fusion proteins (recombinant antigens) can be used in assays which employ a first and second solid support, as follow, for detecting antibody to a specific antigen of an analyte in a test sample. In this assay format, for example, when using CKS recombinant antigens, a first aliquot of a test sample is contacted with a first solid support coated with CKS recombinant protein specific for an analyte for a time and under conditions sufficient to form recombinant protein/analyte antibody complexes. Then, the complexes are contacted with an indicator reagent specific for the recombinant antigen. The indicator reagent is detected to determine the presence of antibody to the recombinant protein in the test sample. Following this, the presence of a different antigenic determinant of the same analyte is determined by contacting a second aliquot of a test sample with a second solid support coated with CKS recombinant protein specific for the second antibody for a time and under conditions sufficient to form recombinant protein/second antibody complexes. The complexes are contacted with a second indicator reagent specific for the antibody of the complex. The signal is detected in order to determine the presence of antibody in the test sample, wherein the presence of antibody to either analyte recombinant protein, or both, indicates the presence of anti-analyte in the test sample. It also is contemplated that the solid supports can be tested simultaneously.
The use of haptens is known in the art. It is contemplated that haptens also can be used in assays employing CKS fusion proteins in order to enhance performance of the assay.
Screening for Anti-Viral Agents For HGBV
The availability of cell culture and animal model systems for HGBV also renders screening for anti-viral agents which inhibit HGBV replication possible, and particularly for those agents which preferentially allow cell growth and multiplication while inhibiting viral replication. These screening methods are known in the art. Generally, the anti-viral agents are tested at a variety of concentrations, for their effect on preventing viral replication in cell culture systems which support viral replication, and then for an inhibition of infectivity or of viral pathogenicity, and a low level of toxicity, in an animal model system. The methods and composition provided herein for detecting HGBV antigens are useful for screening of anti-viral agents because they provide an alternative, and perhaps a more sensitive means, for detecting the agent's effect on viral replication than the cell plaque assay or ID.sub.50 assay. Anti-HGBV antibodies may be used to identify and quantitate HGBV antigen(s) in the cell culture utilizing the immunoassays described herein. Also, since it may be desirable to quantitate HGBV antigens in the infected cell culture by a competition assay, the polypeptides encoded within the HGBV nucleic acid sequences described herein are useful for these assays. Generally, a recombinant HGBV polypeptide derived from the HGBV genomic or cDNA would be labeled, and the inhibition of binding of this labeled polypeptide to an HGBV polypeptide due to the antigen produced in the cell culture system would be monitored. These methods are especially useful in cases where the HGBV may be able to replicate in a cell lines without causing cell death.
Hosts and Expression Control Sequences
Although the following are known in the art, included herein are general techniques used in extracting the genome from a virus, preparing and probing a genomic library, sequencing clones, constructing expression vectors, transforming cells, performing immunological assays, and for growing cell in culture.
Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotics include promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts are commonly derived from the plasmid pBR322 which contains operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers. These markers may be used to obtain successful transformants by selection. Commonly used prokaryotic control sequences include the beta-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 198:1056 [1977]) the tryptophan promoter system (reported by Goeddel et al., Nucleic Acid Res 8:4057 [1980]) and the lambda-derived pL promoter and N gene ribosome binding site (Shimatake et al., Nature 292:128 [1981]) and the hybrid Tac promoter (De Boer et al., Proc. Natl. Acad. Sci. USA 292:128 [1983]) derived from sequences of the trp and lac UV5 promoters. The foregoing systems are particularly compatible with E. coli; however, other prokaryotic hosts such as strains of Bacillus or Pseudomonas may be used if desired, with corresponding control sequences.
Eukaryotic hosts include yeast and mammalian cells in culture systems. Saccharomyces cerevisiae and Saccharomyces carlsbergensis are the most commonly used yeast hosts, and are convenient fungal hosts. Yeast compatible vectors carry markers which permit selection of successful transformants by conferring protrophy to auxotrophic mutants or resistance to heavy metals on wild-type strains. Yeast compatible vectors may employ the 2 micron origin of replication (as described by Broach et al., Meth. Enz. 101:307 [1983]), the combination of CEN3 and ARS1 or other means for assuring replication, such as sequences which will result in incorporation of an appropriate fragment into the host cell genome. Control sequences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic enzymes, including the promoter for 3 phosphophycerate kinase. See, for example, Hess et al., J. Adv. Enzyme Reg. 7: 149 (1968), Holland et al., Biochemistry 17:4900 (1978) and Hitzeman J. Biol. Chem. 255:2073 (1980). Terminators also may be included, such as those derived from the enolase gene as reported by Holland, J. Biol. Chem. 256:1385 (1981). It is contemplated that particularly useful control systems are those which comprise the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) promoter or alcohol dehydrogenase (ADH) regulatable promoter, terminators also derived from GAPDH, and if secretion is desired, leader sequences from yeast alpha factor. In addition, the transcriptional regulatory region and the transcriptional initiation region which are operably linked may be such that they are not naturally associated in the wild-type organism.
Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines which are available from the American Type Culture Collection. These include HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and others. Suitable promoters for mammalian cells also are known in the art and include viral promoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus (BPV), cytomegalovirus (CMV). Mammalian cells also may require terminator sequences and poly A addition sequences; enhancer sequences which increase expression also may be included, and sequences which cause amplification of the gene also may be desirable. These sequences are known in the art. Vectors suitable for replication in mammalian cells may include viral replicons, or sequences which insure integration of the appropriate sequences encoding non-A, non-B, non-C, non-D, non-E epitopes into the host genome. An example of a mammalian expression system for HCV is described in U.S. patent application Ser. No. 07/830,024, filed Jan. 31, 1992.
Baculovirus Expression Systems
Commercially available baculovirus expression systems are widely available for the production of recombinant proteins in large quantities. Baculovirus systems offer an advantage over bacterial expression systems in that they allow for high level expression of glycosylated proteins, although these modifications may not be identical to native glycosylation. Expression in these systems is achieved by cloning the gene of interest into a transfer vector in which the gene is placed under the control of a strong viral promoter, usually the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV). The transfer vector is then co-transfected into an appropriate insect cell line with baculovirus genomic DNA. Infectious viruses which result from recombination between the transfer vector and viral genomic DNA are then isolated. Selection of recombinant viruses has been greatly facilitated by commercially available baculovirus genomic DNA for transfection. One such system, BaculoGold.TM. (PharMingen, San Diego, Calif.), is linearized baculovirus genomic DNA that contains a lethal deletion, thus, genomic DNA cannot make infectious virus particles in transfected cells unless the deletion is complemented by a co-transfected polyhedrin-based Baculovirus transfer vector.
In order to direct secretion of the recombinant protein into cell culture supernatants, commercially available transfer vectors are available which allow fusion proteins to be made between a secreted protein signal sequence and the protein of interest. One such vector, pAcGP67A (PharMingen), utilizes the signal sequence of gp67, the most abundant envelope protein of AcNPV. The gp67 signal sequence directs the recombinant protein into the cellular secretion pathway. In doing so, the signal peptide is removed by cellular proteases present in the endoplasmic reticulum, just as would be the case for gp67. The resulting recombinant protein should be secreted into the cell culture supernatants with 3 gp67 amino acids present on the amino-terminus.
Transformations
Transformation may be by any known method for introducing polynucleotides into a host cell, including packaging the polynucleotide in a virus and transducing a host cell with the virus, and by direct uptake of the polynucleotide. The transformation procedures selected depends upon the host to be transformed. Bacterial transformation by direct uptake generally employs treatment with calcium or rubidium chloride. Cohen, Proc. Natl. Acad. Sci. USA 69:2110 (1972). Yeast transformation by direct uptake may be conducted using the calcium phosphate precipitation method of Graham et al., Virology 52:526 (1978), or modification thereof.
Vector Construction
Vector construction employs methods known in the art. Generally, site-specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions which generally are specified by the manufacturer of these commercially available enzymes. Usually, about 1 microgram (.mu.g) of plasmid or DNA sequence is cleaved by 1-10 units of enzyme in about 20 .mu.l of buffer solution by incubation at 37.degree. C. for 1 to 2 hours. After incubation with the restriction enzyme, protein is removed by phenol/chloroform extraction and the DNA recovered by precipitation with ethanol. The cleaved fragments may be separated using polyacrylamide or agarose gel electrophoresis methods, according to methods known by the routineer.
Sticky end cleavage fragments may be blunt ended using E. coli DNA polymerase 1 (Klenow) in the presence of the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with S1 nuclease also may be used, resulting in the hydrolysis of any single stranded DNA portions.
Ligations are performed using standard buffer and temperature conditions using T4 DNA ligase and ATP. Sticky end ligations require less ATP and less ligase than blunt end ligations. When vector fragments are used as part of a ligation mixture, the vector fragment often is treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase to remove the 5'-phosphate and thus prevent religation of the vector. Or, restriction enzyme digestion of unwanted fragments can be used to prevent ligation. Ligation mixtures are transformed into suitable cloning hosts such as E. coli and successful transformants selected by methods including antibiotic resistance, and then screened for the correct construction.
Verification of Construction and Sequencing
For standard vector constructions, ligation mixtures are transformed into E. coli strain XL-1 Blue or other suitable host, and successful transformants selected by antibiotic resistance or other markers. Plasmids from the transformants then are prepared according to the method of Clewell et al., Proc. Natl. Acad. Sci. USA 62:1159 (1969) usually following chloramphenicol amplification as reported by Clewell et al., J. Bacteriol. 110:667 (1972). The DNA is isolated and analyzed usually by restriction enzyme analysis and/or sequencing. Sequencing may be by the well-known dideoxy method of Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977) as further described by Messing et al., Nucleic Acid Res. 9:309 (1981), or by the method reported by Maxam et al., Methods in Enzymology 65:499 (1980). Problems with band compression, which are sometimes observed in GC rich regions, are overcome by use of T-deazoguanosine according to the method reported by Barr et al., Biotechniques 4:428 (1986).
Enzyme-Linked Immunosorbent Assay
Enzyme-inked immunosorbent assay (ELISA) can be used to measure either antigen or antibody concentrations. This method depends upon conjugation of an enzyme label to either an antigen or antibody, and uses the bound enzyme activity (signal generated) as a quantitative label (measurable generated signal). Methods which utilize enzymes as labels are described herein, as are examples of such enzyme labels.
The present invention will now be described by way of examples, which are meant to illustrate, but not to limit, the spirit and scope of the invention.
EXAMPLES
The initial studies of the transmissibility of HGBV were performed as described in U.S. Ser. No. 08/283,314, U.S. Ser. No. 08/242,654, and U.S. Ser. No. 08/196,030, all of which have been incorporated previously herein by reference. Additional infectivity studies have been disclosed and described in these three preceding applications and in U.S. Ser. Nos. 08/344,185 and 08/344,190, each filed Nov. 23, 1994 and previously incorporated herein by reference. These previous applications also disclosed examples describing the extension of the HGBV clone sequence (generation of HGBV sequences, evidence for the existence of two HCV-like viruses in HGBV, evidence that HGBV-A and HGBV-B represent two distinct RNA species and distinct viruses, and evidence that HGBV-A and HGBV-B are members of the Flaviviridae); an example detailing the CKS-based expression vector system for expression and detection of immunogenic HGBV polypeptides, serological studies which utilized recombinant protein and its purification protocol and included a polystyrene bead coating procedure, the ELISA protocol for detection of antibodies to HGBV, and the detection of HGBV derived RNA in serum from infected individuals including humans and tamarins; an example which detailed the evidence for exposure to HGBV in human populations, including the experimental protocol used, the cutoff determinations, supplemental testing, serological data obtained with low-risk specimens, specimens tested which were from individuals considered "at risk" for hepatitis over various counties of the world, and the statistical significance of serological results obtained from testing; another example detailed additional studies which provided evidence for exposure to HGBV in human populations, including experimental protocol utilized, cutoff determination, supplemental testing, serological data obtained with low-risk specimens, serological data obtained from individuals "at risk" for hepatitis and the statistical significance of serological results; another example set forth the identification of a GB-related virus in humans, and detailed the scientific reasoning to its identification, the detailed cloning of the NS3-like region of HGBV-C, nucleotide sequences totaling 5163 bp in length, the scientific experiments which led to the conclusion that GB-C is exogenous, experiments that GB-C can be detected in additional human serum samples, experiments which detailed the PCR walking technique used to extend the HGBV-C sequence, all of which was presented as a nucleic acid sequence and a six-frame translation of the 5163 bp. These sequences are set forth in U.S. Ser. No. 08/344,190 filed Nov. 23, 1994, which previously has been incorporated herein by reference. The sequence was obtained from clone pHGB-C clone #1, previously deposited at the A.T.C.C. and accorded A.T.C.C. Deposit No. 69711 on Nov. 8, 1994 as described in U.S. Ser. No. 08/344,190; these sequences were identified in U.S. Ser. No. 08/344,190 as SEQUENCE I.D. No. 76 and its six possible reading frames. U.S. Ser. No. 08/377,557 filed Jan. 30, 1995 (previously incorporated herein by reference) extended the 5163 bp sequence to a length of 8087 bp and also provided a translation of the three forward reading frames of the 8087 bp sequence. U.S. Ser. No. 08/424,550 (previously incorporated herein by reference) extended the 8087 bp of HGBV-C to 9034 bp and also provided additional serological data relating to HGBV-A, HGBV-B and HGBV-C. The P.C.T. application corresponding to these applications, PCT/US95/02118, was published Aug. 18, 1995. Ser. No. 08/417,629 extended the HGBV-C sequence 88 bp, thus extending the sequence to 9122 bp, and also updated serological data of HGBV-A, HGBV-B and HGBV-C by correlating antibody detection and PCR results in Western Africa and summarizing PCR results in volunteer blood donors, I.V. drug users and non-A-E hepatitis individuals.
Example 1
CKS-based Expression and Detection of Immunogenic HGBV-C Polypeptides
The HGBV-C sequences obtained from walking experiments previously described in Example 18 (TABLE 21) of U.S. Ser. No. 08/424,550 were cloned into the CKS expression vectors pJO200, pJO201, and pJO202 using the restriction enzymes listed hereinbelow in TABLE 1 and in TABLE 22 of U.S. Ser. No. 08/424,550 (10 units, NEB), following procedures described in Example 13 of U.S. Ser. No. 08/424,550 and known in the art. Two additional PCR clones, designated C.3/2 and C.8/12, were also expressed (FIG. 1). Briefly, PCR product C.3/2 was generated using primers SEQUENCE I.D. NO. 29 and the complement of SEQUENCE I.D NO. 30 and PCR product C.8/12 was generated using primers SEQUENCE I.D. NO. 31 and the complement of SEQUENCE I.D. No. 32, following procedures previously described in Example 9 of U.S. Ser. No. 08/424,550 and known in the art. The PCR products were cloned into pT7Blue as described previously, then liberated with the restriction enzymes listed in TABLE 1 and also in TABLE 22 of U.S. Ser. No. 08/424,550 and cloned into pJO200, pJO201 and pJO202, following known standard methods and as described in Example 13 of U.S. Ser. No. 08/424,550.
TABLE 1______________________________________HGBV-C Samples Reactivity Reactivity PCR Restriction with human with human product.sup.a digest.sup.b G8-81 serum 240D serum______________________________________GB-C KpnI, XbaI + - C.1 EcoRI, XbaI + - C.3/2 EcoRI, XbaI - - C.4 KpnI, XbaI - - C.9 KpnI, PstI ND - C.10 EcoRI, XbaI ND - C.5 KpnI, XbaI +/- - C.6 KpnI, PstI + - C.7 NdeI-fill, BamHI - + C.8/12 KpnI, XbaI + -______________________________________ .sup.a PCR product is as indicated in previous TABLES or Examples. .sup.b Restriction digests used to liberate the PCR fragment from pT7Blue Tvector. ND = not done.
Two human sera which had indicated the presence of antibodies to one or more of the CKS/HGBV-A or CKS/HGBV-B fusion proteins by the 1.7, 4.1 or 2.17 ELISAs (see Examples 15 and 16 of U.S. Ser. No. 08/424,550) were chosen for Western blot analysis. One of these sera (240 D) was from an individual with non A-E hepatitis (Egypt) and the other (G8-81) was from a West African individual "at risk" for exposure to HGBV (see Example 15 of U.S. Ser. No. 08/424,550). The CKS/HGBV-C fusion proteins were expressed and transferred to nitrocellulose sheets as previously described in Example 13 of U.S. Ser. No. 08/424,550, and as known in the art. The blots were preblocked as previously described in Example 13 of U.S. Ser. No. 08/424,550 and known in the art, and incubated overnight with one of the human serum sample diluted 1:100 in blocking buffer containing 10% E. coli lysate and 6 mg/ml XL1-Blue/CKS lysate. The blots were washed two times in TBS, reacted with HRPO-conjugated goat anti-human IgG and developed as indicated in Example 13 of U.S. Ser. No. 08/424,550. The results are shown in TABLE 1 and in TABLE 22 of U.S. Ser. No. 08/424,550.
Several of the HGBV-C proteins showed reactivity with one or the other of the two sera, and three (C.1, C.6 and C.7) were chosen for use in ELISA assays following methods described hereinabove (see Example 20 of U.S. Ser. No. 08/424,550). Thus, samples previously identified as reactive with HGBV-A and/or HGBV-B proteins additionally showed reactivity with HGBV-C proteins. The reactivity with multiple proteins from the 3 HGBV viruses may have been due to cross-reactivity resulting from shared epitopes between the viruses. Alternatively, this may have been a result of infection with multiple viruses, or to other unidentified factors.
Example 2
Epitope Mapping of Immunoreactive HGBV-C Proteins
The reactive fragments GB-C, C.1, C.5, C.6, C.7 and C.8/12 previously identified in Example 19 of U.S. Ser. No. 08/424,550, previously incorporated herein by reference, span a region of the HGBV-C large open reading frame (SEQUENCE I.D. NO. 33) from residues 876 to 2335 (see FIG. 1). A set of fourteen overlapping PCR fragments covering this area of HGBV-C was generated to further localize the antigenic regions. HGBV-C PCR products were generated from cDNA derived from the West African sample used to originally identify GB-C (see Example 18 of U.S. Ser. No. 08/424,550. Nucleic acid extractions and cDNA reactions were performed as described. All PCRs utilized 1 .mu.M primers for 35-40 cycles (94.degree. C., 20-30 sec; 50-55.degree. C., 30 sec; 72.degree. C., 30-120 sec). Primers for each clone are shown in TABLE 2. Each primer had a restriction site added at the 5' end to facilitate cloning into the pJO201 multiple cloning site, preceded by six nucleotides to ensure complete digestion of the PCR product. The products were cloned into pJO201 as described in Example 13 of U.S. Ser. No. 08/424,550. The restriction sites engineered into the primers and the locations of the encoded proteins within The HGBV-C polyprotein are also shown in TABLE 2.
TABLE 2__________________________________________________________________________ Residues Reactivity Reactivity Reactivity PCR in SEQ I.D. with human with human with human product.sup.a Restriction sites.sup.b Primer set No. 404.sup.c G8-81 serum 240E serum M47__________________________________________________________________________ serumC.17 BamHI, HindIII SEQ ID #1/SEQ ID #2 865-974 - - - C.18 EcoRI, HindIII SEQ ID #3/SEQ ID #4 965-1082 - - - C.19 BamHI, HindIII SEQ ID #5/SEQ ID #6 1073-1190 + - - C.20 BamHI, HindIII SEQ ID #7/SEQ ID #8 1181-1298 - - - C.21 BamHI, HindIII SEQ ID #9/SEQ ID #10 1289-1406 - - - C.22 BamHI, HindIII SEQ ID #11/SEQ ID #12 1397-1512 - - - C.23 BamHI, HindIII SEQ ID #13/SEQ ID #14 1503-1622 - - - C.24 BamHI, HindIII SEQ ID #15/SEQ ID #16 1613-1721 + - - C.25 BamHI, HindIII SEQ ID #17/SEQ ID #18 1712-1838 - - - C.26 BamHI, HindIII SEQ ID #19/SEQ ID #20 1829-1948 - - - C.27 BamHI, HindIII SEQ ID #21/SEQ ID #22 1939-2055 - - - C.28 BamHI, HindIII SEQ ID #23/SEQ ID #24 2046-2163 + + + C.29 BamHI, HindIII SEQ ID #25/SEQ ID #26 2154-2270 + - + C.30 BamHI, HindIII SEQ ID #27/SEQ ID #28 2261-2378 - - +__________________________________________________________________________ .sup.a PCR product is as indicated in FIG. X. .sup.b Restriction sites engineered into PCR primers and used to clone th PCR fragment into pJO201. .sup.c From U.S. Ser. No. 08/424,550.
Cultures expressing the new CKS/HGBV-C fusion proteins were grown and proteins were transferred to nitrocellulose as previously described in Example 13 of U.S. Ser. No. 08/424,550 and following standard methods known in the art. The proteins were reacted with the two human sera described in Example 19 of U.S. Ser. No. 08/424,550, except that a different bleed date ten days subsequent to the one used previously was utilized for the Egyptian 240 sera. An additional sera (M47), which was found to be PCR positive for HGBV-C, was reacted with the proteins as well. The reactivities of the proteins with these sera also are indicated in TABLE 2. PCR fragments C.19, C.24, C.28, C.29 and C.30 showed reactivity with at least one of the sera. Fragment C.28 was reactive with all three sera. Fragment C.19 maps the C.1 reactive region to residues 1073-1190 of the HGBV-C large open reading frame (SEQUENCE I.D. NO. 33) and fragment C.24 maps the C.6 reactive region to residues 1613-1721 of SEQUENCE I.D. NO. 33. Fragments C.28, C.29 and C.30, spanning residues 2046-2378 of SEQUENCE I.D. NO. 33, were all reactive indicating the presence of at least two epitopes in this region. Although fragments GB-C and C.5 had shown some reactivity previously with the G8-81 sera, none of the epitope mapping fragments in these regions showed reactivity. It may have been that the new fragments split an epitope such that the epitope is not fully represented in any of the new constructs. Alternatively, the reactivity seen with the larger proteins may have been due to some type of conformational epitope not fully represented in the smaller products.
Strains replicated (clones 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30) from the HGBV nucleic acid sequence library have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, as of Oct. 10, 1995, under the terms of the Budapest Treaty and will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, or for the enforceable period of the U.S. patent, whichever is longer. The deposits and any other deposited material described herein are provided for convenience only, and are not required to practice the present invention in view of the teachings provided herein. The HGBV cDNA sequences in all of the deposited materials are incorporated herein by reference. The plasmids were accorded the following A.T.C.C. deposit numbers: Clone 17 (C.17) was accorded A.T.C.C. Deposit No. 69910; Clone 18 (C.18) was accorded A.T.C.C. Deposit No.69911; Clone 19 (C.19) was accorded A.T.C.C. Deposit No. 69912; Clone 20 (C.20) was accorded A.T.C.C. Deposit No. 69913; Clone 21 (C.21) was accorded A.T.C.C. Deposit No.69914; Clone 22 (C.22) was accorded A.T.C.C. Deposit No. 69915; Clone 23 (C.23) was accorded A.T.C.C. Deposit No. 69916; Clone 24 (C.24) was accorded A.T.C.C. Deposit No. 69917; Clone 25 (C.25) was accorded A.T.C.C. Deposit No. 69918; Clone 26 (C.26) was accorded A.T.C.C. Deposit No. 69919; Clone 27 (C.27) was accorded A.T.C.C. Deposit No. 69920; Clone 28 (C.28) was accorded A.T.C.C. Deposit No. 69921; Clone 29 (C.29) was accorded A.T.C.C. Deposit No. 69922; and Clone 30 (C.30) was accorded A.T.C.C. Deposit No. 69923.
Example 3
Expression of GBV-C Polypeptides in a Permanent Cell Line
A. Construction of GBV-C E2 Expression Plasmid
Plasmid 577, described in U.S. patent application Ser. No. 08/478,073, filed Jun. 7, 1995 and previously incorporated herein by reference, has been constructed for the expression of secreted antigens, particularly hepatitis C E2 protein, in a permanent cell line. This plasmid contains the following DNA segments: (a) a 2.3 Kb fragment of pBR322 containing bacterial beta-lactamase and origin of DNA replication; (b) a 1.8 Kb cassette directing expression of a neomycin resistance gene under control of HSV-1 thymidine kinase promoter and poly-A addition signals; (c) a 1.9 Kb cassette directing expression of a dihydrofolate reductase gene under the control of an SV-40 promoter and poly-A addition signals; (d) a 3.5 Kb cassette directing expression of a rabbit immunoglobulin heavy chain signal sequence fused to a modified hepatitis C virus E2 protein under the control of the Simian Virus 40 T-Ag promoter and transcription enhancer, the hepatitis B virus surface antigen enhancer I followed by a fragment of Herpes Simplex Virus-I genome providing poly-A addition signals; and (e) a residual 0.7 Kb fragment of Simian Virus 40 genome late region of no function in this plasmid.
Plasmid 577 is graphically depicted in FIG. 2. The plasmid in FIG. 2 is disclosed as a series of assembled fragments with sections numbered 1 to 13, and described in the following TABLE 3. The accession numbers of the sections in TABLE 3 refer to GenBank.RTM. accession numbers. Please note that slight sequence variations may occur and may have occurred when constructing the plasmid. All of the segments of the vector were assembled by standard methods known to those skilled in the art of molecular biology.
TABLE 3______________________________________Plasmid Figure Legend Construction Plasmid 577, 10,186 base pairs double stranded DNA SECTION DESCRIPTION______________________________________1 (NT 4361-2067 OF PBR322 ACCESSION J02224) 2 (NT 2249-1624 HSV-1 ACCESSION J02224 NT) 3 (NT 2518-1519 Tn5 ACCESSION NOS U00004 L19385) 4 (NT 460-210 HSV-1 ACCESSION J02224) 5 (NT 272-1, 5243-5173 SV40) 6 (NT 1-701 MOUSE DHFR ACCESSION L26316) 7 (NT 4714-4100 SV40 ACCESSION V08380) 8 (NT 272-1, 5243-5173 SV40) 9 (NT 1-77 DNA Sequence Figure SYNTHETIC DNA RABBIT IgG HEAVY CHAIN LEADER (HCL)) 10 (NT 78-938 DNA Sequence Figure HCV E2 antigen PCR product) 11 (HBV ENHANCER NT 2373-2811 ACCESSION NO. X02763 WITH G AT NT. 2976 AND T AT NT. 2654) 12 (NT 3688-5468 HSV1 ACCESSION NO. NT 3687-5468) 13 (NT 2536-1785 SV40 ACCESSION V08380)______________________________________
Plasmids for the expression of secretable GBV-C proteins were constructed by replacing the hepatitis C virus E2 protein coding sequence in plasmid 577 with those from GBV-C, as follows. All GBV-C inserts were from SEQUENCE I.D. NO. 34, a GBV-C genotype 1 sequence previously described in patent application Ser. No. 08/580,038, previously incorporated herein by reference. Digestion of plasmid 577 with XbaI released the hepatitis C virus E2 gene fragment. The resulting plasmid backbone allowed insertion of GBV-C gene products downstream of the rabbit immunoglobulin heavy chain signal sequence which directs the expressed proteins into the secretory pathway of the cell. All of the GBV-C fragments were generated by PCR using standard procedures. The first PCR fragment encoded a 336 amino acid segment of the GBV-C E2 gene, from aa 221 to 556 of SEQUENCE I.D. NO. 35. Encoded in the sense PCR primer sequence (SEQUENCE I.D. NO. 36) was an Xba 1 site, immediately followed by a 12 nucleotide sequence that encoded the amino acid sequence Ser-Asn-Glu-Leu ("SNEL"), the amino terminal sequence of human pro-urokinase. The amino acid sequence SNEL was intended, as the N-terminus of the protein, to promote signal protease processing, efficient secretion and final product stability in culture fluids. Immediately following this 12 nucleotide sequence the primer contained nucleotides complementary to template sequences encoding amino acids starting at residue 221 of GBV-C. The antisense PCR primer (SEQUENCE I.D. NO. 37) contained sequences homologous to template sequences that encode amino acids ending at residue 556 of GBV-C, followed by two stop codons and an Xba 1 site for cloning purposes. The GBV-C E2 protein was truncated at this position to promote secretion. PCR was performed using GeneAmp.RTM. reagents obtained from Perkin-Elmer-Cetus, essentially as directed by the supplier's instructions. PCR primers were used at a final concentration of 0.5 .mu.M. PCR was performed on plasmid template in a 100 .mu.l reaction for 35 cycles (94.degree. C., 30 seconds; 55.degree. C., 30 seconds; 72.degree. C., 90 seconds) followed by an extension cycle of 72.degree. C. for 10 min.
