Polypeptide originating in haemophilus paragallinarum and process for producing the same

Abstract

A novel peptide obtained from Haemophilus paragallinarum has been found useful for preventing avian infectious coryza. This polypeptide induces production of hemagglutination-inhibition antibody and prevents infection and onset of avian infectious coryza. The invention further provides a gene coding for the polypeptide, a recombinant vector for expression of this gene, a host transformed with this vector, a process for preparing the polypeptide in a host, a vaccine for avian infectious coryza comprising the polypeptide as an active ingredient, a monoclonal antibody obtained using the polypeptide as an immunogen, and a diagnostic agent and a therapeutic agent for avian infectious coryza using the peptide and the antibody.

Description

FIELD OF THE INVENTION

The present invention relates to a polypeptide which can prevent avian infectious coryza. More particularly, the present invention relates to a polypeptide from Haemophilus paragallinarum, the causative agent of avian infectious coryza, a gene coding for said polypeptide and an antibody protein which recognizes said polypeptide. The present invention further relates to a process for preparing said polypeptide and the use of said polypeptide for a vaccine, a diagnostic agent and a therapeutic agent.

BACKGROUND ART

Avian infectious coryza is one of the most important respiratory diseases in poultry, which is an acute respiratory disease caused by infection with Haemophilus paragallinarum (hereinafter also referred to as “HPG”) with cardinal symptoms being a running nose, swelling of the face and epiphora. Avian infectious coryza brings about a great economical damage since it leads to decrease in the breeding rate of poultry, retarding of egg laying, decrease in egg production or failure of egg laying. For prevention of avian infectious coryza, an inactivated vaccine has hitherto been used widely which is obtained by culturing Haemophilus paragalinarum, recovering and inactivating the cells with formalin, thimerosal and the like. However, adverse side effects caused by such an inactivated vaccine has been an issue as it has been reported that local necrotic lesions are formed in the inoculated chicken when the vaccine is administered (M. Matsumoto and R. Yamamoto, Avian Dis., 15: 109-117, 1971), and hence, development of a highly safe vaccine is earnestly desired.

In recent years, laborsaving in breeding and managing poultry is in progress with a scale-up of breeding poultry. As a part of this, laborsaving in vaccination has also been earnestly desired, and as a result, a mixed vaccine has already been developed and widely used in the field so that a frequency of inoculation can be reduced by mixing several kinds of vaccines together.

In order to provide a mixed vaccine showing immunogenicity equivalent to that of each plain vaccine without increase of dosage amount, it is necessary to increase an amount of each antigen contained in a mixed vaccine or to find out and use a more suitable adjuvant. However, in case of gram-negative bacteria such as HPG, a higher amount of antigen is likely to enhance a response to injection such as swelling at the inoculated site. Therefore, in order to reduce such an adverse response, it is preferable to obtain only a protective antigen, i.e. an effective component, from bacterial cells or culture supernatant, or to clone a gene coding for said antigen by the genetic recombination technique, to express said gene in bacteria, yeast, an animal cell, a plant cell, an insect cell and the like, and to purify a product expressed in a large amount, which is then mixed with an appropriate adjuvant together with other vaccines.

Another approach for laborsaving of vaccination is the use of virus or bacteria as a vector. That is, genes coding for protective antigens from one or plural pathogens have been incorporated into an attenuated virus or bacteria to prepare a polyvalent live vaccine. For fowls, poxvirus, Marek's disease virus and the like have been investigated as a vector. A vaccine comprising a viral vector has been put into practice wherein genes coding for HN and F proteins of Newcastle disease virus are incorporated into fowl pox virus.

It is thus most important to identify a protective antigen of HPG for development of a safe and effective vaccine against avian infectious coryza both as a component vaccine and as a vector vaccine.

Among protective antigens of HPG such as hemagglutinin (HA) and outer-membrane protein, HA is considered a most important antigen since immunization of chicken with HPG increases a hemagglutination-inhibition antibody (hereinafter referred to as “HI antibody”) and higher protective effect is observed for chickens with high level of HI antibody (K. Otsuki and Y. Iritani, Avian Dis., 18: 297-304, 1974 and K. Kume et al., Jpn. J. Vet. Sci., 46: 843-850, 1984).

Serotype of HPG is classified into serotypes A, B and C (Page, Am. J. Vet. Res., 23: 85-95, 1962) or into serotypes 1 and 2 (Sawata et al., Jpn. J. Vet. Sci., 40: 645-652, 1978) based on the agglutination test. It is considered that serotype A by Page corresponds to serotype 1 by Sawata et al. whereas serotype C by Page corresponds to serotype 2 by Sawata et al. (K. Kume, et al., Am. J. Vet. Res., 41: 757-760, 1980 and Sawata et al., Am. J. Vet. Res., 41: 1901-1904, 1980).

Kume et al. reported that HPG serotype A (serotype 1) has at least three kinds of HA, i.e. HA-L (heat-labile, trypsin-sensitive), HA-HL (heat-labile, trypsin-resistant) and HA-HS (heast-stable, trypsin-resistant), and that HA-L alone exhibits not only HA activity to usual fresh chicken erythrcytes but also to glutaraldehyde-fixed chicken erythrocytes and is involved in protection against infection with HPG serotype A (K. Kume, Jpn. J. Vet. Sci., 45: 783-792, 1983 and Sawata et al., Jpn. J. Vet. Sci., 46: 21-29, 1984).

Iritani et al. reported that HPG serotype A has two kinds of HA, i.e. type 1 HA (heat-labile, protease-sensitive) and type 2 HA (heat-labile, protease-resistant), and that type 1 HA, which is heat-labile and protease-sensitive and consisted of a polypeptide having a molecular weight of about 39 kd as a subunit, is involved in protection against infection (T. Yamaguchi and Y Iritani, Jpn. J. Vet. Sci., 42: 709-711, 1980 and Y. Iritani et al., Am. J. Vet. Res., 41: 2114-2118, 1980). It is considered that HA-L and HA-HL by Kume et al. correspond to type 1 HA and type 2 HA by Iritani et al., respectively. As to HPG serotype C (serotype 2), Sawata et al. reported that an antigen was found which is heat-labile and trypsin-sensitive and exhibits the HA activity to glutaraldehyde-fixed chicken erythrocytes and that this antigen is distinct from HA of HPG serotype A in their antigenicity (Sawata et al., Am. J. Vet. Res., 43: 1311-1314, 1982). However, to date, a protective antigen of HPG has not yet seen materially identified except for type 1 HA produced by HPG serotype A as reported by Iritani et al.

As mentioned hereinabove, the conventional inactivated vaccine obtained by inactivating Haemophilus paragallinarum cells with thimerosal, formalin and the like has provoked problems in that the adverse side effects as mentioned above are induced when it is applied to fowls in a large amount since it includes various substances from the cells other than the protective antigen.

DISCLOSURE OF INVENTION

The inventor has earnestly studied in order to solve the problems, and as a result, has successfully purified, from a culture supernatant of Haemophilus paragallinarum serotype A, a polypeptide having about 130 kd of molecular weight from Haemophilus paragallinarum serotype A, said polypeptide inducing production of HI antibody and protecting against avian infectious coryza by Haemophilus paragallinarum serotype A.

Furthermore, the present inventor has prepared a genomic DNA library from HPG serotype A, cloned a gene fragment coding for the above 130 Kd polypeptide, expressed said gene fragment in E. coli and has found that the produced polypeptide could prevent avian infectious coryza by Haemophilus paragallinarum serotype A. Said gene fragment coding for the above 130 Kd polypeptide was also used as a probe for cloning a gene fragment hybridizable with said DNA fragment from HPG serotype C to give E. coli which expresses the polypeptide from HPG serotype C.

The present invention provides a safer, effective vaccine against avian infectious coryza, pathogenic bacteria of which is Haemophilus paragallinarum, with less adverse side effects and a process for preparing the same.

That is, an object of the present invention is to provide a novel polypeptide from Haemophilus paragallinarum as well as a peptide which shares at least a portion of the amino acid sequence.

Another object of the present invention is to provide a gene coding for said novel polypeptide from Haemophilus paragallinarum as well as the peptide which shares at least a potion of the amino acid sequence and a recombinant vector for expression of said gene.

Still another object of the present invention is to provide a process for preparing said novel polypeptide from Haemophilus paragallinarum and the polypeptide which shares at least a portion of the amino acid sequence from microorganisms or cells transformed with said recombinant vector.

Still further object of the present invention is to provide a monoclonal or polyclonal antibody which is prepared by using as an immunogen the thus prepared novel peptide from Haemophilus paragallinarum or the polypeptide which shares at least a portion of the amino acid sequence.

Still another object of the present invention is to provide a method for detecting Haemophilus paragallinarum or an antibody thereto by a combination of the above-mentioned peptide, DNA fragment, transformant or antibody.

Still further object of the present invention is to provide a therapeutic agent for avian infectious coryza which comprises as an active ingredient the antibody against the novel polypeptide from Haemophilus paragallinarum.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results obtained by challenging chickens with Haemophilus paragallinarum serotype A strain 221 after passive immunization with monoclonal antibodies (clones HpgA 59-40, HpgA 59-180 and HpgA 59-284) wherein the onset of the disease was retarded in the groups previously administered with the monoclonal antibodies having the HI activity (clones HpgA 59-40 and HpgA 59-180).

FIG. 2 shows the results obtained by challenging chickens with Haemophilus paragallinarum serotype A strain 221 after passive immunization with monoclonal antibodies (clones HpgA 59-33, HpgA 59-48B and HpgA 59-180) wherein the onset of the disease was retarded in the groups previously administered with the monoclonal antibody having the HI activity (clone HpgA 59-180).

FIG. 3 shows the results obtained by challenging chickens with Haemophilus paragallinarum serotype A strain 221 after passive immunization with monoclonal antibodies (clones HpgA 59-48A, HpgA 59-145 and HpgA 59-180) wherein the onset of the disease was retarded in the groups previously administered with the monoclonal antibodies having the HI activity (clones HpgA 59-145 and HpgA 59-180).

FIG. 4 shows the results obtained by challenging chickens with Haemophilus paragallinarum serotype A strain 221 after passive immunization with monoclonal antibodies (clones HpgA 59-188, HpgA 59-236 and HpgA 59-180) wherein the onset of the disease was retarded in the groups previously administered with the monoclonal antibody having the HI activity (clone HpgA 59-180).

FIG. 5 is a photograph showing the result of SDS-PAGE electrophoresis with CBB staining of HPGp130 polypeptide which is purified by affinity chromatography using the monoclonal antibody having the HI activity (clone HpgA 59-180) as a ligand.

FIG. 6 is (a) a photograph showing the results of SDS-PAGE electrophoresis with CBB staining of the purified HPGp130 polypeptide and Haemophilus paragallinarum serotype A strain 221 treated with 2-mercaptoethanol; and (b) a photograph showing the results of detection of proteins reactive with guinea pig antiserum against the purified HPGp130 polypeptide after SDS-PAGE electrophoresis of the purified HPGp130 polypeptide and Haemophilus paragallinarum serotype A strain 221 treated with 2-mercaptoethanol and transferring to a thin membrane (PVDF).

FIG. 7 is a schematic illustration showing the position of HPG1.2 k DNA, HPG3.5 k DNA, HPG4.1 k DNA, HPG6.7 k DNA and HPG2.7 k DNA fragments cloned from the genome of Haemophilus paragallinarum serotype A strain 221.

FIG. 8 is a schematic illustration showing construction of plasmid pSA4.1 by inserting the XhoI-XbaI fragment (HPG4.1 k DNA) from the genome of Haemophilus paragallinarum serotype A strain 221 into plasmid pSP72, followed by construction of plasmid pTA4.1 by inserting the XhoI-KpnI fragment from the plasmid pSA4.1 into plasmid pTrcHisC.

FIG. 9 is a schematic illustration showing construction of plasmid pSA6.7 by inserting the XhoI-PstI fragment (HPG6.7 k DNA) from the genome of Haemophilus paragallinarum serotype A strain 221 into plasmid pSP72, followed by construction of plasmid pSA2.7 by inserting the XbaI fragment from the plasmid pSA6.7 into plasmid pSP72.

FIG. 10 is a photograph showing the results of detection of DNA fragments hybridizable with HPG1.2 k DNA as a probe after agarose electrophoresis of DNA fragments obtained by digesting the genome from Haemophilus paragallinarum serotypes A, B and C with restriction enzyme EcoRI and transferring to a thin membrane (Hybond N+).

FIG. 11 is a schematic illustration showing the position of HPG-C1 DNA, HPG-C2 DNA, HPG-C3 DNA and HPG-C4 DNA fragments cloned from the genome of Haemophilus paragallinarum serotype C strain 53-47.

FIG. 12 is a photograph showing the result of 0.8% agarose gel electrophoresis of PCR products obtained by PCR with primers prepared on the basis of the nucleotide sequences coding for the N-terminal and C-terminal amino acid sequences of HPG serotype A HMTp210 polypeptide and the genome of Haemophilus paragallinarum serotype A, B or C as a template.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is explained in more detail hereinbelow.

The polypeptide from Haemophilus paragallinarum serotype A of the present invention which induces production of the HI antibody is prepared from a culture supernatant of HPG serotype A or a suspension of ruptured cells by affinity chromatography with the monoclonal antibody having the HI activity as a ligand.

The monoclonal antibody having the HI activity (hereinafter also referred to as “HI-MCA”) is obtained by preparing the hybridomas producing the monoclonal antibodies which bind to Haemophilus paragallinarum serotype A by the conventional cell fusion procedure and then screening the hybridoma producing the monoclonal antibody having the HI activity with HI test.

For use as an immunogen for production of the above antibody, Haemophilus paragallinarum serotype A is obtained by the conventional procedure used for usual culture of Haemophilus paragallinarum. For example, the cells of HPG strain 221 can be recovered by shaking culture in a chicken meat infusion medium supplemented with chicken serum (including chicken meat infusion 300 ml, chicken serum 10 ml, polypeptone 5 g, glucose 1 g, casamino acid 1 g, sodium glutamate 5 g, sodium chloride 5 g, nicotinamide adenine dinucleotide 0.025 g in 1000 ml medium) at 37° C. overnight followed by centrifugation.

Immunization can be carried out in a usual manner, for example, after inactivating Haemophilus paragallinarum serotype A with thimerosal, formalin and the like, by administering the inactivated cells together with the conventional adjuvant to BALB/c mouse via intraperitoneal, subcutaneous, intradermal or intravenous administration. The immunogen includes HPG cells per se, or alternatively, the cells treated with potassium rhodanide, sonication or hyaluronidase, or a processed antigen obtained by treatment with a surf actant such as sodium N-lauroylsarcosinate, NONIDET P-40 or TRITON X-100. The adjuvant includes Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide gel, and the like. More specifically, immunization is conducted as follows: Haemophilus paragallinarum serotype A strain 221 cultured in a chicken meat infusion medium supplemented with chicken serum is inactivated with thimerosal and then sonicated. An emulsion obtained by mixing the resultant suspension of ruptured cells with Freund's complete adjuvant is administered subcutaneously to the back of BALB/c mouse, followed by subcutaneous administration of an emulsion prepared from the same amount of the suspension and Freund's incomplete adjuvant at the back every 2 to 4 weeks. A serum antibody level is monitored, and after confirming the elevated level of antibody titer, a suspension of ruptured cells after sonication is further administered intravenously after additional 2 to 4 weeks as a final immunization.

As an immunocyte for preparing a monoclonal antibody, splenocytes removed 2 to 4 days after the final administration is preferably used. Mouse myeloma cells include, for example, NSI-Ag4/1 (Eur. J. Immunol., 6: 511, 1976), P3X63-Ag8.U1 (Curr. Topics Microbiol. Immunol., 81:1, 1978), X63-Ag8.653 (J. Immunol., 123: 1548, 1979), and the like. Fusion of splenocytes with mouse myeloma cells may be carried out in accordance with Milstein et al., Method Enzymol., 73, 3-46,1981. That is, fusion can be carried out with approximately 1 to 10 folds higher amount of splenocytes than mouse myeloma cells. A cell fusion promoting agent may be polyethylene glycol having a molecular weight of 1,000-6,000 at a concentration of 30 to 50% (w/v). More specifically, cell fusion is preferably carried out with about 10 8 splenocytes and about 10 7 P3X63-Ag8.U1 myeloma cells in a culture medium usually used for culture of lymphocytes such as RPMI 1640 medium, containing 45% polyethylene glycol 4,000, which is previously heated at 37° C.

Hybridoma may be obtained by culture in HAT medium for a sufficient period of time so that the non-fused cells cannot survive, usually for several days to several weeks. The thus obtained hybridomas are then used for selection and cloning of strains producing a desired antibody in accordance with the usual limiting dilution procedure, using the culture supernatant of the hybridomas.

Screening of strains producing an antibody recognizing Haemophilus paragallinarum serotype A is carried out in accordance with the usual ELISA, RIA, Western blotting, and the like. An antigen used in these methods may be either a suspension of Haemophilus paragallinarum serotype A cells, the cells treated with potassium rhodanide, sonication, hyaluronidase, and the like, or an extraction of said cells with a surfactant.

Then, strains producing an antibody having the HI activity are screened in accordance with the usual HI test, using a culture supernatant of the above hybridomas or ascites from mouse administered with said hybridomas. HA antigen includes a suspension of Haemophilus paragallinarum cells or the cells treated with potassium rhodanide, sonication, hyaluronidase, and the like. Erythrocytes used for HI test may be either 0.5% fresh chicken erythrocytes, glutaraldehyde-fixed 1% chicken erythrocytes or formalin-fixed chicken erythrocytes, with glutaraldehyde-fixed chicken erythrocytes being preferable.

More specifically, a supernatant obtained after centrifugation of ascites treated with 5 folds amount of a 25% kaolin solution is added to precipitates of glutaraldehyde-fixed chicken erythrocytes, which is then shaken at 37° C. for 60 minutes for sensitization. To a two-fold serial dilution of this supernatant is added the same amount of a suspension of strain 221 cells including 4 hemagglutinin units and the mixture is left to stand for 15 minutes. Thereto is added a suspension of glutaraldehyde-fixed 1% chicken erythrocytes, the mixture is left to stand at room temperature for 60 minutes, and observed at the bottom of microtiter plate. An HI antibody titer is defined as a maximum dilution which can block hemagglutination.

Recovery of monoclonal antibodies having the HI activity from the thus obtained hybridomas is carried out by culturing said hybridomas in a large amount and harvesting said antibodies from the culture supernatant, or by administering said hybridomas to mice compatible with said hybridomas so that said hybridomas are proliferated and harvesting said antibodies from the ascites thereof.

Purification of the monoclonal antibody may be done by the conventional procedures used in the protein chemistry such as, for example, a salting out, ultrafiltration, an isoelectric precipitation, an electrophoresis, an ion exchange chromatography, a gel filtration chromatography, an affinity chromatography, and the like. More specifically, purification of the monoclonal antibody from ascites may be done using Protein A-Sepharose CL-4B (manufactured by Pharmacia) and MAPS-II Mouse Monoclonal Antibody Purification Kit (manufactured by Bio Rad) in accordance with protocol of the manufacturer.

An affinity column with the antibody having the HI activity as a ligand for purification of the polypeptide from Haemophilus paragallinarum serotype A which induces production of the HI antibody may be prepared by a conventional procedure, for example, by binding the above purified antibody to HiTrap NHS-Activated Column (manufactured by Pharmacia) in accordance with protocol of the manufacturer.

Using the thus prepared affinity column, the polypeptide from Haemophilus paragallinarum serotype A which induces production of the HI antibody may be obtained from a culture supernatant of HPG serotype A cells or from a suspension of ruptured cells. Specifically, a polypeptide (hereinafter referred to as “HPGp130”) with a molecular weight of about 130 Kd having a high capacity to produce the HI antibody and the activity to prevent avian infectious coryza was obtained from a culture supernatant of HPG strain 221 cultured in the chicken meat infusion medium supplemented with chicken serum at 37° C. for two days.

An amino acid sequence of the thus obtained polypeptide may be determined by the usual procedures such as Edman degradation (P. Edman, Acta Chem. Scand., 4: 283, 1950). The amino acid sequence at the N-terminal of said polypeptide is shown in SEQ ID NO: 2.

Cloning of a gene or a gene fragment coding for the polypeptide from Haemophilus paragallinarum serotype A may be done by the usual procedures as described by Sambrook et al. (Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, New York, 1989). That is, Haemophilus paragallinarum serotype A strain 221 cells are cultured and recovered by the above procedures, a genomic DNA is extracted and purified with Sepagene kit (manufactured by Sanko Junyaku K.K.) in accordance with protocol attached thereto. The genomic DNA is then cleaved with a commercially available restriction enzyme (preferably EcoRI), the obtained DNA fragments are inserted into a commercially available cloning vector (e.g. λgt11) to prepare a DNA library, among which such clones expressing the antigen that responds to the desired antibody having the HI activity are screened. The antibody having the HI activity includes the culture supernatant of the hybridomas or the ascites of mice obtained as mentioned above. Antisera is preferably used which is obtained by immunization with the polypeptide from Haemophilus paragallinarum serotype A isolated by affinity chromatography with the monoclonal antibody having the HI activity as a ligand. A nucleotide sequence of the exogenous DNA fragment in the thus obtained recombinant λgt11 phage DNA may be determined with a DNA sequencer (for example, Applied Biosystems 377). Novelty of the obtained exogenous DNA fragment may be confirmed by homology search between the whole nucleotide sequence and the existing data base (for example, GeneBank, EMBL, and the like).

As shown in Example 3, for example, ten positive λgt11 phages were obtained from the DNA library and each DNA of these phages included an exogenous DNA fragment of about 1.2 kb (hereinafter also referred to as “HPG1.2 k DNA fragment”) as demonstrated in an agarose electrophoresis. The nucleotide sequence of said exogenous DNA corresponds to the nucleotide sequence of from nucleotides No. 1988 to No. 3157 of SEQ ID NO: 1.

Since an initiation codon and a termination codon are not found in the HPG1.2 k DNA, this DNA fragment is considered to encode a portion of the polypeptide from Haemophilus paragallinarum serotype A. A gene coding for a full length of said polypeptide may be obtained by using the HPG1.2 k DNA as a probe to give a longer DNA fragment, determining a nucleotide sequence of this DNA fragment and finding out an initiation codon and a termination codon.

More specifically, the genomic DNA of Haemophilus paragallinarum serotype A strain 221 is cleaved with a restriction enzyme whose cleavage site is not present in the 1.2 kb DNA (for example, HindIII) and the resulting DNA fragments are separated with an agarose electrophoresis. Using DIG-DNA Labeling Kit (manufactured by Boehringer Mannheim), Southern hybridization is carried out using digoxigenin (DIG)-labeled 1.2 kb DNA fragment as a probe for detecting desired DNA fragments. As a result, there was obtained a HindIII-digested DNA fragment of about 3.5 kb which hybridized with the 1.2 kb DNA fragment (hereinafter also referred to as “HPG3.5 k DNA fragment”). A nucleotide sequence of the HPG3.5 k DNA fragment corresponds to the nucleotide sequence of from nucleotides No. 1 to No. 3450 of SEQ ID NO: 1. An identical sequence to the amino acid sequence at the N-terminal of the above HPGp130 polypeptide was found at the amino acid sequence of from amino acid residues No. 1 to No. 13 (corresponding to nucleotide sequence of from No. 453 to No. 491) in SEQ ID NO: 1.

Since only an initiation codon was found in the HPG3.5 k DNA but a termination codon was not, the 1.2 kb DNA fragment and the 3.5 kb DNA fragment labeled with DIG were used as a probe to give a XhoI-XbaI digested DNA fragment of about 4.1 kb (hereinafter also referred to as “HPG4.1 k DNA fragment”). A nucleotide sequence of the HPG4.1 k DNA fragment corresponds to the nucleotide sequence of from nucleotides No. 2212 to No. 6275 of SEQ ID NO: 1. Since a termination codon was not found in the HPG4.1 k DNA fragment, a XhoI-PstI digested DNA fragment of about 6.7 kb (hereinafter also referred to as “HPG6.7 k DNA fragment”; this fragment encompasses the above HPG4.1 k DNA fragment) was obtained using the 1.2 kb DNA and the 3.5 kb DNA labeled with DIG. A nucleotide sequence of the HPG6.7 k DNA fragment corresponds to the nucleotide sequence of from nucleotides No. 2212 to No. 8930 of SEQ ID NO: 1. There existed a termination codon in the HPG6.7 k DNA fragment.

It was found that the nucleotide sequence of SEQ ID NO: 1, consisting of a total of 8930 nucleotides, included an open reading frame starting from nucleotide No. 243 which can code for 2042 amino acid residues (SEQ ID NO:6). A polypeptide comprising the 2042 amino acid residues is hereinafter also referred to as “serotype A HMTp210”. Homology search with the existing data base (GeneBank and EMBL) revealed no homology with any known nucleotide and amino acid sequences, indicating that the serotype A HMTp120 polypeptide is a novel substance.

The presence of another possible open reading frame in the nucleotide sequence of SEQ ID NO: 1 was also suggested which starts from nucleotide No. 8375 and can code for 185 amino acid residues (SEQ ID NO: 8). No termination codon was found-in this sequence. Homology search with the existing data base (GeneBank and EMBL) revealed no homology with any known nucleotide and amino acid sequences, indicating that the polypeptide coded by this open reading frame is also a novel substance.

The DNA fragments from Haemophilus paragallinarum serotype A can also be used as a probe for obtaining DNA fragments from different serotype of Haemophilus paragallinarum such as serotype B or serotype C as well as polypeptides coded by said DNA fragments.

More specifically, a genomic DNA is extracted and purified from HPG serotype C strain 53-47 and cleaved with a suitable restriction enzyme (preferably HindIII), the obtained DNA fragments are inserted into a commercially available cloning vector (e.g. λDASHII) to prepare a DNA library, among which clones are screened by using the serotype A HPG3.5 k DNA fragment labeled with DIG as a probe.

As shown in Example 5, ten positive λDASHII phages were obtained from the DNA library and each DNA of these phages included an exogenous DNA fragment of about 13.5 kb (hereinafter also referred to as “HPG-C1 DNA”) as demonstrated in an agarose electrophoresis.

Since the HPG-C1 DNA fragment of about 13.5 kb is too large to be subcloned into a plasmid vector, it was cleaved with a suitable restriction enzyme (preferably XbaI) and the resulting DNA fragments were inserted into a commercially available cloning vector (for example, pUC119). As a result, DNA fragments of about 5.6 kb (hereinafter also referred to as “HPG-C2 DNA”), about 0.9 kb (hereinafter also referred to as “HPG-C3 DNA”) and about 6.9 kb (hereinafter also referred to as “HPG-C4 DNA”) were obtained. A nucleotide sequence of a portion of HPG-C2 DNA fragment and HPG-C4 DNA fragment was determined to reveal the presence of an initiation codon and a termination codon in these DNA fragments, respectively.

It was found that the nucleotide sequence of SEQ ID NO: 5, consisting of a total of 7486 nucleotides, included an open reading frame starting from nucleotide No. 848 which can code for 2039 amino acid residues (SEQ ID NO: 7). A polypeptide comprising the 2039 amino acid residues is hereinafter also referred to as “serotype C HMTp210”. Homology search with the existing data base (GeneBank and EMBL) revealed no homology with any known nucleotide and amino acid sequences, indicating that the serotype C HMTp120 polypeptide is a novel substance.

Homology search between the nucleotide sequences coding for the serotype C HMTp120 polypeptide and the serotype A HMTp120 polypeptide revealed about 80% homology. It was further revealed that the region of about 3.4 kb at the 5′ site and the region of about 1.2 kb at the 3′ site exhibited extremely high homology whereas the region of about 1.5 kb between these 5′ and 3′ regions showed low homology. The same was also applicable to the corresponding polypeptides encoded by these genes.

Based on the nucleotide sequence coding for the serotype A HMTp120 polypeptide, there can also be obtained, by PCR, DNA fragments from different serotype of Haemophilus paragallinarum such as serotype B or serotype C as well as polypeptides coded by said DNA fragments.

More specifically, based on the nucleotide sequence coding for the serotype A HMTp120 polypeptide, there were prepared a synthetic DNA having the nucleotide sequence of SEQ ID NO: 3 as an upstream PCR primer and a synthetic DNA having the nucleotide sequence of SEQ ID NO: 4 as a downstream PCR primer. These primers were designed such that BamHI recognition sequences were added at the 5′ sites, respectively, and a full length of translation region of the serotype A HMTp210 polypeptide can be amplified. Using these primers, PCR was carried out using as a template the genomic DNAs from a total of nine strains, i.e. Haemophilus paragallinarum serotype A strains 221, 083, W, Germany and Georgia, HPG serotype B strains Spross and 0222, and HPG serotype C strains Modesto and 53-47. Analysis of the obtained PCR products on 0.8% agarose gel electrophoresis confirmed the amplified fragment of about 6.1 kb in any of these strains.

The thus obtained DNA fragment or a portion thereof may be incorporated into a suitable expression vector, the resulting expression vector is used for transformation of a microorganism or an animal cell, and the transformant is cultured to produce the polypeptide of the present invention from Haemophilus paragallinarum or a peptide which shares at least a portion of the amino acid sequence of said polypeptide. The peptide which shares a portion of the amino acid sequence can also be produced with a peptide synthesizer.

A suitable signal sequence for secretion in a microorganism or an animal cell can also be linked upstream the DNA coding for the polypeptide of the present invention so that said polypeptide can be secreted into a culture medium. The thus modified DNA for secretion is advantageous in that said polypeptide secreted into a culture medium can easily be purified. A signal sequence includes pelB signal (S. P. Lei et al., J. Bacteriology, 169: 4379-4383, 1987) for E. coli, signal from α factor (A. J. Brake, Yeast Genetic Engineering, p269, Butterworth, 1989) for yeast, signal SG-1 from immunoglobulin (H. Maeda et al., Hum. Antibod. Hybridomas, 2: 124-134, 1991), C25 signal (PCT International Publication No. WO94/20632) for an animal cell.

An expression vector includes a plasmid, a viral vector and the like. Any promoter may be included in the expression vector such as lac, tac, pho5, adh, SV40 early, SV40 late, β actin and the like, in consideration of a microorganism or an animal cell used as a host, insofar as the polypeptide having the activity to prevent avian infectious coryza is ultimately obtained. The polypeptide of the present invention can also be expressed as a fusion protein with another protein or peptide such as β-galactosidase, glutathione-S-transferase, maltose binding protein, Protein A, histidine hexamer, and the like. A marker gene includes, in case of an expression vector for a microorganism cell, ampicillin resistant gene, tetracycline resistant gene for E. coli as a host, β-isopropyl malate dehydrogenase (Leu2) gene for yeast as a host, and in case of an expression vector for an animal cell, aminoglycoside 3′ phosphotransferase (neo) gene, dihydrofolate reductase (dhfr) gene, glutamine synthetase (GS) gene, and the like. An additive for selection includes G418, neomycin, methotrexate, and the like.

Transformation of a host cell may be carried out by the known methods including, for example, a calcium chloride method, a calcium phosphate coprecipitation method, a DEAE dextran method, a lipofectin method, a protoplast polyethylene fusion method, an electroporation, and the like, which can suitably be selected depending on a host used.

The novel polypeptide of the present invention from Haemophilus paragallinarum or a peptide which shares at least a portion of the amino acid sequence of said polypeptide may be prepared as described hereinbelow. For example, the HPG3.5 k DNA fragment from HGP serotype A is incorporated into an expression vector pTrcHisC (manufactured by Invitrogen), said expression vector is introduced into E. coli strain JM109 for transformation. Among the resulting transformed cells, those transformants which produce the target novel polypeptide are screened by a dot blotting with an index of reactivity with the antibody against said polypeptide. Chicken immunized with a supernatant obtained after centrifugation of a suspension of the ruptured cells have an elevated protection against challenge with HPG serotype A strain 221.

The novel polypeptide may be purified from an extract of cells or a culture supernatant from a large scale culture of the transformant producing said polypeptide by utilizing the above-mentioned methods used in the field of protein chemistry.

The thus obtained novel polypeptide from Haemophilus paragallinarum has the activity to prevent avian infectious coryza. Said polypeptide from Haemophilus paragallinarum, monoclonal and polyclonal antibodies against said polypeptide and the expression vector as mentioned above may be used as a vaccine or a therapeutic agent for avian infectious coryza either alone or in combination with a suitable carrier, diluent or stabilizing agent in a conventional manner such as injections or oral drugs.

The above novel polypeptide from Haemophilus paragallinarum or a polypeptide which shares at least a portion of the amino acid sequence of said polypeptide may be used as an immunogen for preparing a polyclonal and monoclonal antibodies in accordance with the procedures described hereinabove. Said polypeptide as well as the antibody having the capacity to bind thereto may also be utilized in an antigen or antibody detection system such as Western blot, ELISA, and the like, and may also be a material for constructing a diagnostic agent. In addition, affinity chromatography with a suitable carrier to which the above antibody is bound may be used for purification of the above polypeptide.

In accordance with the present invention, there are provided the novel polypeptide from Haemophilus paragallinarum and the gene fragment coding for said polypeptide for prevention of avian infectious coryza and the antibody having the HI activity which can be used as a therapeutic agent.

The polypeptide from Haemophilus paragallinarum, which the present inventor has found, has a molecular weight of about 130 Kd, has the activity to induce production of the HI antibody, and is the novel, important polypeptide for prevention of avian infectious coryza. Technical problems associated with the obtention of said polypeptide, such as isolation of the gene coding for said polypeptide, construction of the expression vector, preparation of the expression cell, and purification of said polypeptide, are solved by the present invention, which allows for provision of a more effective vaccine than the prior art vaccines. Furthermore, these are useful as a material for providing a rapid, simple diagnostic agent for avian infectious coryza.

The present invention is illustrated in more detail by means of the following Examples but should not be construed to be limited thereto.

EXAMPLE 1

Preparation and Features of Monoclonal Antibody

(1) Preparation of Monoclonal Antibody

Haemophilus paragallinarum serotype A strain 221 cells were inoculated to 100 ml of chicken meat infusion medium supplemented with chicken serum and shake-cultured at 37° C. overnight, followed by centrifugation (8,000 rpm, 20 minutes) to recover cells. The obtained cells were washed with PBS while centrifugation and then suspended in PBS containing 0.01% thimerosal at about 5×10 10 cells/ml. The suspension was sonicated with Branson Sonifier 350 at 20 kHz, 4° C. for 10 minutes (alternative repeat of sonication for 0.5 second and cooling for 0.5 second). The thus obtained suspension of the ruptured cells by sonication was mixed with the same amount of Freund's complete adjuvant and the mixture was well blended till a water-in-oil (w/o) was achieved. Each 0.1 ml of this emulsion was subcutaneously administered to BALB/c mouse at two sites of the back. Four weeks later, each 0.1 ml of an emulsion prepared similarly with Freund's incomplete adjuvant was subcutaneously administered at two sites of the back. After additional 18 days, 0.1 ml of the suspension of the ruptured cells by sonication was intravenously administered.

Three days after the final administration, splenocytes were removed. Said splenocytes (1×10 8 cells) were mixed with mouse myeloma cells P3X63-Ag8.U1 (1×10 7 cells) by padding, thereto was added RPMI1640 medium containing 45% polyethylene glycol previously warmed at 37° C. to conduct cell fusion. The cells after fusion reaction were suspended in HAT medium (RPMI1640 medium containing 5% fetal calf serum supplemented with 1×10 −4 M hypoxanthine, 4×10 −7 M aminopterin and 1.6×10 −5 M thymidine), and after plated on 96-well microtiter plate for cell culture (manufactured by Coning), cultured under the conditions of 37° C. and 5% CO 2 .

For the wells where hybridomas propagated, the presence of the monoclonal antibody recognizing Haemophilus paragallinarum in the culture supernatant was determined with ELISA as described hereinbelow. A suspension of ruptured cells by sonication of Haemophilus paragallinarum serotype A strain 221 prepared as mentioned above was diluted 300 folds with PBS and each 100 μl of the suspension was plated on well of microtiter plate for ELISA (Immulon II manufactured by Dynatech). The microtiter plate was left to stand at 4° C. overnight and masked with PBS containing 5% skim milk at 200 μl per well at room temperature for 2 hours. The microtiter plate was washed with PBS containing 0.05% TWEEN 20 (PBS-T) and thereto was added 100 μl of the culture supernatant of hybridomas diluted 10 folds with PBS-T containing 5% skim milk for reaction at room temperature for 2 hours. After washing with PBS-T, each 100 μl of peroxidase-labeled anti-mouse IgG (manufactured by Bio-Rad) diluted 10,000 folds with PBS-T containing 5% skim milk was added for reaction at room temperature for 2 hours. Then, after washing with PBS-T, each 100 μl of 0.05 M citrate-0.1 M disodium hydrogenphosphate buffer (pH 5.0) containing 6 mg per 11 ml of ortho-phenylene-diamine dihydrochloride (OPD; manufactured by Katayama Kagaku K.K.) and 4.75 μl of hydrogen peroxide (containing H 2 O 2 at 31%; manufactured by Mitsubishi Gasu Kagaku K.K.) was added for reaction at room temperature for 30 minutes. Each 50 μl of 3 M sulfuric acid was added to quench the reaction and absorbance (490 nm) of each well was measured with Autoreader for ELISA.

Hybridomas of the wells where the antibody against Haemophilus paragallinarum serotype A was secreted in the culture supernatant were cloned by a limiting dilution method so that they become monoclonal. Thus, nine clones producing the monoclonal antibody against Haemophilus paragallinarum serotype A were obtained.

(2) HI Activity of Monoclonal Antibodies

These hybridomas were cultured in a large amount and intraperitoneally administered to BALB/c mice, pretreated with an immunosuppressive agent, pristane (2,6,10,14-tetramethyl-pentadecane; manufactured by Aldrich), where the hybridomas propagated. Ten to twenty days later, the mice were sacrificed and the produced ascites were removed therefrom and HI activity of the ascites was determined.

A suspension of Haemophilus paragallinarum serotype A strain 221 cells inactivated with thimerosal was used as an HA antigen for HI test and prepared based on HA titer. First, using a V-shaped microtiter plate (Sanko Junyaku K.K.), a suspension of a glutaraldehyde-fixed 1% chicken erythrocytes (0.05 ml) was added to a 2 folds serial dilution of HA antigen (0.05 ml), and after standing at room temperature for 60 minutes, the bottom of the plate was observed. A maximum dilution which agglutinates erythrocytes was defined as HA titer and, regarding a concentration of HA antigen at this dilution as 1 unit, a stock solution of HA antigen was prepared so that it contains 4 units.

Then, to 0.2 ml of mouse ascites was added 5 folds amount of 25% kaolin solution and the mixture was shaken at 37° C. for 30 minutes for sensitization, followed by centrifugation to give a supernatant. This supernatant of centrifugation after kaolin treatment was added to precipitates obtained by centrifugation of glutaraldehyde-fixed 10% chicken erythrocytes (2 ml) and the mixture was shaken for sensitization at 37° C. for 60 minutes. After sensitization, a supernatant was obtained by centrifugation and used as 5 folds diluted mouse ascites for determination of HI antibody. Using a V-shaped microtiter plate, to 0.025 ml of a 2 folds serial dilution of this supernatant was added the same amount of the suspension of strain 221 cells inactivated with thimerosal containing 4 hemagglutination units and, after mixing, the mixture was left to stand for 15 minutes. After sufficient sensitization, 0.05 ml of a suspension of glutaraldehyde-f ixed 1% chicken erythrocytes was added. After the mixture was left to stand at room temperature for 60 minutes, the bottom of the microtiter plate was observed. A maximum dilution which inhibits hemagglutination was defined as an HI antibody titer. Among nine clones, the monoclonal antibodies from three clones (HpgA 59-40, HpgA 59-145 and HpgA 59-180) exhibited a high HI activity (Table 1). The clone HpgA 59-180 has been deposited by the applicant as FERM BP-6084 at National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken) on Sep. 5, 1996.

TABLE 1
Monoclonal antibody HI antibody titer
HpgA 59-33          <50
HpgA 59-40          25,600
HpgA 59-48A         <50
HpgA 59-48B         <50
HpgA 59-145         1,600
HpgA 59-180         12,800
HpgA 59-188         <50
HpgA 59-236         <50
HpgA 59-284         <50

(3) Protective Activity of Monoclonal Antibodies

A mouse ascites (0.3 ml) containing these antibodies was intraperitoneally administered to SPF white leghorn chickens of 4 to 6 weeks old, each group comprising 8 to 10 chickens, and on the next day, about 10 8 cells of Haemophilus paragallinarum serotype A strain 221 were applied dropwise to the nasal cavity of the chickens for challenge. A control group which was given no mouse ascites was also used and was challenged in the same manner. Each group was observed for the presence of the coryza symptoms (i.e. a running nose, swelling of the face and epiphora) for 10 days. All the groups which previously received the monoclonal antibodies having the HI activity (hereinafter also referred to as “HI-MCA”) were likely to retard the onset as compared to the control group. On the contrary, all the groups administered with the monoclonal antibodies of the other clones showed no significant difference ( FIGS. 1 to 4 ).

EXAMPLE 2

Purification and Property of Antigen Recognized by HI-MCA

(1) Purification of HI-MCA

HI-MCA (HpgA 59-180) was purified from mouse ascites using Protein A-Sepharose CL-4B (manufactured by Pharmacia) and MAPS-II Mouse Monoclonal Antibody Purification Kit (manufactured by Bio-Rad) in accordance with protocol attached thereto. First of all, to 4 ml of mouse ascites was added the same amount of a binding buffer included in the Antibody Purification Kit. After the mixture was filtered with Sterivex filter of 0.45 micron (manufactured by Millipore), it was applied to Protein A-Sepharose CL-4B column (gel bed volume 5 ml) and was thoroughly washed with the binding buffer till less than 0.05 of the absorbance at 280 nm was obtained. Then, the antibodies bound to the column were eluted with an elution buffer included in the kit. The eluted antibodies were dialyzed against 0.2 M sodium hydrogen carbonate (pH 8.3) containing 0.5 M sodium chloride to give 40 mg of purified HI-MCA (HpgA 59-180). Similarly, HI-MCA (HpgA 59-40) was also purified to give 12 mg.

(2) Binding of HI-MCA to Carrier

Then, the purified HI-MCA (HpgA 59-180) as a ligand was bound to HiTrap NHS-activated column (manufactured by Pharmacia) in accordance with protocol attached thereto. First of all, HiTrap NHS-activated column (gel bed volume 1 ml) was washed with 1 mM hydrochloric acid and then circulated with 0.2 M sodium hydrogen carbonate solution (10 ml) containing 0.5 M sodium chloride and 10 mg of the above purified HI-MCA (HpgA 59-180) at room temperature for 30 minutes so that HI-MCA was bound to the column. The obtained HI-MCA-bound HiTrap column was washed each three times alternatively with 0.5 M ethanolamine (pH 8.3) containing 0.5 M sodium chloride, and 0.1 M sodium acetate buffer (pH 4.0) containing 0.5 M sodium chloride and equilibrated with PBS for purification of an antigen recognized by HI-MCA.

(3) Purification of Antigen Recognized by HI-MCA

An antigen was purified from a culture of Haemophilus paragallinarum serotype A strain 221 by an affinity chromatography using HI-MCA as a ligand. An antigen was detected by ELISA method as described hereinbelow.

The above purified HI-MCA (HpgA 59-40) was diluted with 0.05 M sodium carbonate buffer (pH 9.0) to a concentration of 1.6 μg/ml and was placed in a well of microtiter plate for ELISA. The plate was left to stand at 4° C. overnight and masked with PBS containing 5% skim milk at room temperature for 2 hours. After washing with PBS-T, an eluate from the column diluted 10 folds with PBS-T containing 5% skim milk was reacted at room temperature for 2 hours. After washing with PBS-T, peroxidase-labeled HI-MCA (HpgA 59-180) diluted 10,000 folds with PBS-T containing 5% skim milk was reacted at room temperature for 2 hours. Then, after washing with PBS-T, a substrate solution containing OPD and hydrogen peroxide was added for reaction at room temperature for 30 minutes. Peroxidase-labeled HI-MCA (HpgA 59-180) was prepared by binding horseradish peroxidase (manufactured by Toyobo K.K.) to the above purified HI-MCA (HpgA 59-180) as described by Yoshitake et al. (J. Biochem., 92: 1413-1424, 1982).