Three additional GBV-C E2-encoding plasmids were constructed using the same sense primer as above, but utilizing three different antisense primers (SEQUENCE I.D. NOs. 38, 39, and 40), the same plasmid template and the same PCR conditions. These PCR fragments encoded 315, 289 and 222 amino acid segments of GBV-C E2, each beginning at amino acid residue 221 of SEQUENCE I.D. NO. 35. As with the 336 amino acid GBV-C E2 protein, the truncation sites were chosen to promote secretion. The antisense primers SEQUENCE I.D. NOs. 38 and 39 (utilized to make the 315 and 289 amino acid constructs, respectively) each incorporated a sequence encoding the eight amino acids Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys just before the stop codons. Within this sequence is the recognition site for a monoclonal antibody (MAb) designated anti-FLAG M2 (Eastman Kodak, Co.; New Haven, Conn.). It was incorporated to aid in analysis and purification of the GBV-C E2 protein products.
B. Transfection of Dihydrofolate Reductase Deficient Chinese Hamster Ovary Cells
The plasmids described supra were transfected into CHO/dhfr- cells (DXB-111) (Uriacio, et al., Proc. Nat. Acad. Sci. 77, 4451-4466; 1980); these cells are available from the American Type Culture Collection [A.T.C.C.], 12301 Parklawn Drive, Rockville, Md. 20852, under Accession No. CRL 9096), using the cationic liposome-mediated procedure (Felgner, P. L. et al., Proc. Natl. Acad. Sci. 84, 7413-7417; 1987), as follows. CHO/dhfr- cells were cultured in Ham's F-12 media supplemented with 10% fetal calf serum, L-glutamine (1 mM) and freshly seeded into a 25 cm.sup.2 flask at a density of 5-8.times.10.sup.5 cells per flask. The cells were grown to between 60 and 80% confluency for transfection. Twenty micrograms of plasmid DNA was added to 1.5 ml of Opti-MEM I medium and 100 microliters (.mu.l) of Lipofectin Reagent (Gibco-BRL; Grand Island, N.Y.) was added to a second 1.5 ml portion of Opti-MEM I media. The two solutions were mixed and incubated at room temperature for 20 minutes. The culture medium was removed from cells and cells were rinsed three times with 5 ml of Opti-MEM I medium. The Opti-MEM I-Lipofection-plasmid DNA solution was then overlaid onto the cells. The cells were incubated for three hours at 37.degree. C., after which time the Opti-MEM I-Lipofectin-DNA solution was replaced with culture medium for an additional 24 hours prior to selection.
C. Selection and Amplification
One day after transfection, cells were passaged 1:3 and incubated with dhfr/G418 selection medium (hereafter, "F-12 minus medium G"). Selection medium was Ham's F-12 with L-glutamine and without hypoxanthine, thymidine, and glycine (JRH Biosciences, Lenexa, Kans.), and 300 micrograms per ml G418 (Gibco-BRL; Grand Island, N.Y.). Media volume to surface area ratios of 5 ml per 25 cm.sup.2 were maintained.
Colonies showing the presence of dihydrofolate reductase (Ringold, et al., J. Mol Appl. Genet. 1:165-174; 1981) plus aminoglycoside phosphotransferase (Southern, P. J. and Berg, P. J., Mol. Appl. Genet. 1:327-341; 1981) appeared after 4-5 days of incubation of transfected cells with F-12 minus medium G. After approximately two weeks, DHFR/G418 cells were sufficiently expanded to allow passage and continuous maintenance in F-12 minus medium G.
Amplification of each of the transfected GBV-C E2 genes was achieved by stepwise selection of DHFR.sup.+, G418.sup.+ cells with methotrexate (reviewed by Schimke, R., Cell 37, 705-713 (1984). Cells were incubated with F-12 minus medium G containing 150 nM methotrexate (MTX) (Sigma; St. Louis, Mo.) for approximately two weeks until resistant colonies appeared. Further gene amplification was achieved by selection of 150 nM adapted cells with 5 .mu.M MTX. This adaption process also took several weeks. Five .mu.M MTX adapted cells were used for all antigen production.
D. Maintenance and Storage of Cell Lines
Cells in culture and undergoing selection or amplification procedures were re-fed with either F-12 minus medium G, F-12 minus medium G with 150 nm MTX or F-12 minus medium G with 5 .mu.M MTX three times weekly. Cells were passaged 1:4 into 75 cm.sup.2 flasks with 15 ml of media described hereinabove and incubated at 37.degree. C. with 5% CO.sub.2 using standard methods. Cryostorage was accomplished by resuspension of 2-4.times.10.sup.6 cells in 1.8 ml of either F-12 minus medium G, F-12 minus medium G with 150 nm MTX or F-12 minus medium G with 5 .mu.M MTX depending on the stage of selection and amplification of the cell line, which also contained 5% DMSO (Sigma; St. Louis, Mo.). Cells underwent cold storage for 24 hours at -80.degree. C. and then permanent storage at -135.degree. C.
E. GBV-C E2 Antigen Production
F-12 minus medium G supplemented with 5 .mu.M MTX was overlaid onto just confluent monolayers for 12 to 24 hours at 37.degree. C. in 5% CO.sub.2. Then, the growth medium was removed and the cells were rinsed three times with Dulbecco's phosphate buffered saline (PBS) (with calcium and magnesium) (Gibco-BRL; Grand Island, N.Y.), to remove the remaining media/serum which might be present. Cells then were incubated with VAS custom medium (VAS custom formulation with L-glutamine with HEPES without phenol red, available from JRH Bioscience; Lenexa, Kans., product number 52-08678P), for one hour at 37.degree. C. in 5% CO.sub.2. Cells then were overlaid with VAS for production at 5 ml per T 25 cm.sup.2 flask, scaled proportionally for larger flasks or roller bottles). For harvest 1, the medium was removed after 7 days of incubation and then frozen to await purification with harvests 2, 3 and 4. The monolayers were overlaid with VAS for three more 7-day harvests. The cultures were observed daily to determine cell conditions.
F. Analysis of GBV-C E2 Antigen Expression
Aliquots of VAS supernatants from the cells expressing the GBV-C E2-336 amino acid construct were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods and reagents known in the art (Laemmli discontinuous gels) to assess protein expression levels and reactivity with human and rabbit antisera. In some cases, supernatants were first concentrated by ultrafiltration using Amicon concentrator units with 10 kilodalton (kDa) MWCO (Amicon, Inc., Beverly, Mass.). After electrophoresis, proteins were electrophoretically transferred to polyvinylidene fluoride membranes and analyzed by immunoblotting as described in Example 13 of U.S. Ser. No. 08/424,550 and known in the art. An immunoreactive band of approximately 54 kDa was found to be reactive with several sera, including: (1) sera from eight rabbits previously immunized with an E. coli derived GBV-C E2 protein or with peptides whose sequences are found within the GBV-C E2 protein; (2) two GBV-C E2 RT-PCR positive human sera which previously tested seronegative with other GBV-C derived recombinant proteins; (3) two GBV-C RT-PCR positive human sera which previously had tested seropositive with other GBV-C derived recombinant proteins; (4) three RT-PCR negative human sera which previously tested seropositive with other GBV-C derived recombinant proteins; and (5) four RT-PCR negative human sera which previously tested seronegative with other GBV-C derived recombinant proteins. Deglycosylation of this 54 kDa protein with N-Glycanase (Genzyme Corp.; Cambridge, Mass.) was performed after denaturation in the presence of 0.5% SDS, 50 mM b-mercaptoethanol and 50 mM EDTA. Deglycosylation was for 60 hours at 37.degree. C. in pH 7.5 buffer containing 0.17% SDS, 16.7 mM b-mercaptoethanol, 16.7 mM EDTA, 1.25% Nonidet P40 and 0.9 units of N-Glycanase per mg protein. Immunoblotting showed a shift of the immunoreactive band down to 37 kilodaltons, the expected size of the non-glycosylated GBV-C E2-336 protein.
G. Purification of GBV-C E2 Antigens
Protein purification of CHO/E2-315 was performed as follows. For purification of protein, VAS medium from harvests was clarified by centrifugation at 1500.times.g for 15 minutes or by filtration through a 0.45 .mu.m cellulose acetate membrane, followed by concentration up to 100.times. by ultrafiltration using Amicon concentrator units with 10 kDa molecular weight cut-off (Amicon, Inc.; Beverly, Mass.).
Purification of the GBV-C E2-315 amino acid construct containing the eight amino acid FLAG sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) at its C-terminus was performed by immunoaffinity chromatography using an affinity matrix comprising anti-FLAG M2 monoclonal antibody covalently attached to agarose by hydrazide linkage (Eastman Kodak Co., New Haven, Conn.). Prior to affinity purification, protein in pooled VAS medium harvests from roller bottles was exchanged into 50 mM Tris, 150 mM NaCl, pH 7.5 buffer using a Sephadex G-25 (Pharmacia Biotech Inc., Uppsala, Sweden) column. Protein in this buffer was applied to the anti-FLAG M2 antibody affinity column, non-binding protein was eluted by washing the column with 50 mM Tris, 150 mM NaCl, pH 7.5 and bound protein was eluted using an excess of FLAG peptide (in 50 mM Tris, 150 mM NaCl, pH 7.5) over the number of moles of antigen binding sites of anti-FLAG M2 monoclonal antibody on the column. FIG. 7 shows a Coomassie Brilliant Blue R-250 stained SDS polyacrylamide gel of the purification. This procedure provided, in one step, GBV-C E2-315 protein judged homogeneous by scanning densitometry of Coomassie Brilliant Blue R-250 stained SDS polyacrylamide gels of the FLAG peptide - eluted protein. For use in immunoassay, the FLAG peptide used for elution was removed from the GBV-C E2 315 purified stock by gel filtration using Sephadex G-25 (Pharmacia Biotech Inc., Uppsala, Sweden).
Purification of the GBV-C E2-336 amino acid construct was performed using a series of columns which included utilizing two or more of the following types of chromatographies: cation exchange, anion exchange, lectin affinity, hydrophobic interaction.
Example 4
Transient Expression of GBV-C Polypeptides Using the Sindbis and Semliki Forest Virus Vector Systems
A. Construction of Secretion Vectors
Two systems for transient expression of proteins in alphavirus vectors are now commercially available. The Sindbis virus system (Invitrogen Corp., San Diego, Calif.) and Semliki forest virus (SFV) system (Gibco/BRL; Grand Island, N.Y.) allow for high expression levels of proteins in a variety of eukaryotic cell lines. In both cases, the gene of interest is inserted into a plasmid such that it is under the control of the viral subgenomic promoter. This plasmid is used to make a genome length RNA transcript in vitro. This RNA can then be transfected into a eukaryotic cell line and, once inside the cell, this RNA acts as a messenger RNA and directs translation of protein from the inserted gene of interest. Because this subgenomic RNA becomes the most abundant mRNA in the cell, it recruits a large portion of the cell's translational machinery and thus results in production of high levels of the protein of interest. For a more detailed description of the two systems, see Liljestrom and Garoff, Bio/Technology 9:1356-1361 (1991) and Bredenbeek et al., J. Virol. 67:6439-6446 (1993).
In order to direct secretion of proteins not normally secreted, a cassette encoding the human lysozyme secretion signal was inserted into both the Sindbis expression plasmid (pSinRep5) and the SFV expression plasmid (pSFV1). For pSinRep5, two oligonucleotides were generated (SEQUENCE I.D. NOs. 41 and 42) such that when annealed, the duplex had on each end overhangs compatible with cloning into an XbaI site. This oligonucleotide duplex then was ligated into XbaI-digested pSinRep5. When the resulting plasmid, pSinRep-ss, was digested with XbaI, the upstream site was not cut due to a mutation of one base in the XbaI recognition site. This allowed cloning of a gene at the downstream XbaI site such that the first amino acid encoded by the inserted gene followed directly after the signal peptidase cleavage site encoded by the lysozyme signal sequence. The lysozyme signal sequence thus directed the protein encoded by the inserted gene into the cellular secretion pathway. An analogous strategy was used for insertion of the signal sequence into pSFV1, except that the cloning site was BamHI. The oligonucleotides used for this construct were SEQUENCE I.D. NOs. 43 and 44, and the resulting plasmid was designated pSFV-ss.
B. Sindbis Constructs
Following the methods described hereinabove, a number of GBV-C sequences encoding both structural and non-structural proteins were inserted into pSinRep5 or pSinRep-ss. These are listed in TABLE 4. The sequences were derived from one of two GBV-C isolates: SEQUENCE I.D. NO. 34 is the prototype GBV-C sequence previously described in patent application Ser. No. 08/580,038, and belongs to GBV-C genotype 1 based on analysis described in that application. SEQUENCE I.D. NO. 45 belongs to GBV-C genotype 3 based on analysis described in the same application. Four of the fragments expressed in this system were the same GBV-C E2 constructs described hereinabove in Example 3 (designated E2-222, E2-289, E2-315, and E2-336). These E2 constructs were truncated on the N-terminus to delete the E2 signal sequence and truncated to varying degrees on the C-terminus to delete a putative membrane anchoring domain and other hydrophobic regions which may interfere with secretion. E2-289 and E2-315 have the anti-FLAG recognition sequence on the C-teminus as described supra. All four of these were inserted into pSinRep-ss. An additional E2 construct (from the GBV-C genotype 3 isolate) was generated which had the same 3' end as E2-336, but included an additional 16 amino acid residues of GBV-C on the N-terminus. These residues represent the putative GBV-C E2 signal sequence. This fragment, designated E2-352, was cloned into pSinRep5 to allow secretion directed by the putative GBV-C E2 signal sequence. A GBV-C E1 fragment designated E1-168, also from the genotype 3 isolate, was inserted into pSinRep5. It encompassed amino acid residues 12 to 179 of SEQUENCE I.D. NO. 46 and incorporated the putative E1 signal sequence at the 5' end and terminated at the 3' end just upstream of the hydrophobic putative membrane anchoring domain of the E1 protein. Two additional constructs were amplified for expression as non-secreted proteins in pSinRep5. These represented two of the non-structural proteins, NS3 and NS5A, from GBV-C. The NS3 fragment (from the GBV-C genotype 1 isolate) encompassed amino acid residues 912 to 1566 of SEQUENCE I.D. NO. 35 and the NS5A fragment (from the GBV-C genotype 3 isolate) encompassed residues 1877 to 2290 of SEQUENCE I.D. NO. 46.
TABLE 4__________________________________________________________________________ NUCLEOTIDE AMINO ACID NUCLEOTIDE AMINO ACID INSERTED RESIDUES IN RESIDUES IN RESIDUES IN RESIDUES IN FRAGMENT VECTOR PCR PRIMERS SEQ ID #1 SEQ ID #2 SEQ ID #3 SEQ ID__________________________________________________________________________ #4E2-336 pSinRep-ss SEQ ID #5/SEQ ID #6 1143-2150 221-556 N/A N/A E2-315 pSinRep-ss SEQ ID #7/SEQ ID #6 1143-2087 221-535 N/A N/A E2-289 pSinRep-ss SEQ ID #8/SEQ ID #6 1143-2009 221-509 N/A N/A E2-222 pSinRep-ss SEQ ID #9/SEQ ID #6 1143-1808 221-442 N/A N/A E2-352 pSinRep5 SEQ ID #14/SEQ ID #6 N/A N/A 1085-2140 199-550 E1-168 pSinRep5 SEQ ID #15/SEQ ID #16 N/A N/A 524-1027 12-179 NS3 pSinRep5 SEQ ID #17/SEQ ID #18 3216-5180 912-1566 N/A N/A NS5A pSinRep5 SEQ ID #19/SEQ ID #20 N/A N/A 6119-7360 1877-2290__________________________________________________________________________
The sense PCR primers for all constructs inserted into pSinRep5, namely constructs not utilizing the lysozyme secretion signal, included a ribosome binding site sequence and initiator methionine just upstream of the GBV-C sequence. The primers for all constructs except NS5A incorporated XbaI sites for cloning into the Sindbis vectors. The primers for the NS5A construct incorporated AvrII sites due to an XbaI site within the NS5A region of GBV-C. The overhang generated by digestion with AvrII was compatible with cloning into an XbaI site. PCRs were performed using GeneAmp.RTM. reagents obtained from Perkin-Elmer-Cetus, essentially as directed by the supplier's instructions. PCR primers were used at a final concentration of 1 .mu.M. PCRs were performed on plasmid or cDNA templates in 50 .mu.l reactions for 2540 cycles (94.degree. C., 20 seconds; 50.degree. C., 30 seconds; 72.degree. C., 1-2 min) followed by an extension cycle of 72.degree. C. for 10 min. All PCR products were then digested with XbaI (or AvrII for NS5A), ligated into XbaI-digested, de-phosphorylated pSinRep5 or pSinRep-ss and transformed into E. coli by standard methods. All inserts were sequenced using the dideoxynucleotide chain termination technique (Sanger et al., supra) using either a kit (T7 Sequenase 7-deaza-dGTP DNA sequencing kit, Amersham Life Sciences, Inc.; Arlington Heights, Ill.) followed by manual gel electrophoresis or alternatively, using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit and an ABI 373 automated sequencer (Perkin Elmer Corp.; Foster City, Calif.).
C. SFV Constructs
TABLE 5 below lists the fragments generated for cloning into the SFV vectors. All were from the GBV-C genotype 1 isolate (SEQUENCE I.D. NO. 34). The E2-222, E2-336, E2-352, E1-168, NS3 and NS5A fragments are identical to those described above for the Sindbis vectors, except for the isolate of GBV-C from which they were derived. Two additional clones were generated for expression in this system. E1-149 is identical to E1-168, except that the sequence encoding the 19 residue putative E1 secretion signal on the 5' end was eliminated, and the fragment was cloned into pSFV-ss in order to utilize the lysozyme signal sequence. The other, E1-E2-NS2/3, is a large fragment encompassing all of E1, E2, NS2, and the 5' third of NS3. It has been predicted that this fragment will direct co-expression, appropriate processing and interaction of E1 and E2.
TABLE 5__________________________________________________________________________ NUCLEOTIDE AMINO ACID INSERTED RESIDUES IN RESIDUES IN FRAGMENT VECTOR PRIMERS SEQ ID #34 SEQ ID #35__________________________________________________________________________E2-222 pSFV-ss SEQ ID #54/SEQ ID #55 1143-1808 221-442 E2-336 pSFV-ss SEQ ID #56/SEQ ID #55 1143-2150 221-556 E2-352 pSFV1 SEQ ID #57/SEQ ID #58 1095-2150 205-556 E1-149 pSFV-ss SEQ ID #59/SEQ ID #60 591-1037 37-185 E1-168 pSFV1 SEQ ID #61/SEQ ID #60 534-1037 18-185 NS3 pSFV1 SEQ ID #62/SEQ ID #63 3216-5180 912-1566 NS5A pSFV1 SEQ ID #64/SEQ ID #65 6129-7370 1883-2296 E1-E2-NS2/3 pSFV1 SEQ ID #66/SEQ ID #67 534-3755 18-1091__________________________________________________________________________
The PCR primers for all constructs inserted into pSFV1 and pSFV-ss incorporated BglII sites for cloning. The BglII overhang was compatible with cloning into the BamHI site of the vector, and was utilized because there were BamHI sites but not BglII sites within the amplified GBV-C sequences. PCRs were performed as described above for the Sindbis fragments. All PCR products were then digested with BglII, ligated into BamHI-digested, de-phosphorylated pSFV1 or pSFV-ss and transformed into E. coli by employing standard methods. All inserts were sequenced as described above.
D. RNA Synthesis
Prior to in vitro synthesis of RNA, each of the Sindbis and SFV plasmids were linearized at a restriction site downstream of the inserted fragment. The Sindbis constructs were linearized with NotI, while the SFV constructs were linearized with SpeI. After digestion, the DNAs were extracted with phenol-chloroform-isoamyl alcohol (25:24:1), precipitated with ethanol, and resuspended in DEPC-treated H.sub.2 O. RNA was synthesized using the InvitroScript CAP SP6 In Vitro Transcription Kit (Invitrogen Corp.; San Diego, Calif.) as directed by the manufacturer. The synthesized RNA was divided into approximately 5 mg aliquots and stored at -70.degree. C. without any further manipulations.
E. Cell Culture
The cell line BHK-21 (A.T.C.C. CCL-10) was obtained from the American Type Culture Collection (A.T.C.C.) Rockville, Md. Cells were cultured in growth media consisting of Glasgow Minimum Essential Medium with 2 mM glutamine, 10% tryptose phosphate broth, 5% FCS, 10 mM HEPES, 100 U/ml Penicillin/streptomycin (Gibco-BRL; Grand Island, N.Y.) at 37.degree. C. and 5% CO.sub.2. Cultures were routinely split at confluency on 3 or 4 day intervals. For transfection, cells were planted in 850 cm.sup.2 roller bottles and grown to 50% confluency. For cell passage or transfection, cells were harvested by removing the growth media and rinsing three times with Dulbecco's Phosphate Buffered Saline (D-PBS) (Gibco-BRL; Grand Island, N.Y.) without calcium and magnesium. Cells were then overlaid with trypsin EDTA (Gibco-BRL; Grand Island, N.Y.) for 5 minutes, and the trypsin and cell suspension was decanted from the bottle. The trypsin in the cell suspension was neutralized with 10% growth media, and the suspension centrifuged at low speed to pellet the cells. For passage, cells were resuspended in growth media and planted in a vessel with a surface area to volume ratio of 25 cm.sup.2 to 5 ml of growth media.
F. Transfections
Cells were grown to mid-log phase and harvested from the culture vessel as described above. Cell density was adjusted to a final count of 1.times.10.sup.7 cells in 0.8 ml after 3 washes in D-PBS without calcium and magnesium. Cells were kept at room temperature for less than 30 minutes before electroporation. Approximately 5-10 mg of RNA was added to the 0.8 ml cell suspension and immediately electroporated. Electroporation was conducted at 1250 volts, 25 uF with 3 manual pulses using a Bio-Rad Gene Pulser and a 4 cm Gene Pulser cuvette (Hercules, Calif.). After transfection, cells were allowed to rest for 5 minutes at room temperature in the electroporation cuvette. Cells were then transferred to growth media and planted in a culture vessel. Transfection efficiencies were routinely observed to be 95-100% using RNA derived from the Lac-Z positive control plasmids pSFV3-lacZ or pSinRep/lacZ, and b-galactosidase assay reagents as supplied/described by Gibco BRL.
G. Metabolic Labeling of Transfected Cells
After transfection cells were allowed 5 hours in Glasgow Minimum Essential Medium with 2 mM glutamine, 10% tryptose phosphate broth, 5% FCS, 10 mM HEPES, 100 U/ml Penicillin/Streptomycin (Gibco-BRL, Grand Island, N.Y.) at 37.degree. C. and 5% CO.sub.2 to firmly attach to the culture vessel. The growth media was then removed and the cells were washed 3 times with D-PBS with calcium and magnesium. Cells were then overlaid with minimum essential medium without cystine (MEM minus) constructed with a MEM Select-Amine kit (Gibco-BRL; Grand Island, N.Y.). A culture vessel surface area to volume ratio of 25 cm.sup.2 to 2 ml was maintained. Radio-labeled [.sup.35 S]cysteine (Amersham Life Sciences; Arlington Heights, Ill.) was added to a final concentration of 75 mCi per ml and cells were incubated at 37.degree. C. and 5% CO.sub.2 until the cells and supernatants were harvested. Supernatants were harvested and spun at low speed to pellet any non-adherent cells. Clarified supernatant was then ready for analysis. Adherent cells were washed 3 times in D-PBS with calcium and magnesium and overlaid with lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM EDTA, 1 mg/ml PMSF). A surface area to volume ratio of 25 cm.sup.2 to 250 ml of lysis buffer was maintained. Lysis was conducted on wet ice for 10 minutes. Lysates were then harvested from the culture vessel and ready for analysis.
H. Analysis of Protein Expression
Aliquots of labeled cell lysates or supernatants were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods and reagents (Laemmli discontinuous gels) to assess protein expression levels. In some cases, supernatants were first concentrated by ultrafiltration through Microcon filter units (Amicon, Inc.; Beverly, Mass.) in order to analyze lysates and supernatants from equivalent numbers of cells. After electrophoresis, gels were fixed in 10% acetic acid/40% methanol, dried and developed using a Molecular Dynamics PhosphorImager (Sunnyvale, Calif.), or alternatively, proteins in the gel were electrophoretically transferred to nitrocellulose and analyzed by immunoblotting as described in Example 1.
Radioimmune precipitations were performed on labeled cell lysates and supernatants as follows. Lysates or supernatants were first pre-incubated for 1 hour at 4.degree. C. with Pansorbin cells (Calbiochem Corp.; La Jolla, Calif.). After centrifuging for 15 minutes at 10,000.times.g, an aliquot of the pre-adsorbed supernatant or lysate was mixed with 2-8 .mu.l of each sera to be examined and incubated on ice for 2-4 hours. Protein A agarose beads were added and tubes were incubated at 4.degree. C. for 1 hour with rocking. The protein A agarose beads were pelleted and washed 2 times with lysis buffer (0.2% NP40, 50 mM Tris pH 8.0, 150 mM NaCl), 1 time with lysis buffer containing 0.2% SDS, 1 time with lysis buffer containing 500 mM NaCl and finally 1 time with H.sub.2 O. The beads were resuspended in Laemmli sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.1 mg/ml bromophenol blue) and heated at 90.degree. C. for 5 minutes. Supernatants were run on SDS-PAGE, fixed, dried and exposed as above.
I. Immunoreactivity with Sindbis and SFV-expressed GBV-C E2 Proteins
Results obtained by immunoblotting and RIPAs on the GBV-C proteins expressed in the Sindbis system were as follows: GBV-C E2 proteins expressed from pSin-ss/E2-222, pSin-ss/E2-315 and pSin-ss/E2-336 were detected in cell lysates and supernatants (indicating secretion from the cells) as analyzed by immunoblotting using the rabbit anti-GBV-C peptide and E. coli recombinant protein sera described in Example 3. GBV-C E2 protein expressed from pSin-ss/E2-289 was detected only in cell lysate, but not in supernatant.
Results obtained by immunoblotting and RIPAs on the GBV-C proteins expressed in the SFV system are summarized below:
GBV-C E2 proteins expressed from pSFV-ss/E2-222 and pSFV-ss/E2-336 were detected in cell lysates and supernatants (indicating secretion from the cells) as analyzed by immunoblotting using the rabbit anti-GBV-C E2 peptide sera and rabbit anti-E. coli GBV-C E2 recombinant protein serum described in Example 3. RIPAs were performed on pSFV-ss/E2-336 supernatant and lysate using the rabbit anti-E. coli GBV-C E2 recombinant protein serum and various human sera. The GBV-C E2 protein expressed from this construct was detected in both lysates and supernatants with the post-immune rabbit serum, but not with the pre-immune serum. It was also detected in lysates and supernatants with 2 GBV-C RT-PCR positive human sera found thus far to be seronegative against E. coli derived GBV-C proteins (as tested by ELISA as described in patent application Ser. No. 08/424,550 and with 4 GBV-C RT-PCR negative human sera previously found to be immunoreactive with E. coli derived GBV-C proteins.