Haemophilus paragallinarum serotype A strain 221 cells were inoculated to 100 ml of chicken meat infusion culture supplemented with chicken serum and shake-cultured at 37° C. for 2 days. To a culture supernatant obtained after removal of cells by centrifugation at 8,000 rpm for 20 minutes was immediately added a serine protease inhibitor, phenylmethylsulfonyl fluoride, at 1 mM, and the mixture was filtered with 0.45 micron Sterivex filter. The HI-MCA-bound HiTrap column preequilibrated with PBS was added with 60 ml of the above filtrate and washed with PBS. When the absorbance at 280 nm became less than 0.05, an antigen bound to HI-MCA was eluted with 3M sodium thiocyanate. Antigens recognized by HI-MCA were not found in unbound fractions but in most part were recovered in fractions eluted with 3 M sodium thiocyanate. This eluate was dialyzed against 50 mM Tris-HCl buffer (pH 8.0) containing 50 mM sodium chloride.

(4) Amino Acid Sequence Analysis of N-terminal of Antigen Recognized by HI-MCA

After treatment with 2-mercaptoethanol, the eluate from the affinity column was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 5 to 20% polyacrylamide gel in accordance with Laemmli, Nature, 227: 680-685, 1970, which was stained with 0.25% Coomassie Brilliant Blue R250 (CBB) dissolved in 50% methanol-10% acetic acid to reveal a band of a molecular weight about 130 Kd (FIG. 5 ). This polypeptide was referred to as HPGp130 and an amino acid sequence of the N-terminal was determined as described hereinbelow.

First, the purified HPGp130 polypeptide was treated with 2-mercaptoethanol and then subjected to SDS-PAGE using 5% polyacrylamide gel. After electrophoresis, the gel was washed with a transfer buffer (10 mM N-cyclohexyl-3-amino-propanesulfonic acid, 10% methanol, pH 11) and overlaid to polyvinylidene difluoride (PVDF) membrane (manufactured by Millipore), which was previously immersed successively in 100% methanol and a transfer buffer, followed by transfer with TRANS-BLOT CELL (manufactured by Bio Rad) at 20 V overnight. The PVDF membrane after transfer was washed with water and stained with 0.1% Amido Black dissolved in 45% methanol-10% acetic acid for 30 seconds, followed by decolorization with distilled water.

The stained band of a molecular weight 130 Kd was cut out and analyzed with Protein Sequencer (Applied Biosystems 477A). Thirteen amino acid residues at the N-terminal were analyzed, and as a result, the amino acid sequence was found to be Lys-Trp-Leu-Glu-Val-Tyr-Ser-Ser-Ser-Val-Lys-Leu-Ser as shown in SEQ ID NO: 2.

(5) Induction of HI Antibody Production by HPGp130

Whether HPGp130 polypeptide could induce production of HI antibody was investigated. An emulsion (1 ml; about 20 μg of HPGp130 polypeptide per animal) prepared by mixing the HPGp130 polypeptide solution (about 40 μg/ml) with the same amount of Freund's complete adjuvant was subcutaneously injected to guinea pig at two sites of the back for immunization. About three weeks later, 1 ml of an emulsion prepared similarly with Freund's incomplete adjuvant was injected subcutaneously at two sites of the back. Additional two weeks later, the emulsion prepared with Freund's incomplete adjuvant was boosted subcutaneously at two sites of the back and four weeks thereafter the test animals were bled. HI antibody titer of the obtained antisera was determined as described above to reveal a high HI antibody titer (5,120 folds). Thus, it was found that the HPGp130 polypeptide induced production of HI antibody deeply involved in protection against avian infectious. coryza.

(6) Peptide Recognized by Anti-HPGp130 Polypeptide Guinea Pig Sera

A polypeptide recognized by anti-HPGp130 polypeptide guinea pig serum was analyzed by Western blot. First, the purified HPGp130 polypeptide and HPG seritype A strain 221 cells cultured in chicken meat infusion medium supplemented with chicken serum were treated with 2-mercaptoethanol and subjected to SDS-PAGE. After completion of electrophoresis, the gel was immersed in a transfer buffer (25 mM Tris, 192 mM glycine, 20% ethanol, pH 8.3) for 5 minutes and overlaid to PVDF membrane, which was previously immersed in 100% methanol and the transfer buffer in this order, and a transfer was carried out using TRANS-BLOT SD CELL (manufactured by Bio Rad) at 7 V for 1 hour. The membrane was masked with PBS containing 5% skim milk at 4° C. overnight, washed with PBS-T and then reacted with anti-HPGp130 polypeptide guinea pig serum diluted 1,000 folds with PBS-T containing 5% skim milk at room temperature for 2 hours. After washing with PBS-T, peroxidase-labeled anti-guinea pig IgG (manufactured by Zymed) diluted 2,000 folds with PBS-T containing 5% skim milk was reacted at room temperature for 2 hours. After washing with PBS-T, the membrane was immersed in 10 ml of 0.1 M Tris-HCl buffer (pH 7.5) containing 5 mg of 3,3′-diaminobenzidine tetrahydrochloride (DAB; manufactured by Dojin Kagaku K.K.) and 3 μl of hydrogen peroxide for reaction. As a result, anti-HPGp130 polypeptide guinea pig serum recognized the HPGp130 polypeptide and a band of a molecular weight about 160 Kd, possibly a precursor of the polypeptide (FIG. 6 ).

(7) Immunogenicity of HPGp130 Polypeptide

In accordance with the procedures as described hereinabove, ten SPF white leghorn chickens of 5 weeks old were immunized by subcutaneously administering at the leg 0.5 ml of an emulsion (containing about 10 μg of HPGp130 polypeptide) prepared by mixing an HPGp130 polypeptide solution (about 40 μg/ml) and the same amount of Freund's complete adjuvant. Three weeks later, the chickens were subcutaneously administered at the leg with 0.5 ml of an emulsion prepared similarly with Freund's incomplete adjuvant. Two weeks later, the chickens were boosted subcutaneously at the leg with an emulsion prepared similarly with Freund's incomplete adjuvant. Seven weeks after the first immunization, the chickens were challenged with Haemophilus paragallinarum serotype A strain 221. As a control, one group was immunized twice with 0.5 ml of 0.25% formalin-inactivated HPG serotype A strain 221 (cell number prior to inactivation: 4×10 8 cells/ml) supplemented with aluminum hydroxide gel (in terms of aluminum: 0.5 mg/ml) at the interval of three weeks and another group was not immunized and both control groups were challenged similarly. The results are shown in Table 2. Both groups immunized either with HPGp130 polypeptide or formalin-inactivated cells showed protection against the onset of the disease in all the chickens. For the non-immunization group, however, the symptoms were shown in all the chickens.

	TABLE 2
		Tested	Protected	Protection
	Immunization group	chicken	chicken	rate (%)
	Purified HPGp130	10	10	100
	Formalin-inactivated	10	10	100
	strain 221
	Non immunization control	8	0	0

EXAMPLE 3

Cloning of Gene Coding for Polypeptide (Serotype A HMTp210) from Haemophilus paragallinarum Serotype A Strain 221

(1) Screening from Genomic Library

Haemophilus paragallinarum serotype A strain 221 cells were inoculated to 5 ml of chicken meat infusion medium supplemented with chicken serum and shake-cultured at 37° C. overnight and the cells were recovered by centrifugation. After washing the obtained cells with PBS by centrifugation, DNA was extracted and purified from the cells with Sepagene kit (manufactured by Sanko Junyaku K.K.) in accordance with protocol attached thereto. The DNA was dissolved in 50 μl of TE buffer (10 mM Tris-HCl buffer containing 1 mM EDTA, pH 8.0) and the obtained solution was used as a genomic DNA solution. Then, using cDNA Rapid Cloning Module-λgt11 (manufactured by Amersham), 0.2 μg of the genomic DNA digested with restriction enzyme EcoRI was ligated to 0.5 μg of λgt11 arm digested with restriction enzyme EcoRI in accordance with protocol attached thereto. Using λ-DNA In Vitro Packaging Module (manufactured by Amersham), the ligated product was inserted into λ phage in accordance with protocol attached thereto. The obtained solutions of recombinant phage were used as a genomic library.

The above solutions of genomic library were added to a suspension of E.coli strain Y1090 (manufactured by Amersham) about 10 8 cells in an aqueous solution of 10 mM magnesium sulfate for absorption at 37° C. for 15 minutes. Thereto was added LB soft agar medium (containing tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, ampicillin 50 mg, maltose 4 g and agar 8 g in 1000 ml, pH 7) for overlay warmed at 45° C. The mixture was overlaid to LB agar medium (containing tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, ampicillin 50 mg and agar 15 g in 1000 ml, pH 7) and incubated at 42° C. for 3 hours. A nitrocellulose membrane immersed in an aqueous solution of 10 mM isopropyl-β-D-thiogalacto-pyranoside (IPTG) was air-dried, overlaid to the above plate and incubated at 37° C. overnight. The nitrocellulose membrane was then peeled off from the plate, washed with PBS-T and masked with PBS containing 5% skim milk at room temperature for 2 hours. Thereafter, the procedures as described in Example 2 (6) were repeated so that anti-HPGp130 polypeptide guinea pig serum, peroxidase-labeled anti-guinea pig IgG and a substrate were successively reacted. A series of these procedures gave plaques which express an antigen specifically reactive with anti-HPGp130 guinea pig serum from Haemophilus paragallinarum serotype A strain 221. About 5,000 plaques were immunologically screened as described above to give 43 positive plaques. These positive plaques were recovered in an SM buffer (50 mM Tris-HCl buffer containing 0.1 M sodium chloride, 10 mM magnesium sulfate and 0.01% gelatin, pH 7.5) and, after adding several drops of chloroform, stored at 4° C. Ten among the recovered positive plaques were further subjected to second and third screening as in the primary screening.

The recombinant λgt11 phages found positive in the immunological screening were added to a suspension of E. coli strain Y1090 about 10 8 cells in an aqueous solution of 10 mM magnesium sulfate for absorption at 37° C. for 15 minutes. Thereto was added 10 ml of LB liquid medium containing 0.4% maltose, 5 mM calcium chloride and ampicillin 50 μg/ml and the cells were further cultured at 37° C. overnight. After bacteriolysis with addition of several drops of chloroform, the lysis solution was centrifuged to remove the intact E. coli cells and debris. To 5 ml of the obtained culture supernatant was added the same amount of an aqueous solution of 2.5 M sodium chloride containing 20% polyethylene glycol 6,000 and the mixture was left to stand on ice for 1 hour. After centrifugation at 10,000 rpm, precipitated λgt11 phage was subjected to. phenol treatment and isopropanol precipitation to recover phage DNA. About 150 μg of the obtained phage DNA was digested with EcoRI and then electrophoresed on 0.8% agarose gel to separate DNA fragments derived from Haemophilus paragallinarum serotype A strain 221. Using SEPHAGLAS™ BandPrep Kit (manufactured by Pharmacia), the DNA fragments were eluted and recovered from the gel in accordance with protocol attached thereto. All the DNA fragments obtained from ten positive phages had a length of about 1.2 kb. A DNA fragment (hereinafter referred to as “HPG1.2 k DNA”) obtained from the phage of a clone (clone 2) was used in the following test.

(2) Nucleotide Sequence of HPG1.2 k DNA Fragment

Plasmid pUC119 (manufactured by Takara Shuzo K.K.) was digested with EcoRI and then treated with alkaline phosphatase to dephosphorize the 5′ end. The cleaved pUC119 DNA was treated with phenol and chloroform and then harvested by precipitation with ethanol. The cleaved pUC119 and the HPG1.2 k DNA fragment were ligated together with DNA Ligation Kit ver. 2 (manufactured by Takara Shuzo K.K.). Competent cells of E.coli strain JM109 (manufactured by Takara Shuzo K.K.) were transformed with the ligated product and then cultured on Circle Grow agar medium (manufactured by BIO101) containing 50 μg/ml of ampicillin at 37° C. overnight. Colonies grown on the agar medium were inoculated to 0.5 ml of Circle Grow medium containing 50 μg/ml of ampicillin and cultured at 37° C. for 5 hours. Plasmids were extracted from the cells by an alkali method and, after digestion with EcoRI, subjected to 0.8% agarose gel electrophoresis to detect recombinant plasmids containing DNA fragment with the same length as the 1.2 k DNA derived from Haemophilus paragallinarum serotype A strain 221, and thereby transformed E.coli were confirmed.

The obtained transformants of E.coli were cultured on Circle Grow medium containing 50 μg/ml of ampicillin and then the recombinant plasmids (hereinafter referred to as “pUA1.2”) were recovered from the cells by PEG precipitation method. Using a Primer Walking method, a nucleotide sequence of the HPG1.2 k DNA fragment was analyzed using a DNA sequencer (Applied Biosytems 377). As a result, a sequence of 1170 nucleotides was determined. It was found that the nucleotide sequence of the HPG1.2 k DNA corresponds to the sequence of from No. 1988 to No. 3157 in SEQ ID NO: 1, which is a nucleotide sequence coding for serotype A HMTp120 polypeptide as described hereinbelow, and codes for 389 amino acid residues with no initiation codon and termination codon within this region. A corresponding amino acid sequence was also shown which depicts no sequence equivalent to the N-terminal amino acid sequence of HPGp130 polypeptide. Accordingly, it was considered that HPG1.2 k DNA codes for a portion of HPGp130 polypeptide.

(3) Cloning of HPG3.5 k DNA

Using DIG-DNA Labeling Kit (manufactured by Boehringer Mannheim), about 0.3 μg of the above HPG1.2 k DNA was labeled with digoxigenin (DIG) in accordance with protocol attached thereto. After the genomic DNA of Haemophilus paragallinarum serotype A strain 221 was cleaved with several restriction enzymes, a suitable amount of the cleaved products was electrophoresed on 0.8% agarose gel and then transferred to HYBOND N+ membrane (manufactured by Amersham). Using the DIG-labeled HPG1.2 k DNA as a probe, a Southern hybridization was carried out with DIG Nucleic Acid Detection Kit (manufactured by Boehringer Mannheim) in accordance with protocol attached thereto for detection of desired DNAs. As a result, about 3.5 kb fragment obtained by HindIII digestion hybridized to the DIG-labeled HPG1.2 k DNA. Thus, this fragment was separated on 0.8% agarose gel electrophoresis and eluted and recovered from the gel with SEPHAGLAS™ BandPrep Kit in accordance with protocol attached thereto.

On the other hand, plasmid pUC119 was digested with HindIII and then treated with alkaline phosphatase to dephosphorize the 5′ end. The cleaved pUC119 DNA was treated with phenol and chloroform and then recovered by precipitation with ethanol. The cleaved pUC119 and the above HindIII digest (about 3.5 kb) from the genome of Haemophilus paragallinarum serotype A strain 221 were ligated together with DNA Ligation Kit ver. 2. Competent cells of E.coli strain JM109 were transformed with the ligated product and then cultured on Circle Grow agar medium containing 50 μg/ml of ampicillin at 37° C. overnight. To the agar medium where transformed E.coli grown was overlaid Hybond N+ membrane to lift the colonies. Using the DIG-labeled HPG1.2 k DNA as a probe, a colony hybridization was carried out in the conventional manner and positive clones were screened with DIG Nucleic Acid Detection Kit.

The positive clones were cultured on Circle Grow medium containing 50 μg/ml of ampicillin. Plasmids were recovered from the cells by PEG precipitation method. The obtained recombinant plasmid (hereinafter referred to as “pUA3.5”) was digested with HindIII and then electrophoresed on 0.8% agarose gel to separate 3.5 kb DNA fragment derived from Haemophilus paragallinarum serotype A strain 221. Using Seph-ag-l-as BandPrep Kit, this DNA fragment (hereinafter referred to as “HPG3.5 k DNA”) was eluted and recovered in accordance with protocol attached thereto. E.coli UA3.5JM transformed with the recombinant plasmid has been deposited by the applicant as FERM BP-6083 at National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibarakiken) on Sep. 5, 1996.

(4) Expression of HPG3.5 k DNA

The expression vector pTrcHisC (manufactured by Invitrogen) was digested with HindIII and then treated with alkaline phosphatase to dephosphorize the 5′ end. The cleaved pTrcHisC DNA was treated with phenol and chloroform and then recovered by precipitation with ethanol. The cleaved pTrcHisC and the above HPG3.5 k DNA were ligated together with DNA Ligation Kit ver. 2. Competent cells of E.coli strain JM109 were transformed with the ligated product and then cultured on CIRCLE GROW agar medium containing 50 μg/ml of ampicillin at 37° C. overnight. Colonies grown on the agar medium were inoculated to 0.5 ml of CIRCLE GROW medium containing 50 μg/ml of ampicillin and cultured at 37° C. for 5 hours. Plasmids were extracted from the cells by an alkali method and, after digestion with HindIII, subjected to 0.8% agarose gel electrophoresis to detect recombinant plasmids containing DNA fragment with the same length as the 3.5 k DNA derived from Haemophilus paragallinarum serotype A strain 221, and thereby transformed E.coli cells were confirmed.

The obtained transformants of E.coli were plated on 1 ml of CIRCLE GROW medium containing 50 μg/ml of ampicillin and cultured at 37° C. for 3 hours. Thereto was further added IPTG (final concentration of 1 mM) and the transformants were cultured at 37° C. for additional 3 hours. The cells were harvested from the culture by centrifugation and suspended in 50 μl of PBS. The suspension of the cells (10 μl) was mixed with the same amount of 2% SDS and the mixture was boiled for 5 minutes and 2 μl was then spotted on a nitrocellulose membrane. The nitrocellulose membrane was air-dried and then masked with PBS containing 5% skim milk at 4° C. overnight. Thereafter, the procedures as described in Example 2 (6) were repeated so that anti-HPGp130 polypeptide guinea pig serum, peroxidase-labeled anti-guinea pig IgG and a substrate were successively reacted. A series of these procedures gave E.coli which was transformed with a recombinant plasmid wherein HPG3.5 k DNA was ligated in a right direction and expresses an antigen specifically reactive with anti-HPGp130 guinea pig serum.

(5) Immunogenicity of HPG3.5 k-HIS Polypeptide

The obtained transformants of E.coli were inoculated to 200 ml of Circle Grow medium containing 50 μg/ml of ampicillin and cultured at 37° C. for 3 hours. Thereto was added IPTG (final concentration of 1 mM) and the transformants were cultured at 37° C. for additional 3 hours. The cells were harvested from the culture by centrifugation and suspended in 10 ml of PBS. To the suspension was added lysozyme at 100 μg/ml for reaction at 4° C. for 1 hour. The suspension was sonicated with Branson SONIFIER 350 at 4° C. for 10 minutes for bacteriolysis. Intact cells were removed by centrifugation and the obtained supernatant was used as a crude HPG3.5 k-HIS polypeptide.

Ten SPF white leghorn chickens 8 weeks old were immunized by subcutaneously administering at the leg 0.5 ml of an emulsion prepared by thoroughly mixing the crude HPG3.5 k-HIS polypeptide solution with the same amount of Freund's complete adjuvant. Three weeks later, the chickens were subcutaneously administered at the leg with 0.5 ml of an emulsion prepared similarly with Freund's incomplete adjuvant. Two weeks later, the chickens were boosted subcutaneously at the leg with an emulsion prepared similarly with Freund's incomplete adjuvant. Seven weeks after the first immunization, the chicken were challenged with Haemophilus paragallinarum serotype A strain 221. As a control, as described in Example 2 (7), one group was immunized with formalin-inactivated HPG serotype A strain 221 and another group was not immunized and both control groups were challenged similarly. The results are shown in Table 3. The group immunized with the crude HPG3.5 k-HIS polypeptide showed protection against the onset of the disease in seven among ten chicken. The group immunized with the formalin-inactivated cells exhibited protection against the onset of the disease in all the chickens whereas the non-immunization group showed the symptoms in all the chickens.

	TABLE 3
		Tested	Protected	Protection
	Immunization group	chicken	chicken	rate (%)
	Crude HPGp3.5k-HIS	10	7	70
	Formalin-inactivated	10	10	100
	strain 221
	Non immunization control	8	0	0

(6) Nucleotide Sequence of HPG3.5 k DNA Fragment

A nucleotide sequence of HPG3.5 k DNA fragment was analyzed with a DNA sequencer as described above. As a result, a sequence of 3450 nucleotides was determined. The nucleotide sequence of HPG3.5 k DNA fragment corresponds to the nucleotide sequence of from nucleotides No. 1 to No. 3450 in SEQ ID NO: 1. A region was found which codes for an amino acid sequence identical to that of the N-terminal of HPGp130 polypeptide. An open reading frame was obtained from HPG3.5 k DNA in the same frame as that of HPGp130 polypeptide and it was found that translation starts at nucleotide No. 243 to code for 1069 amino acid residues. There was no termination codon within the region and thus it was assumed that HPG3.5 k DNA codes for a portion of HPGp130 polypeptide. A corresponding amino acid sequence is also shown.

(7) Cloning of HPG4.1 k DNA

The above HPG3.5 k DNA fragment was labeled with DIG as described above. After the genomic DNA of Haemophilus paragallinarum serotype A strain 221 was cleaved with restriction enzymes XhoI and XbaI, a Southern hybridization was carried out as described in Example 3 (3) using the DIG-labeled HPG3.5 k DNA or the DIG-labeled HPG1.2 k DNA as a probe. As a result, DNAs of about 5.5 kb, about 4.1 kb and about 1 kb were detected with the DIG-labeled HPG3.5 k DNA as a probe. When the DIG-labeled HPG1.2 k DNA was used as a probe, DNAs of about 4.1 kb and about 1 kb were detected. Since there are two XhoI sites within the HPG3.5 k DNA fragment as shown in FIG. 7 , it was considered that the DNA of about 5.5 kb was a fragment corresponding to the 5′ site from the first XhoI cleavage site, the DNA of about 4.1 kb was a fragment corresponding to the 3′ site from the second XhoI cleavage site and the DNA of about 1 kb was a fragment between these two XhoI sites. Thus, the fragment of about 4.1 kb was separated and recovered on 0.8% agarose gel electrophoresis.