Sera from 27 GBV-C RT-PCR negative intravenous drug users were tested in RIPAs using the pSFV-ss/E2-336 supernatant and all 27 were reactive with the E2 protein. It is known that this population is at very high risk of exposure to parenterally transmitted agents, as evidenced by the seroprevalence of antibodies to hepatitis C virus (99%) and hepatitis B virus (75%). Testing for hepatitis C virus was performed with an HCV EIA second generation assay, with confirmatory testing by synthetic peptides and the antibodies for hepatitis B core antigen were determined with the Corzyme.RTM. test (all available from Abbott Laboratories, North Chicago, Ill.). The seroprevalence level of antibody to GBV-C E2 protein indicated a similar level exposure to GBV-C in this population as to these other parenterally transmitted hepatitis agents.
RIPAs were performed with the pSFV-ss/E2-336 supernatant using serial bleeds from 7 patients who were all PCR negative for GBV-C prior to being transfused. Subsequent to transfusion with blood containing GBV-C, each of these 7 individuals developed elevated liver enzymes and became GBV-C RNA positive as detected by RT-PCR. The PCR and RIPA results from this study are summarized in TABLE 6. FIG. 6 shows the RIPA results from patient 2 (TABLE 6).
TABLE 6__________________________________________________________________________ PCR pSFV-ss/E2-336 E2-315 SAMPLE STATUS RIPA STATUS ELISA S/N__________________________________________________________________________Patient 1 Donor + - 0.80 Recipient, pre-inoculation - - 0.80 Recipient, 10 days post-inoculation - - 0.91 Recipient, 18 days post-inoculation + - 0.86 Recipient, 33 days post-inoculation + - 0.68 Recipient, 46 days post-inoculation + - 0.92 Recipient, 104 days post-inoculation + - 0.83 Recipient, 117 days post-inoculation + weak + 1.28 Recipient, 154 days post-inoculation + + 25.76 Recipient, 184 days post-inoculation - + 27.07 Recipient, 207 days post-inoculation - + 44.49 Recipient, 284 days post-inoculation - + 38.43 Patient 2 Donor 1 - - 0.32 Donor 2 - - 0.54 Donor 3 - - 0.80 Donor 4 + - 1.43 Recipient, pre-inoculation - - 0.26 Recipient, 8 days post-inoculation + - 0.34 Recipient, 27 days post-inoculation + - 0.67 Recipient, 48 days post-inoculation + - 0.80 Recipient, 88 days post-inoculation + weak + 2.22 Recipient, 113 days post-inoculation + + 6.55 Recipient, 185 days post-inoculation + + 71.97 Recipient, 283 days post-inoculation - + 79.79 Patient 3 Donor 1 - + 9.88 Donor 2 + - 0.76 Recipient, 6 days post-inoculation - - 1.74 Recipient, 17 days post-inoculation - - 1.63 Recipient, 33 days post-inoculation - - 1.55 Recipient, 45 days post-inoculation - - 1.05 Recipient, 62 days post-inoculation - - 1.29 Recipient, 75 days post-inoculation + - 1.32 Recipient, 90 days post-inoculation + - 1.01 Recipient, 97 days post-inoculation + - 1.12 Recipient, 151 days post-inoculation + - 1.30 Recipient, 339 days post-inoculation - + 31.32 Patient 4 Donor 1 - - ND Donor 2 + - ND Donor 3 - + ND Recipient, pre-inoculation - - ND Recipient, 8 days post-inoculation - - ND Recipient, 15 days post-inoculation + - ND Recipient, 43 days post-inoculation + - ND Recipient, 64 days post-inoculation + - ND Recipient, 85 days post-inoculation + - ND Recipient, 125 days post-inoculation + - ND Recipient, 145 days post-inoculation + - ND Recipient, 173 days post-inoculation + - ND Recipient, 278 days post-inoculation - + ND Patient 5 Donor + - 4.09 Recipient, pre-inoculation - weak + 0.92 Recipient, 35 days post-inoculation + weak + 1.43 Recipient, 80 days post-inoculation + + 10.22 Recipient, 106 days post-inoculation + + 14.67 Recipient, 134 days post-inoculation - + 14.51 Recipient, 169 days post-inoculation - + 10.09 Recipient, 184 days post-inoculation - + 12.62 Recipient, 212 days post-inoculation + + 10.53 Recipient, 239 days post-inoculation - + 13.36 Recipient, 339 days post-inoculation - + 12.17 Recipient, 428 days post-inoculation - + 10.32 Recipient, 522 days post-inoculation - + 7.33 Patient 6 Donor 1 + - 0.39 Donor 2 - - 0.43 Donor 3 - + 11.79 Donor 4 - - 0.49 Donor 5 + - 0.79 Donor 6 - - 1.00 Donor 7 - - 0.76 Donor 8 - - 0.57 Donor 9 - - 0.58 Donor 10 - - 0.46 Recipient, pre-inoculation - - 1.18 Recipient, 14 days post-inoculation - weak + 2.12 Recipient, 41 days post-inoculation - weak + 1.54 Recipient, 68 days post-inoculation - - 1.41 Recipient, 83 days post-inoculation - - 1.53 Recipient, 89 days post-inoculation + - 1.17 Recipient, 105 days post-inoculation + - 0.97 Recipient, 118 days post-inoculation + - 1.00 Recipient, 166 days post-inoculation + - 1.24 Recipient, 280 days post-inoculation + + 3.16 Patient 7 Donor 1 - - 0.79 Donor 2 - - 1.43 Donor 3 + - 0.34 Donor 4 + - 0.29 Recipient, pre-inoculation - - 0.32 Recipient, 16 days post-inoculation + - 0.66 Recipient, 44 days post-inoculation + - 0.51 Recipient, 85 days post-inoculation + - 0.53 Recipient, 127 days post-inoculation + - 0.46 Recipient, 161 days post-inoculation + - 0.36 Recipient, 265 days post-inoculation + - 0.41__________________________________________________________________________
J. Results
Six of the 7 patients (patients 1-6) developed immunoreactivity against the GBV-C E2 protein during the study period, and all but one of these individuals (patient 6) was GBV-C RT-PCR negative by their last bleed date. Patients 6 and 7 were still GBV-C RT-PCR positive on the last date of sample availability and patient 7 exhibited no immunoreactivity to the GBV-C E2 protein.
These results indicate that, as has been found for other viruses including some flavi- and pesti-viruses, there may be a neutralizing epitope or epitopes found on the GBV-C E2 envelope protein which signifies or predicts that the viremic stage of infection is ending. The presence of antibodies to the E2 protein appears to be a good marker of recovery from GBV-C infection. In addition, this protein is a good candidate for potential vaccines to protect against exposure to GBV-C. Further, the vaccine could be used to treat chronically infected patients to assist in viral clearance. Moreover, the use of antisera harboring antibodies against this protein may be an efficacious treatment for patients infected with GBV-C who have not mounted an immune response sufficient to clear the virus, or to eliminate cells harboring E2 proteins. Such cells may contribute to tissue damage or even carcinogenesis.
Example 5
ELISA for Detection of Antibodies to GBV-C E2 315
A. Polystyrene Bead Coating Procedure
The GBV-C E2 315 expressed in CHO cells as described hereinabove in preceding examples was evaluated for antigenicity on polystyrene coated beads, and an enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies to GBV-C E2. In the first study, one-quarter inch polystyrene beads were coated with purified protein (approximately 60 beads per lot) and evaluated in an ELISA test (described below). Briefly, polystyrene beads were coated with the purified proteins by adding the washed beads to a scintillation vial and immersing the beads (approximately 0.233 ml per bead) in a buffered solution containing the GBV-C E2 protein. The GBV-C E2 protein was approximately 125 ug/ml. The protein was diluted into bead coating buffer (0.1 M sodium phosphate pH 7.5) at 2 and 4 ug/ml. The vials were then placed on a rotating device in a 40.degree. C. incubator for 2 hours, after which the fluids were aspirated and the beads were washed three times in phosphate buffered saline (PBS), pH 6.8. The beads were then treated with 0.1% Triton X-100.RTM. for 1 hour at 40.degree. C. and washed three times in PBS. Next, the beads were overcoated with 5% bovine serum albumin and incubated at 40.degree. C. for 1 hour with agitation. After additional washing steps with PBS, the beads were overcoated with 5% sucrose for 20 minutes at room temperature and the fluids were aspirated. Finally, the beads were air dried and then utilized for developing ELISA's for detection of antibodies to GBV-C E2.
B. ELISA Protocol for Detection of Antibodies to HGBV
An indirect assay format was utilized for the ELISA. Briefly, specimens (sera or plasma) being tested were diluted in specimen diluent (available from Abbott Laboratories, Abbott Park, Ill.) and reacted with a GBV-C E2 antigen coated solid phase (bead) having the GBV-C E2 antigen grown in CHO cells, all as described hereinabove. After a washing step, the beads were reacted with horseradish-peroxidase (HRPO) labeled antibodies directed against human immunoglobulins to detect human antibodies bound to the solid phase. Specimens which produced signals above a cutoff value were considered reactive. Additional details pertaining to the ELISA's are described below.
The format for the ELISAs entailed contacting the GBV-C E2 antigen-coated solid phase (prepared in CHO cells as described hereinabove and coated on beads as described hereinabove) with human serum pre-diluted in specimen diluent (buffered solution containing animal sera and non-ionic detergents, available from Abbott Laboratories, Abbott Park, Ill.). This specimen diluent was formulated to reduce background signals obtained from non-specific binding of immunoglobulins to the solid phase while enhancing the binding of specific antibodies to the antigen-coated solid phase. Specifically, 10 .mu.l of human serum was diluted in 150 .mu.l of specimen diluent and vortexed. Ten microliters of this pre-diluted specimen was then added to the well of a reaction tray, followed by the addition of 200 .mu.l of specimen diluent and an antigen coated polystyrene bead. The reaction tray was then incubated in a Dynamic Incubator (Abbott Laboratories, Abbott Park, Ill.) set for constant agitation at room temperature. After a 1 hour incubation, the fluids were aspirated, and the wells containing the beads were washed three times in distilled water (5 ml per wash). Next, 200 .mu.l of HRPO-labeled goat anti-human immunoglobulin (available from Kirkegaard and Perry, Gaithersburg, Md.) diluted in a conjugate diluent (buffered solution containing animal sera and non-ionic detergents, available from Abbott Laboratories, Abbott Park, Ill.) was added to each well and the reaction tray was incubated again as described hereinabove for 1 hour. The fluids then were aspirated, and the wells containing the beads were washed three times in distilled water as described above. The beads containing complexed antigen and bound immunoglobulins were removed from the wells, each bead was placed in a test tube and reacted with 300 .mu.l of a solution of 0.3% o-phenylenediamine-2 HCl in 0.1 M citrate buffer (pH 5.5) with 0.02% H.sub.2 0.sub.2 (available from Abbott Laboratories, Abbott Park, Ill.). After incubation for 30 minutes at room temperature, the reaction was terminated by the addition of 1 N H.sub.2 SO.sub.4. The absorbance at 492 nm was read on a spectrophotometer. The color produced was directly proportional to the amount of antibody present in the test sample.
A small panel was tested to determine whether the GBV-C E2 ELISA might be useful. Serum from volunteer donors who were prescreened as negative for GBV-C RNA by RT-PCR and negative for GBV-C proteins tested in other ELISAs utilizing proteins other than GBV-C E2 proteins were used as the negative controls. Positive controls included specimens identified as having antibodies to GBV-C E2 either by Western blotting (as described in Example 3F) or by SDS-PAGE RIPA on GBV-C E2 pSFV-ss/E2-336 proteins (as described in Example 4I). The results obtained with these first coated beads indicated that low absorbance values were obtained with the presumed negative controls and higher absorbance values were noted for the specimens which were presumed to contain antibodies to GBV-C E2. Further experiments then were undertaken.
C. Separation of GBV-E2 Protein from Free FLAG Peptides
The first preparation of GBV-C E2 315 purified protein used to coat beads was likely to also contain free FLAG peptide used for elution from the anti-FLAG M2 affinity column. Since the possibility existed that FLAG peptide might interfere with the ability of the GBV-C E2 protein to coat the solid phase, the protein was further purified for the scaled-up bead coating procedure to remove the free FLAG. This was performed using Sephadex G-25.RTM. as described above.
D. Utility of GBV-C E2 ELISA
The bead coating was scaled up to 500 beads utilizing the same procedure described above. Five hundred beads coated with the E2-315 protein were prepared as described hereinabove and utilized in ELISA testing as described hereinabove. The results were as follows.
1. Volunteer Blood Donors. A population of 100 volunteer blood donors assumed to be at "low risk" for exposure to GBV-C were tested for antibodies to GBV-C E2 utilizing the GBV-C E2 ELISA. (These specimens were considered as low risk since the serum had been screened as negative for evidence of active infection with known hepatotropic agents (hepatitis B virus [HBV], hepatitis C virus [HCV]) utilizing appropriate tests (hepatitis B surface antigen test, anti-HCV test, available from Abbott Laboratories, Abbott Park, Ill.) and were screened as having normal serum alanine aminotransferase levels (ALT), suggesting that there was no current evidence of active liver disease). Three specimens among the 100 samples tested had relatively high absorbance values and were considered as likely to contain antibodies to GBV-C E2. The other 97 specimens had relatively low absorbance values and appeared to represent a typical profile of seronegative individuals. A preliminary cutoff was determined by calculating the population mean of the 97 negative specimens and determining the standard deviation (FIG. 3). The cutoff was determined as the sum of the population mean and seven standard deviations from the population mean. Utilizing this cutoff, three of the volunteer blood donors were positive for antibodies to GBV-C E2. In general, the cutoff corresponded to sample to negative control ratio (S/N) of 7.5.
2. GBV-C RNA Positive Blood Transfusion Recipients. Serial specimens were obtained from several individuals who were exposed to GBV-C via blood transfusion. In all cases, each individual was negative for GBV-C RNA by RT-PCR prior to transfusion and became GBV-C RNA positive following transfusion with blood from at least one GBV-C RNA positive blood donor. A typical profile showing the PCR reactivity and the ELISA values is depicted in FIG. 4. This individual (Patient #1 in TABLE 6) was PCR negative for GBV-C RNA prior to exposure to GBV-C and on day 10 post exposure. GBV-C RNA was detected on days 18, 33, 46, 104, 117 and 154 post exposure. Antibodies to GBV-C E2 were first detected by RIPA against the GBV-C E2-336 protein generated via the SFV vector (described in Example 4J, above) on day 117 post exposure and continued to be detected on all follow-up specimens (days 154, 184, 207 and 284 post exposure). The GBV-C E2 ELISA detected antibodies to GBV-C E2 on days 154, 184, 207 and 284 post exposure. These data suggest that the antibodies to GBV-C E2 are associated with viral clearance and further suggest that, once antibodies to GBV-C E2 are present, the individual's viremia will resolve in a short period of time. An example of a second profile from a second individual is depicted in FIG. 5. This individual (Patient #7 in TABLE 6) was negative for GBV-C RNA prior to transfusion, became GBV-C RNA positive on day 16 and remained PCR positive on all of the follow-up specimens (days 44, 85, 127, 161 and 265 post exposure). No antibody response was noted either by the ELISA or RIPA. These data support the hypothesis that antibodies to GBV-C E2 must be generated in order to clear GBV-C virus from the bloodstream.
3. Other Sera. Several other different classifications of individuals were tested for antibodies to GBV-C E2. These data are presented in TABLE 7. As noted previously, only three of 100 volunteer blood donors were antibody positive in the GBV-C E2 ELISA test. In stark contrast, however, 25 of 25 Intravenous drug users (IVDU's) who were negative for GBV-C RNA were antibody positive in the ELISA. These data indicate that the IVDU's indeed represent a population of individuals who are now conclusively shown to be "at high risk" for exposure to GBV-C. Previous studies had indicated that GBV-C RNA can be detected in about 15% of IVDU's. Combined, the PCR data and antibody data indicate that within the limits of the study, 100% of the IVDU's from this cohort have been exposed to GBV-C. Further, four of 20 West Africans who were GBV-C RNA negative were antibody positive in the GBV-C E2 ELISA. (Previous data had indicated that GBV-C RNA was detected in 10-15% of the West African residents; these antibody results indicate that the exposure to GBV-C is actually much higher than noted with the GBV-C RNA studies alone). Moreover, among 50 PCR positive individuals from different patient categories (IVDU's, West Africans, non A-E hepatitis patients), only seven of the 50 (14.0%) were antibody positive to GBV-C. These data lend additional support to previous studies which indicated that antibodies to GBV-C E2 may be more readily detected in individuals recovering or having recovered from GBV-C infection.
TABLE 7______________________________________Preliminary Anti-GBV-C E2 Prevalence in Various Groups Number Number Category Tested Positive Percentage______________________________________Volunteer Blood Donors 100 3 3.0 PCR Negative Individuals Intravenous Drug Users 25 25 100.0 West African Residents 20 4 20.0 PCR Positive Individuals 50 7 14.0______________________________________
Example 6
Expression of GBV-C Polypeptides Using a Baculovirus Vector System
A. Baculovirus Transfer Vector Construction
A transfer vector for the expression of GBV-C E2 protein was constructed by restricting pAcGP67A with both Bam HI and Pst I restriction endonucleases. A GBV-C E2 gene fragment with compatible Bam HI and Pst I ends was generated by PCR as described supra using a plasmid template containing a portion of GBV-C. Vector assembly was performed by standard methods known to those skilled in the art and termed pAcGP67A/E2C. The fragment encoded a 336 amino acid segment of the GBV-C E2 gene (amino acids 221 to 556 of SEQUENCE I.D. NO. 35). Contained within the sense PCR primer sequence (SEQUENCE I.D. NO. 68) was a Bam HI site, followed immediately by nucleotides corresponding to template sequences encoding amino acids starting at residue 221 of GBV-C. The antisense PCR primer (SEQUENCE I.D. NO. 69) contained nucleotides complementary to template sequences that encode amino acids ending at residue 556 of GBV-C, followed by a sequence encoding the 8 amino acids Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (see anti-FLAG recognition site supra), two stop codons and a Pst I restriction enzyme site compatible with the cloning vector.
B. Transfection and Amplification of Recombinant Baculovirus
The transfer vector described above was transfected into shaker flask adapted Sf9 cells (available from Gibco-BRL; Grand Island, N.Y.) using the BaculoGold.TM. Transfection Kit (PharMingen). Briefly, 3 ml of Sf9 cells diluted to 1.times.10.sup.6 /ml in Sf-900 II SFM medium (Gibco-BRL) were dispensed into T25 cell culture flasks. After being allowed to adhere at room temperature for 10 minutes, medium was removed and 1 ml of Buffer A (obtained from the BaculoGold.TM. Transfection Kit described hereinabove) was added. 4 .mu.g of pAcGP67A-E2C was mixed with 0.5 .mu.g BaculoGold DNA and 1 ml of Buffer B (obtained from the BaculoGold.TM. Transfection Kit described hereinabove). After 5 minutes at room temperature, DNA mix was added drop-by-drop to the Sf9 cells in Buffer A. Cultures were incubated at 28.degree. C. for 4 hours, at which time the precipitate was removed, the cells were washed with Sf-900 II SFM and cultured with 3 ml of fresh medium at 28.degree. C. After 4 days, 0.5 ml of the transfected cell supernatant was added to Sf9 cells to expand the concentration of recombinant virus. Upon observing viral CPE, both cells and cell supernatants were evaluated for successful expression of the recombinant protein. It was observed that both cells and cell supernatants contained a band of approximately 40-50 kD that was immunoreactive with anti-FLAG BioM2 monoclonal antibody (Eastman Kodak Co., New Haven, Conn.) reactive with the 8 amino acid FLAG peptide engineered to the carboxyl-terminus. Additionally, this band was reactive with sera from rabbits immunized with an E. coli derived GBV-C E2 antigen or with a synthetic peptide "GE2-1" having the amino acid sequence "QGAPASVLGSRPFE" (SEQUENCE ID NO. 70) corresponding to sequences within GBV-C E2. Recombinant virus was plaque-purified in Sf9 cells under an agarose-overlay using methods known to those skilled in the art. Plaque-pure viruses were isolated and expanded in Sf9 cells as described hereinabove, from which high-titered stocks were made (Invitrogen, San Diego, Calif.). Titering was conducted with Sf9 cells utilizing standard methodologies, and stocks were stored at 4.degree. C. Analysis and purification of GBV-C E2 was performed as described supra.
D. Maintenance and Storage of Cell Lines
Sf9 cells maintained in culture or being used for infection purposes were passaged twice weekly in Sf-900 II SFM medium. Cells were diluted to 3-4.times.10.sup.5 /ml (100 ml volumes) in 250 ml shaker flasks and incubated at 28.degree. C. on a shaker platform rotating at 150 rpm. Cryostorage was by resuspension of 1.times.10.sup.7 cells into 1.0 ml of Sf-900 II SFM medium supplemented with 10% FBS (Sigma Chem. Co., St. Louis, Mo.) and 7.5% DMSO (Sigma) and cold storage for 24-48 hours at -80.degree. C. Permanent storage was in liquid nitrogen.
Strains replicated from the HGBV nucleic acid sequence library have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, as of May 31, 1996, under the terms of the Budapest Treaty and will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, or for the enforceable period of the U.S. patent, whichever is longer. The deposits and any other deposited material described herein are provided for convenience only, and are not required to practice the present invention in view of the teachings provided herein. The HGBV cDNA sequences in all of the deposited materials are incorporated herein by reference. The plasmids were accorded the following A.T.C.C. deposit numbers: Clone pSinRep5/NS5A was accorded A.T.C.C. deposit No. 98073; Clone pCHO/E2-336 was accorded A.T.C.C. deposit No. CRL12111; Clone pSFV-ss/E2-336 was accorded A.T.C.C. deposit No. 98070; Clone pSFV-ss/NS3 was accorded A.T.C.C. deposit No. 98071; Clone pCHO/E2-315 was accorded A.T.C.C. deposit No. CRL12110; and Clone pAcGP67A-E2C was accorded A.T.C.C. deposit No. 98072.