As shown in FIG. 8 , plasmid pSP72 (manufactured by Promega) was digested with XhoI and XbaI and, after dephosphorizing the 5′ end, ligated with the above XhoI-XbaI digest (about 4.1 kb) derived from the genome of Haemophilus paragallinarum serotype A strain 221. E.coli strain JM109 cells were transformed with the ligated product. For the obtained E.coli transformants, a colony hybridization was carried out using the DIG-labeled HPG3.5 k DNA as a probe to screen positive clones.

The positive clones were cultured on Circle Grow medium containing 50 μg/ml of ampicillin. Plasmids were recovered from the cells by PEG precipitation method. The obtained plasmid (hereinafter referred to as “pSA4.1”), in which the XhoI-XbaI digest fragment (hereinafter referred to as “HPG4.1 k DNA”) derived from Haemophilus paragallinarum serotype A strain 221 was incorporated, was digested with XhoI and XpnI and then electrophoresed on 0.8% agarose gel to separate and recover a DNA fragment of about 4.1 kb which was the above HPG4.1 k DNA added with XbaI-KpnI fragment from the plasmid pSP72.

(8) Expression of HPG4.1 k DNA

As described in Example 3 (4), the expression vector pTrcHisC was digested with XhoI and XpnI and, after dephosphorizing the 5′ end, ligated with the above XhoI-XpnI digest of about 4.1 kb. E.coli strain JM109 cells were transformed with the ligated product. From the obtained transformants of E.coli , there was obtained E.coli which was transformed with a recombinant plasmid wherein HPG4.1 k DNA was ligated in a right direction and expresses an antigen specifically reactive with anti-HPGp130 guinea pig serum.

(9) Immunogenicity of HPG4.1 k-HIS Polypeptide

The obtained transformants of E.coli were inoculated to 200 ml of Circle Grow medium containing 50 μg/ml of ampicillin and cultured at 37° C. for 3 hours. Thereto was added IPTG (final concentration of 1 mM) and the transformants were cultured at 37° C. for additional 3 hours. The cells were harvested from the culture by centrifugation and suspended in 10 ml of PBS. To the suspension was added lysozyme at 100 μg/ml for reaction at 4° C. for 1 hour. The suspension was sonicated at 4° C. for 10 minutes for bacteriolysis. Intact cells were removed by centrifugation and the obtained supernatant was used as a crude HPG4.1 k-HIS polypeptide.

Ten SPF white leghorn chickens 5 weeks old were immunized by subcutaneously administering at the leg 0.5 ml of an emulsion prepared by thoroughly mixing the crude HPG4.1 k-HIS polypeptide solution with the same amount of Freund's complete adjuvant. About three weeks later, the chickens were subcutaneously administered at the leg with 0.5 ml of an emulsion prepared similarly with Freund's incomplete adjuvant. Two weeks later, the chickens were boosted subcutaneously at the leg with an emulsion prepared similarly with Freund's incomplete adjuvant. Seven weeks after the first immunization, the chickens were challenged with Haemophilus paragallinarum serotype A strain 221. As a control, as described in Example 2 (7), one group was immunized with formalin-inactivated HPG serotype A strain 221 and another group was not immunized and both control groups were challenged similarly. The results are shown in Table 4. The group immunized with the crude HPG4.1 k-HIS polypeptide showed protection against the onset of the disease in every ten among the tested chickens. The group immunized with the formalin-inactivated cells exhibited protection against the onset of the disease in all the chickens whereas the non-immunization group showed the symptoms in all the chickens.

	TABLE 4
		Tested	Protected	Protection
	Immunization group	chicken	chicken	rate (%)
	Crude HPGp4.1k-HIS	10	10	100
	Formalin-inactivated	10	10	100
	strain 221
	Non immunization control	10	0	0

(10) Nucleotide Sequence of HPG4.1 k DNA Fragment

A nucleotide sequence of a region in HPG4.1 k DNA fragment which does not overlap with HPG3.5 k DNA fragment, i.e. a region ranging from the HindIII cleavage site to the XbaI cleavage site, was analyzed with a DNA sequencer as described above. As a result, a sequence of 2831 nucleotides was determined. The analyzed nucleotide sequence of HPG4.1 k DNA fragment corresponds to the nucleotide sequence of from nucleotides No. 3445 to No. 6275 in SEQ ID NO: 1. No termination codon was found within the region of said DNA fragment. A corresponding amino acid sequence is also shown.

(11) Cloning of HPG6.7 k DNA

After the genomic DNA of Haemophilus paragallinarum serotype A strain 221 was cleaved with XhoI and PstI, a Southern hybridization was carried out as described in Example 3 (3) using the DIG-labeled HPG3.5 k DNA or the DIG-labeled HPG1.2 k DNA as a probe. As a result, DNAs of about 9.4 kb, about 6.7 kb and about 1 kb were detected with the DIG-labeled HPG3.5 k DNA as a probe. When the DIG-labeled HPG1.2 k DNA was used as a probe, DNAs of about 6.7 kb and about 1 kb were detected. Since there are two XhoI cleavage sites within the HPG3.5 k DNA fragment as described above, it was considered that the DNA of about 9.4 kb was a fragment corresponding to the 5′ site from the first XhoI cleavage site, the DNA of about 6.7 kb was a fragment corresponding to the 3′ site from the second XhoI cleavage site and the DNA of about 1 kb was a fragment between these two XhoI sites. Thus, the fragment of about 6.7 kb was separated and recovered on 0.8% agarose gel electrophoresis.

As shown in FIG. 9 , plasmid pSP72 was digested with XhoI and PstI and, after dephosphorizing the 5′ end, ligated with the above XhoI-PstI digest (about 6.7 kb) derived from the genome of Haemophilus paragallinarum serotype A strain 221. E.coli strain JM109 cells were transformed with the ligated product. For the obtained E.coli transformants, a colony hybridization was carried out using the DIG-labeled HPG3.5 k DNA as a probe to screen positive clones.

The positive clones were cultured on CIRCLE GROW medium containing 50 μg/ml of ampicillin. Plasmids were recovered from the cells by PEG precipitation method. The obtained recombinant plasmid is hereinafter referred to as “pSA6.7”. E.coli SA6.7JM transformed with the recombinant plasmid has been deposited by the applicant as FERM BP-6081 at National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken) on Aug. 27, 1997.

(12) Cloning of HPG2.7 k DNA

Since the DNA fragment of about 6.7 kb (hereinafter referred to as “HPG6.7 k DNA”) incorporated in the obtained recombinant plasmid (pSA6.7) encompasses the above HPG4.1 k DNA, a fragment of about 2.7 kb (hereinafter referred to as “HPG2.7 k DNA”) was subcloned which is a subtraction of HPG4.1 k DNA from HPG6.7 k DNA. pSA6.7 was digested with XbaI and then electrophoresed on 0.8% agarose gel to separate and recover a DNA fragment of about 2.7 kb which was the above HPG2.7 k DNA added with PstI-XbaI fragment from the plasmid pSP72.

Plasmid pSP72 was then digested with XbaI and, after dephosphorizing the 5′ end, ligated with the above XbaI digest of about 2.7 kb. E.coli strain JM109 cells were transformed with the ligated product. The obtained E.coli transformants were cultured on CIRCLE GROW medium containing 50 μg/ml of ampicillin. Plasmids were recovered from the cells by PEG precipitation method. The obtained recombinant plasmid is hereinafter referred to as “pSA2.7”.

(13) Nucleotide Sequence of HPG2.7 k DNA

A nucleotide sequence of HPG2.7 k DNA fragment was analyzed with a DNA sequencer as described above. As a result, a sequence of 2661 nucleotides was determined. The nucleotide sequence of HPG2.7 k DNA fragment corresponds to the nucleotide sequence of from nucleotides No. 6270 to No. 8930 in SEQ ID NO: 1. A termination codon was found within the region. A corresponding amino acid sequence is also shown.

It was found that the nucleotide sequence of SEQ ID NO: 1, consisting of a total of 8930 nucleotides, included an open reading frame starting from nucleotide No. 243 which can code for 2042 amino acid residues. A polypeptide comprising the 2042 amino acid residues is hereinafter referred to as “serotype A HMTp210”. Homology search with the existing data base (GeneBank and EMBL) revealed no homology with any known nucleotide and amino acid sequences, indicating that the serotype A HMTp120 polypeptide is a novel substance.

The presence of another possible open reading frame in the nucleotide sequence of SEQ ID NO: 1 was also suggested which starts from nucleotide No. 8375 and can code for 185 amino acid residues. No termination codon was found in this sequence. Homology search with the existing data base (GeneBank and EMBL) revealed no homology with any known nucleotide and amino acid sequences, indicating that the polypeptide coded by this open reading frame is also a novel substance.

EXAMPLE 4

Search for DNA Fragment Hybridizable to HPG1.2 k DNA from Other Strains than Haemophilus paragallinarum Serotype A strain 221

As described in Example 3 (1), genomic DNAs were prepared from a total of nine strains, i.e. HPG serotype A strains 221, 083, W, Germany and Georgia, HPG serotype B strains Spross and 0222, and HPG serotype C strains Modesto and 53-47. After the prepared genomic DNAs were cleaved with restriction enzyme EcoRI, a Southern hybridization was carried out using the DIG-labeled HPG1.2 k DNA as a probe as described in Example 3 (3). As a result, fragments hybridizable with HPG1.2 k DNA were detected in every strains although size of each fragment was varied depending on the strains (FIG. 10 ).

EXAMPLE 5

Cloning of Gene Coding for Polypeptide (Serotype C HMTp210) from Haemophilus paragallinarum Serotype C

(1) Screening from Genomic Library

A genomic library of Haemophilus paragallinarum serotype C strain 53-47 was prepared in the same manner as described in Example 3 (1). That is, a genomic DNA of HPG serotype C strain 53-47 digested with restriction enzyme HindIII was ligated to λDASHII (manufactured by STRATAGENE) arm digested with restriction enzyme HindIII using cDNA Rapid Cloning Module-λgt11. Using λ-DNA in vitro packaging module, the ligated product was inserted into λ phage. The obtained solutions of recombinant phage were used as a genomic library.

The above solutions of genomic library were added to a suspension of E.coli strain XL1-Blue MRA (P2) (manufactured by STRATAGENE) about 10 8 cells in an aqueous solution of 10 mM magnesium sulfate for absorption at 37° C. for 15 minutes. Thereto was added LB soft agarose medium (containing tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, ampicillin 50 mg, maltose 4 g and agarose 8 g in 1000 ml, pH 7) for overlay warmed at 45° C. The mixture was overlaid to LB agar medium and incubated at 37° C. overnight. To the agar medium where transformed E.coli grown was overlaid Hybond N+ membrane to lift the phage plaques. Using the DIG-labeled serotype A HPG3.5 k DNA as a probe, a plaque hybridization was carried out in the conventional manner and positive clones were screened. About 1,000 plaques were immunologically screened as described above to give 37 positive plaques. Ten among the obtained positive plaques were further subjected to second and third screening as in the primary screening.

The recombinant λDASHII phages found positive in the plaque hybridization were added to a suspension of E. coli strain XL1-Blue MRA (manufactured by STRATAGENE) about 10 8 cells in an aqueous solution of 10 mM magnesium sulfate for absorption at 37° C. for 15 minutes. As described in Example 3 (1), the phage DNA was recovered. The obtained phage DNA was digested with HindIII and then electrophoresed on 0.8% agarose gel to separate and recover DNA fragments derived from Haemophilus paragallinarum serotype C strain 53-47. All the DNA fragments obtained from ten positive phages had a length of about 13.5 kb. A DNA fragment (hereinafter referred to as “HPG-C1 DNA”) obtained from the phage of a clone (clone 1) was used in the following test.

(2) Fragmentation and Subcloning of HPG-C1 DNA

Since the HPG-C1 DNA of about 13.5 kb is too large to be subcloned into a plasmid vector, it was cleaved with several restriction enzymes and a suitable amount of the resulting DNA fragments was electrophoresed on 0.8% agarose gel. As a result, DNA fragments of about 6.9 kb, about 5.6 kb and about 0.9 kb were detected when digested with XbaI.

Plasmid pUC119 was digested with HindIII and XbaI and, after dephosphorizing the 5′ end, ligated with the above XbaI digests of HPG-C1 DNA. E.coli strain JM109 cells were transformed with the ligated products. Furthermore, E.coli cells transformed with the recombinant plasmid containing either DNA fragment of about 5.6 kb or about 0.9 kb were cultured and the plasmids were recovered from the cells by PEG precipitation method. The obtained recombinant plasmids (hereinafter referred to as “pU-C2” and “pU-C3”, containing either DNA fragment of about 5.6 kb and about 0.9 kb, respectively) was digested with HindIII-XbaI and then electrophoresed on 0.8% agarose gel to separate and recover DNA fragments of about 5.6 kb and about 0.9 kb (hereinafter referred to as “HPG-C2 DNA” and “HPG-C3 DNA” respectively). E.coli U-C2JM transformed with the recombinant plasmid pU-C2 has been deposited by the applicant as FERM BP-6082 at National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken) on Aug. 27, 1997.

Plasmid pUC119 was digested with XbaI and, after dephosphorizing the 5′ end, ligated with the above XbaI digests of HPG-C1 DNA. E.coli strain JM109 cells were transformed with the ligated products. Furthermore, E.coli cells transformed with the recombinant plasmid containing DNA fragment of about 6.9 kb were cultured and the plasmid was recovered from the cells by PEG precipitation method. The obtained recombinant plasmid (hereinafter referred to as “pU-C4”) was digested with XbaI and then electrophoresed on 0.8% agarose gel to separate and recover DNA fragment of about 6.9 kb (hereinafter referred to as “HPG-C4 DNA”). E.coli U-C4JM transformed with the recombinant plasmid pU-C4 has been deposited by the applicant as FERM BP-6080 at National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken) on Aug. 27, 1997.

Each of the obtained DNA fragments HPG-C2, HPG-C3 and HPG-C4 was spotted on Hybond N+ membrane. Then, a dot hybridization was carried out using as a probe either the above DIG-labeled HPG3.5 k DNA or HPG4.1 k or HPG2.7 k DNA labeled similarly with DIG. When the DIG-labeled HPG3.5 k DNA or DIG-labeled HPG4.1 k DNA was used as a probe, HPG-C4 DNA was detected. On the other hand, when HPG2.7 k DNA was used as a probe, HPG-C2 DNA was detected. From this, it was assumed that HPG-C3, HPG-C4 and HPG-C2 were positioned in this order from the 5′ site and HPG-C4 mainly encompasses a region coding for the polypeptide as shown in FIG. 11 .

(3) Nucleotide Sequence of HPG-C4 DNA Fragment

A nucleotide sequence of HPG-C4 DNA fragment was analyzed with a DNA sequencer as described above. As a result, a sequence of 6871 nucleotides was determined. The nucleotide sequence of HPG-C4 DNA fragment corresponds to the nucleotide sequence of from nucleotides No. 1 to No. 6871 in SEQ ID NO: 5. Based on high homology with the gene coding for serotype A HMTp210, an open reading frame was obtained from HPG-C4 DNA in the same frame as that of the gene coding for serotype A HMTp120 and it was found that translation starts at nucleotide No. 848 to code for 2008 amino acid residues. However, no termination codon was found within the region of said DNA fragment. A corresponding amino acid sequence was also shown.

(4) Nucleotide Sequence of a Portion of HPG-C2 DNA Fragment

Since no termination codon was found within the region of HPG-C4 DNA fragment, a nucleotide sequence at the 5′ site of HPG-C2 DNA fragment, which is at the 3′ site of HPG-C4 DNA fragment, was analyzed. As shown in FIG. 11 , there are three AccI cleavage sites within HPG-C2 DNA fragment. It was also revealed that a fragment ranging from the cloning site, i.e. XbaI cleavage site, to the first AccI cleavage site is of size about 0.6 Kb as demonstrated in an agarose gel electrophoresis. Thus, a nucleotide sequence of this fragment of about 0.6 Kb was analyzed with a DNA sequencer as described above. As a result, a sequence of 621 nucleotides was determined. The nucleotide sequence of a portion of HPG-C2 DNA fragment corresponds to the nucleotide sequence of from nucleotides No. 6866 to No. 7486 in SEQ ID NO: 5. A termination codon was found within the region of this portion of HPG-C2 DNA fragment. A corresponding amino acid sequence was also shown.

It was found that the nucleotide sequence of SEQ ID NO: 5, consisting of a total of 7486 nucleotides, included an open reading frame starting from nucleotide No. 848 which can code for 2039 amino acid residues. A polypeptide comprising the 2039 amino acid residues is hereinafter referred to as “serotype C HMTp210”. Homology search with the existing data base (GeneBank and EMBL) revealed no homology with any known nucleotide and amino acid sequences, indicating that the serotype C HMTp120 polypeptide is a novel substance.

Homology search between the nucleotide sequences coding for the serotype C HMTp120 polypeptide and the serotype A HMTp120 polypeptide revealed about 80% homology. It was further revealed that the region of about 3.4 kb at the 5′ site and the region of about 1.2 kb at the 3′ site exhibited extremely high homology whereas the region of about 1.5 kb between these 5′ and 3′ regions showed low homology. The same was also applicable to the corresponding polypeptides encoded by these genes.

EXAMPLE 6

PCR Amplification of HMTp120 Gene from Genomic DNA of HPG Serotypes A, B and C Cells

As described in Example 3 (1), genomic DNAs were prepared from a total of nine strains, i.e. HPG serotype A strains 221, 083, W, Germany and Georgia, HPG serotype B strains Spross and 0222, and HPG serotype C strains Modesto and 53-47. Based on the nucleotide sequence coding for the Type A HMTp120 polypeptide, there were prepared a synthetic DNA having the nucleotide sequence of SEQ ID NO: 3 as an upstream PCR primer and a synthetic DNA having the nucleotide sequence of SEQ ID NO: 4 as a downstream PCR primer. These primers were designed such that BamHI recognition sequences were added at the 5′ site, respectively, and a full length of translation region of the serotype A HMTp120 polypeptide can be amplified. Using these primers, PCR was carried out using the genomic DNAs prepared as mentioned above as a template. PCR was carried out with LA PCR Kit ver. 2 (manufactured by Takara Shuzo K.K.) under the following conditions: after reaction at 94° C. for 1 minute, 30 cycles of reactions at 98° C. for 40 seconds and at 60° C. for 10 minutes, followed by reaction at 72° C. for 10 minutes. Analysis of the obtained PCR products on 0.8% agarose gel electrophoresis confirmed the amplified fragment of about 6.1 Kb in any of these strains (FIG. 12 ).

EXAMPLE 7

Expression of Full-length Serotypes A and C HMTp210 Polypeptides

(1) Expression of Serotype A HMTp120 Polypeptide

The PCR product obtained in Example 6 with the genomic DNA from Haemophilus paragallinarum serotype A strain 221 as a template was digested with BamHI. After separation on 0.8% agarose gel electrophoresis, the amplified fraction of about 6.1 Kb was eluted and recovered with Sephaglas™ BandPrep Kit.

Plasmid pUC119 was digested with BamHI and, after dephosphorizing the 5′ end, ligated with the above amplified fragment of about 6.1 kb. E.coli strain JM109 cells were transformed with the ligated product. Furthermore, E.coli cells transformed with the recombinant plasmid containing DNA fragment of about 6.1 kb were cultured and the plasmid was recovered from the cells by PEG precipitation method. The obtained recombinant plasmid (hereinafter referred to as “pU-AP1”) was digested with BamHI and then electrophoresed on 0.8% agarose gel to separate and recover DNA fragment of about 6.1 kb (hereinafter referred to as “HPG-AP1 DNA”).

As described in Example 3 (4), the expression vector pTrcHisA (manufactured by Invitrogen) was digested with BamHI and, after dephosphorizing the 5′ end, ligated with the above HPG-AP1 DNA. E.coli strain JM109 cells were transformed with the ligated product. From the obtained transformants of E.coli , there was obtained E.coli which was transformed with a recombinant plasmid wherein HPG-AP1 DNA was ligated in a right direction and expressed an antigen specifically reactive with anti-HPGp130 guinea pig serum.

(2) Expression of Serotype C HMTp120 Polypeptide

The PCR product obtained in Example 6 with the genomic DNA from Haemophilus paragallinarum serotype C strain 53-47 as a template was digested with BamHI. After separation on 0.8% agarose gel electrophoresis, the amplified fraction of about 6.1 Kb was recovered.

Plasmid pUC119 was digested with BamHI and, after dephosphorizing the 5′ end, ligated with the above amplified fragment of about 6.1 kb. E.coli strain JM109 cells were transformed with the ligated product. Furthermore, E.coli cells transformed with the recombinant plasmid containing DNA fragment of about 6.1 kb were cultured and the plasmid was recovered from the cells by PEG precipitation method. The obtained recombinant plasmid (hereinafter referred to as “pU-CP1”) was digested with BamHI and then electrophoresed on 0.8% agarose gel to separate and recover DNA fragment of about 6.1 kb (hereinafter referred to as “HPG-CP1 DNA”).