The present invention is intended to be limited only by the appended claims.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 70 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - GGAAAAGGAT CCCGGCTTGT GTCCAAGATG TGCG - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - GGGGAAAAGC TTAAGCCTTA GTGACCCCGA GGAAG - # -# 35 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - GGGGAAGAAT TCGGAAAGGG CTTCCTCGGG GT - # - # 32 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - GGCGGCAAGC TTACACGTCC AGTTCTACCT TGTCCC - # -# 36 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - GGAAAAGGAT CCAGCAAGGG GGACAAGGTA GA - # - # 32 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - GGGGAAAAGC TTACATGGCC CTCACAGTGG CAA - # - # 33 - - - - (2) INFORMATION FOR SEQ ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - - GGAAAAGGAT CCAACCCGTC GGTTGCCACT GTGA - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - - GGCGGCAAGC TTAGTCCAGC TTTGTCTCAA TTATGG - #- # 36 - - - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - - GGAAAAGGAT CCCATCCATC CATAATTGAG AC - # - # 32 - - - - (2) INFORMATION FOR SEQ ID NO:10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: - - GGGGAAAAGC TTAAGGGACA GTCCGCAAGG AAAT - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: - - GGTTTTGGAT CCACCATTAC CATTTCCTTG CGGAC - #- # 35 - - - - (2) INFORMATION FOR SEQ ID NO:12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: - - GGGAAAAAGC TTACCAATTG ACGCCGCGAA CTTTTG - #- # 36 - - - - (2) INFORMATION FOR SEQ ID NO:13: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: - - GGGAAAGGAT CCAGCTGGGC AAAAGTTCGC GGCGGCG - #- # 37 - - - - (2) INFORMATION FOR SEQ ID NO:14: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: - - GGCGGCAAGC TTAAAGGGGA TTGCCACCTC CCTTC - # -# 35 - - - - (2) INFORMATION FOR SEQ ID NO:15: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: - - GGAAAAGGAT CCTGGGATGT GAAGGGAGGT GGC - # - # 33 - - - - (2) INFORMATION FOR SEQ ID NO:16: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: - - GGCGGAAAGC TTAACCCCCG GCGAAGAGCT TGTCAAC - #- # 37 - - - - (2) INFORMATION FOR SEQ ID NO:17: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: - - GGTTTTGGAT CCTCAATTGT TGACAAGCTC TTCG - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:18: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: - - GGCGGAAAGC TTAAAGGTCT TCTGTTGAAA GTTTCC - # -# 36 - - - - (2) INFORMATION FOR SEQ ID NO:19: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: - - GGAAAAGGAT CCATGGCTGG GAAACTTTCA ACAG - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:20: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: - - GGGGAAAAGC TTATCTAGAC ATCACCATGC GCACCTC - #- # 37 - - - - (2) INFORMATION FOR SEQ ID NO:21: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: - - GGAAAAGGAT CCATGCGCCA GGTGCGCATG GTGATG - # -# 36 - - - - (2) INFORMATION FOR SEQ ID NO:22: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: - - GGGGAAAAAA GCTTAGATCA CCACGTGGGT AGGGGTCAC - # - # 39 - - - - (2) INFORMATION FOR SEQ ID NO:23: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: - - GGAAAAGGAT CCGTGGTGGT GACCCCTACC CAC - # - # 33 - - - - (2) INFORMATION FOR SEQ ID NO:24: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: - - GGCGGCAAGC TTATCTCGCA GCATTCTCTA TCGC - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:25: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: - - GGAAAAGGAT CCGCCCTCCA AGCGATAGAG AATG - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:26: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: - - GGCGGCAAGC TTACTTAACC GTTAGCTTTC GTG - # - # 33 - - - - (2) INFORMATION FOR SEQ ID NO:27: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: - - GGAAAAGGAT CCAGCGATGC CACACGAAAG CTAAC - # -# 35 - - - - (2) INFORMATION FOR SEQ ID NO:28: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: - - GGGGAAAAGC TTAGGTGGTG TCTGCCACCA ACAAG - # -# 35 - - - - (2) INFORMATION FOR SEQ ID NO:29: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: - - CTCGTATGAT GCGACAGTCC GTCC - # - # 24 - - - - (2) INFORMATION FOR SEQ ID NO:30: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: - - GATTATGGCC TTTGTGCTTC CACCC - # - # 25 - - - - (2) INFORMATION FOR SEQ ID NO:31: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: - - GCATCGAGAT CGGGACGGAG ACTG - # - # 24 - - - - (2) INFORMATION FOR SEQ ID NO:32: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: - - GAATGTGACA AGTGTGAGGC ACG - # - # 23 - - - - (2) INFORMATION FOR SEQ ID NO:33: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2905 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: - - Pro Ile Gly Val Arg Arg Val Asp Lys Asp Gl - #n Trp Gly Pro Gly Gly 1 5 - # 10 - # 15 - - Arg Gly Lys Asp Pro His Arg Cys Pro Ser Ar - #g Gly Gly Gly Lys Cys 20 - # 25 - # 30 - - Met Gly Pro Pro Ser Ser Ala Ala Ala Tyr Se - #r Arg Gly Ser Pro Arg 35 - # 40 - # 45 - - Thr Ser Gly Glu Gly Gly Trp His Phe Phe Se - #r Tyr Thr Asp His Gly 50 - # 55 - # 60 - - Ser Pro Ser Ala Pro Thr Arg Gly Gly Ala Gl - #y Ala Ile Leu Ala Pro 65 - # 70 - # 75 - # 80 - - Ala Thr His Ala Cys Ser Ala Lys Gly Gln Ty - #r Xaa Leu Thr Asn Cys 85 - # 90 - # 95 - - Cys Ala Leu Glu Asp Ile Gly Phe Cys Leu Gl - #u Gly Gly Cys Leu Val 100 - # 105 - # 110 - - Ala Leu Gly Cys Thr Ile Cys Thr Asp Arg Cy - #s Trp Pro Leu Tyr Gln 115 - # 120 - # 125 - - Ala Gly Leu Ala Val Arg Pro Gly Lys Ser Al - #a Ala Gln Leu Val Gly 130 - # 135 - # 140 - - Glu Leu Gly Ser Leu Tyr Gly Pro Leu Ser Va - #l Ser Ala Tyr Val Ala 145 1 - #50 1 - #55 1 -#60 - - Gly Ile Leu Gly Leu Gly Glu Val Tyr Ser Gl - #y Val Leu Thr ValGly 165 - # 170 - # 175 - - Val Ala Leu Thr Arg Arg Val Tyr Pro Val Pr - #o Asn Leu Thr Cys Ala 180 - # 185 - # 190 - - Val Glu Cys Glu Leu Lys Trp Glu Ser Glu Ph - #e Trp Arg Trp Thr Glu 195 - # 200 - # 205 - - Gln Leu Ala Ser Asn Tyr Trp Ile Leu Glu Ty - #r Leu Trp Lys Val Pro 210 - # 215 - # 220 - - Phe Asp Phe Trp Arg Gly Val Met Ser Leu Th - #r Pro Leu Leu Val Cys 225 2 - #30 2 - #35 2 -#40 - - Val Ala Ala Leu Leu Leu Leu Glu Gln Arg Il - #e Val Met Val PheLeu 245 - # 250 - # 255 - - Leu Val Thr Met Ala Gly Met Ser Gln Gly Al - #a Pro Ala Ser Ser Val 260 - # 265 - # 270 - - Gly Val Thr Ala Phe Arg Gly Gly Phe Asp Le - #u Ala Val Leu Phe Leu 275 - # 280 - # 285 - - Gln Val Glu Arg Val Pro Arg Ala Asp Arg Gl - #u Arg Val Trp Glu Arg 290 - # 295 - # 300 - - Gly Asn Val Thr Leu Leu Cys Asp Cys Pro As - #n Gly Pro Trp Val Trp 305 3 - #10 3 - #15 3 -#20 - - Val Pro Ala Leu Cys Gln Ala Ile Gly Trp Gl - #y Asp Pro Ile ThrHis 325 - # 330 - # 335 - - Trp Ser His Gly Gln Asn Gln Trp Pro Leu Se - #r Cys Pro Gln Phe Val 340 - # 345 - # 350 - - Tyr Gly Ala Val Ser Val Thr Cys Val Trp Gl - #y Ser Val Ser Trp Phe 355 - # 360 - # 365 - - Ala Ser Thr Gly Gly Arg Asp Ser Lys Val As - #p Val Trp Ser Leu Val 370 - # 375 - # 380 - - Pro Val Gly Ser Ala Ser Cys Thr Ile Ala Al - #a Leu Gly Ser Ser Asp 385 3 - #90 3 - #95 4 -#00 - - Arg Asp Thr Val Val Glu Leu Ser Glu Trp Gl - #y Ile Pro Cys AlaThr 405 - # 410 - # 415 - - Cys Ile Leu Asp Arg Arg Pro Ala Ser Cys Gl - #y Thr Cys Val Arg Asp 420 - # 425 - # 430 - - Cys Trp Pro Glu Thr Gly Ser Val Arg Phe Pr - #o Phe His Arg Cys Gly 435 - # 440 - # 445 - - Ala Gly Pro Arg Leu Thr Arg Asp Leu Glu Al - #a Val Pro Phe Val Asn 450 - # 455 - # 460 - - Arg Thr Thr Pro Phe Thr Ile Arg Gly Pro Le - #u Gly Asn Gln Gly Arg 465 4 - #70 4 - #75 4 -#80 - - Gly Asn Pro Val Arg Ser Pro Leu Gly Phe Gl - #y Ser Tyr Thr MetThr 485 - # 490 - # 495 - - Lys Ile Arg Asp Ser Leu His Leu Val Lys Cy - #s Pro Thr Pro Ala Ile 500 - # 505 - # 510 - - Glu Pro Pro Thr Gly Thr Phe Gly Phe Phe Pr - #o Gly Val Pro Pro Leu 515 - # 520 - # 525 - - Asn Asn Cys Met Leu Leu Gly Thr Glu Val Se - #r Glu Val Leu Gly Gly 530 - # 535 - # 540 - - Ala Gly Leu Thr Gly Gly Phe Tyr Glu Pro Le - #u Val Arg Arg Cys Ser 545 5 - #50 5 - #55 5 -#60 - - Glu Leu Met Gly Arg Arg Asn Pro Val Cys Pr - #o Gly Phe Ala TrpLeu 565 - # 570 - # 575 - - Ser Ser Gly Arg Pro Asp Gly Phe Ile His Va - #l Gln Gly His Leu Gln 580 - # 585 - # 590 - - Glu Val Asp Ala Gly Asn Phe Ile Pro Pro Pr - #o Arg Trp Leu Leu Leu 595 - # 600 - # 605 - - Asp Phe Val Phe Val Leu Leu Tyr Leu Met Ly - #s Leu Ala Glu Ala Arg 610 - # 615 - # 620 - - Leu Val Pro Leu Ile Leu Leu Leu Leu Trp Tr - #p Trp Val Asn Gln Leu 625 6 - #30 6 - #35 6 -#40 - - Ala Val Leu Xaa Val Xaa Ala Xaa Xaa Ala Al - #a Val Ala Gly GluVal 645 - # 650 - # 655 - - Phe Ala Gly Pro Ala Leu Ser Trp Cys Leu Gl - #y Leu Pro Phe Val Ser 660 - # 665 - # 670 - - Met Ile Leu Gly Leu Ala Asn Leu Val Leu Ty - #r Phe Arg Trp Met Gly 675 - # 680 - # 685 - - Pro Gln Arg Leu Met Phe Leu Val Leu Trp Ly - #s Leu Ala Arg Gly Ala 690 - # 695 - # 700 - - Phe Pro Leu Ala Leu Leu Met Gly Ile Ser Al - #a Thr Arg Gly Arg Thr 705 7 - #10 7 - #15 7 -#20 - - Ser Val Leu Gly Ala Glu Phe Cys Phe Asp Va - #l Thr Phe Glu ValAsp 725 - # 730 - # 735 - - Thr Ser Val Leu Gly Trp Val Val Ala Ser Va - #l Val Ala Trp Ala Ile 740 - # 745 - # 750 - - Ala Leu Leu Ser Ser Met Ser Ala Gly Gly Tr - #p Lys His Lys Ala Ile 755 - # 760 - # 765 - - Ile Tyr Arg Thr Trp Cys Lys Gly Tyr Gln Xa - #a Leu Arg Gln Arg Val 770 - # 775 - # 780 - - Val Arg Ser Pro Leu Gly Glu Gly Arg Pro Th - #r Lys Pro Leu Thr Ile 785 7 - #90 7 - #95 8 -#00 - - Ala Trp Cys Leu Ala Ser Tyr Ile Trp Pro As - #p Ala Val Met LeuVal 805 - # 810 - # 815 - - Val Val Ala Met Val Leu Leu Phe Gly Leu Ph - #e Asp Ala Leu Asp Trp 820 - # 825 - # 830 - - Ala Leu Glu Glu Leu Leu Val Ser Arg Pro Se - #r Leu Arg Arg Leu Ala 835 - # 840 - # 845 - - Arg Val Val Glu Cys Cys Val Met Ala Gly Gl - #u Lys Ala Thr Thr Val 850 - # 855 - # 860 - - Arg Leu Val Ser Lys Met Cys Ala Arg Gly Al - #a Tyr Leu Phe Asp His 865 8 - #70 8 - #75 8 -#80 - - Met Gly Ser Phe Ser Arg Ala Val Lys Glu Ar - #g Leu Leu Glu TrpAsp 885 - # 890 - # 895 - - Ala Ala Leu Glu Xaa Leu Ser Phe Thr Arg Th - #r Asp Cys Arg Ile Ile 900 - # 905 - # 910 - - Arg Asp Ala Ala Arg Thr Leu Ser Cys Gly Gl - #n Cys Val Met Gly Leu 915 - # 920 - # 925 - - Pro Val Val Ala Arg Arg Gly Asp Glu Val Le - #u Ile Gly Val Phe Gln 930 - # 935 - # 940 - - Asp Val Asn His Leu Pro Pro Gly Phe Xaa Pr - #o Thr Ala Pro Val Val 945 9 - #50 9 - #55 9 -#60 - - Ile Arg Arg Cys Gly Lys Gly Phe Leu Gly Va - #l Thr Lys Ala AlaLeu 965 - # 970 - # 975 - - Thr Gly Arg Asp Pro Asp Leu His Pro Gly As - #n Val Met Val Leu Gly 980 - # 985 - # 990 - - Thr Ala Thr Ser Arg Ser Met Gly Thr Cys Le - #u Asn Gly Leu Leu Phe 995 - # 1000 - # 1005 - - Thr Thr Phe His Gly Ala Ser Ser Arg Thr Il - #e Ala Thr Pro Val Gly 1010 - # 1015 - # 1020 - - Ala Leu Asn Pro Arg Trp Trp Ser Ala Ser As - #p Asp Val Thr Val Tyr 1025 1030 - # 1035 - # 1040 - - Pro Leu Pro Asp Gly Ala Asn Ser Leu Val Pr - #o Cys Ser Cys Gln Ala 1045 - # 1050 - # 1055 - - Glu Ser Cys Trp Val Xaa Arg Ser Asp Gly Al - #a Leu Cys His Gly Leu 1060 - # 1065 - # 1070 - - Ser Lys Gly Asp Lys Val Glu Leu Asp Val Al - #a Met Glu Val Ala Asp 1075 - # 1080 - # 1085 - - Phe Arg Gly Ser Ser Gly Ser Pro Val Leu Cy - #s Asp Glu Gly His Ala 1090 - # 1095 - # 1100 - - Val Gly Met Leu Val Ser Val Leu His Ser Gl - #y Gly Arg Val Thr Ala 1105 1110 - # 1115 - # 1120 - - Ala Arg Phe Thr Arg Pro Trp Thr Gln Val Pr - #o Thr Asp Ala Lys Thr 1125 - # 1130 - # 1135 - - Thr Thr Glu Pro Pro Pro Val Pro Ala Lys Gl - #y Val Phe Lys Glu Ala 1140 - # 1145 - # 1150 - - Pro Leu Phe Met Pro Thr Gly Ala Gly Lys Se - #r Thr Arg Val Pro Leu 1155 - # 1160 - # 1165 - - Glu Tyr Gly Asn Met Gly His Lys Val Leu Il - #e Leu Asn Pro Ser Val 1170 - # 1175 - # 1180 - - Ala Thr Val Arg Ala Met Gly Pro Tyr Met Gl - #u Arg Leu Ala Gly Lys 1185 1190 - # 1195 - # 1200 - - His Pro Ser Ile Phe Cys Gly His Asp Thr Th - #r Ala Phe Thr Arg Ile 1205 - # 1210 - # 1215 - - Thr Asp Ser Pro Leu Thr Tyr Ser Thr Tyr Gl - #y Arg Phe Leu Ala Asn 1220 - # 1225 - # 1230 - - Pro Arg Gln Met Leu Arg Gly Val Ser Val Va - #l Ile Cys Asp Glu Cys 1235 - # 1240 - # 1245 - - His Ser His Asp Ser Thr Val Leu Leu Gly Il - #e Gly Arg Val Arg Asp 1250 - # 1255 - # 1260 - - Val Ala Arg Gly Cys Gly Val Gln Leu Val Le - #u Tyr Ala Thr Ala Thr 1265 1270 - # 1275 - # 1280 - - Pro Pro Gly Ser Pro Met Thr Gln His Pro Se - #r Ile Ile Glu Thr Lys 1285 - # 1290 - # 1295 - - Leu Asp Val Gly Glu Ile Pro Phe Tyr Gly Hi - #s Gly Ile Pro Leu Glu 1300 - # 1305 - # 1310 - - Arg Met Arg Thr Gly Arg His Leu Val Phe Cy - #s His Ser Lys Ala Glu 1315 - # 1320 - # 1325 - - Cys Glu Arg Leu Ala Gly Gln Phe Ser Ala Ar - #g Gly Val Asn Ala Ile 1330 - # 1335 - # 1340 - - Ala Tyr Tyr Arg Gly Lys Asp Ser Ser Ile Il - #e Lys Asp Gly Asp Leu 1345 1350 - # 1355 - # 1360 - - Val Val Cys Ala Thr Asp Ala Leu Ser Thr Gl - #y Tyr Thr Gly Asn Phe 1365 - # 1370 - # 1375 - - Asp Ser Val Thr Asp Cys Gly Leu Val Val Gl - #u Glu Val Val Glu Val 1380 - # 1385 - # 1390 - - Thr Leu Asp Pro Thr Ile Thr Ile Ser Leu Ar - #g Thr Val Pro Ala Ser 1395 - # 1400 - # 1405 - - Ala Glu Leu Ser Met Gln Arg Arg Gly Arg Th - #r Gly Arg Gly Arg Ser 1410 - # 1415 - # 1420 - - Gly Arg Tyr Tyr Tyr Ala Gly Val Gly Lys Al - #a Pro Ala Gly Val Val 1425 1430 - # 1435 - # 1440 - - Arg Ser Gly Pro Val Trp Ser Ala Val Glu Al - #a Gly Val Thr Trp Tyr 1445 - # 1450 - # 1455 - - Gly Met Glu Pro Asp Leu Thr Ala Asn Leu Le - #u Arg Leu Tyr Asp Asp 1460 - # 1465 - # 1470 - - Cys Pro Tyr Thr Ala Ala Val Ala Ala Asp Il - #e Gly Glu Ala Ala Val 1475 - # 1480 - # 1485 - - Phe Phe Ala Gly Leu Ala Pro Leu Arg Met Hi - #s Pro Asp Val Ser Trp 1490 - # 1495 - # 1500 - - Ala Lys Val Arg Gly Val Asn Trp Pro Leu Le - #u Val Gly Val Gln Arg 1505 1510 - # 1515 - # 1520 - - Thr Met Cys Arg Glu Thr Leu Ser Pro Gly Pr - #o Ser Asp Asp Pro Gln 1525 - # 1530 - # 1535 - - Trp Ala Gly Leu Lys Gly Pro Asn Pro Val Pr - #o Leu Leu Leu Arg Trp 1540 - # 1545 - # 1550 - - Gly Asn Asp Leu Pro Ser Lys Val Ala Gly Hi - #s His Ile Val Asp Asp 1555 - # 1560 - # 1565 - - Leu Val Arg Arg Leu Gly Val Ala Glu Gly Ty - #r Val Arg Cys Asp Ala 1570 - # 1575 - # 1580 - - Xaa Pro Ile Leu Met Val Gly Leu Ala Ile Al - #a Gly Gly Met Ile Tyr 1585 1590 - # 1595 - # 1600 - - Ala Ser Tyr Thr Gly Ser Leu Val Val Val Th - #r Asp Trp Asp Val Lys 1605 - # 1610 - # 1615 - - Gly Gly Gly Asn Pro Leu Tyr Arg Ser Gly As - #p Gln Ala Thr Pro Gln 1620 - # 1625 - # 1630 - - Pro Val Val Gln Val Pro Pro Val Asp His Ar - #g Pro Gly Gly Glu Ser 1635 - # 1640 - # 1645 - - Ala Pro Arg Asp Ala Lys Thr Val Thr Asp Al - #a Val Ala Ala Ile Gln 1650 - # 1655 - # 1660 - - Val Asn Cys Asp Trp Ser Val Met Thr Leu Se - #r Ile Gly Glu Val Leu 1665 1670 - # 1675 - # 1680 - - Thr Leu Ala Gln Ala Lys Thr Ala Glu Ala Ty - #r Ala Ala Thr Ser Arg 1685 - # 1690 - # 1695 - - Trp Leu Ala Gly Cys Tyr Thr Gly Thr Arg Al - #a Val Pro Thr Val Ser 1700 - # 1705 - # 1710 - - Ile Val Asp Lys Leu Phe Ala Gly Gly Trp Al - #a Ala Val Val Gly His 1715 - # 1720 - # 1725 - - Cys His Ser Val Ile Ala Ala Ala Val Ala Al - #a Tyr Gly Ala Ser Arg 1730 - # 1735 - # 1740 - - Ser Pro Pro Leu Ala Ala Ala Ala Ser Tyr Le - #u Met Gly Leu Gly Val 1745 1750 - # 1755 - # 1760 - - Gly Gly Asn Ala Gln Ala Arg Leu Ala Ser Al - #a Leu Leu Leu Gly Ala 1765 - # 1770 - # 1775 - - Ala Gly Thr Ala Leu Gly Thr Pro Val Val Gl - #y Leu Thr Met Ala Gly 1780 - # 1785 - # 1790 - - Ala Phe Met Gly Gly Ala Ser Val Ser Pro Se - #r Leu Val Thr Val Leu 1795 - # 1800 - # 1805 - - Leu Gly Ala Val Gly Gly Trp Glu Gly Val Va - #l Asn Ala Ala Ser Leu 1810 - # 1815 - # 1820 - - Val Phe Asp Phe Met Ala Gly Lys Leu Ser Th - #r Glu Asp Leu Trp Tyr 1825 1830 - # 1835 - # 1840 - - Ala Ile Pro Val Leu Thr Ser Pro Xaa Ala Gl - #y Leu Ala Gly Ile Ala 1845 - # 1850 - # 1855 - - Leu Gly Leu Val Leu Tyr Ser Ala Asn Asn Se - #r Gly Thr Thr Thr Trp 1860 - # 1865 - # 1870 - - Leu Asn Arg Leu Leu Thr Thr Leu Pro Arg Se - #r Ser Cys Ile Pro Asp 1875 - # 1880 - # 1885 - - Ser Tyr Phe Gln Gln Ala Asp Tyr Cys Asp Ly - #s Val Ser Ala Ile Val 1890 - # 1895 - # 1900 - - Arg Arg Leu Ser Leu Thr Arg Thr Val Val Al - #a Leu Val Asn Arg Glu 1905 1910 - # 1915 - # 1920 - - Pro Lys Val Asp Glu Val Gln Val Gly Tyr Va - #l Trp Asp Leu Trp Glu 1925 - # 1930 - # 1935 - - Trp Val Met Arg Gln Val Arg Met Val Met Se - #r Arg Leu Arg Ala Leu 1940 - # 1945 - # 1950 - - Cys Pro Val Val Ser Leu Pro Leu Trp His Cy - #s Gly Glu Gly Trp Ser 1955 - # 1960 - # 1965 - - Gly Glu Trp Leu Leu Asp Gly His Val Glu Se - #r Arg Cys Leu Cys Gly 1970 - # 1975 - # 1980 - - Cys Val Ile Thr Gly Asp Val Leu Asn Gly Gl - #n Leu Lys Asp Pro Val 1985 1990 - # 1995 - # 2000 - - Tyr Ser Thr Lys Leu Cys Arg His Tyr Trp Me - #t Gly Thr Val Pro Val 2005 - # 2010 - # 2015 - - Asn Met Leu Gly Tyr Gly Glu Thr Ser Pro Le - #u Leu Ala Ser Asp Thr 2020 - # 2025 - # 2030 - - Pro Lys Val Val Pro Phe Gly Thr Ser Gly Tr - #p Ala Glu Val Val Val 2035 - # 2040 - # 2045 - - Thr Pro Thr His Val Val Ile Arg Arg Thr Se - #r Cys Tyr Lys Leu Leu 2050 - # 2055 - # 2060 - - Arg Gln Gln Ile Leu Ser Ala Ala Val Ala Gl - #u Pro Tyr Tyr Val Asp 2065 2070 - # 2075 - # 2080 - - Gly Ile Pro Val Ser Trp Glu Ala Asp Ala Ar - #g Ala Pro Ala Met Val 2085 - # 2090 - # 2095 - - Tyr Gly Pro Gly Gln Ser Val Thr Ile Asp Gl - #y Glu Arg Tyr Thr Leu 2100 - # 2105 - # 2110 - - Pro His Gln Leu Arg Met Arg Asn Val Ala Pr - #o Ser Glu Val Ser Ser 2115 - # 2120 - # 2125 - - Glu Val Ser Ile Glu Ile Gly Thr Glu Thr Gl - #u Asp Ser Glu Leu Thr 2130 - # 2135 - # 2140 - - Glu Ala Asp Leu Pro Pro Ala Ala Ala Ala Le - #u Gln Ala Ile Glu Asn 2145 2150 - # 2155 - # 2160 - - Ala Ala Arg Ile Leu Glu Pro His Ile Asp Va - #l Xaa Met Glu Asp Cys 2165 - # 2170 - # 2175 - - Ser Thr Pro Ser Leu Cys Gly Ser Ser Arg Gl - #u Met Pro Val Trp Gly 2180 - # 2185 - # 2190 - - Glu Asp Ile Pro Arg Thr Pro Ser Pro Ala Le - #u Ile Ser Val Thr Glu 2195 - # 2200 - # 2205 - - Ser Ser Ser Asp Glu Lys Thr Leu Ser Val Th - #r Ser Ser Gln Glu Asp 2210 - # 2215 - # 2220 - - Thr Pro Ser Ser Asp Ser Phe Glu Val Ile Gl - #n Glu Ser Asp Thr Ala 2225 2230 - # 2235 - # 2240 - - Glu Ser Glu Glu Ser Val Phe Asn Val Ala Le - #u Ser Val Leu Lys Ala 2245 - # 2250 - # 2255 - - Leu Phe Pro Gln Ser Asp Ala Thr Arg Lys Le - #u Thr Val Lys Met Ser 2260 - # 2265 - # 2270 - - Cys Cys Val Glu Lys Ser Val Thr Arg Phe Ph - #e Ser Leu Gly Leu Thr 2275 - # 2280 - # 2285 - - Val Ala Asp Val Ala Ser Leu Cys Glu Met Gl - #u Ile Gln Asn His Thr 2290 - # 2295 - # 2300 - - Ala Tyr Cys Asp Lys Val Arg Thr Pro Leu Gl - #u Leu Gln Val Gly Cys 2305 2310 - # 2315 - # 2320 - - Leu Val Gly Asn Glu Leu Thr Phe Glu Cys As - #p Lys Cys Glu Ala Arg 2325 - # 2330 - # 2335 - - Gln Glu Thr Leu Ala Ser Phe Ser Tyr Ile Tr - #p Ser Gly Val Pro Leu 2340 - # 2345 - # 2350 - - Thr Arg Ala Thr Pro Ala Lys Pro Pro Val Va - #l Arg Pro Val Gly Ser 2355 - # 2360 - # 2365 - - Leu Leu Val Ala Asp Thr Thr Lys Val Tyr Va - #l Thr Asn Pro Asp Asn 2370 - # 2375 - # 2380 - - Val Gly Arg Arg Val Asp Lys Val Thr Phe Tr - #p Arg Ala Pro Arg Val 2385 2390 - # 2395 - # 2400 - - His Asp Lys Phe Leu Val Asp Ser Ile Glu Ar - #g Ala Arg Arg Ala Ala 2405 - # 2410 - # 2415 - - Gln Gly Cys Leu Ser Met Gly Tyr Thr Tyr Gl - #u Glu Ala Ile Arg Thr 2420 - # 2425 - # 2430 - - Val Arg Pro His Ala Ala Met Gly Trp Gly Se - #r Lys Val Ser Val Lys 2435 - # 2440 - # 2445 - - Asp Leu Ala Thr Pro Ala Gly Lys Met Ala Va - #l His Asp Arg Leu Gln 2450 - # 2455 - # 2460 - - Glu Ile Leu Glu Gly Thr Pro Val Pro Phe Th - #r Leu Thr Val Lys Lys 2465 2470 - # 2475 - # 2480 - - Glu Val Phe Phe Lys Asp Arg Lys Glu Glu Ly - #s Ala Pro Arg Leu Ile 2485 - # 2490 - # 2495 - - Val Phe Pro Pro Leu Asp Phe Arg Ile Ala Gl - #u Lys Leu Ile Leu Gly 2500 - # 2505 - # 2510 - - Asp Pro Gly Arg Val Ala Lys Ala Gly Val Gl - #y Gly Ala Tyr Ala Phe 2515 - # 2520 - # 2525 - - Gln Tyr Thr Pro Asn Gln Arg Val Lys Glu Me - #t Leu Lys Leu Trp Glu 2530 - # 2535 - # 2540 - - Ser Lys Lys Thr Pro Cys Ala Ile Cys Val As - #p Ala Thr Cys Phe Asp 2545 2550 - # 2555 - # 2560 - - Ser Ser Ile Thr Xaa Glu Asp Val Ala Leu Gl - #u Thr Glu Leu Tyr Ala 2565 - # 2570 - # 2575 - - Leu Ala Ser Asp His Pro Glu Trp Val Arg Al - #a Leu Gly Lys Tyr Xaa 2580 - # 2585 - # 2590 - - Ala Ser Gly Thr Met Val Thr Pro Glu Gly Va - #l Pro Val Gly Glu Arg 2595 - # 2600 - # 2605 - - Tyr Cys Arg Ser Ser Gly Val Leu Thr Thr Se - #r Ala Ser Asn Cys Leu 2610 - # 2615 - # 2620 - - Thr Cys Tyr Ile Lys Val Arg Ala Ala Cys Gl - #u Arg Ile Gly Leu Lys 2625 2630 - # 2635 - # 2640 - - Asn Val Ser Leu Leu Ile Ala Gly Asp Asp Cy - #s Leu Ile Val Cys Glu 2645 - # 2650 - # 2655 - - Arg Pro Val Cys Asp Pro Cys Glu Ala Leu Gl - #y Arg Thr Leu Ala Ser 2660 - # 2665 - # 2670 - - Tyr Gly Tyr Ala Cys Glu Pro Ser Tyr His Al - #a Ser Leu Asp Thr Ala 2675 - # 2680 - # 2685 - - Pro Phe Cys Ser Thr Trp Leu Ala Glu Cys As - #n Ala Asp Gly Xaa Arg 2690 - # 2695 - # 2700 - - His Phe Phe Leu Thr Thr Asp Phe Arg Arg Pr - #o Leu Ala Arg Met Ser 2705 2710 - # 2715 - # 2720 - - Ser Glu Tyr Ser Asp Pro Met Ala Ser Ala Il - #e Gly Tyr Ile Leu Leu 2725 - # 2730 - # 2735 - - Tyr Pro Trp Xaa Pro Ile Thr Arg Trp Val Il - #e Ile Pro His Val Leu 2740 - # 2745 - # 2750 - - Thr Cys Ala Ser Ser Arg Gly Gly Gly Thr Xa - #a Ser Asp Pro Val Trp 2755 - # 2760 - # 2765 - - Cys Gln Val His Gly Asn Tyr Tyr Lys Phe Pr - #o Leu Asp Lys Leu Pro 2770 - # 2775 - # 2780 - - Asn Ile Ile Val Ala Leu His Gly Pro Ala Al - #a Leu Arg Val Thr Ala 2785 2790 - # 2795 - # 2800 - - Asp Thr Thr Lys Thr Lys Met Glu Ala Gly Ly - #s Val Leu Ser Asp Leu 2805 - # 2810 - # 2815 - - Lys Leu Pro Gly Leu Ala Val His Arg Lys Ly - #s Ala Gly Ala Leu Arg 2820 - # 2825 - # 2830 - - Thr Arg Met Leu Arg Ser Arg Gly Trp Ala Gl - #u Leu Ala Arg Gly Leu 2835 - # 2840 - # 2845 - - Leu Trp His Pro Gly Leu Arg Leu Pro Pro Pr - #o Glu Ile Ala Gly Ile 2850 - # 2855 - # 2860 - - Pro Gly Gly Phe Pro Leu Ser Pro Pro Tyr Me - #t Gly Val Val His Gln 2865 2870 - # 2875 - # 2880 - - Leu Asp Phe Thr Xaa Gln Arg Ser Arg Trp Ar - #g Trp Leu Gly Phe Leu 2885 - # 2890 - # 2895 - - Ala Leu Leu Ile Val Ala Leu Phe Gly 2900 - # 2905 - - - - (2) INFORMATION FOR SEQ ID NO:34: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:9126 base pa - #irs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: - - CCCCCCCCCC GGCACTGGGT GCAAGCCCCA GAAACCGACG CCTACTGAAG TA -#GACGTAAT 60 - - GGCCCCGCGC CGAACCGGCG ACCGGCCAAA AGGTGGTGGA TGGGTGATGA CA -#GGGTTGGT 120 - - AGGTCGTAAA TCCCGGTCAT CCTGGTAGCC ACTATAGGTG GGTCTTAAGG GG -#AGGCTACG 180 - - GTCCCTCTTG CGCATATGGA GGAAAAGCGC ACGGTCCACA GGTGTTGGTC CT -#ACCGGTGT 240 - - AATAAGGACC CGGCGCTAGG CACGCCGTTA AACCGAGCCC GTTACTCCCC TG -#GGCAAACG 300 - - ACGCCCACGT ACGGTCCACG TCGCCCTTCA ATGTCTCTCT TGACCAATAG GC -#GTAGCCGG 360 - - CGAGTTGACA AGGACCAGTG GGGGCCGGGC GGGAGGGGGA AGGACCCCCA CC -#GCTGCCCT 420 - - TCCCGGGGAG GCGGGAAATG CATGGGGCCA CCCAGCTCCG CGGCGGCCTA CA -#GCCGGGGT 480 - - AGCCCAAGAA CCTTCGGGTG AGGGCGGGTG GCATTTCTTT TCCTATACCG AT -#CATGGCAG 540 - - TCCTTCTGCT CCTACTCGTG GTGGAGGCCG GGGCTATTTT AGCCCCGGCC AC -#CCATGCTT 600 - - GTAGCGCGAA AGGGCAATAT TTBCTCACAA ACTGTTGCGC CCTGGAGGAC AT -#AGGCTTCT 660 - - GCCTGGAGGG CGGATGCCTG GTGGCTCTGG GGTGCACCAT TTGCACCGAC CG -#CTGCTGGC 720 - - CACTGTATCA GGCGGGTTTG GCCGTGCGGC CCGGCAAGTC CGCCGCCCAG TT -#GGTGGGGG 780 - - AACTCGGTAG TCTCTACGGG CCCTTGTCGG TCTCGGCTTA TGTGGCCGGG AT -#CCTGGGGC 840 - - TTGGGGAGGT CTACTCGGGG GTCCTCACCG TCGGGGTGGC GTTGACGCGC AG -#GGTCTACC 900 - - CGGTCCCGAA CCTGACGTGT GCAGTAGAGT GTGAGTTGAA GTGGGAAAGT GA -#GTTTTGGA 960 - - GATGGACTGA ACAGCTGGCC TCAAACTACT GGATTCTGGA ATACCTCTGG AA -#GGTGCCTT 1020 - - TCGACTTTTG GCGGGGAGTG ATGAGCCTTT CTCCTCTCTT GGTGTGCGTG GC -#GGCCCTCC 1080 - - TCCTGCTGGA GCAGCGTATT GTCATGGTCT TCCTCCTGGT CACTATGGCG GG -#CATGTCAC 1140 - - AAGGCGCGCC CGCCTCAGTG TTGGGGTCAC GGCCTTTCGA GGCCGGGCTG AC -#TTGGCAGT 1200 - - CTTGTTCTTG CAGGTCGAAC GGGTCCCGCG CGCCGACAGG GGAGAGGGTT TG -#GGAACGTG 1260 - - GGAACGTCAC ACTTTTGTGT GACTGCCCCA ACGGTCCTTG GGTGTGGGTC CC -#GGCCCTTT 1320 - - GCCAGGCAAT CGGATGGGGC GACCCTATCA CTCATTGGAG CCACGGACGA AA -#TCAGTGGC 1380 - - CCCTTTCTTG TCCCCAATTT GTCTACGGCG CCGTTTCAGT GACCTGCGTG TG -#GGGTTCTG 1440 - - TGTCTTGGTT TGCTTCCACT GGGGGTCGCG ACTCCAAGGT TGATGTGTGG AG -#TTTGGTTC 1500 - - CAGTTGGCTC TGCCAGCTGT ACCATAGCCG CACTGGGATC TTCGGATCGC GA -#CACAGTGG 1560 - - TTGAGCTCTC CGAATGGGGA ATCCCCTGCG CCACTTGTAT CCTGGACAGG CG -#GCCTGCCT 1620 - - CGTGTGGCAC CTGTGTGAGG GACTGCTGGC CCGAGACCGG GTCGGTACGT TT -#CCCATTCC 1680 - - ACAGGTGTGG CGCGGGACCG AGGCTGACCA GAGACCTTGA GGCTGTGCCC TT -#CGTCAATA 1740 - - GGACAACTCC CTTCACCATA AGGGGGCCCC TGGGCAACCA GGGGCGAGGC GA -#CCCGGTGC 1800 - - GGTCGCCCTT GGGTTTTGGG TCCTACACCA TGACCAAGAT CCGAGACTCC TT -#ACACTTGG 1860 - - TGAAATGTCC CACCCCAGCC ATTGAGCCTC CCACCGGAAC GTTTGGGATC TT -#CCCAGGAG 1920 - - TCCCCCCCCT TAACAACTGC ATGCTTCTCG GCACTGAGGT GTCAGAGGTA TT -#GGGTGGGG 1980 - - CGGGCCTCAC TGGGGGGTTT TACGAACCTC TGGTGCGGCG GTGTTCAGAG CT -#GATGGGTC 2040 - - GGCGGAATCC GGTCTGCCCG GGGTTTGCAT GGCTCTCTTC GGGACGGCCT GA -#TGGGTTCA 2100 - - TACATGTACA GGGCCACTTG CAGGAGGTGG ATGCGGGCAA CTTCATTCCG CC -#CCCACGCT 2160 - - GGTTGCTCTT GGACTTTGTA TTTGTCCTGT CATACCTGAT GAAGCTGGCA GA -#GGCACGGT 2220 - - TGGTCCCGCT GATCCTCCTC CTGCTATGGT GGTGGGTGAA CCAGTTGGCG GT -#CCTTGKAC 2280 - - TGSCGGCTGC KCRCGCCGCC GTGGCTGGAG AGGTGTTTGC GGGCCCTGCC TT -#GTCCTGGT 2340 - - GTCTGGGCCT ACCCTTCGTG AGTATGATCC TGGGGCTAGC AAACCTGGTG TT -#GTACTTCC 2400 - - GCTGGATGGG TCCTCAACGC CTGATGTTCC TCGTGTTGTG GAAGCTCGCT CG -#GGGGGCTT 2460 - - TCCCGCTGGC ATTACTGATG GGGATTTCCG CCACTCGCGG CCGCACCTCT GT -#GCTTGGCG 2520 - - CCGAATTCTG CTTTGATGTC ACCTTTGAAG TGGACACGTC AGTCTTGGGT TG -#GGTGGTTG 2580 - - CTAGTGTGGT GGCTTGGGCC ATAGCGCTCC TGAGCTCTAT GAGCGCGGGG GG -#GTGGAAGC 2640 - - ACAAAGCCAT AATCTATAGG ACGTGGTGTA AAGGGTACCA GGCYCTTCGC CA -#GCGCGTGG 2700 - - TGCGTAGCCC CCTCGGGGAG GGGCGGCCCA CCAAGCCGCT GACGATAGCC TG -#GCGTCTGG 2760 - - CCTCTTACAT CTGGCCGGAC GCTGTGATGT TGGTGGTTGT GGCCATGGTC CT -#CCTCTTCG 2820 - - GCCTTTTCGA CGCGCTCGAT TGGGCCTTGG AGGAGCTCCT TGTGTCGCGG CC -#TTCGTTGC 2880 - - GTCGTTTGGC AAGGGTGGTG GAGTGTTGTG TGATGGCGGG CGAGAAGGCC AC -#TACCGTCC 2940 - - GGCTTGTGTC CAAGATGTGC GCGAGAGGGG CCTACCTGTT TGACCACATG GG -#GTCGTTCT 3000 - - CGCGCGCGGT CAAGGAGCGC TTGCTGGAGT GGGACGCGGC TTTGGAGMCC CT -#GTCATTCA 3060 - - CTAGGACGGA CTGCCGCATC ATACGAGACG CCGCCAGGAC TCTGAGCTGC GG -#CCAATGCG 3120 - - TCATGGGCTT GCCCGTGGTG GCTAGGCGCG GCGATGAGGT CCTGGTTGGG GT -#CTTTCAGG 3180 - - ATGTGAACCA CTTGCCTCCG GGGTTTGYTC CTACAGCGCC TGTTGTCATC CG -#TCGGTGCG 3240 - - GAAAGGGCTT CCTCGGGGTC ACTAAGGCTG CCTTGACTGG TCGGGATCCT GA -#CTTACACC 3300 - - CAGGAAACGT CATGGTTTTG GGGACGGCTA CCTCGCGCAG CATGGGAACG TG -#CTTAAACG 3360 - - GGTTGCTGTT CACGACATTC CATGGGGCTT CTTCCCGAAC CATTGCGACA CC -#TGTGGGGG 3420 - - CCCTTAACCC AAGGTGGTGG TCGGCCAGTG ATGACGTCAC GGTCTATCCC CT -#CCCCGATG 3480 - - GAGCTAACTC GTTGGTTCCC TGCTCGTGTC AGGCTGAGTC CTGTTGGGTC AT -#YCGATCCG 3540 - - ATGGGGCTCT TTGCCATGGC TTGAGCAAGG GGGACAAGGT AGAACTGGAC GT -#GGCCATGG 3600 - - AGGTTGCTGA CTTTCGTGGG TCGTCTGGGT CTCCTGTCCT ATGCGACGAG GG -#GCACGCTG 3660 - - TAGGAATGCT CGTGTCCGTC CTTCATTCGG GGGGGAGGGT GACCGCGGCT CG -#ATTCACTC 3720 - - GGCCGTGGAC CCAAGTCCCA ACAGACGCCA AGACTACCAC TGAGCCACCC CC -#GGTGCCAG 3780 - - CTAAAGGGGT TTTCAAAGAG GCTCCTCTTT TCATGCCAAC AGGGGCGGGG AA -#AAGCACAC 3840 - - GCGTCCCTTT GGAATATGGA AACATGGGGC ACAAGGTCCT GCTTCTCAAC CC -#GTCGGTTG 3900 - - CCACTGTGAG GGCCATGGGC CCTTACATGG AGAAGCTGGC GGGGAAACAT CC -#TAGCATTT 3960 - - TCTGTGGACA CGACACAACA GCTTTCACAC GGATCACGGA CTCTCCATTG AC -#GTACTCTA 4020 - - CCTATGGGAG GTTTCTGGCC AACCCGAGGC AGATGCTGAG GGGAGTTTCC GT -#GGTCATCT 4080 - - GTGATGAGTG CCACAGTCAT GACTCAACTG TGTTGCTGGG TATAGGCAGG GG -#CAGGGAGC 4140 - - TGGCGCGGGG GTGTGGAGTG CAATTAGTGC TCTACGCTAC TGCGACTCCC CC -#GGGCTCGC 4200 - - CTATGACTCA GCATCCATCC ATAATTGAGA CAAAGCTGGA CGTCGGTGAG AT -#CCCCTTTT 4260 - - ATGGGCATGG TATCCCCCTC GAGCGTATGA GGACTGGTCG CCACCTTGTA TT -#CTGCCATT 4320 - - CCAAGGCGGA GTGCGAGAGA TTGGCCGGCC AGTTCTCCGC GCGGGGGGTT AA -#TGCCATCG 4380 - - CCTATTATAG GGGTAAGGAC AGTTCCATCA TCAAAGACGG AGACCTGGTG GT -#TTGTGCGA 4440 - - CAGACGCGCT CTCTACCGGG TACACAGGAA ACTTCGATTC TGTCACCGAC TG -#TGGGTTAG 4500 - - TGGTGGAGGA GGTCGTTGAG GTGACCCTTG ATCCCACCAT TACCATTTCC TT -#GCGGACTG 4560 - - TCCCTGCTTC GGCTGAATTG TCGATGCAGC GGCGCGGACG CACGGGGAGA GG -#TCGGTCGG 4620 - - GCCGCTACTA CTACGCTGGG GTCGGTAAGG CTCCCGCGGG GGTGGTGCGG TC -#TGGTCCGG 4680 - - TCTGGTCGGC AGTGGAAGCT GGAGTGACCT GGTATGGAAT GGAACCTGAC TT -#GACAGCAA 4740 - - ACCTTCTGAG ACTTTACGAC GACTGCCCTT ACACCGCAGC CGTCGCAGCT GA -#CATTGGTG 4800 - - AAGCCGCGGT GTTCTTTGCG GGCCTCGCGC CCCTCAGGAT GCATCCCGAT GT -#TAGCTGGG 4860 - - CAAAAGTTCG CGGCGTCAAT TGGCCCCTCC TGGTGGGTGT TCAGCGGACG AT -#GTGTCGGG 4920 - - AAACACTGTC TCCCGGCCCG TCGGACGACC CTCAGTGGGC AGGTCTGAAA GG -#CCCGAATC 4980 - - CTGCCCCACT ACTGCTGAGG TGGGGCAATG ATTTGCCATC AAAAGTGGCC GG -#CCACCACA 5040 - - TAGTTGACGA TCTGGTCCGT CGGCTCGGTG TGGCGGAGGG ATACGTGCGC TG -#TGATGCTG 5100 - - GRCCCATCCT CATGGTGGGC TTGGCCATAG CGGGCGGCAT GATCTACGCC TC -#TTACACTG 5160 - - GGTCGCTAGT GGTGGTAACA GACTGGAATG TGAAGGGAGG TGGCAATCCC CT -#TTATAGGA 5220 - - GTGGTGACCA GGCCACCCCT CAACCCGTGG TGCAGGTCCC CCCGGTAGAC CA -#TCGGCCGG 5280 - - GGGGGGAGTC TGCGCCAGCG GATGCCAAGA CAGTGACAGA TGCGGTGGCA GC -#CATCCAGG 5340 - - TGAACTGCGA TTGGTCTGTG ATGACCCTGT CGATCGGGGA AGTCCTCACC TT -#GGCTCAGG 5400 - - CTAAGACAGC CGAGGCCTAC GCAGCTACTT CCAGGTGGCT CGCTGGCTGC TA -#CACGGGGA 5460 - - CGCGGGCCGT CCCCACTGTA TCAATTGTTG ACAAGCTCTT CGCCGGGGGT TG -#GGCCGCCG 5520 - - TGGTGGGTCA CTGTCACAGC GTCATTGCTG CGGTGGTGGC TGCCTATGGG GT -#TTCTCGAA 5580 - - GTCCTCCACT GGCCGCGGCG GCATCCTACC TCATGGGGTT GGGCGTCGGA GG -#CAACGCAC 5640 - - AGGCGCGCTT GGCTTCAGCT CTTCTACTGG GGGCTGCTGG TACGGCTCTG GG -#GACCCCTG 5700 - - TCGTGGGACT CACCATGGCG GGGGCCTTCA TGGGCGGTGC CAGCGTGTCC CC -#CTCCCTCG 5760 - - TCACTGTCCT ACTTGGGGCT GTGGGAGGTT GGGAGGGCGT TGTCAACGCT GC -#CAGTCTCG 5820 - - TCTTCGACTT CATGGCTGGG AAACTTTCAA CAGAAGACCT TTGGTATGCC AT -#CCCGGTAC 5880 - - TCACTAGTCC TGGRGCGGGC CTCGCGGGGA TTGCCCTTGG TCTGGTTTTG TA -#CTCAGCAA 5940 - - ACAACTCTGG CACTACCACA TGGCTGAACC GTCTGCTGAC GACGTTGCCA CG -#GTCATCTT 6000 - - GCATACCCGA CAGCTACTTC CAACAGGCTG ACTACTGCGA CAAGGTCTCG GC -#AATGCTGC 6060 - - GCCGCCTGAG CCTTACTCGC ACCGTGGTGG CCCTGGTCAA CAGGGAGCCT AA -#GGTGGATG 6120 - - AGGTCCAGGT GGGGTACGTC TGGGATCTGT GGGAGTGGGT AATGCGCCAG GT -#GCGCATGG 6180 - - TGATGTCTAG ACTCCGGGCC CTCTGCCCTG TGGTGTCACT CCCCTTGTGG CA -#CCGCGGGG 6240 - - AGGGGTGGTC CGGTGAATGG CTTCTCGATG GGCACGTGGA GAGTCGTTGT CT -#GTGCGGGT 6300 - - GTGTAATCAC CGGCGACGTC CTCAATGGGC AACTCAAAGA TCCAGTTTAC TC -#TACCAAGC 6360 - - TGTGCAGGCA CTACTGGATG GGAACTGTGC CGGTCAACAT GCTGGGCTAC GG -#GGAAACCT 6420 - - CACCTCTTCT CGCCTCTGAC ACCCCGAAGG TGGTACCCTT CGGGACGTCG GG -#GTGGGCTG 6480 - - AGGTGGTGGT GACCCCTACC CACGTGGTGA TCAGGCGCAC GTCCTGTTAC AA -#ACTGCTTC 6540 - - GCCAGCAAAT TCTTTCAGCA GCTGTAGCTG AGCCCTACTA CGTTGATGGC AT -#TCCGGTCT 6600 - - CTTGGGAGGC TGACGCGAGA GCGCCGGCCA TGGTCTACGG TCCGGGCCAA AG -#TGTTACCA 6660 - - TTGATGGGGA GCGCTACACC CTTCCGCACC AGTTGCGGAT GCGGAATGTG GC -#GCCCTCTG 6720 - - AGGTTTCATC CGAGGTCAGC ATCGAGATCG GGACGGAGAC TGAAGACTCA GA -#ACTGACTG 6780 - - AGGCCGATTT GCCACCAGCG GCTGCTGCCC TCCAAGCGAT AGAGAATGCT GC -#GAGAATTC 6840 - - TCGAACCGCA CATCGATGTC AYCATGGAGG ATTGCAGTAC ACCCTCTCTC TG -#TGGTAGTA 6900 - - GCCGAGAGAT GCCTGTGTGG GGAGAAGACA TACCCCGCAC TCCATCGCCT GC -#ACTTATCT 6960 - - CGGTTACGGA GAGCAGCTCA GATGAGAAGA CCCTGTCGGT GACCTCCTCG CA -#GGAGGACA 7020 - - CCCCGTCCTC AGACTCATTT GAAGTCATCC AAGAGTCTGA TACTGCTGAA TC -#AGAGGAAA 7080 - - GCGTCTTCAA CGTGGCTCTT TCCGTACTAA AAGCATTATT TCCACAGAGC GT -#TGCCACAC 7140 - - GAAAGCTAAC GGTTAAGATG TCTTGCTGTG TTGAGAAGAG CGTAACACGC TT -#CTTTTCTT 7200 - - TAGGGTTGAC CGTGGCTGAC GTGGCTAGCC TGTGTGAGAT GGAGATCCAG AA -#CCATACAG 7260 - - CCTATTGTGA CAAGGTGCGC ACTCCGCTCG AATTGCAAGT TGGGTGCTTG GT -#GGGCAATG 7320 - - AACTTACCTT TGAATGTGAC AAGTGTGAGG CACGCCAAGA GACCCTTGCC TC -#CTTCTCCT 7380 - - ACATATGGTC CGGGGTCCCA CTTACTCGGG CCACTCCGGC CAAACCACCA GT -#GGTGAGGC 7440 - - CGGTGGGGTC CTTGTTGGTG GCAGACACCA CCAAGGTCTA CGTGACCAAT CC -#GGACAATG 7500 - - TTGGGAGGAG GGTTGACAAG GTGACTTTCT GGCGCGCTCC TCGGGTACAC GA -#CAAGTTCC 7560 - - TCGTGGACTC GATCGAGCGC GCTCGGAGAG CTGCTCAAGG CTGCCTAAGC AT -#GGGTTACA 7620 - - CTTATGAGGA GGCAATAAGG ACTGTTAGGC CGCATGCTGC CATGGGCTGG GG -#ATCTAAGG 7680 - - TGTCGGTCAG GGACTTGGCC ACCCCTGCGG GGAAGATGGC TGTTCATGAC CG -#GCTTCAGG 7740 - - AGATACTTGA AGGGACTCCA GTCCCTTTTA CCCTGACTGT CAAAAAGGAG GT -#GTTCTTCA 7800 - - AAGATCGTAA GGAGGAGAAG GCCCCCCGCC TCATTGTGTT CCCCCCCCTG GA -#CTTCCGGA 7860 - - TAGCTGAAAA GCTCATTCTG GGAGACCCGG GGCGGGTTGC AAAGGCGGTG TG -#GGGGGGGG 7920 - - CTTACGCCTT CCAGTACACC CCCAACCAGC GGGTTAAGGA GATGCTAAAG CT -#GTGGGAAT 7980 - - CAAAGAAGAC CCCGTGCGCC ATCTGTGTGG ATGCCACTTG CTTCGACAGT AG -#CATTACTG 8040 - - ARGAGGACGT GGCACTAGAG ACAGAGCTTT ACGCCCTGGC CTCGGACCAT CC -#AGAATGGG 8100 - - TGCGCGCCCT GGGGAAATAC TRTGCCTCTG GCACAATGGT GACCCCGGAA GG -#GGTGCCAG 8160 - - TGGGCGAGAG GTATTGTAGG TCCTCGGGTG TGTTAACCAC AAGTGCTAGC AA -#CTGTTTGA 8220 - - CCTGCTACAT CAAAGTGAGA GCCGCCTGTG AGAGGATCGG ACTGAAAAAT GT -#CTCGCTTC 8280 - - TCATCGCGGG CGATGACTGC TTAATTGTGT GCGAGAGGCC TGTATGCGAC CC -#TTGCGAGG 8340 - - CCCTGGGCCG AGCCCTGGCT TCGTACGGGT ACGCGTGTGA GCCCTCGTAT CA -#CGCTTCAC 8400 - - TGGACACAGC CCCCTTCTGC TCCACTTGGC TTGCTGAGTG CAATGCGGAT GG -#GRAAAGGC 8460 - - ATTTCTTCCT GACCACGGAC TTTCGGAGAC CACTCGCTCG CATGTCGAGC GA -#GTACAGTG 8520 - - ACCCTATGGC TTCGGCCATT GGTTACATTC TCCTCTATCC CTGGCRTCCC AT -#CACACGGT 8580 - - GGGTCATCAT CCCGCATGTG CTAACATGCG CTTCTTTCCG GGGTGGTGGC AC -#ACSGTCTG 8640 - - ATCCGGTTTG GTGTCAGGTT CATGGTAACT ACTACAAGTT TCCCCTGGAC AA -#ACTGCCTA 8700 - - ACATCATCGT GGCCCTCCAC GGACCAGCAG CGTTGAGGGT TACCGCAGAC AC -#AACCAAAA 8760 - - CAAAGATGGA GGCTGGGAAG GTTCTGAGCG ACCTCAAGCT CCCTGGTCTA GC -#CGTCCACC 8820 - - GCAAGAAGGC CGGGGCATTG CGAACACGCA TGCTCCGGTC GCGCGGTTGG GC -#GGAGTTGG 8880 - - CTAGGGGCCT GTTGTGGCAT CCAGGACTCC GGCTTCCTCC CCCTGAGATT GC -#TGGTATCC 8940 - - CAGGGGGTTT CCCTCTGTCC CCCCCCTACA TGGGGGTGGT TCATCAATTG GA -#TTTCACAG 9000 - - CSCAGCGGAG TCGCTGGCGG TGGTTGGGGT TCTTAGCCCT GCTCATCGTA GC -#GCTCTTTG 9060 - - GGTGAACTAA ATTCATCTGT TGCGGCCGGA GTCAGACCTG AGCCCCGTTC AA -#AAGGGGAT 9120 - - TGAGAC - # - # -# 9126 - - - - (2) INFORMATION FOR SEQ ID NO:35: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2860 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: - - Pro Lys Asn Leu Arg Val Arg Ala Gly Gly Il - #e Ser Phe Pro Ile Pro - #5 - #10 - #15 - - Ile Met Ala Val Leu Leu Leu Leu Leu Val Va - #l Glu Ala Gly Ala Ile 20 - # 25 - # 30 - - Leu Ala Pro Ala Thr His Ala Cys Ser Ala Ly - #s Gly Gln Tyr Xaa Leu 35 - # 40 - # 45 - - Thr Asn Cys Cys Ala Leu Glu Asp Ile Gly Ph - #e Cys Leu Glu Gly Gly 50 - # 55 - # 60 - - Cys Leu Val Ala Leu Gly Cys Thr Ile Cys Th - #r Asp Arg Cys Trp Pro 65 - # 70 - # 75 - # 80 - - Leu Tyr Gln Ala Gly Leu Ala Val Arg Pro Gl - #y Lys Ser Ala Ala Gln 85 - # 90 - # 95 - - Leu Val Gly Glu Leu Gly Ser Leu Tyr Gly Pr - #o Leu Ser Val Ser Ala 100 - # 105 - # 110 - - Tyr Val Ala Gly Ile Leu Gly Leu Gly Glu Va - #l Tyr Ser Gly Val Leu 115 - # 120 - # 125 - - Thr Val Gly Val Ala Leu Thr Arg Arg Val Ty - #r Pro Val Pro Asn Leu 130 - # 135 - # 140 - - Thr Cys Ala Val Glu Cys Glu Leu Lys Trp Gl - #u Ser Glu Phe Trp Arg 145 1 - #50 1 - #55 1 -#60 - - Trp Thr Glu Gln Leu Ala Ser Asn Tyr Trp Il - #e Leu Glu Tyr LeuTrp 165 - # 170 - # 175 - - Lys Val Pro Phe Asp Phe Trp Arg Gly Val Me - #t Ser Leu Ser Pro Leu 180 - # 185 - # 190 - - Leu Val Cys Val Ala Ala Leu Leu Leu Leu Gl - #u Gln Arg Ile Val Met 195 - # 200 - # 205 - - Val Phe Leu Leu Val Thr Met Ala Gly Met Se - #r Gln Gly Ala Pro Ala 210 - # 215 - # 220 - - Ser Val Leu Gly Ser Arg Pro Phe Glu Ala Gl - #y Leu Thr Trp Gln Ser 225 2 - #30 2 - #35 2 -#40 - - Cys Ser Cys Arg Ser Asn Gly Ser Arg Ala Pr - #o Thr Gly Glu ArgVal 245 - # 250 - # 255 - - Trp Glu Arg Gly Asn Val Thr Leu Leu Cys As - #p Cys Pro Asn Gly Pro 260 - # 265 - # 270 - - Trp Val Trp Val Pro Ala Leu Cys Gln Ala Il - #e Gly Trp Gly Asp Pro 275 - # 280 - # 285 - - Ile Thr His Trp Ser His Gly Arg Asn Gln Tr - #p Pro Leu Ser Cys Pro 290 - # 295 - # 300 - - Gln Phe Val Tyr Gly Ala Val Ser Val Thr Cy - #s Val Trp Gly Ser Val 305 3 - #10 3 - #15 3 -#20 - - Ser Trp Phe Ala Ser Thr Gly Gly Arg Asp Se - #r Lys Val Asp ValTrp 325 - # 330 - # 335 - - Ser Leu Val Pro Val Gly Ser Ala Ser Cys Th - #r Ile Ala Ala Leu Gly 340 - # 345 - # 350 - - Ser Ser Asp Arg Asp Thr Val Val Glu Leu Se - #r Glu Trp Gly Ile Pro 355 - # 360 - # 365 - - Cys Ala Thr Cys Ile Leu Asp Arg Arg Pro Al - #a Ser Cys Gly Thr Cys 370 - # 375 - # 380 - - Val Arg Asp Cys Trp Pro Glu Thr Gly Ser Va - #l Arg Phe Pro Phe His 385 3 - #90 395 - # 400 - - Arg Cys Gly Ala Gly Pro Arg Leu Thr Arg As - #p Leu Glu Ala Val Pro 405 - # 410 - # 415 - - Phe Val Asn Arg Thr Thr Pro Phe Thr Ile Ar - #g Gly Pro Leu Gly Asn 420 - # 425 - # 430 - - Gln Gly Arg Gly Asp Pro Val Arg Ser Pro Le - #u Gly Phe Gly Ser Tyr 435 - # 440 - # 445 - - Thr Met Thr Lys Ile Arg Asp Ser Leu His Le - #u Val Lys Cys Pro Thr 450 - # 455 - # 460 - - Pro Ala Ile Glu Pro Pro Thr Gly Thr Phe Gl - #y Ile Phe Pro Gly Val 465 4 - #70 4 - #75 4 -#80 - - Pro Pro Leu Asn Asn Cys Met Leu Leu Gly Th - #r Glu Val Ser GluVal 485 - # 490 - # 495 - - Leu Gly Gly Ala Gly Leu Thr Gly Gly Phe Ty - #r Glu Pro Leu Val Arg 500 - # 505 - # 510 - - Arg Cys Ser Glu Leu Met Gly Arg Arg Asn Pr - #o Val Cys Pro Gly Phe 515 - # 520 - # 525 - - Ala Trp Leu Ser Ser Gly Arg Pro Asp Gly Ph - #e Ile His Val Gln Gly 530 - # 535 - # 540 - - His Leu Gln Glu Val Asp Ala Gly Asn Phe Il - #e Pro Pro Pro Arg Trp 545 5 - #50 5 - #55 5 -#60 - - Leu Leu Leu Asp Phe Val Phe Val Leu Ser Ty - #r Leu Met Lys LeuAla 565 - # 570 - # 575 - - Glu Ala Arg Leu Val Pro Leu Ile Leu Leu Le - #u Leu Trp Trp Trp Val 580 - # 585 - # 590 - - Asn Gln Leu Ala Val Leu Xaa Leu Xaa Ala Al - #a Xaa Ala Ala Val Ala 595 - # 600 - # 605 - - Gly Glu Val Phe Ala Gly Pro Ala Leu Ser Tr - #p Cys Leu Gly Leu Pro 610 - # 615 - # 620 - - Phe Val Ser Met Ile Leu Gly Leu Ala Asn Le - #u Val Leu Tyr Phe Arg 625 6 - #30 6 - #35 6 -#40 - - Trp Met Gly Pro Gln Arg Leu Met Phe Leu Va - #l Leu Trp Lys LeuAla 645 - # 650 - # 655 - - Arg Gly Ala Phe Pro Leu Ala Leu Leu Met Gl - #y Ile Ser Ala Thr Arg 660 - # 665 - # 670 - - Gly Arg Thr Ser Val Leu Gly Ala Glu Phe Cy - #s Phe Asp Val Thr Phe 675 - # 680 - # 685 - - Glu Val Asp Thr Ser Val Leu Gly Trp Val Va - #l Ala Ser Val Val Ala 690 - # 695 - # 700 - - Trp Ala Ile Ala Leu Leu Ser Ser Met Ser Al - #a Gly Gly Trp Lys His 705 7 - #10 7 - #15 7 -#20 - - Lys Ala Ile Ile Tyr Arg Thr Trp Cys Lys Gl - #y Tyr Gln Ala LeuArg 725 - # 730 - # 735 - - Gln Arg Val Val Arg Ser Pro Leu Gly Glu Gl - #y Arg Pro Thr Lys Pro 740 - # 745 - # 750 - - Leu Thr Ile Ala Trp Arg Leu Ala Ser Tyr Il - #e Trp Pro Asp Ala Val 755 - # 760 - # 765 - - Met Leu Val Val Val Ala Met Val Leu Leu Ph - #e Gly Leu Phe Asp Ala 770 - # 775 - # 780 - - Leu Asp Trp Ala Leu Glu Glu Leu Leu Val Se - #r Arg Pro Ser Leu Arg 785 7 - #90 7 - #95 8 -#00 - - Arg Leu Ala Arg Val Val Glu Cys Cys Val Me - #t Ala Gly Glu LysAla 805 - # 810 - # 815 - - Thr Thr Val Arg Leu Val Ser Lys Met Cys Al - #a Arg Gly Ala Tyr Leu 820 - # 825 - # 830 - - Phe Asp His Met Gly Ser Phe Ser Arg Ala Va - #l Lys Glu Arg Leu Leu 835 - # 840 - # 845 - - Glu Trp Asp Ala Ala Leu Glu Xaa Leu Ser Ph - #e Thr Arg Thr Asp Cys 850 - # 855 - # 860 - - Arg Ile Ile Arg Asp Ala Ala Arg Thr Leu Se - #r Cys Gly Gln Cys Val 865 8 - #70 8 - #75 8 -#80 - - Met Gly Leu Pro Val Val Ala Arg Arg Gly As - #p Glu Val Leu ValGly 885 - # 890 - # 895 - - Val Phe Gln Asp Val Asn His Leu Pro Pro Gl - #y Phe Xaa Pro Thr Ala 900 - # 905 - # 910 - - Pro Val Val Ile Arg Arg Cys Gly Lys Gly Ph - #e Leu Gly Val Thr Lys 915 - # 920 - # 925 - - Ala Ala Leu Thr Gly Arg Asp Pro Asp Leu Hi - #s Pro Gly Asn Val Met 930 - # 935 - # 940 - - Val Leu Gly Thr Ala Thr Ser Arg Ser Met Gl - #y Thr Cys Leu Asn Gly 945 9 - #50 9 - #55 9 -#60 - - Leu Leu Phe Thr Thr Phe His Gly Ala Ser Se - #r Arg Thr Ile AlaThr 965 - # 970 - # 975 - - Pro Val Gly Ala Leu Asn Pro Arg Trp Trp Se - #r Ala Ser Asp Asp Val 980 - # 985 - # 990 - - Thr Val Tyr Pro Leu Pro Asp Gly Ala Asn Se - #r Leu Val Pro Cys Ser 995 - # 1000 - # 1005 - - Cys Gln Ala Glu Ser Cys Trp Val Ile Arg Se - #r Asp Gly Ala Leu Cys 1010 - # 1015 - # 1020 - - His Gly Leu Ser Lys Gly Asp Lys Val Glu Le - #u Asp Val Ala Met Glu 1025 1030 - # 1035 - # 1040 - - Val Ala Asp Phe Arg Gly Ser Ser Gly Ser Pr - #o Val Leu Cys Asp Glu 1045 - # 1050 - # 1055 - - Gly His Ala Val Gly Met Leu Val Ser Val Le - #u His Ser Gly Gly Arg 1060 - # 1065 - # 1070 - - Val Thr Ala Ala Arg Phe Thr Arg Pro Trp Th - #r Gln Val Pro Thr Asp 1075 - # 1080 - # 1085 - - Ala Lys Thr Thr Thr Glu Pro Pro Pro Val Pr - #o Ala Lys Gly Val Phe 1090 - # 1095 - # 1100 - - Lys Glu Ala Pro Leu Phe Met Pro Thr Gly Al - #a Gly Lys Ser Thr Arg 1105 1110 - # 1115 - # 1120 - - Val Pro Leu Glu Tyr Gly Asn Met Gly His Ly - #s Val Leu Leu Leu Asn 1125 - # 1130 - # 1135 - - Pro Ser Val Ala Thr Val Arg Ala Met Gly Pr - #o Tyr Met Glu Lys Leu 1140 - # 1145 - # 1150 - - Ala Gly Lys His Pro Ser Ile Phe Cys Gly Hi - #s Asp Thr Thr Ala Phe 1155 - # 1160 - # 1165 - - Thr Arg Ile Thr Asp Ser Pro Leu Thr Tyr Se - #r Thr Tyr Gly Arg Phe 1170 - # 1175 - # 1180 - - Leu Ala Asn Pro Arg Gln Met Leu Arg Gly Va - #l Ser Val Val Ile Cys 1185 1190 - # 1195 - # 1200 - - Asp Glu Cys His Ser His Asp Ser Thr Val Le - #u Leu Gly Ile Gly Arg 1205 - # 1210 - # 1215 - - Gly Arg Glu Leu Ala Arg Gly Cys Gly Val Gl - #n Leu Val Leu Tyr Ala 1220 - # 1225 - # 1230 - - Thr Ala Thr Pro Pro Gly Ser Pro Met Thr Gl - #n His Pro Ser Ile Ile 1235 - # 1240 - # 1245 - - Glu Thr Lys Leu Asp Val Gly Glu Ile Pro Ph - #e Tyr Gly His Gly Ile 1250 - # 1255 - # 1260 - - Pro Leu Glu Arg Met Arg Thr Gly Arg His Le - #u Val Phe Cys His Ser 1265 1270 - # 1275 - # 1280 - - Lys Ala Glu Cys Glu Arg Leu Ala Gly Gln Ph - #e Ser Ala Arg Gly Val 1285 - # 1290 - # 1295 - - Asn Ala Ile Ala Tyr Tyr Arg Gly Lys Asp Se - #r Ser Ile Ile Lys Asp 1300 - # 1305 - # 1310 - - Gly Asp Leu Val Val Cys Ala Thr Asp Ala Le - #u Ser Thr Gly Tyr Thr 1315 - # 1320 - # 1325 - - Gly Asn Phe Asp Ser Val Thr Asp Cys Gly Le - #u Val Val Glu Glu Val 1330 - # 1335 - # 1340 - - Val Glu Val Thr Leu Asp Pro Thr Ile Thr Il - #e Ser Leu Arg Thr Val 1345 1350 - # 1355 - # 1360 - - Pro Ala Ser Ala Glu Leu Ser Met Gln Arg Ar - #g Gly Arg Thr Gly Arg 1365 - # 1370 - # 1375 - - Gly Arg Ser Gly Arg Tyr Tyr Tyr Ala Gly Va - #l Gly Lys Ala Pro Ala 1380 - # 1385 - # 1390 - - Gly Val Val Arg Ser Gly Pro Val Trp Ser Al - #a Val Glu Ala Gly Val 1395 - # 1400 - # 1405 - - Thr Trp Tyr Gly Met Glu Pro Asp Leu Thr Al - #a Asn Leu Leu Arg Leu 1410 - # 1415 - # 1420 - - Tyr Asp Asp Cys Pro Tyr Thr Ala Ala Val Al - #a Ala Asp Ile Gly Glu 1425 1430 - # 1435 - # 1440 - - Ala Ala Val Phe Phe Ala Gly Leu Ala Pro Le - #u Arg Met His Pro Asp 1445 - # 1450 - # 1455 - - Val Ser Trp Ala Lys Val Arg Gly Val Asn Tr - #p Pro Leu Leu Val Gly 1460 - # 1465 - # 1470 - - Val Gln Arg Thr Met Cys Arg Glu Thr Leu Se - #r Pro Gly Pro Ser Asp 1475 - # 1480 - # 1485 - - Asp Pro Gln Trp Ala Gly Leu Lys Gly Pro As - #n Pro Ala Pro Leu Leu 1490 - # 1495 - # 1500 - - Leu Arg Trp Gly Asn Asp Leu Pro Ser Lys Va - #l Ala Gly His His Ile 1505 1510 - # 1515 - # 1520 - - Val Asp Asp Leu Val Arg Arg Leu Gly Val Al - #a Glu Gly Tyr Val Arg 1525 - # 1530 - # 1535 - - Cys Asp Ala Gly Pro Ile Leu Met Val Gly Le - #u Ala Ile Ala Gly Gly 1540 - # 1545 - # 1550 - - Met Ile Tyr Ala Ser Tyr Thr Gly Ser Leu Va - #l Val Val Thr Asp Trp 1555 - # 1560 - # 1565 - - Asn Val Lys Gly Gly Gly Asn Pro Leu Tyr Ar - #g Ser Gly Asp Gln Ala 1570 - # 1575 - # 1580 - - Thr Pro Gln Pro Val Val Gln Val Pro Pro Va - #l Asp His Arg Pro Gly 1585 1590 - # 1595 - # 1600 - - Gly Glu Ser Ala Pro Ala Asp Ala Lys Thr Va - #l Thr Asp Ala Val Ala 1605 - # 1610 - # 1615 - - Ala Ile Gln Val Asn Cys Asp Trp Ser Val Me - #t Thr Leu Ser Ile Gly 1620 - # 1625 - # 1630 - - Glu Val Leu Thr Leu Ala Gln Ala Lys Thr Al - #a Glu Ala Tyr Ala Ala 1635 - # 1640 - # 1645 - - Thr Ser Arg Trp Leu Ala Gly Cys Tyr Thr Gl - #y Thr Arg Ala Val Pro 1650 - # 1655 - # 1660 - - Thr Val Ser Ile Val Asp Lys Leu Phe Ala Gl - #y Gly Trp Ala Ala Val 1665 1670 - # 1675 - # 1680 - - Val Gly His Cys His Ser Val Ile Ala Ala Va - #l Val Ala Ala Tyr Gly 1685 - # 1690 - # 1695 - - Val Ser Arg Ser Pro Pro Leu Ala Ala Ala Al - #a Ser Tyr Leu Met Gly 1700 - # 1705 - # 1710 - - Leu Gly Val Gly Gly Asn Ala Gln Ala Arg Le - #u Ala Ser Ala Leu Leu 1715 - # 1720 - # 1725 - - Leu Gly Ala Ala Gly Thr Ala Leu Gly Thr Pr - #o Val Val Gly Leu Thr 1730 - # 1735 - # 1740 - - Met Ala Gly Ala Phe Met Gly Gly Ala Ser Va - #l Ser Pro Ser Leu Val 1745 1750 - # 1755 - # 1760 - - Thr Val Leu Leu Gly Ala Val Gly Gly Trp Gl - #u Gly Val Val Asn Ala 1765 - # 1770 - # 1775 - - Ala Ser Leu Val Phe Asp Phe Met Ala Gly Ly - #s Leu Ser Thr Glu Asp 1780 - # 1785 - # 1790 - - Leu Trp Tyr Ala Ile Pro Val Leu Thr Ser Pr - #o Gly Ala Gly Leu Ala 1795 - # 1800 - # 1805 - - Gly Ile Ala Leu Gly Leu Val Leu Tyr Ser Al - #a Asn Asn Ser Gly Thr 1810 - # 1815 - # 1820 - - Thr Thr Trp Leu Asn Arg Leu Leu Thr Thr Le - #u Pro Arg Ser Ser Cys 1825 1830 - # 1835 - # 1840 - - Ile Pro Asp Ser Tyr Phe Gln Gln Ala Asp Ty - #r Cys Asp Lys Val Ser 1845 - # 1850 - # 1855 - - Ala Met Leu Arg Arg Leu Ser Leu Thr Arg Th - #r Val Val Ala Leu Val 1860 - # 1865 - # 1870 - - Asn Arg Glu Pro Lys Val Asp Glu Val Gln Va - #l Gly Tyr Val Trp Asp 1875 - # 1880 - # 1885 - - Leu Trp Glu Trp Val Met Arg Gln Val Arg Me - #t Val Met Ser Arg Leu 1890 - # 1895 - # 1900 - - Arg Ala Leu Cys Pro Val Val Ser Leu Pro Le - #u Trp His Arg Gly Glu 1905 1910 - # 1915 - # 1920 - - Gly Trp Ser Gly Glu Trp Leu Leu Asp Gly Hi - #s Val Glu Ser Arg Cys 1925 - # 1930 - # 1935 - - Leu Cys Gly Cys Val Ile Thr Gly Asp Val Le - #u Asn Gly Gln Leu Lys 1940 - # 1945 - # 1950 - - Asp Pro Val Tyr Ser Thr Lys Leu Cys Arg Hi - #s Tyr Trp Met Gly Thr 1955 - # 1960 - # 1965 - - Val Pro Val Asn Met Leu Gly Tyr Gly Glu Th - #r Ser Pro Leu Leu Ala 1970 - # 1975 - # 1980 - - Ser Asp Thr Pro Lys Val Val Pro Phe Gly Th - #r Ser Gly Trp Ala Glu 1985 1990 - # 1995 - # 2000 - - Val Val Val Thr Pro Thr His Val Val Ile Ar - #g Arg Thr Ser Cys Tyr 2005 - # 2010 - # 2015 - - Lys Leu Leu Arg Gln Gln Ile Leu Ser Ala Al - #a Val Ala Glu Pro Tyr 2020 - # 2025 - # 2030 - - Tyr Val Asp Gly Ile Pro Val Ser Trp Glu Al - #a Asp Ala Arg Ala Pro 2035 - # 2040 - # 2045 - - Ala Met Val Tyr Gly Pro Gly Gln Ser Val Th - #r Ile Asp Gly Glu Arg 2050 - # 2055 - # 2060 - - Tyr Thr Leu Pro His Gln Leu Arg Met Arg As - #n Val Ala Pro Ser Glu 2065 2070 - # 2075 - # 2080 - - Val Ser Ser Glu Val Ser Ile Glu Ile Gly Th - #r Glu Thr Glu Asp Ser 2085 - # 2090 - # 2095 - - Glu Leu Thr Glu Ala Asp Leu Pro Pro Ala Al - #a Ala Ala Leu Gln Ala 2100 - # 2105 - # 2110 - - Ile Glu Asn Ala Ala Arg Ile Leu Glu Pro Hi - #s Ile Asp Val Xaa Met 2115 - # 2120 - # 2125 - - Glu Asp Cys Ser Thr Pro Ser Leu Cys Gly Se - #r Ser Arg Glu Met Pro 2130 - # 2135 - # 2140 - - Val Trp Gly Glu Asp Ile Pro Arg Thr Pro Se - #r Pro Ala Leu Ile Ser 2145 2150 - # 2155 - # 2160 - - Val Thr Glu Ser Ser Ser Asp Glu Lys Thr Le - #u Ser Val Thr Ser Ser 2165 - # 2170 - # 2175 - - Gln Glu Asp Thr Pro Ser Ser Asp Ser Phe Gl - #u Val Ile Gln Glu Ser 2180 - # 2185 - # 2190 - - Asp Thr Ala Glu Ser Glu Glu Ser Val Phe As - #n Val Ala Leu Ser Val 2195 - # 2200 - # 2205 - - Leu Lys Ala Leu Phe Pro Gln Ser Val Ala Th - #r Arg Lys Leu Thr Val 2210 - # 2215 - # 2220 - - Lys Met Ser Cys Cys Val Glu Lys Ser Val Th - #r Arg Phe Phe Ser Leu 2225 2230 - # 2235 - # 2240 - - Gly Leu Thr Val Ala Asp Val Ala Ser Leu Cy - #s Glu Met Glu Ile Gln 2245 - # 2250 - # 2255 - - Asn His Thr Ala Tyr Cys Asp Lys Val Arg Th - #r Pro Leu Glu Leu Gln 2260 - # 2265 - # 2270 - - Val Gly Cys Leu Val Gly Asn Glu Leu Thr Ph - #e Glu Cys Asp Lys Cys 2275 - # 2280 - # 2285 - - Glu Ala Arg Gln Glu Thr Leu Ala Ser Phe Se - #r Tyr Ile Trp Ser Gly 2290 - # 2295 - # 2300 - - Val Pro Leu Thr Arg Ala Thr Pro Ala Lys Pr - #o Pro Val Val Arg Pro 2305 2310 - # 2315 - # 2320 - - Val Gly Ser Leu Leu Val Ala Asp Thr Thr Ly - #s Val Tyr Val Thr Asn 2325 - # 2330 - # 2335 - - Pro Asp Asn Val Gly Arg Arg Val Asp Lys Va - #l Thr Phe Trp Arg Ala 2340 - # 2345 - # 2350 - - Pro Arg Val His Asp Lys Phe Leu Val Asp Se - #r Ile Glu Arg Ala Arg 2355 - # 2360 - # 2365 - - Arg Ala Ala Gln Gly Cys Leu Ser Met Gly Ty - #r Thr Tyr Glu Glu Ala 2370 - # 2375 - # 2380 - - Ile Arg Thr Val Arg Pro His Ala Ala Met Gl - #y Trp Gly Ser Lys Val 2385 2390 - # 2395 - # 2400 - - Ser Val Arg Asp Leu Ala Thr Pro Ala Gly Ly - #s Met Ala Val His Asp 2405 - # 2410 - # 2415 - - Arg Leu Gln Glu Ile Leu Glu Gly Thr Pro Va - #l Pro Phe Thr Leu Thr 2420 - # 2425 - # 2430 - - Val Lys Lys Glu Val Phe Phe Lys Asp Arg Ly - #s Glu Glu Lys Ala Pro 2435 - # 2440 - # 2445 - - Arg Leu Ile Val Phe Pro Pro Leu Asp Phe Ar - #g Ile Ala Glu Lys Leu 2450 - # 2455 - # 2460 - - Ile Leu Gly Asp Pro Gly Arg Val Ala Lys Al - #a Val Trp Gly Gly Ala 2465 2470 - # 2475 - # 2480 - - Tyr Ala Phe Gln Tyr Thr Pro Asn Gln Arg Va - #l Lys Glu Met Leu Lys 2485 - # 2490 - # 2495 - - Leu Trp Glu Ser Lys Lys Thr Pro Cys Ala Il - #e Cys Val Asp Ala Thr 2500 - # 2505 - # 2510 - - Cys Phe Asp Ser Ser Ile Thr Glu Glu Asp Va - #l Ala Leu Glu Thr Glu 2515 - # 2520 - # 2525 - - Leu Tyr Ala Leu Ala Ser Asp His Pro Glu Tr - #p Val Arg Ala Leu Gly 2530 - # 2535 - # 2540 - - Lys Tyr Xaa Ala Ser Gly Thr Met Val Thr Pr - #o Glu Gly Val Pro Val 2545 2550 - # 2555 - # 2560 - - Gly Glu Arg Tyr Cys Arg Ser Ser Gly Val Le - #u Thr Thr Ser Ala Ser 2565 - # 2570 - # 2575 - - Asn Cys Leu Thr Cys Tyr Ile Lys Val Arg Al - #a Ala Cys Glu Arg Ile 2580 - # 2585 - # 2590 - - Gly Leu Lys Asn Val Ser Leu Leu Ile Ala Gl - #y Asp Asp Cys Leu Ile 2595 - # 2600 - # 2605 - - Val Cys Glu Arg Pro Val Cys Asp Pro Cys Gl - #u Ala Leu Gly Arg Ala 2610 - # 2615 - # 2620 - - Leu Ala Ser Tyr Gly Tyr Ala Cys Glu Pro Se - #r Tyr His Ala Ser Leu 2625 2630 - # 2635 - # 2640 - - Asp Thr Ala Pro Phe Cys Ser Thr Trp Leu Al - #a Glu Cys Asn Ala Asp 2645 - # 2650 - # 2655 - - Gly Xaa Arg His Phe Phe Leu Thr Thr Asp Ph - #e Arg Arg Pro Leu Ala 2660 - # 2665 - # 2670 - - Arg Met Ser Ser Glu Tyr Ser Asp Pro Met Al - #a Ser Ala Ile Gly Tyr 2675 - # 2680 - # 2685 - - Ile Leu Leu Tyr Pro Trp Xaa Pro Ile Thr Ar - #g Trp Val Ile Ile Pro 2690 - # 2695 - # 2700 - - His Val Leu Thr Cys Ala Ser Phe Arg Gly Gl - #y Gly Thr Xaa Ser Asp 2705 2710 - # 2715 - # 2720 - - Pro Val Trp Cys Gln Val His Gly Asn Tyr Ty - #r Lys Phe Pro Leu Asp 2725 - # 2730 - # 2735 - - Lys Leu Pro Asn Ile Ile Val Ala Leu His Gl - #y Pro Ala Ala Leu Arg 2740 - # 2745 - # 2750 - - Val Thr Ala Asp Thr Thr Lys Thr Lys Met Gl - #u Ala Gly Lys Val Leu 2755 - # 2760 - # 2765 - - Ser Asp Leu Lys Leu Pro Gly Leu Ala Val Hi - #s Arg Lys Lys Ala Gly 2770 - # 2775 - # 2780 - - Ala Leu Arg Thr Arg Met Leu Arg Ser Arg Gl - #y Trp Ala Glu Leu Ala 2785 2790 - # 2795 - # 2800 - - Arg Gly Leu Leu Trp His Pro Gly Leu Arg Le - #u Pro Pro Pro Glu Ile 2805 - # 2810 - # 2815 - - Ala Gly Ile Pro Gly Gly Phe Pro Leu Ser Pr - #o Pro Tyr Met Gly Val 2820 - # 2825 - # 2830 - - Val His Gln Leu Asp Phe Thr Ala Gln Arg Se - #r Arg Trp Arg Trp Leu 2835 - # 2840 - # 2845 - - Gly Phe Leu Ala Leu Leu Ile Val Ala Leu Ph - #e Gly 2850 - # 2855 - # 2860 - - - - (2) INFORMATION FOR SEQ ID NO:36: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: - - GATCGTCTAG AAGCAATGAA CTTGGCGCGC CCGCCTCAGT GTTG - # - # 44 - - - - (2) INFORMATION FOR SEQ ID NO:37: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: - - ATATATCTAG ACTATCACGG AATGAAGTTG CCCGCATCCA C - # - # 41 - - - - (2) INFORMATION FOR SEQ ID NO:38: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: - - ATATATCTAG ACTATCACTT GTCGTCGTCG TCCTTGTAGT CCCGTCCCGA AG -#AGAGCCA 59 - - - - (2) INFORMATION FOR SEQ ID NO:39: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: - - ATATATCTAG ACTATCACTT GTCGTCGTCG TCCTTGTAGT CAGGTTCGTA AA -#ACCCCCCA 60 - - - - (2) INFORMATION FOR SEQ ID NO:40: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: - - AATATATCTA GACTATCAGG GCGACCGCAC CGGGTTGCCT - # - # 40 - - - - (2) INFORMATION