As described in Example 3 (4), the expression vector pTrcHis A (manufactured by Invitrogen) was digested with BamHI and, after dephosphorizing the 5′ end, ligated with the above HPG-CP1 DNA. E.coli strain JM109 cells were transformed with the ligated product. From the obtained transformants of E.coli, there was obtained E.coli which was transformed with a recombinant plasmid wherein HPG-CP1 DNA was ligated in a right direction and expressed an antigen specifically reactive with anti-HPGp130 guinea pig serum.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 8
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8930 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 8374..8929
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
AAGCTTTTTC GGGCGATTGA AGACGGAATG TTACTTTGGC AAGCGGTTTG AAACCTTTGA     60
ACAGCTTGAA AAAGTGATTC ACGAGTACAT TCATTACTAC AACAATGAGC GTATTCAAG     120
GAAGCTCAAA GGACTAAGCC CTGTGGAATA CAGAACTCAG TCCTTGAATG AAATTAGAA     180
ATAGTCTAAC TTTTTGGGGC AGATCAACAC TCATTTTTAA TATTAATATA GGAAAATGA     240
TT ATG AAT AAA GTT TTT AAA ATT AAA TAT TCT GTT GTA AAA CAA GAA       287
Met Asn Lys Val Phe Lys Ile Lys Tyr Ser Val Val Lys Gln Glu
-70                 -65                 -60
ATG ATT GTG GTT TCA GAG CTA GCA AAT AAT AAA GAT AAA ACA GCT AGC      335
Met Ile Val Val Ser Glu Leu Ala Asn Asn Lys Asp Lys Thr Ala Ser
-55                 -50                 -45                 -40
CAA AAA AAC ACA CAT AAT ACT GCA TTT TTT CAA CCG CTA TTT ACA AAG      383
Gln Lys Asn Thr His Asn Thr Ala Phe Phe Gln Pro Leu Phe Thr Lys
-35                 -30                 -25
TGT ACA TAT CTT GCT CTT CTC ATT AAT ATC GCA CTA GGA GCA TCA TTA      431
Cys Thr Tyr Leu Ala Leu Leu Ile Asn Ile Ala Leu Gly Ala Ser Leu
-20                 -15                 -10
TTC CCT CAA TTA GCT AAT GCG AAG TGG TTA GAG GTT TAT AGT AGC TCC      479
Phe Pro Gln Leu Ala Asn Ala Lys Trp Leu Glu Val Tyr Ser Ser Ser
-5                  1               5
GTA AAA CTA TCT ACT GTT AGT GCA CAA AGT AAT AGT GTT AAT CTT AAT      527
Val Lys Leu Ser Thr Val Ser Ala Gln Ser Asn Ser Val Asn Leu Asn
10                  15                  20                  25
CCA TCG GGA GCT GAG AGT GTT GGC ACA AAT AGC CCA CAA GGG GTT GCT      575
Pro Ser Gly Ala Glu Ser Val Gly Thr Asn Ser Pro Gln Gly Val Ala
30                  35                  40
ATT GGC TAT GGT GCA ACC AAC GAT AGA TCT GCA ACA GGA GCT ATT GCT      623
Ile Gly Tyr Gly Ala Thr Asn Asp Arg Ser Ala Thr Gly Ala Ile Ala
45                  50                  55
CTT GGG GTT GGG GTA AAA AAT GAA ACT TTA GCG AAA GAC TCT ATT GCC      671
Leu Gly Val Gly Val Lys Asn Glu Thr Leu Ala Lys Asp Ser Ile Ala
60                  65                  70
ATT GGT TAT GGG GCA AAA AAT GAA AGC ACA GCA CCA AGT TCT GTG ACT      719
Ile Gly Tyr Gly Ala Lys Asn Glu Ser Thr Ala Pro Ser Ser Val Thr
75                  80                  85
ATT GGA AAA CAG GCG ATT AAC CGT TTT GAA AAA TCT ATT GTG ATG GGT      767
Ile Gly Lys Gln Ala Ile Asn Arg Phe Glu Lys Ser Ile Val Met Gly
90                  95                  100                 105
CTT AAT GCT TAT ACA CAA TTA GAT CCC CGT GGA ACT AGT AAA GAA ACC      815
Leu Asn Ala Tyr Thr Gln Leu Asp Pro Arg Gly Thr Ser Lys Glu Thr
110                 115                 120
CGT CAA GGT TCT GTA GTG ATT GGG GAA AAT GCG AAA AGT GCT GGG AAT      863
Arg Gln Gly Ser Val Val Ile Gly Glu Asn Ala Lys Ser Ala Gly Asn
125                 130                 135
CAA TCT GTT TCT TTA GGG CAA AAT TCG TGG TCA AAA ACC AAT TCT ATT      911
Gln Ser Val Ser Leu Gly Gln Asn Ser Trp Ser Lys Thr Asn Ser Ile
140                 145                 150
TCT ATT GGG GCA GGA ACC TTT GCG GAA GGA AAA TCA AGC ATT GCT ATA      959
Ser Ile Gly Ala Gly Thr Phe Ala Glu Gly Lys Ser Ser Ile Ala Ile
155                 160                 165
GGG ACT GAT AAA ATA TCA GGG ACT AAG TAT AAT GAC AAA TTG CCT GCT     1007
Gly Thr Asp Lys Ile Ser Gly Thr Lys Tyr Asn Asp Lys Leu Pro Ala
170                 175                 180                 185
ACT GCT TGG AAT GGA ACA GGC ACT GTT CCG AAA AAC TCC ATT TGG GAT     1055
Thr Ala Trp Asn Gly Thr Gly Thr Val Pro Lys Asn Ser Ile Trp Asp
190                 195                 200
ATA TTT TCT GAG TTA TAT ATG GGG AAA CAG ACT AAC GGC AGA GAT TAT     1103
Ile Phe Ser Glu Leu Tyr Met Gly Lys Gln Thr Asn Gly Arg Asp Tyr
205                 210                 215
GAT ACA ACT ACT CGA GAC CCT AAT AAA CCG GAG GCA TTT TAT AAA TTT     1151
Asp Thr Thr Thr Arg Asp Pro Asn Lys Pro Glu Ala Phe Tyr Lys Phe
220                 225                 230
AGC GAT TTT AAA GGA AAA TAT GTC AAT ACC CCA ACT GCT TCA CCT ACT     1199
Ser Asp Phe Lys Gly Lys Tyr Val Asn Thr Pro Thr Ala Ser Pro Thr
235                 240                 245
TAT GCA GGG AAA TTA GGG GCA ATT GCT CTA GGT TCC CGC ACC ATT GCC     1247
Tyr Ala Gly Lys Leu Gly Ala Ile Ala Leu Gly Ser Arg Thr Ile Ala
250                 255                 260                 265
GCG GGG GAA ATG TCC ACC GCA GTG GGT TCG TTA GCC TTT GCA TTG GCA     1295
Ala Gly Glu Met Ser Thr Ala Val Gly Ser Leu Ala Phe Ala Leu Ala
270                 275                 280
GAT AGA TCC ACC GCA ATG GGG TTA CGT TCT TTT GTT GCT AAA GAC GCC     1343
Asp Arg Ser Thr Ala Met Gly Leu Arg Ser Phe Val Ala Lys Asp Ala
285                 290                 295
GTA GGT GGA ACG GCG ATC GGG GAA GAA TCT CGA ACC TTT GCT AAA GAT     1391
Val Gly Gly Thr Ala Ile Gly Glu Glu Ser Arg Thr Phe Ala Lys Asp
300                 305                 310
TCC GTT GCC ATT GGT AAT AAA ACT GAA GCC TCA AAT GCT GGC TCA ATG     1439
Ser Val Ala Ile Gly Asn Lys Thr Glu Ala Ser Asn Ala Gly Ser Met
315                 320                 325
GCT TAT GGT TAT AAG GCG AAA GCA GTA GGT GCG GGA GCA ATC GCA ATT     1487
Ala Tyr Gly Tyr Lys Ala Lys Ala Val Gly Ala Gly Ala Ile Ala Ile
330                 335                 340                 345
GGG ACA GAA GTC GCA GCA GGG GCT AAA TTT AAT AGC CAT CAA ACA GGA     1535
Gly Thr Glu Val Ala Ala Gly Ala Lys Phe Asn Ser His Gln Thr Gly
350                 355                 360
AAT TTA CTA CAG GAT AAT AAT GCT TAT GCT ACC TTA AAA AAT GCC GAT     1583
Asn Leu Leu Gln Asp Asn Asn Ala Tyr Ala Thr Leu Lys Asn Ala Asp
365                 370                 375
AAA TCA GAT GAT ACT AAA ACC GGA AAT GCG ATT ACT GTA TTT ACC CAG     1631
Lys Ser Asp Asp Thr Lys Thr Gly Asn Ala Ile Thr Val Phe Thr Gln
380                 385                 390
TCT TTT GAT AAT ATG CTT ACT AAT GGA TTA CCG CTG GTA AGT GAA AAC     1679
Ser Phe Asp Asn Met Leu Thr Asn Gly Leu Pro Leu Val Ser Glu Asn
395                 400                 405
GAA ACC TAT TTA ACG ACC TCA GCG GGA GCA ATT AAA AAA ACT GCA ACA     1727
Glu Thr Tyr Leu Thr Thr Ser Ala Gly Ala Ile Lys Lys Thr Ala Thr
410                 415                 420                 425
ACA GAC AGC AGT GCG GGG GGA GGT AAA AAT GCC ATT GCA ATT GGT AGT     1775
Thr Asp Ser Ser Ala Gly Gly Gly Lys Asn Ala Ile Ala Ile Gly Ser
430                 435                 440
AAA ACC TTT GCC TCT AAA GCA AAT TCT GTG GCA TTA GGG AGC TAT GCC     1823
Lys Thr Phe Ala Ser Lys Ala Asn Ser Val Ala Leu Gly Ser Tyr Ala
445                 450                 455
TTA GCC GAT GCC CAA AAT GCC TTT GCA CTA GGT TCT TAT TCT TTT GTG     1871
Leu Ala Asp Ala Gln Asn Ala Phe Ala Leu Gly Ser Tyr Ser Phe Val
460                 465                 470
GAA TCT TCA GCA ACA AAT ACA ATC ACA ATT GGT GTG GGA AGT TAT GCC     1919
Glu Ser Ser Ala Thr Asn Thr Ile Thr Ile Gly Val Gly Ser Tyr Ala
475                 480                 485
AAA GGG AAA AAC AGT TTC TTA GGG GGG ACT TGG GCA TCA ACC CTT TCA     1967
Lys Gly Lys Asn Ser Phe Leu Gly Gly Thr Trp Ala Ser Thr Leu Ser
490                 495                 500                 505
GAT CGG ACA GTT GTG CTA GGG AAT TCC ACT TCA ATT AGC TCA GGT TCT     2015
Asp Arg Thr Val Val Leu Gly Asn Ser Thr Ser Ile Ser Ser Gly Ser
510                 515                 520
CAG AAT GCA TTA GCA ATC GGG GTG AAT GTC TTT ATT GGT AAT GAT AGT     2063
Gln Asn Ala Leu Ala Ile Gly Val Asn Val Phe Ile Gly Asn Asp Ser
525                 530                 535
GCT TCT TCA TTG GCA TTA GGT ATG GGT TCT ACT ATT GCG AAA AGT GCC     2111
Ala Ser Ser Leu Ala Leu Gly Met Gly Ser Thr Ile Ala Lys Ser Ala
540                 545                 550
AAA TCC CCT GAC AGC TTA GCC ATT GGT AAA GAG GCA CGA ATT GAC GCT     2159
Lys Ser Pro Asp Ser Leu Ala Ile Gly Lys Glu Ala Arg Ile Asp Ala
555                 560                 565
AAA GAT ACA GAT AAT GGT ACT TTG TAT CAG CCT CAA GTT TAT GAT GAA     2207
Lys Asp Thr Asp Asn Gly Thr Leu Tyr Gln Pro Gln Val Tyr Asp Glu
570                 575                 580                 585
ACT ACT CGA GCC TTT AGA AAC TTT AAT GAA AGT AGC GAT TAT ATG CGT     2255
Thr Thr Arg Ala Phe Arg Asn Phe Asn Glu Ser Ser Asp Tyr Met Arg
590                 595                 600
CAA GCA ATG GCA TTA GGT TTT AAT GCT AAA GTT TCG CGT GGG GTG GGC     2303
Gln Ala Met Ala Leu Gly Phe Asn Ala Lys Val Ser Arg Gly Val Gly
605                 610                 615
AAA ATG GAA ACG GGG ATT AAC TCG ATG GCG ATT GGT GCT TAT GCT CAA     2351
Lys Met Glu Thr Gly Ile Asn Ser Met Ala Ile Gly Ala Tyr Ala Gln
620                 625                 630
GCA ACT TTG CAA AAT TCC ACC GCA CTT GGG GTA GGC TCT AAA ACA GAT     2399
Ala Thr Leu Gln Asn Ser Thr Ala Leu Gly Val Gly Ser Lys Thr Asp
635                 640                 645
TAC ACT TGG GAA CAG TTA GAA ACC GAT CCT TGG GTA TCT GAA GGG GCA     2447
Tyr Thr Trp Glu Gln Leu Glu Thr Asp Pro Trp Val Ser Glu Gly Ala
650                 655                 660                 665
ATC AGT ATC CCA ACT TCA GGT AAA ACT GGG GTT ATC TCT GTG GGT TCA     2495
Ile Ser Ile Pro Thr Ser Gly Lys Thr Gly Val Ile Ser Val Gly Ser
670                 675                 680
AAA GGT TCA GAA CGT CGT ATT GTG AAT CTT GCT TCG GGT TCT TCT GAT     2543
Lys Gly Ser Glu Arg Arg Ile Val Asn Leu Ala Ser Gly Ser Ser Asp
685                 690                 695
ACT GAT GCC GTG AAT GTT GCT CAG TTA AAA ACC GTT GAA GAA CGT TTC     2591
Thr Asp Ala Val Asn Val Ala Gln Leu Lys Thr Val Glu Glu Arg Phe
700                 705                 710
CTA TCT GAA ATT AAT TTA TTA CAA AAT GGC GGT GGG GTG AAA TAT CTC     2639
Leu Ser Glu Ile Asn Leu Leu Gln Asn Gly Gly Gly Val Lys Tyr Leu
715                 720                 725
TCT GTT GAA AAA ACG AAT ATC AAT GGA CAA TCG GGG AGA GTG GCT AGC     2687
Ser Val Glu Lys Thr Asn Ile Asn Gly Gln Ser Gly Arg Val Ala Ser
730                 735                 740                 745
CAA ATT CGT AAA GGG GAA AAT TAT GAG CGA TAT GTG AAA TTA AAA ACA     2735
Gln Ile Arg Lys Gly Glu Asn Tyr Glu Arg Tyr Val Lys Leu Lys Thr
750                 755                 760
CAA TTG CTC TAT TTA GAT GCA CGA GGA AAA TTA AAT GGA GAG AAG TTT     2783
Gln Leu Leu Tyr Leu Asp Ala Arg Gly Lys Leu Asn Gly Glu Lys Phe
765                 770                 775
GAT CAA AAT TCA TTA AAC AAA ATT CGT GCG GTA GTG CAA GAA CTT GAA     2831
Asp Gln Asn Ser Leu Asn Lys Ile Arg Ala Val Val Gln Glu Leu Glu
780                 785                 790
GCG GAA TAT AGT GGC GAG TTA AAA ACA ACC GCG TCA GCT CTC AAT CAG     2879
Ala Glu Tyr Ser Gly Glu Leu Lys Thr Thr Ala Ser Ala Leu Asn Gln
795                 800                 805
GTT GCA ACA CAA TTA GAG CAA GAA GTA ACC ACA AAT AAC TTC GAC AAA     2927
Val Ala Thr Gln Leu Glu Gln Glu Val Thr Thr Asn Asn Phe Asp Lys
810                 815                 820                 825
TTT AAT CAA TAT AAA ACG CAG ATT GAG AAT GCA AGC AAT GCG GAT TCA     2975
Phe Asn Gln Tyr Lys Thr Gln Ile Glu Asn Ala Ser Asn Ala Asp Ser
830                 835                 840
GCA AGA AAT GTA GGC GGC TTA ACC CCT CAA GCA ATT GCA CAG TTA AAA     3023
Ala Arg Asn Val Gly Gly Leu Thr Pro Gln Ala Ile Ala Gln Leu Lys
845                 850                 855
GCC AAT AAT AAC TAT CTT AAT GAT GGT GCA AAA GGG CAA GAC AGT ATT     3071
Ala Asn Asn Asn Tyr Leu Asn Asp Gly Ala Lys Gly Gln Asp Ser Ile
860                 865                 870
GCA TTT GGC TGG CAG GCA AAA ACC TCA GGA GCT AAT AAT GGA TTA GCA     3119
Ala Phe Gly Trp Gln Ala Lys Thr Ser Gly Ala Asn Asn Gly Leu Ala
875                 880                 885
GGG AAA CAA GCC ATT GCG ATT GGT TTC CAA GCG AAT TCT TCC GCT GAA     3167
Gly Lys Gln Ala Ile Ala Ile Gly Phe Gln Ala Asn Ser Ser Ala Glu
890                 895                 900                 905
AAT GCC ATT TCA ATC GGC ACG AAT TCG GAT ACC TCA ATG ACA GGG GCA     3215
Asn Ala Ile Ser Ile Gly Thr Asn Ser Asp Thr Ser Met Thr Gly Ala
910                 915                 920
GTG GCG ATT GGT AAA GGT GCA ACG GTT ACT GCG GGT GGA AAA CCT TCC     3263
Val Ala Ile Gly Lys Gly Ala Thr Val Thr Ala Gly Gly Lys Pro Ser
925                 930                 935
ATT GCA TTG GGG CAA GAT TCG ACG GTT GCC AAT TCC GCA ATT AGC CGT     3311
Ile Ala Leu Gly Gln Asp Ser Thr Val Ala Asn Ser Ala Ile Ser Arg
940                 945                 950
ACA AGT TCA CCG ATG ATA AAT GGT TTA ATA TTC AAT AAT TTT GCA GGT     3359
Thr Ser Ser Pro Met Ile Asn Gly Leu Ile Phe Asn Asn Phe Ala Gly
955                 960                 965
TCC CCT GAA ACA CTC GGT GTG TTA AGT ATC GGA ACG GCT GGG AGA GAG     3407
Ser Pro Glu Thr Leu Gly Val Leu Ser Ile Gly Thr Ala Gly Arg Glu
970                 975                 980                 985
CGT AAA ATT GTT AAT GTT GCA GCA GGC GAT GTT TCG CAA GCT TCT ACT     3455
Arg Lys Ile Val Asn Val Ala Ala Gly Asp Val Ser Gln Ala Ser Thr
990                 995                 1000
GAA GCC ATT AAC GGC TCA CAG CTT TAT GCA ACG AAC TTT ATG TTG AGC     3503
Glu Ala Ile Asn Gly Ser Gln Leu Tyr Ala Thr Asn Phe Met Leu Ser
1005                1010                1015
AAA GTG GCT CAA TCT GTT AAG AGC AAC TTT GGT GGC AAT GTA AAT CTT     3551
Lys Val Ala Gln Ser Val Lys Ser Asn Phe Gly Gly Asn Val Asn Leu
1020                1025                1030
GGC ACT GAT GGC ACA ATT ACA TTT ACA AAT ATT GGC GGC ACA GGG CAA     3599
Gly Thr Asp Gly Thr Ile Thr Phe Thr Asn Ile Gly Gly Thr Gly Gln
1035                1040                1045
GCT ACA ATC CAC GAT GCG ATT AAT AAT GTT CTC ACT AAA GGG ATC TAC     3647
Ala Thr Ile His Asp Ala Ile Asn Asn Val Leu Thr Lys Gly Ile Tyr
1050                1055                1060                1065
CTT AAA GCG GAT CAG AAT GAT CCA ACA GGA AAT CAA GGT CAG AAA GTG     3695
Leu Lys Ala Asp Gln Asn Asp Pro Thr Gly Asn Gln Gly Gln Lys Val
1070                1075                1080
GAA CTT GGT AAT GCA ATA ACG CTT TCG GCA ACA AAT CAA TGG GCG AAT     3743
Glu Leu Gly Asn Ala Ile Thr Leu Ser Ala Thr Asn Gln Trp Ala Asn
1085                1090                1095
AAC GGC GTA AAT TAT AAA ACG AAC AAT TTA ACC ACT TAT AAT TCA CAA     3791
Asn Gly Val Asn Tyr Lys Thr Asn Asn Leu Thr Thr Tyr Asn Ser Gln
1100                1105                1110
AAT GGC ACG ATT TTA TTT GGA ATG CGT GAA GAT CCA AGT GTA AAA CAA     3839
Asn Gly Thr Ile Leu Phe Gly Met Arg Glu Asp Pro Ser Val Lys Gln
1115                1120                1125
ATT ACA GCG GGA ACC TAT AAT ACA ACG GGT GAT GCG AAC AAT AAA AAT     3887
Ile Thr Ala Gly Thr Tyr Asn Thr Thr Gly Asp Ala Asn Asn Lys Asn
1130                1135                1140                1145
CAA CTA AAT AAT ACA CTT CAA CAA ACC ACG CTT GAA GCA ACT GGG ATC     3935
Gln Leu Asn Asn Thr Leu Gln Gln Thr Thr Leu Glu Ala Thr Gly Ile
1150                1155                1160
ACC AGT AGC GTA GGT TCA ACT AAC TAC GCT GGC TTT AGC TTA GGG GCA     3983
Thr Ser Ser Val Gly Ser Thr Asn Tyr Ala Gly Phe Ser Leu Gly Ala
1165                1170                1175
GAC AGC GTC ACC TTC TCG AAA GGT GGA GCT GGC ACG GTG AAA CTT TCT     4031
Asp Ser Val Thr Phe Ser Lys Gly Gly Ala Gly Thr Val Lys Leu Ser
1180                1185                1190
GGC GTA AGC GAT GCC ACA GCC GAC ACC GAC GCT GCC ACT CTA AAA CAA     4079
Gly Val Ser Asp Ala Thr Ala Asp Thr Asp Ala Ala Thr Leu Lys Gln
1195                1200                1205
GTG AAA GAA TAC CGC ACA ACA TTA GTG GGT GAT AAT GAC ATC ACC GCA     4127
Val Lys Glu Tyr Arg Thr Thr Leu Val Gly Asp Asn Asp Ile Thr Ala
1210                1215                1220                1225
GCA GAT CGT AGT GGC GGC ACA AGC AAT GGC ATT ACC TAC AAC TTA AGC     4175
Ala Asp Arg Ser Gly Gly Thr Ser Asn Gly Ile Thr Tyr Asn Leu Ser
1230                1235                1240
CTT AAT AAA GGT ACG GTT TCG GCA ACA GAA GAA AAA GTG GTG TCA GGG     4223
Leu Asn Lys Gly Thr Val Ser Ala Thr Glu Glu Lys Val Val Ser Gly
1245                1250                1255
AAA ACT GTC TAT GAA GCC ATT AGA AAT GCC ATC ACA GGC AAC ATC TTC     4271
Lys Thr Val Tyr Glu Ala Ile Arg Asn Ala Ile Thr Gly Asn Ile Phe
1260                1265                1270
ACA ATT GGC TTA GAC GAT ACC ACC TTG AAC AAA ATC AAC AAT CCC GCG     4319
Thr Ile Gly Leu Asp Asp Thr Thr Leu Asn Lys Ile Asn Asn Pro Ala
1275                1280                1285
GAT CAA GAT CTT TCA AAC CTC AGT GAA AGT GGC AAA AAT GCC ATT ACG     4367
Asp Gln Asp Leu Ser Asn Leu Ser Glu Ser Gly Lys Asn Ala Ile Thr
1290                1295                1300                1305
GGC TTA GTG GAT GTG GTG AAA AAA ACA AAT TCA CCG ATC ACA GTT GAG     4415
Gly Leu Val Asp Val Val Lys Lys Thr Asn Ser Pro Ile Thr Val Glu
1310                1315                1320
CCT TCT ACC GAT AGC AAC AAG AAA AAA ACC TTC ACT GTA GGC GTG GAT     4463
Pro Ser Thr Asp Ser Asn Lys Lys Lys Thr Phe Thr Val Gly Val Asp
1325                1330                1335
TTC ACC GAT ACC ATT ACG GAA GGT GAC GCA ACG GAT GAT AAA AAA CTG     4511
Phe Thr Asp Thr Ile Thr Glu Gly Asp Ala Thr Asp Asp Lys Lys Leu
1340                1345                1350
ACG ACT TCA AAA TCC GTT GAA AGC TAT GTC ACA AAC AAA CTC GCG AAC     4559
Thr Thr Ser Lys Ser Val Glu Ser Tyr Val Thr Asn Lys Leu Ala Asn
1355                1360                1365
TTC TCT ACA GAT ATT TTG TTA TCG GAT GGG CGT TCT GGT AAC GCA ACA     4607
Phe Ser Thr Asp Ile Leu Leu Ser Asp Gly Arg Ser Gly Asn Ala Thr
1370                1375                1380                1385
ACG GCA AAT GAT GGG GTG GGT AAA CGT CGT TTG TCT GAT GGC TTT ACG     4655
Thr Ala Asn Asp Gly Val Gly Lys Arg Arg Leu Ser Asp Gly Phe Thr
1390                1395                1400
ATC AAA TCT GAA AAC TTT ACG CTA GGT TCA AAA CAA TAT AAT GGC TCT     4703
Ile Lys Ser Glu Asn Phe Thr Leu Gly Ser Lys Gln Tyr Asn Gly Ser
1405                1410                1415
GAT AGC TTA GGG GTA ATG TAT GAC GAT CAA AAT GGG GTC TTT AAA TTA     4751
Asp Ser Leu Gly Val Met Tyr Asp Asp Gln Asn Gly Val Phe Lys Leu
1420                1425                1430
AGC CTA AAT ATG ACC GCA CTT ACC ACT TCA TTG GCT AAT ACT TTC GCG     4799
Ser Leu Asn Met Thr Ala Leu Thr Thr Ser Leu Ala Asn Thr Phe Ala
1435                1440                1445
AAG TTG GAT GCC TCT AAC CTT ACT GAT GAT AGC AAT AAA GAG AAA TGG     4847
Lys Leu Asp Ala Ser Asn Leu Thr Asp Asp Ser Asn Lys Glu Lys Trp
1450                1455                1460                1465
CGT ACT GCG TTG AAT GTG TAT TCA AAA ACA GAA GTA GAT GCA GAA ATT     4895
Arg Thr Ala Leu Asn Val Tyr Ser Lys Thr Glu Val Asp Ala Glu Ile
1470                1475                1480
CAA AAA TCC AAG GTA ACA CTC ACA CCA GAT TCG GGT TTG ATC TTT GCG     4943
Gln Lys Ser Lys Val Thr Leu Thr Pro Asp Ser Gly Leu Ile Phe Ala
1485                1490                1495
ACC AAA CAA GCT GGG AGT GGT AAT AAC GCA GGT ATT GAT GCT GGG AAT     4991
Thr Lys Gln Ala Gly Ser Gly Asn Asn Ala Gly Ile Asp Ala Gly Asn
1500                1505                1510
AAG AAA ATT AGT AAT GTC GCC GAT GGG GAT ATT TCT CCA ACC AGT GGT     5039
Lys Lys Ile Ser Asn Val Ala Asp Gly Asp Ile Ser Pro Thr Ser Gly
1515                1520                1525
GAT GTA GTG ACA GGT CGT CAG CTC TAC GCC TTA ATG CAG AAA GGT ATT     5087
Asp Val Val Thr Gly Arg Gln Leu Tyr Ala Leu Met Gln Lys Gly Ile
1530                1535                1540                1545
CGC GTG TAT GGT GAT GAA GTT AGT CCA ACG AAG ACT CAA ACA ACA GCA     5135
Arg Val Tyr Gly Asp Glu Val Ser Pro Thr Lys Thr Gln Thr Thr Ala
1550                1555                1560
CCT ACA AAT GCA AAC CCA ACT GCG ACG ACA GCA CCT ACA GCA TCT AGC     5183
Pro Thr Asn Ala Asn Pro Thr Ala Thr Thr Ala Pro Thr Ala Ser Ser
1565                1570                1575
ACT CAA GGT TGG GCG ACA ACG GCG AAT ACG GCG GGT GGT GTA GCA CCA     5231
Thr Gln Gly Trp Ala Thr Thr Ala Asn Thr Ala Gly Gly Val Ala Pro
1580                1585                1590
GCA GGT AAT GTA GCA ACG GGG GAT ATT GCG CCG ACA CAG CCA ACA TTG     5279
Ala Gly Asn Val Ala Thr Gly Asp Ile Ala Pro Thr Gln Pro Thr Leu
1595                1600                1605
CCA GAG ATG AAT ACG GCA TTG GTT GAT GAT CAC TTG GCT GTG CCG TTA     5327
Pro Glu Met Asn Thr Ala Leu Val Asp Asp His Leu Ala Val Pro Leu
1610                1615                1620                1625
GGT GGA AGC CTC AAG ATT CAC GGA GAT CAT AAT GTG AAA ACA ACG ATT     5375
Gly Gly Ser Leu Lys Ile His Gly Asp His Asn Val Lys Thr Thr Ile
1630                1635                1640
TCT GCG GAT AAT CAA GTG GGG ATT TCA TTA CAG CCA AAT ATT TCT ATT     5423
Ser Ala Asp Asn Gln Val Gly Ile Ser Leu Gln Pro Asn Ile Ser Ile
1645                1650                1655
GAG AAT AAC TTG GTA ATT GGT TCA AAT GAT CCT GAG AAG GCA AAA TTA     5471
Glu Asn Asn Leu Val Ile Gly Ser Asn Asp Pro Glu Lys Ala Lys Leu
1660                1665                1670
GCC GCA CAA GAA GGT AAT GCT TTG GTT ATC ACT AAC AAA GAT GAC GGG     5519
Ala Ala Gln Glu Gly Asn Ala Leu Val Ile Thr Asn Lys Asp Asp Gly
1675                1680                1685
AAT GCG GCG ATG GTC TTT AAT AAC GAG AAA AAT ATG CTT GTT CTC AGT     5567
Asn Ala Ala Met Val Phe Asn Asn Glu Lys Asn Met Leu Val Leu Ser
1690                1695                1700                1705
GAT AAA GAG GCG AAA CCA AGA GTG CTT CTT GAT GGA CAA AAT GGG GCA     5615
Asp Lys Glu Ala Lys Pro Arg Val Leu Leu Asp Gly Gln Asn Gly Ala
1710                1715                1720
TTA ACT TTA GTC GGC AAT GAT GAT TCT CAA GTC ACC CTT TCC TCT AAG     5663
Leu Thr Leu Val Gly Asn Asp Asp Ser Gln Val Thr Leu Ser Ser Lys
1725                1730                1735
AAA GGT AAA GAT ATT GAT GGA AAT GAT TTG AGC CGT CTC TCT GTG ACG     5711
Lys Gly Lys Asp Ile Asp Gly Asn Asp Leu Ser Arg Leu Ser Val Thr
1740                1745                1750
ACT GAA AGA ACA AAT GCT GAT GGG CAA CTT GAA AAA GTG GAA ACC TCA     5759
Thr Glu Arg Thr Asn Ala Asp Gly Gln Leu Glu Lys Val Glu Thr Ser
1755                1760                1765
TTT GCT ACA ATG GAT GAT GGC TTG AAG TTC AAA GCC GAC GGG GAT AAA     5807
Phe Ala Thr Met Asp Asp Gly Leu Lys Phe Lys Ala Asp Gly Asp Lys
1770                1775                1780                1785
GTG ATT AAT AAG AAA CTT AAT GAA ACC GTT GAA ATT GTT GGT GAT GAG     5855
Val Ile Asn Lys Lys Leu Asn Glu Thr Val Glu Ile Val Gly Asp Glu
1790                1795                1800
AAT GTG ACA ACA TCT ATT ACT GAT GAT AAT AAG GTG AAA GTT TCA CTG     5903
Asn Val Thr Thr Ser Ile Thr Asp Asp Asn Lys Val Lys Val Ser Leu
1805                1810                1815
AAT AAG AAA ATC GCG ATT GAT GAG GTT AAG ATT CCA AAT ACA GAT CCT     5951
Asn Lys Lys Ile Ala Ile Asp Glu Val Lys Ile Pro Asn Thr Asp Pro
1820                1825                1830
GAT GCT CAA AAG GGA GAT AGC ATT GTA ATC AAC AAT GGT GGA ATC CAC     5999
Asp Ala Gln Lys Gly Asp Ser Ile Val Ile Asn Asn Gly Gly Ile His
1835                1840                1845
GCA GGT AAT AAA GTG ATT ACT GGC GTT AAA GCG AGT GAT GAC CCA ACC     6047
Ala Gly Asn Lys Val Ile Thr Gly Val Lys Ala Ser Asp Asp Pro Thr
1850                1855                1860                1865
AGT GCA GTG AAT CGA GGT CAA TTA AAT ACT GTG ATT GAT AAT GTT CAA     6095
Ser Ala Val Asn Arg Gly Gln Leu Asn Thr Val Ile Asp Asn Val Gln
1870                1875                1880
AAT AAT TTC AAT CAA GTT AAT CAA CGT ATT GGC GAT TTA ACA CGG GAG     6143
Asn Asn Phe Asn Gln Val Asn Gln Arg Ile Gly Asp Leu Thr Arg Glu
1885                1890                1895
TCG CGT GCA GGT ATT GCA GGT GCA ATG GCG ACG GCA AGC CTA CAA AAT     6191
Ser Arg Ala Gly Ile Ala Gly Ala Met Ala Thr Ala Ser Leu Gln Asn
1900                1905                1910
GTT GCT TTA CCA GGG AAA ACA ACG ATT TCC GTA GGT ACA GCA ACG TTC     6239
Val Ala Leu Pro Gly Lys Thr Thr Ile Ser Val Gly Thr Ala Thr Phe
1915                1920                1925
AAA GGG GAG AAT GCT GTT GCA ATA GGG ATG TCT AGA CTC TCT GAT AAT     6287
Lys Gly Glu Asn Ala Val Ala Ile Gly Met Ser Arg Leu Ser Asp Asn
1930                1935                1940                1945
GGA AAA GTA GGT ATC CGT TTA TCT GGT ATG AGT ACG AGT AAC GGA GAT     6335
Gly Lys Val Gly Ile Arg Leu Ser Gly Met Ser Thr Ser Asn Gly Asp
1950                1955                1960
AAA GGG GCA GCA ATG AGT GTT GGA TTT AGC TTT TAGCCTTAAT CCATAAAT     6388
Lys Gly Ala Ala Met Ser Val Gly Phe Ser Phe
1965                1970
GCAAAAAGCG AATCACCTTT GATTCGCTTT TTTTATCAGA TTATGTGCCG TAAAACTC     6448
TCCTTCAGGG CGGAGATATA AGGCACAAAC GGCGTAAGCC GTTTCAAACC TAACTAAT     6508
GGTGTTTGTT GTTGCTCAAT GTATTGGCGA ATAATGGAAA TTGGAGCGCC ACCACAAC     6568
CCTGCAAAAT AAGACGGAGA CCAAAGCTGA TTACCCCAAA GTTTTTTGCG GATGTTCG     6628
TAGTTTTTCT TCCTAATCAT TCGGCTTGAT ACACCTTTTA AACTGTTTAC AAGTGTAG     6688
ACAGCCACTT TCGGTGGATA TTCCACAAGT AAATGAACAT GATCGTCTTC ACCGTCAA     6748
TCAACTAATT TTGCTTTAAA ATCATTGCAG ACGCTTTCAA AAATCAATTT GAGTTCGT     6808
AAAATAGCTT TCGTAAAAAC ATCACGGCGA TATTTTGTTA CAAAGACTAA GTGAACAT     6868
ATATTAAAAA CACAATGTCT ACCGTGCCTA ATTTCTGTTT CTTTTTGCAT AGACCAAG     6928
TAAAATGTTG AAAACTTACA TTCTAAACCT TGTCAATGCA ACTACGCAAA GCCTTTAA     6988
TCGAGATAAT GCCGAATGGC GAACAAACCC GTAAAATCAA GCAATTTTGC GGTTGTTC     7048
GTTTTGTGTT CAATCGGGCA TTGGCTTGGC AAAATGAACA ATACGGGCAA GATAACAG     7108
TTAAGTTCAG TTACACTAAA ATCGCCAACT TGCTTCCACA ATGGAAAAAA GAATTAGT     7168
GGCTAAAAGA ATGCCATTCT CAAGTGCTTC AACAGTCGCT AAAAGATCTT GAGAGTGC     7228
TCAAAAATTT CTTTCAGAAA CGTGCCGACT TTCCAAAATT CAAGAAAAAA GGCGTGAA     7288
AGAGCTTTCG TTTTCCGCAA GGTTGCAAAT TAGAACAGGA AAATGACCGC TTATTTTT     7348
CAAAAATCGG CTGGATTCGC TATCGCAACA GCCGAGATAT CGTTGGTGAA ATCAAAAA     7408
TTACCGTCAG CCAAAAGTGC GGTCACTATT TTGTCAGTAT TCAAACTGAA TTTGAGTA     7468
AAATCCCGAC ACATAAAGGC GGTGAAATCG GTATTGATAT GGGCGTTGCA CGTTTTGC     7528
CATTGTCAAA TGGTGAATAT TTTGAACCGG TTAACGCCTT TAAAACTTAC AAAGGAAA     7588
TGGCTAAACT GCAAAAGAGG CTTAAAAATA AAGTAAAATT TAGCCAAAAT TGGCAGAA     7648
TAAAGGCGAA AATCGCCAAA CTGCATCATA AAATTGCTAA TTGTCGCAAA GACTTCTT     7708
ATCAGACTTC AAGCAAAATC AGCAAAAACC ACGCCATGAT CTATATTGAA GATTTGCA     7768
TGTCAAATAT GTCAAAATCA GCCAAAGGTA CGGCGGAAAC ACCAGGCAAA AATGTTGC     7828
CAAAATCAGG GTTGAACCAA GCGATATTAG ATCAATCTTG GTTTGAGTTT CGCCGTCA     7888
TGGACTACAA AACGCAATGG CAAGGTGGAT TTTTAGTGGC AGTGCCAGCG CAAAATAC     7948
GTCGAACTTG CCCTTGTTGT GGTCATGTAG CAAAAGAAAA TCGCCAAACA CAGGCTAA     8008
TTGAGTGTGT AGAATGTGGC TACACAGAAA ATGCCGATGT GGTTGGAGCG TTAAATGT     8068
TGGGGCGTGG GCGAGCTATC GTCCACGCGT AATAAAATGT CAGGGCAGGA CATGCCCG     8128
GAGCTTGTGA AGTGAACTTC ATTGAGAGGT CAGCAACAAG AACCCACCGA GAGTAGCC     8188
TTGCTTGCCA ATTGGCACTA GTAGGAATCC CCATCCTTTA GGGCGGGGAG GATGTCAA     8248
ACATCATTAA TATTTAATGA AAAATATTAT AACTAATTGA TTTTTATATT ATTATTTG     8308
TATTTGGGCG GTGGGACATA ATTTTGACAG ACAGAATGAT ATCGTTTATA TTTCCGAA     8368
TCTGAT ATG TTA TTT AGT AAA ATA TCA GAT AAG AAA AAT TTA TTT TTC      8416
Met Leu Phe Ser Lys Ile Ser Asp Lys Lys Asn Leu Phe Phe
1               5                   10
TTT ATA TAT AGC TCA ATT AAA AGG AAA TTT ATT ATG AAA AAG ACA CTT     8464
Phe Ile Tyr Ser Ser Ile Lys Arg Lys Phe Ile Met Lys Lys Thr Leu
15                  20                  25                  30
ATC GCT TTA GCT GTA ATA ACA ATG TTT TCA AGT GCA GCA AAT GCT GCG     8512
Ile Ala Leu Ala Val Ile Thr Met Phe Ser Ser Ala Ala Asn Ala Ala
35                  40                  45
GTC ATT TAT GAA AAA GAA GGT ACG AAA ATT GAT ATT GAT GGT CGT ATG     8560
Val Ile Tyr Glu Lys Glu Gly Thr Lys Ile Asp Ile Asp Gly Arg Met
50                  55                  60
CAT TTT GAA TTA CGT AAT GAT TCA GGC AAA CGT TCT GAT TTA CAA GAT     8608
His Phe Glu Leu Arg Asn Asp Ser Gly Lys Arg Ser Asp Leu Gln Asp
65                  70                  75
GCA GGC TCT CGT GTC CGC GTA AGA GCT TTT CAA GAA ATT GGC AAT GGC     8656
Ala Gly Ser Arg Val Arg Val Arg Ala Phe Gln Glu Ile Gly Asn Gly
80                  85                  90
TTT TCT ACC TAT GGG GCT GTT GAG TTT CGT TTT TCT ACT AAG AAA GAT     8704
Phe Ser Thr Tyr Gly Ala Val Glu Phe Arg Phe Ser Thr Lys Lys Asp
95                  100                 105                 110
GGC TCA GAA CAA AGT ATT GGA TCT GAC TTA AGA GCT CAC CGC TTT TTT     8752
Gly Ser Glu Gln Ser Ile Gly Ser Asp Leu Arg Ala His Arg Phe Phe
115                 120                 125
GCA GGA ATT AAA CAA AAA GAC ATA GGG GAA TTA ACT TTC GGT AAA CAA     8800
Ala Gly Ile Lys Gln Lys Asp Ile Gly Glu Leu Thr Phe Gly Lys Gln
130                 135                 140
CTC CAT TTA GGT GAT CTT GTC CCG AAA GCA AAT TAT TCT TAT GAT TTA     8848
Leu His Leu Gly Asp Leu Val Pro Lys Ala Asn Tyr Ser Tyr Asp Leu
145                 150                 155
GGG GCG AAC TCT TTT TTT GGT GCA CAT AGT AAA GTA GCA CAT TTT ATT     8896
Gly Ala Asn Ser Phe Phe Gly Ala His Ser Lys Val Ala His Phe Ile
160                 165                 170
TCT GTA CCA TTT AAT GGT GTG AGG GTG TCT GCA G                       8930
Ser Val Pro Phe Asn Gly Val Arg Val Ser Ala
175                 180                 185
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Lys Trp Leu Glu Val Tyr Ser Ser Ser Val Lys Leu Ser
1               5                   10
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (synthetic DNA)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CGCGGATCCA TGAATAAAGT TTTTAAAATT AAATATTCTG TTG                       43
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (synthetic DNA)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CGCGGATCCT TAAGGCTAAA AGCTAAATCC AACACTCAT                            39
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7486 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 848..6964
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TCTAGAATAT AAATCTTCAG TATCAACATA CAAGGGGCGT ATCGCATACG CCCCTGTGCT     60
GAATTTTTAC TTACAGAACA CCGTACTTTT GTTCTGTCAT TTTTGGATAT TTGGGCTGAT    120
GGCTTTTTTC TTTGGTAGTT ATAACGGGTT TCGCTCGTTG AGCGACTTAC TTTCTTTTAC    180
ATTCCCAAAA GAAAGTAAGC AAAGAAAAGG GAACCCGACT AAATTGCTGT TCCTCATTCC    240
AATAAAATTT TCTTCATGAA AAGTAAGCCT GATGTTCGCT TCGCTCTCGC TCGGCGTTAC    300
TTTTCTTAAA ATTTTATTTC CATTCGGGCA ATTTACACGG GAAAGGGCGA TTTTTAAAAG    360
TGCGGTGGTT TTTGAAGGAT ATTTTTGTAT TTGGAAAAAT CAAGAAAATG GCTTTAGAAA    420
ACACCTCACT TATTTTAACT GTAGGTATTG CATTTTTAAT AATACAAATT TTTCTTGAAA    480
TGATGAAATA ACCAATCAAA TTAGTCAGTT ATAAGTGGAG AAACTTAAAG AAAATGATTA    540
AATTAGGCTC ACTCATTAGA CCAGTAAGGG AATTAAAATA GTATTTTTAA TTGCATTTAG    600
TTATTAAGTG TTAGAAATTA CCTATTGCAT CAATAAATGA GGTGTTTTTA TTTGTAATCT    660
CTAATTAATT AGAGTAGTAT TAAGTGGAGT TTTATCTTTA CTAAATTAAT GGTATCACCT    720
CTCAGAGAGG GAGAAGCAAA TTCCCCCCCC CTAGAAATAC CTAATAAGAG TTACATTAAG    780
GGCATATTAT AAAAGTAATT TATCAATAAT GATTAACGCT CATTATTATT AACAAAGGCA    840
AATGATT ATG AAT AAA GTT TTT AAA ATT AAA TAT TCT GTT GTA AAA CAA      889
Met Asn Lys Val Phe Lys Ile Lys Tyr Ser Val Val Lys Gln
-70                 -65                 -60
GAA ATG ATT GTG GTT TCA GAG CTA GCA AAT AAT AAA GAT AAA ACA GCT      937
Glu Met Ile Val Val Ser Glu Leu Ala Asn Asn Lys Asp Lys Thr Ala