FOR SEQ ID NO:41: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 64 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: - - CTAGCCACCA TGAAGGCTCT CATTGTTCTG GGGCTTGTCC TCCTTTCTGT TA -#CGGTCCAG 60 - - GGCT - # - # - # 64 - - - - (2) INFORMATION FOR SEQ ID NO:42: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 64 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: - - CTAGAGCCCT GGACCGTAAC AGAAAGGAGG ACAAGCCCCA GAACAATGAG AG -#CCTTCATG 60 - - GTGG - # - # - # 64 - - - - (2) INFORMATION FOR SEQ ID NO:43: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 79 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: - - GATCATAGCG CACTATTATA GCACCATGAA GGCTCTCATT GTTCTGGGGC TT -#GTCCTCCT 60 - - TTCTGTTACG GTCCAGGGG - # - # - # 79 - - - - (2) INFORMATION FOR SEQ ID NO:44: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 79 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: - - GATCCCCCTG GACCGTAACA GAAAGGAGGA CAAGCCCCAG AACAATGAGA GC -#CTTCATGG 60 - - TGCTATAATA GTGCGCTAT - # - # - # 79 - - - - (2) INFORMATION FOR SEQ ID NO:45: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9014 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: - - CACTGGGTGC AAGCCCCAGA AACCGACGCC TATCTAAGTA GACGCAATGA CT -#CGGCGCCG 60 - - ACCCGGCGAC CGGCCAAAAG GTGGTGGATG GGTGATGCCA GGGTTGGTAG GT -#CGTAAATC 120 - - CCGGTCATCT TGGTAGCCAC TATAGGTGGG TCTTAAGGGA AGGTTAAGAT TC -#CTCTTGTG 180 - - CCTGTGGCGA GACAGCGCAC GGTCCACAGG TGTTGGCCCT ACCGGTGGGA AT -#AAGGGCCC 240 - - GACGTCAGGC TCGTCGTTAA ACCGAGACCG ACACCCACCT GGGCAAACGA CG -#CCCACGTA 300 - - CGGTCCACGT CGCCCTTCAA TGTCTCTCTT GACCAATAGG CTTTGCCGGC GA -#GTTGACAA 360 - - GGACCAGTGG GGGCCGGGGG TGGAGGGAAG GACCCTCTCA CCCTGYCCTT CC -#CGGTGGGA 420 - - CGGGAAATGC ATGGGGCCAC CCAGCTCCGC GGCGGCCTGC AGCCGGGGTA GC -#CCAAGAGC 480 - - CTTCGGGTGA GGGCGGGTGG CATTTTTCTT TTCTATACCG ATCATGGCAG TC -#CTTCTGCT 540 - - CTTCTTCGTG GTTGAGGCCG GGGCCATTCT GGCCCCGGCC ACCCACGCTT GC -#CGAGCTGA 600 - - TGGGCAATAT TTCCTCACAA ACTGTTGCGC CCCGGAGGAC ATTGGGTTCT GC -#CTGGAAGG 660 - - TGGATGCCTG GTGGCCTTGG GTTGCACTAT TTGTACTGAC CGATGCTGGC CA -#CTGTATCA 720 - - GGCGGGTTTG GCTGTGCGGC CTGGCAAGTC CGCGGCCCAG CTGGTGGGGG AA -#CTGGGGAG 780 - - CCTTTATGGG CCCTTGTCGG TCTCSGCYTA CGTGGCGGGG ATCTTGGGCC TA -#GGAGAGGT 840 - - TTACTCCGGT GTCCTCACGG TTGGGGTCGC GTTGACGCGC CGGGTCTACC CG -#GCCCCTAA 900 - - CCTGACGTGC ACCGTAGAGT GTGAGTTAAA GTGGGAAAGT GAGTTTTGGA GA -#TGGACTGA 960 - - ACAGCTGGCC TCCAACTACT GGATTCTGGA ATATCTCTGG AAAGTGCCTT TT -#GACTTCTG 1020 - - GCGGGGAGTG ATGAGCTTGA CCCCCTTGTT GGTGTGTGTC GCGGCCCTCC TC -#CTGCTTGA 1080 - - GCAGAGGCTC GTCATGGTCT TCCTGTTGGT GACGATGGCT GGGATGTCGC AA -#GGCGCTCC 1140 - - GGCCTCCGTT TTGGGGTCAC GCCCCTTTGA CCGCGGGTTG ACATGGCAGT CT -#TGTTCTTG 1200 - - TAGGGCGAAT GGCTCACGCA TTCCTACTGG GGAGAAAGTT TGGGACCGAG GG -#AACGTCAC 1260 - - ACTTCTGTGT GACTGCCCCA ATGGCCCCTG GGTCTGGCTG CCGGCCGTGT RC -#CAAGCGAT 1320 - - CGGCTGGGGC GACCCTATCA CTCATTGGAG CCATGGACAA AATCAATGGC CC -#CTATCATG 1380 - - CCCCCAGTAC GTCTACGGTG CTGTTTCAGT GACATGCGTT TGGGGTTCCG TG -#TCTTGGTT 1440 - - TGCAACCACA GGTGGCCGCG ATTCCAAGCT CGACGTGTGG AGTTTGGTAC CA -#GTTGGCTC 1500 - - TGCCAGCTGC ACCATAGCCG CTCTCGGGTC ATCGGATCGT GACACCGTGG TG -#GAGCTCTC 1560 - - CGAGTGGGGG ATCCCTTGCG CTACCTGCAT TCTGGATCGT CGACCGGCCT CG -#TGTGGCAC 1620 - - CTGTGTGCGG GACTGCTGGC CCGAGACCGG GTCTGTCAGA TTCCCTTTCC AT -#CGGTGCGG 1680 - - CGCGGGGCCT AGGTTGACAA AGGACTTGGA AGCTGTGCCC TTCGTCAATA GG -#ACAACTCC 1740 - - CTTCACCATA AGGGGCCCCC TGGGCAACCA GGGGAGAGGC AACCCGGTGC GG -#TCGCCCCT 1800 - - GGGTTTTGGG TCCTACACCA TGACCAAGAT CCGGGACTCC TTACATCTGG TG -#AAATGTCC 1860 - - CACACCAGCC ATTGAGCCTY CCACCGGGAC GTTTGGGTTC TTCCCTGGAG TG -#CCGCCCCT 1920 - - TAACAACTGC ATGCTCCTGG GCACTGAGGT GTCAGAAGCA TTGGGTGGGG CT -#GGCCTCAC 1980 - - GGGGGGGTTC TATGAACCCC TGGTGCGCAG GTGTTCGGAG CTGATGGGGC GC -#CGAAATCC 2040 - - GGTTTGCCCG GGGTACGCAT GGCTGTCCTC GGGTAGGCCT GATGGGTTCA TA -#CATGTTCA 2100 - - GGGCCACTTG CAGGAGGTGG ATGCGGGCAA CTTCATCCCG CCTCCTTGCT GG -#TTGCTTTT 2160 - - GGACTTTGTA TTTGTCCTGT TATACCTGAT GAAGCTGGCG GAGGCACGGC TG -#GTCCCGCT 2220 - - GATACTTCTC CTGTTGTGGT GGTGGGTTAA CCAGCTGGCG GTCTTAGGAT TG -#CCGGCTGC 2280 - - TCACGCCGCC GTGGCGGGGG AGGTGTTTGC GGGTCCAGCC CTGTCGTGGT GT -#TTGGGGCT 2340 - - CCCCTTCGTT AGTATGATAC TCGGTCTAGC AAACCTGGTG TTGTATTTCC GA -#TGGATGGG 2400 - - GCCTCAACGT CTCATGTTCC TTGTGTTGTG GAAGCTCGCT CGGGGGGCTT TC -#CCGCTGGC 2460 - - TCYCTTGATG GGGATTTCGG CTACCCGTGG TCGCACCTCA GTGCTCGGGG CC -#GAGTTCCG 2520 - - CTTCGACATG ACATTCGAGG TTGACACGTC GGTCTTGGGA TGGGTGGTTG CT -#AGTGTGGT 2580 - - TGCTTGGGCC ATAGCGCTCC TGAGCTCGAT GAGCGCAGGG GGGTGGAAGC AC -#AAAGCTGT 2640 - - GATCTACAGG ACGTGGTGCA AGGGGTACCA GGCCATACGC CAGAGGGTTG TG -#CGGAGCCC 2700 - - CCTCGGGGAG GGGCGTYCCA CAAAGCCTTT GACCATTGCT TGGTGCTTGG CC -#TCATACAT 2760 - - CTGGCCGGAC GCTGTGATGA TGGTGGTGGT CGCCATGGTT CTTCTCTTCG GC -#CTGTTCGA 2820 - - CGCGTTGGAC TGGGCTTTGG AGGAGCTCCT GGTGGCTAGG CCCTCGCTGC GG -#CGCCTTKC 2880 - - CCGGGTGGTT GAGTGTTGTG TKATGGCTGG CGAAAGGGCG ACCACCATAA GG -#CTGGTGTC 2940 - - CAAGATGTGT GCAAGAGGGG CCTACTTGTT CGATCACATG GGCTCTTTTT CG -#CGCGCTGT 3000 - - CAAGGAACGC TTGCTAGAAT GGGATGCCGC TCTCGAGCCT CTTTCATTCA CC -#AGGACGGA 3060 - - CTGTCGCATC ATTCGGGACG CCGCCAGGAC CTTGTCCTGC GGGCAGTGCG TT -#ATGGGTTT 3120 - - ACCTGTAGTC GCGCGCCGTG GTGATGAAGT TTTGATCGGC GTTTTCCAAG AT -#GTGAATCA 3180 - - TCTGCCTCCC GGGTTTGTCC CGACCGCGCC TGTTGTCATC CGTCGGTGCG GG -#AAGGGCTT 3240 - - TCTCGGAGTC ACAAAGGCTG CCTTGACTGG ACGGGATCCT GACTTACACC CA -#GGGAACGT 3300 - - CATGGTGCTG GGGACGGCAA CGTCGCGAAG CATGGGAACA TGTCTGAACG GC -#TTGTTGTT 3360 - - CACAACATTC CATGGGGCTT CTTCCCGAAC CATCGCCACA CCCGTGGGGG CC -#CTAAATCC 3420 - - AAGATGGTGG TCGGCCAGTG ATGACGTAAC CGTGTACCCC CTACCTGATG GT -#GCTACCTC 3480 - - GCTGACCCCT TGCACATGCC AGGCGGAGTC CTGCTGGGTC ATCAGATCCG AC -#GGGGCTTT 3540 - - GTGCCATGGC TTGTCCAAGG GAGACAAGGT TGAGCTCGAT GTGGCCATGG AG -#GTTTCTGA 3600 - - CTTTCGTGGT TCGTCGGGCT CGCCCGTCTT GTGCGACGAG GGACACGCAG TG -#GGAATGCT 3660 - - GGTGTCAGTG CTCCATTCAG GTGGTAGGGT GACGGCGGCC CGATTCACAC GG -#CCTTGGAC 3720 - - CCAAGTTCCA ACAGACGCCA AGACCACCAC TGAACCCCCG CCGGTTCCGG CA -#AAAGGAGT 3780 - - TTTCAAAGAG GCCCCGTTGT TTATGCCTAC GGGAGCCGGG AAGAGCACCC GG -#GTCCCGCT 3840 - - GGAGTACGGC AACATGGGTC ACAAGGTTCT GATTCTCAAC CCGTCGGTAG CT -#ACTGTGAG 3900 - - GGCCATGGGC CCTTACATGG AGCGGTTGGC GGGCAAACAC CCAAGCATTT AC -#TGTGGACA 3960 - - CGATACAACA GCTTTCACGA GGATCACTGA CTCGCCCCTC ACGTACTCAA CC -#TACGGAAG 4020 - - GTTTTTGGCC AACCCTAGGC AGATGCTGCG GGGCGTATCG GTGGTAATCT GT -#GATGAGTG 4080 - - CCACAGTCAT GACTCTACGG TGTTGCTGGG TATTGGGCGG GTCCGGGAGC TG -#GCGCGGGG 4140 - - GTGTGGAGTT CAACTGGTTC TCTACGCCAC CGCCACGCCT CCGGGCTCGC CC -#ATGACCCA 4200 - - GCACCCCTCA ATTATTGAGA CAAAGCTGGA YGTTGGWGAG ATCCCCTTTT AT -#GGGCATGG 4260 - - CATCCCCCTG GAGCGGATGA GGACCGGTAG GCACCTGGTA TTCTGCCACT CA -#AAGGCGGA 4320 - - GTGTGAGAGG CTGGCCGGCC AATTCTCCTC ACGGGGGGTT AATGCTGTTG CC -#TATTATAG 4380 - - GGGTAAGGAC AGTTCAATCA TCAAGGATGG TGACCTGGTG GTGTGCGCTA CT -#GACGCGCT 4440 - - ATCTACCGGK TACACAGGAA ACTTTGACTC CGTGACCGAC TGTGGTTTAG TG -#GTGGAGGA 4500 - - GGTCGTTGAG GTGACCCTTG ATCCCACCAT AACCATCTCC CTGCGGACAG TG -#CCTGCGTC 4560 - - GGCAGAATTG TCGATGCAAC GGCGAGGACG CACGGGTAGA GGCCGGTCTG GG -#CGTTACTA 4620 - - CTACGCGGGG GTGGGCAAAG CCCCTGCTGG TGTGGTGAGG TCCGGGCCGG TC -#TGGTCGGC 4680 - - GGTGGAGGCC GGAGTGACCT GGTACGGAAT GGAACCTGAC CTGACAGCAA AC -#CTTCTGAG 4740 - - ACTGTACGAC GACTGCCCTT ACACCGCAGC CGTCGCGGCG GACATCGGGG AA -#GCCGCGGT 4800 - - GTTCTTTGCG GGGCTCGCCC CCCTTAGGAT GCATCCTGAT GTCAGCTGGG CA -#AAAGTGCG 4860 - - CGGCGTCAAC TGGCCCCTCC TGGTGGGCGT TCAGCGGACC ATGTGCCGGG AA -#ACACTGTC 4920 - - TCCCGGTCCA TCGGATGACC CCCAGTGGGC AGGTCTGAAG GGCCCAAATC CA -#GTCCCATT 4980 - - ACTGCTGAGG TGGGGCAATG ATTTACCATC GAAAGTGGCC GGCCATCACA TA -#GTGGACGA 5040 - - CTTGGTCCGT CGCCTGGGTG TGGCCGAGGG TTATGTCCGC TGTGACGCGG GC -#CCCATCTT 5100 - - GATGGTGGGG CTTGCGATCG CGGGGGGGAT GATCTACGCG TCATACACCG GG -#TCACTTGT 5160 - - GGTGGTTACA GACTGGGATG TGAAGGGGGG TGGCAACCCC CTTTATAGGA GT -#GGAGACCA 5220 - - AGCCACCCCA CAGCCGGTTG TGCAGGTCCC TCCGGTAGAC CATCGGCCGG GG -#GGGGAATC 5280 - - TGCGCCATCG GATGCCAAGA CAGTGACAGA TGCGGTGGCA GCCATCCAAG TG -#GACTGTGA 5340 - - TTGGTCAGTC ATGACCCTGT CGATCGGGGA AGTGCTTTCC TTGGCTCAGG CT -#AAGACAGC 5400 - - CGAGGCCTAC ACAGCAACCG CAAAGTGGCT CGCTGGCTGC TATACGGGGA CG -#CGGGCCGT 5460 - - CCCCACAGTA TCAATTGTGG ACAAGCTCTT CGCCGGCGGG TGGGCGGCCG TG -#GTGGGTCA 5520 - - CTGCCACAGC GTCATAGCTG CGGCGGTGGC TGCTTACGGG GCTTCTAGGA GT -#CCCCCGTT 5580 - - GGCTGCCGCG GCTTCCTACC TAATGGGGTT GGGCGTCGGA GGCAATGCGC AG -#ACGCGTCT 5640 - - GGCTTCCGCC CTTCTTCTGG GGGCTGCTGG GACCGCCCTG GGCACCCCGG TC -#GTTGGTTT 5700 - - GACCATGGCC GGGGCGTTCA TGGGGGGGGC CAGCGTCTCC CCGTCCTTGG TC -#ACCATTTT 5760 - - ACTGGGGGCC GTGGGCGGTT GGGAGGGCGT GGTTAACGCT GCTAGCCTCG TC -#TTCGACTT 5820 - - CATGGCAGGG AAATTATCAT CAGAAGACCT GTGGTATGCC ATCCCGGTAC TC -#ACTAGCCC 5880 - - GGGCGCGGGC CTCGCGGGGA TCGCGCTCGG GTTGGTGTTG TATTCAGCTA AC -#AACTCTGG 5940 - - CACTACCACT TGGCTGAATC GTCTGCTAAC CACCCTGCCA CCTTCATCGT GC -#ATCCCTGA 6000 - - CAGTTACTTC CAGCAAGCCG ATTACTGTAA CAAGGTCTCG GCCGTGCTCC GG -#CGCCTGAG 6060 - - CCTCACCCGA ACAGTGGTTG CCCTTGTGAA CAGGGAACCA AAGGTTGACG AG -#GTTCAGGT 6120 - - CGGATACGTC TGGGACTTGT GGGAGTGGAT CATGCGTCAA GTGCGCATGG TC -#ATGGCCAG 6180 - - ACTCCGRGCC CTCTGTCCCG TGGTGTCACT ACCCTTGTGG CACTGCGGTG AG -#GGATGGTC 6240 - - CGGGGAGTGG CTGCTGGATG GGCATGTTGA GAGCCGCTGT CTTTGTGGGT GC -#GTGATAAC 6300 - - TGGTGACGTA CTGAATGGAC AACTCAAGGA GCCAGTTTAC TCTACAAAGC TG -#TGCCGGCA 6360 - - CTACTGGATG GGGACCGTAC CTGTGAACAT GTTGGGCTAC GGTGAAACCT CA -#CCTCTCTT 6420 - - GGCCTCAGAC ACCCCGAAGG TGGTACCTTT TGGGACGTCG GGCTGGGCTG AG -#GTGGTGGT 6480 - - GACGCCGACC CATGTGGTAA TCAGGCGGAC CTCTTCCTAC AAGTTGCTGC GC -#CAGCAAAT 6540 - - CCTATCAGCT GCTGTTGCTG AGCCCTACTA TGTCGACGGC ATTCCGGTCT CG -#TGGGACGC 6600 - - AGATGCGAGA GCACCTGCCA TGGTCTATGG CCCTGGACAA AGTGTTACCA TT -#GACGGGGA 6660 - - GCGCTACACC TTGCCGCACC AACTGCGGCT TAGGAACGTA GCGCCCTCTG AG -#GTTTCATC 6720 - - CGAGGTGTCC ATAGACATTG GGACGGAGAC TGAAGACTCA GAACTGACTG AG -#GCCGACTT 6780 - - GCCGCCGGCA GCTGCAGCCC TTCAAGCGAT CGAGAATGCT GCGAGAATTC TT -#GAACCGCA 6840 - - TATTGATGTC ATCATGGAGG ATTGCAGTAC ACCCTCTCTC TGTGGTAGTA GC -#CGAGAGAT 6900 - - GCCTGTGTGG GGAGAAGACA TCCCCSGCAC TCCATCGCCT GCACTTATCT CG -#GTTACGGA 6960 - - GAGCAGCTCA GATGAGAAGA CCCCGTCGGT GTCCTCCTCG CAGGAGGATA CC -#CCGTCCTC 7020 - - AGACTCATTC GAAGTCATCC AAGAGTCAGA GACAGCTGAG GGGGAGGACA GT -#GTCTTCAA 7080 - - CGTGGCTCTT TCTGTACTAA AAGCCTTGTT TCCACAGAGC GATGCCACTC GG -#AAACTGAC 7140 - - GGTCAAGATG TCATGCTGCG TGGAGAAGAG CGTCACTCGC TTCTTTTCCT TG -#GGGTTGAC 7200 - - GGTGGCCGAT GTGGCTAGCC TGTGTGAAAT GGAAATCCAG AACCATACAG CC -#TATTGTGA 7260 - - CAAGGTGCGC ACTCCGCTTG AATTGCAGGT TGGGTGCTTG GTGGGCAATG AA -#CTTACCTT 7320 - - TGAATGTGAC AAGTGTGAGG CAAGGCAAGA GACCTTGGCC TCCTTCTCCT AC -#ATATGGTC 7380 - - CGGTGTCCCG CTGACGCGGG CCACTCCGGC CAAACCCCCA GTGGTGAGGC CG -#GTTGGTTC 7440 - - GCTGTTGGTG GCCGACACCA CCAAGGTGTA TGTGACCAAC CCGGACAACG TG -#GGGAGAAG 7500 - - AGTGGACAAG GTGACCTTCT GGCGTGCCCC TCGAGTCCAT GACAAATTTC TC -#GTGGACTC 7560 - - GATCGAGCGC GCTAGAAAGG CAGCTCAAGC ATGCCTAAGC ATGGGTTACA CT -#TATGAGGA 7620 - - GGCAATAAGG ACTGTTAGGC CACATGCTGC CATGGGCTGG GGATCTAAGG TG -#TCGGTTAA 7680 - - AGACTTGGCC ACCCCTGCGG GGAAGATGGC TGTCCACGAC CGACTTCAGG AG -#ATACTTGA 7740 - - GGGGACTCCG GTTCCCTTTA CCCTGACTGT GAAAAAGGAG GTGTTCTTCA AA -#GACCGAAA 7800 - - GGAGGAGAAG GCCCCCCGCC TCATTGTGTT CCCCCCTCTG GACTTCCGGA TA -#GCTGAAAA 7860 - - GCTTATTTTG GGAGACCCGG GGCGGGTAGC CAAGGCGGTG TTGGGGGGGG CT -#TACGCCTT 7920 - - CCAGTACACC CCAAACCAGC GCGTCAAGGA GATGCTCAAG CTATGGGAGT CA -#AAGAAAAC 7980 - - ACCATGTGCC ATCTGTGTGG ACGCCACGTG CTTCGACAGT AGCATTACTG AA -#GAGGACGT 8040 - - GGCCTTGGAG ACAGAGCTTT ATGCCCTGGC TTCGGACCAT CCAGAGTGGG TG -#CGGGCCCT 8100 - - AGGGAAATAC TATGCCTCAG GCACCATGGT CACTCCGGAA GGGGTGCCCG TC -#GGTGAGAG 8160 - - GTATTGTAGA TCCTCTGGGG TCTTGACCAC TAGCGCGACG AATTGCTTGA CC -#TGCTACAT 8220 - - TAAGGTGTCA GCTGCCTGTC AGCGGGTGGG GCTGAAAAAT GTCTCGCTAC TG -#ATAGCAGG 8280 - - TGACGACTGT CTGATCATAT GCGAACGGCC AGTGTGCGAC CCTAGCGAAG CC -#TTGGGCCG 8340 - - AGCCCTCGCT AGCTATGGGT ATGCATGCGA GCCTTCGTAT CATGCATCAC TG -#GACACGGC 8400 - - CCCCTTCTGC TCCACTTGGC TCGCCGAGTG TAATGCAGAT GGGAAGCGCC AT -#TTCTTCCT 8460 - - GACAACGGAC TTTCGGAGGC CGCTCGCTCG CATGTCGAGT GAGTACAGTG AC -#CCGATGGC 8520 - - TTCGGCCATC GGTTACATCC TCCTTTACCC CTGGCATCCC ATCACACGGT GG -#GTCATCAT 8580 - - TCCACACGTG CTGACTTGCG CGTTTAGGGG TGGTGGTACA CCGTCTGATC CG -#GTCTGGTG 8640 - - CCAGGTTCAT GGAAATTACT ACAAGTTTCC CCTGGACAAA CYGCCAAACA TC -#ATCGTGGC 8700 - - CCTCCATGGA CCAGCAGCAT TGAGGGTTAC CGCAGACACA ACYAAAACAA AG -#ATGGAAGC 8760 - - CGGCAAGGTG CTGAGCGACC TCAAGCTCCC GGGCTTAGCG GTCCACCGCA AG -#AAGGCCGG 8820 - - AGCACTGCGA ACTCGCATGC TTCGGTCGCG CGGTTGGGCT GAGTTGGCTC GG -#GGCCTGTT 8880 - - GTGGCATCCA GGCCWACGGC TCCCACCTCC GGAGATTGCT GGTATCCCCG GG -#GGTTTCCC 8940 - - CCTGTCCCCC CCCTACATGG GGGTGGTCCA TCAATTGGAT TTCACAAGCC AG -#AGGAGTCG 9000 - - CTGGCGGTGG TTGG - # - # - # 9014 - - - - (2) INFORMATION FOR SEQ ID NO:46: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2841 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: - - Gly Arg Val Ala Phe Phe Phe Ser Ile Pro Il - #e Met Ala Val Leu Leu 1 5 - # 10 - # 15 - - Leu Phe Phe Val Val Glu Ala Gly Ala Ile Le - #u Ala Pro Ala Thr His 20 - # 25 - # 30 - - Ala Cys Arg Ala Asp Gly Gln Tyr Phe Leu Th - #r Asn Cys Cys Ala Pro 35 - # 40 - # 45 - - Glu Asp Ile Gly Phe Cys Leu Glu Gly Gly Cy - #s Leu Val Ala Leu Gly 50 - # 55 - # 60 - - Cys Thr Ile Cys Thr Asp Arg Cys Trp Pro Le - #u Tyr Gln Ala Gly Leu 65 - # 70 - # 75 - # 80 - - Ala Val Arg Pro Gly Lys Ser Ala Ala Gln Le - #u Val Gly Glu Leu Gly 85 - # 90 - # 95 - - Ser Leu Tyr Gly Pro Leu Ser Val Ser Ala Ty - #r Val Ala Gly Ile Leu 100 - # 105 - # 110 - - Gly Leu Gly Glu Val Tyr Ser Gly Val Leu Th - #r Val Gly Val Ala Leu 115 - # 120 - # 125 - - Thr Arg Arg Val Tyr Pro Ala Pro Asn Leu Th - #r Cys Thr Val Glu Cys 130 - # 135 - # 140 - - Glu Leu Lys Trp Glu Ser Glu Phe Trp Arg Tr - #p Thr Glu Gln Leu Ala 145 1 - #50 1 - #55 1 -#60 - - Ser Asn Tyr Trp Ile Leu Glu Tyr Leu Trp Ly - #s Val Pro Phe AspPhe 165 - # 170 - # 175 - - Trp Arg Gly Val Met Ser Leu Thr Pro Leu Le - #u Val Cys Val Ala Ala 180 - # 185 - # 190 - - Leu Leu Leu Leu Glu Gln Arg Leu Val Met Va - #l Phe Leu Leu Val Thr 195 - # 200 - # 205 - - Met Ala Gly Met Ser Gln Gly Ala Pro Ala Se - #r Val Leu Gly Ser Arg 210 - # 215 - # 220 - - Pro Phe Asp Arg Gly Leu Thr Trp Gln Ser Cy - #s Ser Cys Arg Ala Asn 225 2 - #30 2 - #35 2 -#40 - - Gly Ser Arg Ile Pro Thr Gly Glu Lys Val Tr - #p Asp Arg Gly AsnVal 245 - # 250 - # 255 - - Thr Leu Leu Cys Asp Cys Pro Asn Gly Pro Tr - #p Val Trp Leu Pro Ala 260 - # 265 - # 270 - - Val Xaa Gln Ala Ile Gly Trp Gly Asp Pro Il - #e Thr His Trp Ser His 275 - # 280 - # 285 - - Gly Gln Asn Gln Trp Pro Leu Ser Cys Pro Gl - #n Tyr Val Tyr Gly Ala 290 - # 295 - # 300 - - Val Ser Val Thr Cys Val Trp Gly Ser Val Se - #r Trp Phe Ala Thr Thr 305 3 - #10 3 - #15 3 -#20 - - Gly Gly Arg Asp Ser Lys Leu Asp Val Trp Se - #r Leu Val Pro ValGly 325 - # 330 - # 335 - - Ser Ala Ser Cys Thr Ile Ala Ala Leu Gly Se - #r Ser Asp Arg Asp Thr 340 - # 345 - # 350 - - Val Val Glu Leu Ser Glu Trp Gly Ile Pro Cy - #s Ala Thr Cys Ile Leu 355 - # 360 - # 365 - - Asp Arg Arg Pro Ala Ser Cys Gly Thr Cys Va - #l Arg Asp Cys Trp Pro 370 - # 375 - # 380 - - Glu Thr Gly Ser Val Arg Phe Pro Phe His Ar - #g Cys Gly Ala Gly Pro 385 3 - #90 3 - #95 4 -#00 - - Arg Leu Thr Lys Asp Leu Glu Ala Val Pro Ph - #e Val Asn Arg ThrThr 405 - # 410 - # 415 - - Pro Phe Thr Ile Arg Gly Pro Leu Gly Asn Gl - #n Gly Arg Gly Asn Pro 420 - # 425 - # 430 - - Val Arg Ser Pro Leu Gly Phe Gly Ser Tyr Th - #r Met Thr Lys Ile Arg 435 - # 440 - # 445 - - Asp Ser Leu His Leu Val Lys Cys Pro Thr Pr - #o Ala Ile Glu Pro Xaa 450 - # 455 - # 460 - - Thr Gly Thr Phe Gly Phe Phe Pro Gly Val Pr - #o Pro Leu Asn Asn Cys 465 4 - #70 4 - #75 4 -#80 - - Met Leu Leu Gly Thr Glu Val Ser Glu Ala Le - #u Gly Gly Ala GlyLeu 485 - # 490 - # 495 - - Thr Gly Gly Phe Tyr Glu Pro Leu Val Arg Ar - #g Cys Ser Glu Leu Met 500 - # 505 - # 510 - - Gly Arg Arg Asn Pro Val Cys Pro Gly Tyr Al - #a Trp Leu Ser Ser Gly 515 - # 520 - # 525 - - Arg Pro Asp Gly Phe Ile His Val Gln Gly Hi - #s Leu Gln Glu Val Asp 530 - # 535 - # 540 - - Ala Gly Asn Phe Ile Pro Pro Pro Cys Trp Le - #u Leu Leu Asp Phe Val 545 5 - #50 5 - #55 5 -#60 - - Phe Val Leu Leu Tyr Leu Met Lys Leu Ala Gl - #u Ala Arg Leu ValPro 565 - # 570 - # 575 - - Leu Ile Leu Leu Leu Leu Trp Trp Trp Val As - #n Gln Leu Ala Val Leu 580 - # 585 - # 590 - - Gly Leu Pro Ala Ala His Ala Ala Val Ala Gl - #y Glu Val Phe Ala Gly 595 - # 600 - # 605 - - Pro Ala Leu Ser Trp Cys Leu Gly Leu Pro Ph - #e Val Ser Met Ile Leu 610 - # 615 - # 620 - - Gly Leu Ala Asn Leu Val Leu Tyr Phe Arg Tr - #p Met Gly Pro Gln Arg 625 6 - #30 6 - #35 6 -#40 - - Leu Met Phe Leu Val Leu Trp Lys Leu Ala Ar - #g Gly Ala Phe ProLeu 645 - # 650 - # 655 - - Ala Xaa Leu Met Gly Ile Ser Ala Thr Arg Gl - #y Arg Thr Ser Val Leu 660 - # 665 - # 670 - - Gly Ala Glu Phe Arg Phe Asp Met Thr Phe Gl - #u Val Asp Thr Ser Val 675 - # 680 - # 685 - - Leu Gly Trp Val Val Ala Ser Val Val Ala Tr - #p Ala Ile Ala Leu Leu 690 - # 695 - # 700 - - Ser Ser Met Ser Ala Gly Gly Trp Lys His Ly - #s Ala Val Ile Tyr Arg 705 7 - #10 7 - #15 7 -#20 - - Thr Trp Cys Lys Gly Tyr Gln Ala Ile