-55                 -50                 -45
AGC CAA AAA AAC ACA CAT AAT ACT GCC TTT TTT CAA CCG CTA TTT ACA      985
Ser Gln Lys Asn Thr His Asn Thr Ala Phe Phe Gln Pro Leu Phe Thr
-40                 -35                 -30                 -25
AAG TGT ACA TAT CTT GCT CTT CTC ATT AAT ATC GCA CTA GGA ACA TCA     1033
Lys Cys Thr Tyr Leu Ala Leu Leu Ile Asn Ile Ala Leu Gly Thr Ser
-20                 -15                 -10
TTA TTC CCT CAA TTA GCT AAT GCG AAA TTT TTA GAG GTT TAT AAT AGC     1081
Leu Phe Pro Gln Leu Ala Asn Ala Lys Phe Leu Glu Val Tyr Asn Ser
-5                  1               5
TCC GTA AAA CTA CAG CAT GTT AAT AGT GGC GTA CCA AGT GAT AGT GTT     1129
Ser Val Lys Leu Gln His Val Asn Ser Gly Val Pro Ser Asp Ser Val
10                  15                  20
AAT CTT AAT CCA TCG GGA GGT GAG AAT GTT GGC ATG AAT AGC AAT CAA     1177
Asn Leu Asn Pro Ser Gly Gly Glu Asn Val Gly Met Asn Ser Asn Gln
25                  30                  35                  40
GGG GTC GCT ATT GGC CGT GGT GCA GTA AAT AAT TAT TCG GCG ACG GGA     1225
Gly Val Ala Ile Gly Arg Gly Ala Val Asn Asn Tyr Ser Ala Thr Gly
45                  50                  55
TCA ATT GCT ATT GGT CAG GGG GCA AAA AAT GAT AAT TGG GCG ACG AGA     1273
Ser Ile Ala Ile Gly Gln Gly Ala Lys Asn Asp Asn Trp Ala Thr Arg
60                  65                  70
TCA ATT GCT ATT GGT CAG GGG GCA AAA AAT GAA AGT ATA GCA TCA GAT     1321
Ser Ile Ala Ile Gly Gln Gly Ala Lys Asn Glu Ser Ile Ala Ser Asp
75                  80                  85
TCT GTG GCT ATT TCC AAC GCG ATT AAC CGT TTT AAA AAA TCT ATT GTG     1369
Ser Val Ala Ile Ser Asn Ala Ile Asn Arg Phe Lys Lys Ser Ile Val
90                  95                  100
ATA GGT CTT AAT ACT TAT ACA CAA TTA GAT CCC CGT AGA GCT CCA GAA     1417
Ile Gly Leu Asn Thr Tyr Thr Gln Leu Asp Pro Arg Arg Ala Pro Glu
105                 110                 115                 120
TCC CGT CAA GGT TCT GTG GTG ATT GGG GAA AAT GCG AAA AGT GCT GGG     1465
Ser Arg Gln Gly Ser Val Val Ile Gly Glu Asn Ala Lys Ser Ala Gly
125                 130                 135
AAT CAA TCT GTT TCT TTA GGG CAA AAT GCG TGG TCA AAA ACC AAT TCT     1513
Asn Gln Ser Val Ser Leu Gly Gln Asn Ala Trp Ser Lys Thr Asn Ser
140                 145                 150
ATT TCT ATT GGG GCA GGA ACC TTT GCG GAA GGG AAA TCA ACC ATT GCT     1561
Ile Ser Ile Gly Ala Gly Thr Phe Ala Glu Gly Lys Ser Thr Ile Ala
155                 160                 165
ATA GGG ACT GAT AAA ATA CTA GGG ACT AAT TAT AAT GAC AAA TTG CCT     1609
Ile Gly Thr Asp Lys Ile Leu Gly Thr Asn Tyr Asn Asp Lys Leu Pro
170                 175                 180
GCT CCT AGT TGG GAT GGA AGA ACA GGT AAG GCA CCT ACT AAT TCC ATT     1657
Ala Pro Ser Trp Asp Gly Arg Thr Gly Lys Ala Pro Thr Asn Ser Ile
185                 190                 195                 200
TGG GAT ATA TTT TCT GAG TTA TAT ATG GGG AAA AAG ACT AAC GGC ACA     1705
Trp Asp Ile Phe Ser Glu Leu Tyr Met Gly Lys Lys Thr Asn Gly Thr
205                 210                 215
GAT TAT GAT GCA AAA AAA AAT GAC CGC GAT CCA AAT AAG CCA GAG GCT     1753
Asp Tyr Asp Ala Lys Lys Asn Asp Arg Asp Pro Asn Lys Pro Glu Ala
220                 225                 230
TTT TAT ACC TAT TCT GAT TTT AAA AGC AGA TAT GTT AAT AAC CCA AGT     1801
Phe Tyr Thr Tyr Ser Asp Phe Lys Ser Arg Tyr Val Asn Asn Pro Ser
235                 240                 245
ACC TCT CCC ACT TAT GCC GCT AAA TTA GGG GCA ATT GCC CTA GGT TCC     1849
Thr Ser Pro Thr Tyr Ala Ala Lys Leu Gly Ala Ile Ala Leu Gly Ser
250                 255                 260
CGC ACC ATT GCT GCG GGG GAA ATG TCC ACT GCG GTC GGT TCC TTA GCC     1897
Arg Thr Ile Ala Ala Gly Glu Met Ser Thr Ala Val Gly Ser Leu Ala
265                 270                 275                 280
TTT GCA TTG GCA GAT AAA TCC ACC GCA ATG GGG TTA CGT TCT TTT GTT     1945
Phe Ala Leu Ala Asp Lys Ser Thr Ala Met Gly Leu Arg Ser Phe Val
285                 290                 295
GCT AAA GAT GCC GTA GGT GGA ACG GCA ATC GGG GAA GAA TCG CGA ACC     1993
Ala Lys Asp Ala Val Gly Gly Thr Ala Ile Gly Glu Glu Ser Arg Thr
300                 305                 310
TTT GCT AAA GAT TCC GTT GCC ATT GGT AAT AAA ACT GAA GCC TCA AAT     2041
Phe Ala Lys Asp Ser Val Ala Ile Gly Asn Lys Thr Glu Ala Ser Asn
315                 320                 325
GCT GGC TCA ATG GCT TAT GGT TAT AAG GCG AAA GCG GTA GGT GCG GGG     2089
Ala Gly Ser Met Ala Tyr Gly Tyr Lys Ala Lys Ala Val Gly Ala Gly
330                 335                 340
GCA ATC GCA ATT GGT GCA GAA GTC GCA GCA GGG GCT GAA TTT GAT AGC     2137
Ala Ile Ala Ile Gly Ala Glu Val Ala Ala Gly Ala Glu Phe Asp Ser
345                 350                 355                 360
AGT CAA GCA GGA AAT TTA TTA CTA AAT AGA GGT GCT TAT GCT ACT TTA     2185
Ser Gln Ala Gly Asn Leu Leu Leu Asn Arg Gly Ala Tyr Ala Thr Leu
365                 370                 375
AAA AGT GCC GAT AAA TCA GAT GAT ATT AAA GCT GGA GAT GCG ATT AAC     2233
Lys Ser Ala Asp Lys Ser Asp Asp Ile Lys Ala Gly Asp Ala Ile Asn
380                 385                 390
GTA TTT ACC CAG TTT TTT GAT AAT ATG CTT ACT CAA GGC TCA CAC CTG     2281
Val Phe Thr Gln Phe Phe Asp Asn Met Leu Thr Gln Gly Ser His Leu
395                 400                 405
ACA TAT GAA AAT ACC ACC TAT TTA ACC ACT TCA GCA GGT GAT ATC AAG     2329
Thr Tyr Glu Asn Thr Thr Tyr Leu Thr Thr Ser Ala Gly Asp Ile Lys
410                 415                 420
AAA ACA TTA GCT GCA GTT GGA GAT GGC GGG AAA AAT GCC ATT GCC ATT     2377
Lys Thr Leu Ala Ala Val Gly Asp Gly Gly Lys Asn Ala Ile Ala Ile
425                 430                 435                 440
GGT AAT AAA ACC TTT GCA TCT AAA GCA AAT TCT GTG GCA TTA GGG AGC     2425
Gly Asn Lys Thr Phe Ala Ser Lys Ala Asn Ser Val Ala Leu Gly Ser
445                 450                 455
TAT GCC TTA GCG AGT GCC CAA AAT GCC TTT GCA CTA GGT TCT TAT TCT     2473
Tyr Ala Leu Ala Ser Ala Gln Asn Ala Phe Ala Leu Gly Ser Tyr Ser
460                 465                 470
TTA GTG TCC CCT TTA GCA GCC AAT ACA ATC GTA ATT GGT GTG GGA GGT     2521
Leu Val Ser Pro Leu Ala Ala Asn Thr Ile Val Ile Gly Val Gly Gly
475                 480                 485
TAT GCC ACA GGA TCA AAC AGT TTC GTA GGG GGT TCT TGG GTA TCA ACC     2569
Tyr Ala Thr Gly Ser Asn Ser Phe Val Gly Gly Ser Trp Val Ser Thr
490                 495                 500
CTT TCA GCT CGG ACA GTT GTG CTA GGG TAT TCC GCT TCA ATT AGC TCA     2617
Leu Ser Ala Arg Thr Val Val Leu Gly Tyr Ser Ala Ser Ile Ser Ser
505                 510                 515                 520
GAT TCT CAT GAT TCA TTA GCA ATG GGG GTG AAT GCC TTT ATT GGT AAT     2665
Asp Ser His Asp Ser Leu Ala Met Gly Val Asn Ala Phe Ile Gly Asn
525                 530                 535
GGT AGT AAT TCT TCA TTG GCA TTA GGT ACG GGA TCT ACT ATT GCG AAA     2713
Gly Ser Asn Ser Ser Leu Ala Leu Gly Thr Gly Ser Thr Ile Ala Lys
540                 545                 550
AAT GCC AAA TCT CCT GAC AGC TTA GCC ATT GGT AAA GAC TCA CGA ATT     2761
Asn Ala Lys Ser Pro Asp Ser Leu Ala Ile Gly Lys Asp Ser Arg Ile
555                 560                 565
GAC GCT AAA GAT ACA GAT AAT GGT GTT TTG TAT ACC CCT CAA GTT TAT     2809
Asp Ala Lys Asp Thr Asp Asn Gly Val Leu Tyr Thr Pro Gln Val Tyr
570                 575                 580
GAT GAA ACT ACT CGA GCC TTT AGA ACC TTT GAT GAA AAC AAA GAT TAT     2857
Asp Glu Thr Thr Arg Ala Phe Arg Thr Phe Asp Glu Asn Lys Asp Tyr
585                 590                 595                 600
ATG CGT CAA GCA ATG GCA TTA GGT TTT AAT GCG AAG GTT TCG CGT GGG     2905
Met Arg Gln Ala Met Ala Leu Gly Phe Asn Ala Lys Val Ser Arg Gly
605                 610                 615
AAG GGC AAA ATG GAA ACG GGG ATT AAC TCG ATG GCG ATT GGT GCT CGT     2953
Lys Gly Lys Met Glu Thr Gly Ile Asn Ser Met Ala Ile Gly Ala Arg
620                 625                 630
TCT CAA GCA ACT TTG CAA AAT TCC ACC GCA CTT GGG GTA AAC GCT AAA     3001
Ser Gln Ala Thr Leu Gln Asn Ser Thr Ala Leu Gly Val Asn Ala Lys
635                 640                 645
ACA GAT TAC ACT TGG GAA CAG TTA GAA GCC GAT CCT TGG GTA TCT AAA     3049
Thr Asp Tyr Thr Trp Glu Gln Leu Glu Ala Asp Pro Trp Val Ser Lys
650                 655                 660
GGG GCA ATC AGT ATC CCA ACT TCA GGC AAA ATT GGG GTT ATC TCT GTG     3097
Gly Ala Ile Ser Ile Pro Thr Ser Gly Lys Ile Gly Val Ile Ser Val
665                 670                 675                 680
GGC TCA AAA GGC TCA GAA CGT CGT ATT GTG AAT GTT GCT TCG GGT TCT     3145
Gly Ser Lys Gly Ser Glu Arg Arg Ile Val Asn Val Ala Ser Gly Ser
685                 690                 695
CTT GAT ACC GAT GCC GTG AAT GTT GCC CAA TTA AAA ACT ATT GAA GAA     3193
Leu Asp Thr Asp Ala Val Asn Val Ala Gln Leu Lys Thr Ile Glu Glu
700                 705                 710
CGT TTC CAA TCT GAA ATT GAT TTA TTA CAA AAT GGC GGT GGG GTG CAA     3241
Arg Phe Gln Ser Glu Ile Asp Leu Leu Gln Asn Gly Gly Gly Val Gln
715                 720                 725
TAT CTC TCT GTT GAA AAA ACG AAT ATC AAT GGA GAA GCG GGG AGA GTG     3289
Tyr Leu Ser Val Glu Lys Thr Asn Ile Asn Gly Glu Ala Gly Arg Val
730                 735                 740
GCT AGC CAA ATT CGT AAA GGG GAA AGT TAT AAG CGA TAT GTG AAA TTA     3337
Ala Ser Gln Ile Arg Lys Gly Glu Ser Tyr Lys Arg Tyr Val Lys Leu
745                 750                 755                 760
AAA ACA CAA TTG CTC TAT TTA GAT GCA CGA AAA AAA TTA AAT GGA GAG     3385
Lys Thr Gln Leu Leu Tyr Leu Asp Ala Arg Lys Lys Leu Asn Gly Glu
765                 770                 775
AAG TTT GAT CAA ACT TCA TTA GAC AAA ATT AGT AAG GCA GTG CAA GAA     3433
Lys Phe Asp Gln Thr Ser Leu Asp Lys Ile Ser Lys Ala Val Gln Glu
780                 785                 790
CTT GAA GCG GAA TAT AGT GGC GAG TTA AAA ACA ACT GCG TCA GAA CTT     3481
Leu Glu Ala Glu Tyr Ser Gly Glu Leu Lys Thr Thr Ala Ser Glu Leu
795                 800                 805
AAT AGA GTT GCA ATG CAA TTG AAT GCT GAG ACA ACT GTA AAT GAC TTC     3529
Asn Arg Val Ala Met Gln Leu Asn Ala Glu Thr Thr Val Asn Asp Phe
810                 815                 820
GGG AAA TTT AAT CAA TAT AAA ACG CAG ATT GAG AAT GCA ACC AAT GCG     3577
Gly Lys Phe Asn Gln Tyr Lys Thr Gln Ile Glu Asn Ala Thr Asn Ala
825                 830                 835                 840
GAT TCA GAA AAA AAT GTA GGC GGC TTA TCC CCT CAA GTA ATT GCA CAG     3625
Asp Ser Glu Lys Asn Val Gly Gly Leu Ser Pro Gln Val Ile Ala Gln
845                 850                 855
TTA AAA GCC AAT AAT AAC TAT CTT AAT GAT GGT GCA AAA GGG CAA GAC     3673
Leu Lys Ala Asn Asn Asn Tyr Leu Asn Asp Gly Ala Lys Gly Gln Asp
860                 865                 870
AGT ATA GCA TTT GGC TGG CAG GCA AAA ACC TCA GAA GCT AAT AAT GGA     3721
Ser Ile Ala Phe Gly Trp Gln Ala Lys Thr Ser Glu Ala Asn Asn Gly
875                 880                 885
TTA GCA GGG AAA CAA GCC ATT GCG ATT GGT TTC CAA GCG AAT TCT TCC     3769
Leu Ala Gly Lys Gln Ala Ile Ala Ile Gly Phe Gln Ala Asn Ser Ser
890                 895                 900
GCT GAA AAT GCC ATT TCT ATC GGT ACG AAT TCG GAT ACC TCA ATG ACA     3817
Ala Glu Asn Ala Ile Ser Ile Gly Thr Asn Ser Asp Thr Ser Met Thr
905                 910                 915                 920
GGG GCA GTG GCG ATT GGT AAA GGT GCA ACG GTT ACT GCG GGT GGA AAA     3865
Gly Ala Val Ala Ile Gly Lys Gly Ala Thr Val Thr Ala Gly Gly Lys
925                 930                 935
CCT TCC ATT GCA TTG GGG CAA GAT TCG ACG GTT GCC AAT TCC GCA ATT     3913
Pro Ser Ile Ala Leu Gly Gln Asp Ser Thr Val Ala Asn Ser Ala Ile
940                 945                 950
AGC CGT ACA AGT TCA GTG ATG ATA AAT GGT TTA ACA TTC AAT AAT TTT     3961
Ser Arg Thr Ser Ser Val Met Ile Asn Gly Leu Thr Phe Asn Asn Phe
955                 960                 965
GCA GGT TCC CCT GAA ACA CTC GGT GTG TTA AGT ATC GGA ACG GCT GGG     4009
Ala Gly Ser Pro Glu Thr Leu Gly Val Leu Ser Ile Gly Thr Ala Gly
970                 975                 980
AAA GAG CGT AAA ATT GTT AAT GTT GCA GCA GGC GAT ATT TCG CAA ACT     4057
Lys Glu Arg Lys Ile Val Asn Val Ala Ala Gly Asp Ile Ser Gln Thr
985                 990                 995                 1000
TCT ACT GAA GCC ATT AAC GGC TCA CAG CTT TAT GCA ACG AAC TTT ATG     4105
Ser Thr Glu Ala Ile Asn Gly Ser Gln Leu Tyr Ala Thr Asn Phe Met
1005                1010                1015
TTG AAC AAA CTG GCT CAA TCC GTT AAA ACG AAT TTT GGC GGT AAT GCA     4153
Leu Asn Lys Leu Ala Gln Ser Val Lys Thr Asn Phe Gly Gly Asn Ala
1020                1025                1030
AAC CTT GCC ACT GAT GGC ACA ATT ACA TTT ACA AAT ATT GGC GGC ACA     4201
Asn Leu Ala Thr Asp Gly Thr Ile Thr Phe Thr Asn Ile Gly Gly Thr
1035                1040                1045
GGG CAA GAT ACA ATC CAC GAT GCG ATT AAT AAT GTT CTC ACC AAA TTG     4249
Gly Gln Asp Thr Ile His Asp Ala Ile Asn Asn Val Leu Thr Lys Leu
1050                1055                1060
ATC TCG CTT TCG GCA ACA GAA GAA GAA GAA GTG GTG TCA GGG GAA GCT     4297
Ile Ser Leu Ser Ala Thr Glu Glu Glu Glu Val Val Ser Gly Glu Ala
1065                1070                1075                1080
GTC TAT GAT GCA CTT AAA GGT GCA AAA CCA ACG GTT TCA GCA GAA GCC     4345
Val Tyr Asp Ala Leu Lys Gly Ala Lys Pro Thr Val Ser Ala Glu Ala
1085                1090                1095
AAC AAA GGC ATT ACT GGC TTG GTG GAT GTG GTG AAA AAA GCA AAT TCA     4393
Asn Lys Gly Ile Thr Gly Leu Val Asp Val Val Lys Lys Ala Asn Ser
1100                1105                1110
CCG ATC ACA GTT GAG CCT TCT ACC GAT AAC AAC AAG AAA AAA ACC TTC     4441
Pro Ile Thr Val Glu Pro Ser Thr Asp Asn Asn Lys Lys Lys Thr Phe
1115                1120                1125
ACT GTC GGC TTA ATG AAA GAC ATT GAA GGG GTA AAC AGC ATT ACC TTT     4489
Thr Val Gly Leu Met Lys Asp Ile Glu Gly Val Asn Ser Ile Thr Phe
1130                1135                1140
GAT AAG TCA GGG CAA GAT CTA AAT CAA GTT ACG GGC AGA ATG AGC AGT     4537
Asp Lys Ser Gly Gln Asp Leu Asn Gln Val Thr Gly Arg Met Ser Ser
1145                1150                1155                1160
GCG GGT TTA ACC TTC AAA AAA GGC GAC ACA ACA AAT GGT TCA ACC ACC     4585
Ala Gly Leu Thr Phe Lys Lys Gly Asp Thr Thr Asn Gly Ser Thr Thr
1165                1170                1175
ACT TTT GCA GAA GAT GGC TTA ACC ATT GAT AGC ACA ACA AAT TCT GCT     4633
Thr Phe Ala Glu Asp Gly Leu Thr Ile Asp Ser Thr Thr Asn Ser Ala
1180                1185                1190
CAA ACA AAC TTA GTG AAA GTA AGT CGT GAT GGC TTC TCG GTG AAA AAT     4681
Gln Thr Asn Leu Val Lys Val Ser Arg Asp Gly Phe Ser Val Lys Asn
1195                1200                1205
GGC AGC GAT GAA AGC AAA TTA GCC TCG ACA AAA TTA TCT ATC GGT GCG     4729
Gly Ser Asp Glu Ser Lys Leu Ala Ser Thr Lys Leu Ser Ile Gly Ala
1210                1215                1220
GAA AAT GCA GAA CAC GTT GAA GTA ACT AAA TCG GGC ATA GCC TTA AAA     4777
Glu Asn Ala Glu His Val Glu Val Thr Lys Ser Gly Ile Ala Leu Lys
1225                1230                1235                1240
GCG GAT AAC ACC TCC GAT AAA TCT AGC ATC ACC TTA GCC CAA GAT GCG     4825
Ala Asp Asn Thr Ser Asp Lys Ser Ser Ile Thr Leu Ala Gln Asp Ala
1245                1250                1255
ATT ACT CTT GCG GGG AAC GCA ACC GGA ACG GCG ATT AAA TTG ACT GGT     4873
Ile Thr Leu Ala Gly Asn Ala Thr Gly Thr Ala Ile Lys Leu Thr Gly
1260                1265                1270
GTT GCA GAT GGC AAC ATT ACG GTA AAT TCA AAA GAT GCG GTA AAT GGG     4921
Val Ala Asp Gly Asn Ile Thr Val Asn Ser Lys Asp Ala Val Asn Gly
1275                1280                1285
GGG CAG TTG CGT ACC TTA TTA GGG GTT GAT AGC GGG GCT AAA ATT GGC     4969
Gly Gln Leu Arg Thr Leu Leu Gly Val Asp Ser Gly Ala Lys Ile Gly
1290                1295                1300
GGT ACT GAG AAA ACA ACG ATC AGT GAA GCC ATT TCT GAT GTG AAG CAA     5017