Arg Gl - #n Arg Val Val ArgSer 725 - # 730 - # 735 - - Pro Leu Gly Glu Gly Arg Xaa Thr Lys Pro Le - #u Thr Ile Ala Trp Cys 740 - # 745 - # 750 - - Leu Ala Ser Tyr Ile Trp Pro Asp Ala Val Me - #t Met Val Val Val Ala 755 - # 760 - # 765 - - Met Val Leu Leu Phe Gly Leu Phe Asp Ala Le - #u Asp Trp Ala Leu Glu 770 - # 775 - # 780 - - Glu Leu Leu Val Ala Arg Pro Ser Leu Arg Ar - #g Leu Xaa Arg Val Val 785 7 - #90 7 - #95 8 -#00 - - Glu Cys Cys Val Met Ala Gly Glu Arg Ala Th - #r Thr Ile Arg LeuVal 805 - # 810 - # 815 - - Ser Lys Met Cys Ala Arg Gly Ala Tyr Leu Ph - #e Asp His Met Gly Ser 820 - # 825 - # 830 - - Phe Ser Arg Ala Val Lys Glu Arg Leu Leu Gl - #u Trp Asp Ala Ala Leu 835 - # 840 - # 845 - - Glu Pro Leu Ser Phe Thr Arg Thr Asp Cys Ar - #g Ile Ile Arg Asp Ala 850 - # 855 - # 860 - - Ala Arg Thr Leu Ser Cys Gly Gln Cys Val Me - #t Gly Leu Pro Val Val 865 8 - #70 8 - #75 8 -#80 - - Ala Arg Arg Gly Asp Glu Val Leu Ile Gly Va - #l Phe Gln Asp ValAsn 885 - # 890 - # 895 - - His Leu Pro Pro Gly Phe Val Pro Thr Ala Pr - #o Val Val Ile Arg Arg 900 - # 905 - # 910 - - Cys Gly Lys Gly Phe Leu Gly Val Thr Lys Al - #a Ala Leu Thr Gly Arg 915 - # 920 - # 925 - - Asp Pro Asp Leu His Pro Gly Asn Val Met Va - #l Leu Gly Thr Ala Thr 930 - # 935 - # 940 - - Ser Arg Ser Met Gly Thr Cys Leu Asn Gly Le - #u Leu Phe Thr Thr Phe 945 9 - #50 9 - #55 9 -#60 - - His Gly Ala Ser Ser Arg Thr Ile Ala Thr Pr - #o Val Gly Ala LeuAsn 965 - # 970 - # 975 - - Pro Arg Trp Trp Ser Ala Ser Asp Asp Val Th - #r Val Tyr Pro Leu Pro 980 - # 985 - # 990 - - Asp Gly Ala Thr Ser Leu Thr Pro Cys Thr Cy - #s Gln Ala Glu Ser Cys 995 - # 1000 - # 1005 - - Trp Val Ile Arg Ser Asp Gly Ala Leu Cys Hi - #s Gly Leu Ser Lys Gly 1010 - # 1015 - # 1020 - - Asp Lys Val Glu Leu Asp Val Ala Met Glu Va - #l Ser Asp Phe Arg Gly 1025 1030 - # 1035 - # 1040 - - Ser Ser Gly Ser Pro Val Leu Cys Asp Glu Gl - #y His Ala Val Gly Met 1045 - # 1050 - # 1055 - - Leu Val Ser Val Leu His Ser Gly Gly Arg Va - #l Thr Ala Ala Arg Phe 1060 - # 1065 - # 1070 - - Thr Arg Pro Trp Thr Gln Val Pro Thr Asp Al - #a Lys Thr Thr Thr Glu 1075 - # 1080 - # 1085 - - Pro Pro Pro Val Pro Ala Lys Gly Val Phe Ly - #s Glu Ala Pro Leu Phe 1090 - # 1095 - # 1100 - - Met Pro Thr Gly Ala Gly Lys Ser Thr Arg Va - #l Pro Leu Glu Tyr Gly 1105 1110 - # 1115 - # 1120 - - Asn Met Gly His Lys Val Leu Ile Leu Asn Pr - #o Ser Val Ala Thr Val 1125 - # 1130 - # 1135 - - Arg Ala Met Gly Pro Tyr Met Glu Arg Leu Al - #a Gly Lys His Pro Ser 1140 - # 1145 - # 1150 - - Ile Tyr Cys Gly His Asp Thr Thr Ala Phe Th - #r Arg Ile Thr Asp Ser 1155 - # 1160 - # 1165 - - Pro Leu Thr Tyr Ser Xaa Tyr Gly Arg Phe Le - #u Ala Asn Pro Arg Gln 1170 - # 1175 - # 1180 - - Met Leu Arg Gly Val Ser Val Val Ile Cys As - #p Glu Cys His Ser His 1185 1190 - # 1195 - # 1200 - - Asp Ser Thr Val Leu Leu Gly Ile Gly Arg Va - #l Arg Glu Leu Ala Arg 1205 - # 1210 - # 1215 - - Gly Cys Gly Val Gln Leu Val Leu Tyr Ala Th - #r Ala Thr Pro Pro Gly 1220 - # 1225 - # 1230 - - Ser Pro Met Thr Gln His Pro Ser Ile Ile Gl - #u Thr Lys Leu Asp Val 1235 - # 1240 - # 1245 - - Gly Glu Ile Pro Phe Tyr Gly His Gly Ile Pr - #o Leu Glu Arg Met Arg 1250 - # 1255 - # 1260 - - Thr Gly Arg His Leu Val Phe Cys His Ser Ly - #s Ala Glu Cys Glu Arg 1265 1270 - # 1275 - # 1280 - - Leu Ala Gly Gln Phe Ser Ser Arg Gly Val As - #n Ala Val Ala Tyr Tyr 1285 - # 1290 - # 1295 - - Arg Gly Lys Asp Ser Ser Ile Ile Lys Asp Gl - #y Asp Leu Val Val Cys 1300 - # 1305 - # 1310 - - Ala Thr Asp Ala Leu Ser Thr Gly Tyr Thr Gl - #y Asn Phe Asp Ser Val 1315 - # 1320 - # 1325 - - Thr Asp Cys Gly Leu Val Val Glu Glu Val Va - #l Glu Val Thr Leu Asp 1330 - # 1335 - # 1340 - - Pro Thr Ile Thr Ile Ser Leu Arg Thr Val Pr - #o Ala Ser Ala Glu Leu 1345 1350 - # 1355 - # 1360 - - Ser Met Gln Arg Arg Gly Arg Thr Gly Arg Gl - #y Arg Ser Gly Arg Tyr 1365 - # 1370 - # 1375 - - Tyr Tyr Ala Gly Val Gly Lys Ala Pro Ala Gl - #y Val Val Arg Ser Gly 1380 - # 1385 - # 1390 - - Pro Val Trp Ser Ala Val Glu Ala Gly Val Th - #r Trp Tyr Gly Met Glu 1395 - # 1400 - # 1405 - - Pro Asp Leu Thr Ala Asn Leu Leu Arg Leu Ty - #r Asp Asp Cys Pro Tyr 1410 - # 1415 - # 1420 - - Thr Ala Ala Val Ala Ala Asp Ile Gly Glu Al - #a Ala Val Phe Phe Ala 1425 1430 - # 1435 - # 1440 - - Gly Leu Ala Pro Leu Arg Met His Pro Asp Va - #l Ser Trp Ala Lys Val 1445 - # 1450 - # 1455 - - Arg Gly Val Asn Trp Pro Leu Leu Val Gly Va - #l Gln Arg Thr Met Cys 1460 - # 1465 - # 1470 - - Arg Glu Thr Leu Ser Pro Gly Pro Ser Asp As - #p Pro Gln Trp Ala Gly 1475 - # 1480 - # 1485 - - Leu Lys Gly Pro Asn Pro Val Pro Leu Leu Le - #u Arg Trp Gly Asn Asp 1490 - # 1495 - # 1500 - - Leu Pro Ser Lys Val Ala Gly His His Ile Va - #l Asp Asp Leu Val Arg 1505 1510 - # 1515 - # 1520 - - Arg Leu Gly Val Ala Glu Gly Tyr Val Arg Cy - #s Asp Ala Gly Pro Ile 1525 - # 1530 - # 1535 - - Leu Met Val Gly Leu Ala Ile Ala Gly Gly Me - #t Ile Tyr Ala Ser Tyr 1540 - # 1545 - # 1550 - - Thr Gly Ser Leu Val Val Val Thr Asp Trp As - #p Val Lys Gly Gly Gly 1555 - # 1560 - # 1565 - - Asn Pro Leu Tyr Arg Ser Gly Asp Gln Ala Th - #r Pro Gln Pro Val Val 1570 - # 1575 - # 1580 - - Gln Val Pro Pro Val Asp His Arg Pro Gly Gl - #y Glu Ser Ala Pro Ser 1585 1590 - # 1595 - # 1600 - - Asp Ala Lys Thr Val Thr Asp Ala Val Ala Al - #a Ile Gln Val Asp Cys 1605 - # 1610 - # 1615 - - Asp Trp Ser Val Met Thr Leu Ser Ile Gly Gl - #u Val Leu Ser Leu Ala 1620 - # 1625 - # 1630 - - Gln Ala Lys Thr Ala Glu Ala Tyr Thr Ala Th - #r Ala Lys Trp Leu Ala 1635 - # 1640 - # 1645 - - Gly Cys Tyr Thr Gly Thr Arg Ala Val Pro Th - #r Val Ser Ile Val Asp 1650 - # 1655 - # 1660 - - Lys Leu Phe Ala Gly Gly Trp Ala Ala Val Va - #l Gly His Cys His Ser 1665 1670 - # 1675 - # 1680 - - Val Ile Ala Ala Ala Val Ala Ala Tyr Gly Al - #a Ser Arg Ser Pro Pro 1685 - # 1690 - # 1695 - - Leu Ala Ala Ala Ala Ser Tyr Leu Met Gly Le - #u Gly Val Gly Gly Asn 1700 - # 1705 - # 1710 - - Ala Gln Thr Arg Leu Ala Ser Ala Leu Leu Le - #u Gly Ala Ala Gly Thr 1715 - # 1720 - # 1725 - - Ala Leu Gly Thr Pro Val Val Gly Leu Thr Me - #t Ala Gly Ala Phe Met 1730 - # 1735 - # 1740 - - Gly Gly Ala Ser Val Ser Pro Ser Leu Val Th - #r Ile Leu Leu Gly Ala 1745 1750 - # 1755 - # 1760 - - Val Gly Gly Trp Glu Gly Val Val Asn Ala Al - #a Ser Leu Val Phe Asp 1765 - # 1770 - # 1775 - - Phe Met Ala Gly Lys Leu Ser Ser Glu Asp Le - #u Trp Tyr Ala Ile Pro 1780 - # 1785 - # 1790 - - Val Leu Thr Ser Pro Gly Ala Gly Leu Ala Gl - #y Ile Ala Leu Gly Leu 1795 - # 1800 - # 1805 - - Val Leu Tyr Ser Ala Asn Asn Ser Gly Thr Th - #r Thr Trp Leu Asn Arg 1810 - # 1815 - # 1820 - - Leu Leu Thr Thr Leu Pro Pro Ser Ser Cys Il - #e Pro Asp Ser Tyr Phe 1825 1830 - # 1835 - # 1840 - - Gln Gln Ala Asp Tyr Cys Asn Lys Val Ser Al - #a Val Leu Arg Arg Leu 1845 - # 1850 - # 1855 - - Ser Leu Thr Arg Thr Val Val Ala Leu Val As - #n Arg Glu Pro Lys Val 1860 - # 1865 - # 1870 - - Asp Glu Val Gln Val Gly Tyr Val Trp Asp Le - #u Trp Glu Trp Ile Met 1875 - # 1880 - # 1885 - - Arg Gln Val Arg Met Val Met Ala Arg Leu Ar - #g Ala Leu Cys Pro Val 1890 - # 1895 - # 1900 - - Val Ser Leu Pro Leu Trp His Cys Gly Glu Gl - #y Trp Ser Gly Glu Trp 1905 1910 - # 1915 - # 1920 - - Leu Leu Asp Gly His Val Glu Ser Arg Cys Le - #u Cys Gly Cys Val Ile 1925 - # 1930 - # 1935 - - Thr Gly Asp Val Leu Asn Gly Gln Leu Lys Gl - #u Pro Val Tyr Ser Thr 1940 - # 1945 - # 1950 - - Lys Leu Cys Arg His Tyr Trp Met Gly Thr Va - #l Pro Val Asn Met Leu 1955 - # 1960 - # 1965 - - Gly Tyr Gly Glu Thr Ser Pro Leu Leu Ala Se - #r Asp Thr Pro Lys Val 1970 - # 1975 - # 1980 - - Val Pro Phe Gly Thr Ser Gly Trp Ala Glu Va - #l Val Val Thr Pro Thr 1985 1990 - # 1995 - # 2000 - - His Val Val Ile Arg Arg Thr Ser Ser Tyr Ly - #s Leu Leu Arg Gln Gln 2005 - # 2010 - # 2015 - - Ile Leu Ser Ala Ala Val Ala Glu Pro Tyr Ty - #r Val Asp Gly Ile Pro 2020 - # 2025 - # 2030 - - Val Ser Trp Asp Ala Asp Ala Arg Ala Pro Al - #a Met Val Tyr Gly Pro 2035 - # 2040 - # 2045 - - Gly Gln Ser Val Thr Ile Asp Gly Glu Arg Ty - #r Thr Leu Pro His Gln 2050 - # 2055 - # 2060 - - Leu Arg Leu Arg Asn Val Ala Pro Ser Glu Va - #l Ser Ser Glu Val Ser 2065 2070 - # 2075 - # 2080 - - Ile Asp Ile Gly Thr Glu Thr Glu Asp Ser Gl - #u Leu Thr Glu Ala Asp 2085 - # 2090 - # 2095 - - Leu Pro Pro Ala Ala Ala Ala Leu Gln Ala Il - #e Glu Asn Ala Ala Arg 2100 - # 2105 - # 2110 - - Ile Leu Glu Pro His Ile Asp Val Ile Met Gl - #u Asp Cys Ser Thr Pro 2115 - # 2120 - # 2125 - - Ser Leu Cys Gly Ser Ser Arg Glu Met Pro Va - #l Trp Gly Glu Asp Ile 2130 - # 2135 - # 2140 - - Pro Xaa Thr Pro Ser Pro Ala Leu Ile Ser Va - #l Thr Glu Ser Ser Ser 2145 2150 - # 2155 - # 2160 - - Asp Glu Lys Thr Pro Ser Val Ser Ser Ser Gl - #n Glu Asp Thr Pro Ser 2165 - # 2170 - # 2175 - - Ser Asp Ser Phe Glu Val Ile Gln Glu Ser Gl - #u Thr Ala Glu Gly Glu 2180 - # 2185 - # 2190 - - Asp Ser Val Phe Asn Val Ala Leu Ser Val Le - #u Lys Ala Leu Phe Pro 2195 - # 2200 - # 2205 - - Gln Ser Asp Ala Thr Arg Lys Leu Thr Val Ly - #s Met Ser Cys Cys Val 2210 - # 2215 - # 2220 - - Glu Lys Ser Val Thr Arg Phe Phe Ser Leu Gl - #y Leu Thr Val Ala Asp 2225 2230 - # 2235 - # 2240 - - Val Ala Ser Leu Cys Glu Met Glu Ile Gln As - #n His Thr Ala Tyr Cys 2245 - # 2250 - # 2255 - - Asp Lys Val Arg Thr Pro Leu Glu Leu Gln Va - #l Gly Cys Leu Val Gly 2260 - # 2265 - # 2270 - - Asn Glu Leu Thr Phe Glu Cys Asp Lys Cys Gl - #u Ala Arg Gln Glu Thr 2275 - # 2280 - # 2285 - - Leu Ala Ser Phe Ser Tyr Ile Trp Ser Gly Va - #l Pro Leu Thr Arg Ala 2290 - # 2295 - # 2300 - - Thr Pro Ala Lys Pro Pro Val Val Arg Pro Va - #l Gly Ser Leu Leu Val 2305 2310 - # 2315 - # 2320 - - Ala Asp Thr Thr Lys Val Tyr Val Thr Asn Pr - #o Asp Asn Val Gly Arg 2325 - # 2330 - # 2335 - - Arg Val Asp Lys Val Thr Phe Trp Arg Ala Pr - #o Arg Val His Asp Lys 2340 - # 2345 - # 2350 - - Phe Leu Val Asp Ser Ile Glu Arg Ala Arg Ly - #s Ala Ala Gln Ala Cys 2355 - # 2360 - # 2365 - - Leu Ser Met Gly Tyr Thr Tyr Glu Glu Ala Il - #e Arg Thr Val Arg Pro 2370 - # 2375 - # 2380 - - His Ala Ala Met Gly Trp Gly Ser Lys Val Se - #r Val Lys Asp Leu Ala 2385 2390 - # 2395 - # 2400 - - Thr Pro Ala Gly Lys Met Ala Val His Asp Ar - #g Leu Gln Glu Ile Leu 2405 - # 2410 - # 2415 - - Glu Gly Thr Pro Val Pro Phe Thr Leu Thr Va - #l Lys Lys Glu Val Phe 2420 - # 2425 - # 2430 - - Phe Lys Asp Arg Lys Glu Glu Lys Ala Pro Ar - #g Leu Ile Val Phe Pro 2435 - # 2440 - # 2445 - - Pro Leu Asp Phe Arg Ile Ala Glu Lys Leu Il - #e Leu Gly Asp Pro Gly 2450 - # 2455 - # 2460 - - Arg Val Ala Lys Ala Val Leu Gly Gly Ala Ty - #r Ala Phe Gln Tyr Thr 2465 2470 - # 2475 - # 2480 - - Pro Asn Gln Arg Val Lys Glu Met Leu Lys Le - #u Trp Glu Ser Lys Lys 2485 - # 2490 - # 2495 - - Thr Pro Cys Ala Ile Cys Val Asp Ala Thr Cy - #s Phe Asp Ser Ser Ile 2500 - # 2505 - # 2510 - - Thr Glu Glu Asp Val Ala Leu Glu Thr Glu Le - #u Tyr Ala Leu Ala Ser 2515 - # 2520 - # 2525 - - Asp His Pro Glu Trp Val Arg Ala Leu Gly Ly - #s Tyr Tyr Ala Ser Gly 2530 - # 2535 - # 2540 - - Thr Met Val Thr Pro Glu Gly Val Pro Val Gl - #y Glu Arg Tyr Cys Arg 2545 2550 - # 2555 - # 2560 - - Ser Ser Gly Val Leu Thr Thr Ser Ala Thr As - #n Cys Leu Thr Cys Tyr 2565 - # 2570 - # 2575 - - Ile Lys Val Ser Ala Ala Cys Gln Arg Val Gl - #y Leu Lys Asn Val Ser 2580 - # 2585 - # 2590 - - Leu Leu Ile Ala Gly Asp Asp Cys Leu Ile Il - #e Cys Glu Arg Pro Val 2595 - # 2600 - # 2605 - - Cys Asp Pro Ser Glu Ala Leu Gly Arg Ala Le - #u Ala Ser Tyr Gly Tyr 2610 - # 2615 - # 2620 - - Ala Cys Glu Pro Ser Tyr His Ala Ser Leu As - #p Thr Ala Pro Phe Cys 2625 2630 - # 2635 - # 2640 - - Ser Thr Trp Leu Ala Glu Cys Asn Ala Asp Gl - #y Lys Arg His Phe Phe 2645 - # 2650 - # 2655 - - Leu Thr Thr Asp Phe Arg Arg Pro Leu Ala Ar - #g Met Ser Ser Glu Tyr 2660 - # 2665 - # 2670 - - Ser Asp Pro Met Ala Ser Ala Ile Gly Tyr Il - #e Leu Leu Tyr Pro Trp 2675 - # 2680 - # 2685 - - His Pro Ile Thr Arg Trp Val Ile Ile Pro Hi - #s Val Leu Thr Cys Ala 2690 - # 2695 - # 2700 - - Phe Arg Gly Gly Gly Thr Pro Ser Asp Pro Va - #l Trp Cys Gln Val His 2705 2710 - # 2715 - # 2720 - - Gly Asn Tyr Tyr Lys Phe Pro Leu Asp Lys Xa - #a Pro Asn Ile Ile Val 2725 - # 2730 - # 2735 - - Ala Leu His Gly Pro Ala Ala Leu Arg Val Th - #r Ala Asp Thr Thr Lys 2740 - # 2745 - # 2750 - - Thr Lys Met Glu Ala Gly Lys Val Leu Ser As - #p Leu Lys Leu Pro Gly 2755 - # 2760 - # 2765 - - Leu Ala Val His Arg Lys Lys Ala Gly Ala Le - #u Arg Thr Arg Met Leu 2770 - # 2775 - # 2780 - - Arg Ser Arg Gly Trp Ala Glu Leu Ala Arg Gl - #y Leu Leu Trp His Pro 2785 2790 - # 2795 - # 2800 - - Gly Xaa Arg Leu Pro Pro Pro Glu Ile Ala Gl - #y Ile Pro Gly Gly Phe 2805 - # 2810 - # 2815 - - Pro Leu Ser Pro Pro Tyr Met Gly Val Val Hi - #s Gln Leu Asp Phe Thr 2820 - # 2825 - # 2830 - - Ser Gln Arg Ser Arg Trp Arg Trp Leu 2835 - # 2840 - - - - (2) INFORMATION FOR SEQ ID NO:47: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: - - AAAATTTCTA GAGCCACCAT GAGGCTCGTC ATGGTCTTCC - # - # 40 - - - - (2) INFORMATION FOR SEQ ID NO:48: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: - - AAAACCTCTA GAGCCACCAT GGCAGTCCTT CTGCTC - # -# 36 - - - - (2) INFORMATION FOR SEQ ID NO:49: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: - - AAAATTTCTA GATCACTATC CCCGCCARAA GTCRAAAGG - # - # 39 - - - - (2) INFORMATION FOR SEQ ID NO:50: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: - - AAAATTTCTA GAGCCACCAT GGCGCCTGTT GTCATCCGTC GG - # - # 42 - - - - (2) INFORMATION FOR SEQ ID NO:51: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO::51: - - AAAACCTCTA GATCACTATG TWACCACCAC WAGYGACCC - # - # 39 - - - - (2) INFORMATION FOR SEQ ID NO:52: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: - - AAAATTCCTA GGGCCACCAT GGTSGGRTAC GTCTGGGAYY TGTG - # - # 44 - - - - (2) INFORMATION FOR SEQ ID NO:53: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: - - AAAACCCCTA GGTCACTAGG CMARGGTCTC TTGSCKTGCC - # - # 40 - - - - (2) INFORMATION FOR SEQ ID NO:54: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: - - AAAATTGGAT CCAGGCGCGC CCGCCTCAGT GTTG - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:55: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: - - AAAATTGGAT CCTTAGGGCG ACCGCACCGG GTTGCCT - #- # 37 - - - - (2) INFORMATION FOR SEQ ID NO:56: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: - - AAAACCGGAT CCTTACGGAA TGAAGTTGCC CGCATCC - #- # 37 - - - - (2) INFORMATION FOR SEQ ID NO:57: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: - - ATATAGATCT GCCACCATGA GGCTCGTCAT GGTCTTCC - # - # 38 - - - - (2) INFORMATION FOR SEQ ID NO:58: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: - - GATTAGATCT TCACTACGGA ATGAAGTTGC CCGCATCC - #- # 38 - - - - (2) INFORMATION FOR SEQ ID NO:59: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: - - ATATAGATCT AACCCATGCT TGTAGCGCGA AAGG - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:60: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: - - GATTAGATCT TCACTATCCC CGCCARAAGT CRAAAGG - #- # 37 - - - - (2) INFORMATION FOR SEQ ID NO:61: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: - - ATATAGATCT GCCACCATGG CAGTCCTTCT GCTC - # -# 34 - - - - (2) INFORMATION FOR SEQ ID NO:62: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: - - ATATAGATCT GCCACCATGG CGCCTGTTGT CATCCGTCGG - # - # 40 - - - - (2) INFORMATION FOR SEQ ID NO:63: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: - - GATTAGATCT TCACTATGTW ACCACCACWA GYGACCC - #- # 37 - - - - (2) INFORMATION FOR SEQ ID NO:64: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: - - ATATAGATCT GCCACCATGG TSGGRTACGT CTGGGAYYTG TG - # - # 42 - - - - (2) INFORMATION FOR SEQ ID NO:65: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: - - GATTAGATCT TCACTAGGCM ARGGTCTCTT GSCKTGCC - #- # 38 - - - - (2) INFORMATION FOR SEQ ID NO:66: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: - - ATATAGATCT GCCACCATGG CAGTCCTTCT GCTCCTAC - #- # 38 - - - - (2) INFORMATION FOR SEQ ID NO:67: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: - - GATTAGATCT TCACTAAGTC TTGGCGTCTG TTGGGACT - #- # 38 - - - - (2) INFORMATION FOR SEQ ID NO:68: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: - - GATCGGGATC CTGGCGCGCC CGCCTCAGTG T - # - # 31 - - - - (2) INFORMATION FOR SEQ ID NO:69: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: - - ATATACTGCA GCTATCACTT GTCGTCGTCG TCCTTGTAGT CCGGAATGAA GT -#TGCCCGCA 60 - - TCC - # - # - # 63 - - - - (2) INFORMATION FOR SEQ ID NO:70: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: - - Gln Gly Ala Pro Ala Ser Val Leu Gly Ser Ar - #g Pro Phe Gln - #5 - #10__________________________________________________________________________

Number	Name	Date
4743535	Carrico	May 1988
4876187	Duck et al.	Oct 1989
5275947	Arima et al.	Jan 1994
5399346	Anderson et al.	Mar 1995
5527669	Resnick et al.	Jun 1996
5576302	Cook et al.	Nov 1996
5766840	Kim et al.	Jun 1998

Number	Date	Country
0318216	May 1989	EPX
9000597	Jan 1990	WOX
9408002	Apr 1994	WOX
9418217	Aug 1994	WOX
9532290	Nov 1995	WOX
9532292	Nov 1995	WOX
9532291	Nov 1995	WOX
WO 9532291	Nov 1995	WOX
9506266	May 1996	WOX

	Number	Date	Country
Parent	480995	Jun 1995
Parent	417629	Apr 1995

Hepatitis GB virus recombinant proteins and uses thereof

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US Referenced Citations (7)

Foreign Referenced Citations (9)

Non-Patent Literature Citations (82)

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Entry
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