Gly Thr Glu Lys Thr Thr Ile Ser Glu Ala Ile Ser Asp Val Lys Gln
1305                1310                1315                1320
GCT CTT ACC GAT GCG ACA TTG GCA TAT AAA GCG GAC AAT AAA AAC GGT     5065
Ala Leu Thr Asp Ala Thr Leu Ala Tyr Lys Ala Asp Asn Lys Asn Gly
1325                1330                1335
AAA ACA GTT AAA TTG ACT GAC GGA TTG AAT TTT ACT AGC ACG ACC AAT     5113
Lys Thr Val Lys Leu Thr Asp Gly Leu Asn Phe Thr Ser Thr Thr Asn
1340                1345                1350
ATT GAT GCT TCA GTG GAA GAT AAC GGT GTG GTG AAA TTC ACC TTA AAA     5161
Ile Asp Ala Ser Val Glu Asp Asn Gly Val Val Lys Phe Thr Leu Lys
1355                1360                1365
GAT AAA TTA ACA GGC TTA AAA ACT ATC GCA ACT GAA TCT TTG AAT GCT     5209
Asp Lys Leu Thr Gly Leu Lys Thr Ile Ala Thr Glu Ser Leu Asn Ala
1370                1375                1380
TCT CAA AAT ATC ATC GCT GGC GGT ACG GTA ACA GTG GGC GGC GAG ACA     5257
Ser Gln Asn Ile Ile Ala Gly Gly Thr Val Thr Val Gly Gly Glu Thr
1385                1390                1395                1400
GAG GGC ATT GTG CTA ACA AAA TCT GGC TCA GGA AAT GAC CGC ACT TTA     5305
Glu Gly Ile Val Leu Thr Lys Ser Gly Ser Gly Asn Asp Arg Thr Leu
1405                1410                1415
TCT TTA TCT GGT GCA GGC AAT GCA GCA ACA GAT GGC ATT AAA GTC TCT     5353
Ser Leu Ser Gly Ala Gly Asn Ala Ala Thr Asp Gly Ile Lys Val Ser
1420                1425                1430
GGC GTG AAA GCA GGG ACG GCA GAC ACC GAT GCG GTG AAT AAA GGT CAG     5401
Gly Val Lys Ala Gly Thr Ala Asp Thr Asp Ala Val Asn Lys Gly Gln
1435                1440                1445
TTA GAT AAA CTT TTT AAA GCG ATC AAT GAC GCA TTA GGC ACA ACA GAT     5449
Leu Asp Lys Leu Phe Lys Ala Ile Asn Asp Ala Leu Gly Thr Thr Asp
1450                1455                1460
TTA GCG GTA ACC AAA AAT CCA AAT CAA ACC TCT ATC TTT AAT CCG ATA     5497
Leu Ala Val Thr Lys Asn Pro Asn Gln Thr Ser Ile Phe Asn Pro Ile
1465                1470                1475                1480
AAC GGC ACG GCT CCA ACC ACC TTT AAA GAC GCG GTG GAT AAA TTA ACC     5545
Asn Gly Thr Ala Pro Thr Thr Phe Lys Asp Ala Val Asp Lys Leu Thr
1485                1490                1495
ACC GCT GTG AAT ACA GGT TGG GGA TCA AAG GTA GGT ATT TTG GCA ACA     5593
Thr Ala Val Asn Thr Gly Trp Gly Ser Lys Val Gly Ile Leu Ala Thr
1500                1505                1510
GGT ATT GAT GGT ATT GAT GCT GGG AAT AAG AAA ATT AGT AAT GTC GCC     5641
Gly Ile Asp Gly Ile Asp Ala Gly Asn Lys Lys Ile Ser Asn Val Ala
1515                1520                1525
GAT GGG GAT ATT TCT CCA ACC AGT GGT GAT GTA GTG ACA GGT CGT CAG     5689
Asp Gly Asp Ile Ser Pro Thr Ser Gly Asp Val Val Thr Gly Arg Gln
1530                1535                1540
CTC TAC GCC TTA ATG CAG AAA GGT ATT CGC GTG TAT GGT GAT GAA GTT     5737
Leu Tyr Ala Leu Met Gln Lys Gly Ile Arg Val Tyr Gly Asp Glu Val
1545                1550                1555                1560
AGT CCA ACG AAG ACT CAA ACA ACA GCA CCT ACA GCA TCT AGC ACT CAA     5785
Ser Pro Thr Lys Thr Gln Thr Thr Ala Pro Thr Ala Ser Ser Thr Gln
1565                1570                1575
GGT GGG GCG ACA ACG GCG AAT ACG GCG GGT GGT GTA GCA CCA GCA GGT     5833
Gly Gly Ala Thr Thr Ala Asn Thr Ala Gly Gly Val Ala Pro Ala Gly
1580                1585                1590
AAT GTA GCA ACG GGG GAT ATT GCG CCG ACA CAG CCA GCA TTG CCA GAG     5881
Asn Val Ala Thr Gly Asp Ile Ala Pro Thr Gln Pro Ala Leu Pro Glu
1595                1600                1605
ATG AAA ACG GCA TTG GTT GGT GAT CAC TTG GCT GTG CCG TTA GGT GGA     5929
Met Lys Thr Ala Leu Val Gly Asp His Leu Ala Val Pro Leu Gly Gly
1610                1615                1620
AGC CTC AAG ATT CAC GGA GAT CAT AAT GTG AAA ACA ACG ATT TCT GCG     5977
Ser Leu Lys Ile His Gly Asp His Asn Val Lys Thr Thr Ile Ser Ala
1625                1630                1635                1640
GGT AAT CAA GTG GGG ATT TCA TTA CAG CCA AAT ATT TCT ATT GAG AAT     6025
Gly Asn Gln Val Gly Ile Ser Leu Gln Pro Asn Ile Ser Ile Glu Asn
1645                1650                1655
AAC TTG GTA ATT GGT TCA AAT AAG CCT GAG AAG GCA AAA TTA GCC GCA     6073
Asn Leu Val Ile Gly Ser Asn Lys Pro Glu Lys Ala Lys Leu Ala Ala
1660                1665                1670
CAA GAA GGT AAT GCT TTG GTT ATC ACT AAC AAA GAT GAC GGG AAT GCG     6121
Gln Glu Gly Asn Ala Leu Val Ile Thr Asn Lys Asp Asp Gly Asn Ala
1675                1680                1685
GCG ATG GTC TTT AAT AAC GAG AAA AAT ATG CTT GTT CTC AGT GAT AAA     6169
Ala Met Val Phe Asn Asn Glu Lys Asn Met Leu Val Leu Ser Asp Lys
1690                1695                1700
AAG GCA AAA CCA AGA GCG GTT CTT GAT GGA CAA AAT GGG GCA TTA ACT     6217
Lys Ala Lys Pro Arg Ala Val Leu Asp Gly Gln Asn Gly Ala Leu Thr
1705                1710                1715                1720
TTA GTC GGC AAT GAT GAT TCT CAA GTC ACC CTT TCC TCT AAG AAA GGT     6265
Leu Val Gly Asn Asp Asp Ser Gln Val Thr Leu Ser Ser Lys Lys Gly
1725                1730                1735
AAA GAT ATT GAT GGA AAT GAT TTG AGC CGT CTC TCT GTG ACG ACT GAA     6313
Lys Asp Ile Asp Gly Asn Asp Leu Ser Arg Leu Ser Val Thr Thr Glu
1740                1745                1750
AGA ACA AAT GCT GAT GGG CAA CTT GAA AAA GTG GAA ACC TCA TTT GCT     6361
Arg Thr Asn Ala Asp Gly Gln Leu Glu Lys Val Glu Thr Ser Phe Ala
1755                1760                1765
ACA ATG GAT GAT GGC TTG AAG TTC AAA GCC GAC GGG GAT AAA GTG ATT     6409
Thr Met Asp Asp Gly Leu Lys Phe Lys Ala Asp Gly Asp Lys Val Ile
1770                1775                1780
AAT AAG AAA CTT AAT GAA ACC GTT GAA ATT GTT GGT GAT GAG AAT GTG     6457
Asn Lys Lys Leu Asn Glu Thr Val Glu Ile Val Gly Asp Glu Asn Val
1785                1790                1795                1800
ACA ACA TCT ATT ACT GAT GAT AAT AAG GTG AAA GTT TCA CTG AAT AAG     6505
Thr Thr Ser Ile Thr Asp Asp Asn Lys Val Lys Val Ser Leu Asn Lys
1805                1810                1815
AAA ATC GCG ATT GAT GAG GTT AAG ATT CCA AAT ACA GAT CCT GAT GCT     6553
Lys Ile Ala Ile Asp Glu Val Lys Ile Pro Asn Thr Asp Pro Asp Ala
1820                1825                1830
CAA AAG GGA GAT AGC ATT GTA ATC AAC AAT GGT GGA ATC CAC GCA GGT     6601
Gln Lys Gly Asp Ser Ile Val Ile Asn Asn Gly Gly Ile His Ala Gly
1835                1840                1845
AAT AAA GTG ATT ACT GGC GTT AAA GCG AGT GAT GAC CCA ACC AGT GCG     6649
Asn Lys Val Ile Thr Gly Val Lys Ala Ser Asp Asp Pro Thr Ser Ala
1850                1855                1860
GTG AAT CGA GGT CAA TTA AAT ACT GTG ATT GAT AAT GTT CAA AAT AAT     6697
Val Asn Arg Gly Gln Leu Asn Thr Val Ile Asp Asn Val Gln Asn Asn
1865                1870                1875                1880
TTC AAT CAA GTT AAT CAA CGT ATT GGC GAT TTA ACA CGG GAG TCG CGT     6745
Phe Asn Gln Val Asn Gln Arg Ile Gly Asp Leu Thr Arg Glu Ser Arg
1885                1890                1895
GCA GGT ATT GCA GGT GCA ATG GCG ACG GCA AGC CTA CAA AAT GTT GCT     6793
Ala Gly Ile Ala Gly Ala Met Ala Thr Ala Ser Leu Gln Asn Val Ala
1900                1905                1910
TTA CCA GGG AAA ACA ACG ATT TCC GTA GGT ACA GCA ACG TTC AAA GGG     6841
Leu Pro Gly Lys Thr Thr Ile Ser Val Gly Thr Ala Thr Phe Lys Gly
1915                1920                1925
GAG AAT GCT GTT GCA ATA GGG ATG TCT AGA CTC TCT GAT AAT GGA AAA     6889
Glu Asn Ala Val Ala Ile Gly Met Ser Arg Leu Ser Asp Asn Gly Lys
1930                1935                1940
GTA GGT ATC CGT TTA TCT GGT ATG AGT ACA AGT AAC GGA GAT AAA GGG     6937
Val Gly Ile Arg Leu Ser Gly Met Ser Thr Ser Asn Gly Asp Lys Gly
1945                1950                1955                1960
GCA GCA ATG AGT GTT GGA TTT ACC TTT TAGCCTTAAT CCATAAATAA GCAAA     6991
Ala Ala Met Ser Val Gly Phe Thr Phe
1965
GCGAATCACC TTTGATTCGC TTTTTTTATC AGATTATGTG CCGTAAAACT CCGTCCTT     7051
GGGCGGAGAT ATAAGGCACA AACGGCGTAA GCCGTTTCAA ACCTAACTAA TCAGGTGT     7111
GTTGTTGCTC AATGTATTGG CGAATAATGG AAATTGGAGT GCCACCACAA CTCCCTGC     7171
AATAAGACGG AGACCAAAGC TGATTACCCC AAAGTTTTTT GCGGATGTTC GAGTAGTT     7231
TCTTCCTAAT CATTCGGCTT GATACACCTT TTAAACTGTT TACAAGTGTA GATACAGC     7291
CTTTCGGTGG ATATTCCACA AGTAAATGAA CATGATCGTC TTCACCGTCA AATTCAAC     7351
ATTTTGCTTT AAAATCATTG CAGACGCTTT CAAAAATCAA TTTGAGTTCG TCTAAAAT     7411
CTTTCGTAAA AACATCACAG CGATATTTTG TTACAAAGAC TAAGTGAACA TGCATATT     7471
AAACACAATG TCTAC                                                    7486
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2042 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Asn Lys Val Phe Lys Ile Lys Tyr Ser Val Val Lys Gln Glu Met
1               5                  10                  15
Ile Val Val Ser Glu Leu Ala Asn Asn Lys Asp Lys Thr Ala Ser Gln
20                  25                  30
Lys Asn Thr His Asn Thr Ala Phe Phe Gln Pro Leu Phe Thr Lys Cys
35                  40                  45
Thr Tyr Leu Ala Leu Leu Ile Asn Ile Ala Leu Gly Ala Ser Leu Phe
50                  55                  60
Pro Gln Leu Ala Asn Ala Lys Trp Leu Glu Val Tyr Ser Ser Ser Val
65                  70                  75                  80
Lys Leu Ser Thr Val Ser Ala Gln Ser Asn Ser Val Asn Leu Asn Pro
85                  90                  95
Ser Gly Ala Glu Ser Val Gly Thr Asn Ser Pro Gln Gly Val Ala Ile
100                 105                 110
Gly Tyr Gly Ala Thr Asn Asp Arg Ser Ala Thr Gly Ala Ile Ala Leu
115                 120                 125
Gly Val Gly Val Lys Asn Glu Thr Leu Ala Lys Asp Ser Ile Ala Ile
130                 135                 140
Gly Tyr Gly Ala Lys Asn Glu Ser Thr Ala Pro Ser Ser Val Thr Ile
145                 150                 155                 160
Gly Lys Gln Ala Ile Asn Arg Phe Glu Lys Ser Ile Val Met Gly Leu
165                 170                 175
Asn Ala Tyr Thr Gln Leu Asp Pro Arg Gly Thr Ser Lys Glu Thr Arg
180                 185                 190
Gln Gly Ser Val Val Ile Gly Glu Asn Ala Lys Ser Ala Gly Asn Gln
195                 200                 205
Ser Val Ser Leu Gly Gln Asn Ser Trp Ser Lys Thr Asn Ser Ile Ser
210                 215                 220
Ile Gly Ala Gly Thr Phe Ala Glu Gly Lys Ser Ser Ile Ala Ile Gly
225                 230                 235                 240
Thr Asp Lys Ile Ser Gly Thr Lys Tyr Asn Asp Lys Leu Pro Ala Thr
245                 250                 255
Ala Trp Asn Gly Thr Gly Thr Val Pro Lys Asn Ser Ile Trp Asp Ile
260                 265                 270
Phe Ser Glu Leu Tyr Met Gly Lys Gln Thr Asn Gly Arg Asp Tyr Asp
275                 280                 285
Thr Thr Thr Arg Asp Pro Asn Lys Pro Glu Ala Phe Tyr Lys Phe Ser
290                 295                 300
Asp Phe Lys Gly Lys Tyr Val Asn Thr Pro Thr Ala Ser Pro Thr Tyr
305                 310                 315                 320
Ala Gly Lys Leu Gly Ala Ile Ala Leu Gly Ser Arg Thr Ile Ala Ala
325                 330                 335
Gly Glu Met Ser Thr Ala Val Gly Ser Leu Ala Phe Ala Leu Ala Asp
340                 345                 350
Arg Ser Thr Ala Met Gly Leu Arg Ser Phe Val Ala Lys Asp Ala Val
355                 360                 365
Gly Gly Thr Ala Ile Gly Glu Glu Ser Arg Thr Phe Ala Lys Asp Ser
370                 375                 380
Val Ala Ile Gly Asn Lys Thr Glu Ala Ser Asn Ala Gly Ser Met Ala
385                 390                 395                 400
Tyr Gly Tyr Lys Ala Lys Ala Val Gly Ala Gly Ala Ile Ala Ile Gly
405                 410                 415
Thr Glu Val Ala Ala Gly Ala Lys Phe Asn Ser His Gln Thr Gly Asn
420                 425                 430
Leu Leu Gln Asp Asn Asn Ala Tyr Ala Thr Leu Lys Asn Ala Asp Lys
435                 440                 445
Ser Asp Asp Thr Lys Thr Gly Asn Ala Ile Thr Val Phe Thr Gln Ser
450                 455                 460
Phe Asp Asn Met Leu Thr Asn Gly Leu Pro Leu Val Ser Glu Asn Glu
465                 470                 475                 480
Thr Tyr Leu Thr Thr Ser Ala Gly Ala Ile Lys Lys Thr Ala Thr Thr
485                 490                 495
Asp Ser Ser Ala Gly Gly Gly Lys Asn Ala Ile Ala Ile Gly Ser Lys
500                 505                 510
Thr Phe Ala Ser Lys Ala Asn Ser Val Ala Leu Gly Ser Tyr Ala Leu
515                 520                 525
Ala Asp Ala Gln Asn Ala Phe Ala Leu Gly Ser Tyr Ser Phe Val Glu
530                 535                 540
Ser Ser Ala Thr Asn Thr Ile Thr Ile Gly Val Gly Ser Tyr Ala Lys
545                 550                 555                 560
Gly Lys Asn Ser Phe Leu Gly Gly Thr Trp Ala Ser Thr Leu Ser Asp
565                 570                 575
Arg Thr Val Val Leu Gly Asn Ser Thr Ser Ile Ser Ser Gly Ser Gln
580                 585                 590
Asn Ala Leu Ala Ile Gly Val Asn Val Phe Ile Gly Asn Asp Ser Ala
595                 600                 605
Ser Ser Leu Ala Leu Gly Met Gly Ser Thr Ile Ala Lys Ser Ala Lys
610                 615                 620
Ser Pro Asp Ser Leu Ala Ile Gly Lys Glu Ala Arg Ile Asp Ala Lys
625                 630                 635                 640
Asp Thr Asp Asn Gly Thr Leu Tyr Gln Pro Gln Val Tyr Asp Glu Thr
645                 650                 655
Thr Arg Ala Phe Arg Asn Phe Asn Glu Ser Ser Asp Tyr Met Arg Gln
660                 665                 670
Ala Met Ala Leu Gly Phe Asn Ala Lys Val Ser Arg Gly Val Gly Lys
675                 680                 685
Met Glu Thr Gly Ile Asn Ser Met Ala Ile Gly Ala Tyr Ala Gln Ala
690                 695                 700
Thr Leu Gln Asn Ser Thr Ala Leu Gly Val Gly Ser Lys Thr Asp Tyr
705                 710                 715                 720
Thr Trp Glu Gln Leu Glu Thr Asp Pro Trp Val Ser Glu Gly Ala Ile
725                 730                 735
Ser Ile Pro Thr Ser Gly Lys Thr Gly Val Ile Ser Val Gly Ser Lys
740                 745                 750
Gly Ser Glu Arg Arg Ile Val Asn Leu Ala Ser Gly Ser Ser Asp Thr
755                 760                 765
Asp Ala Val Asn Val Ala Gln Leu Lys Thr Val Glu Glu Arg Phe Leu
770                 775                 780
Ser Glu Ile Asn Leu Leu Gln Asn Gly Gly Gly Val Lys Tyr Leu Ser
785                 790                 795                 800
Val Glu Lys Thr Asn Ile Asn Gly Gln Ser Gly Arg Val Ala Ser Gln
805                 810                 815
Ile Arg Lys Gly Glu Asn Tyr Glu Arg Tyr Val Lys Leu Lys Thr Gln
820                 825                 830
Leu Leu Tyr Leu Asp Ala Arg Gly Lys Leu Asn Gly Glu Lys Phe Asp
835                 840                 845
Gln Asn Ser Leu Asn Lys Ile Arg Ala Val Val Gln Glu Leu Glu Ala
850                 855                 860
Glu Tyr Ser Gly Glu Leu Lys Thr Thr Ala Ser Ala Leu Asn Gln Val
865                 870                 875                 880
Ala Thr Gln Leu Glu Gln Glu Val Thr Thr Asn Asn Phe Asp Lys Phe
885                 890                 895
Asn Gln Tyr Lys Thr Gln Ile Glu Asn Ala Ser Asn Ala Asp Ser Ala
900                 905                 910
Arg Asn Val Gly Gly Leu Thr Pro Gln Ala Ile Ala Gln Leu Lys Ala
915                 920                 925
Asn Asn Asn Tyr Leu Asn Asp Gly Ala Lys Gly Gln Asp Ser Ile Ala
930                 935                 940
Phe Gly Trp Gln Ala Lys Thr Ser Gly Ala Asn Asn Gly Leu Ala Gly
945                 950                 955                 960
Lys Gln Ala Ile Ala Ile Gly Phe Gln Ala Asn Ser Ser Ala Glu Asn
965                 970                 975
Ala Ile Ser Ile Gly Thr Asn Ser Asp Thr Ser Met Thr Gly Ala Val
980                 985                 990
Ala Ile Gly Lys Gly Ala Thr Val Thr Ala Gly Gly Lys Pro Ser Ile
995                1000                1005
Ala Leu Gly Gln Asp Ser Thr Val Ala Asn Ser Ala Ile Ser Arg Thr
1010                1015                1020
Ser Ser Pro Met Ile Asn Gly Leu Ile Phe Asn Asn Phe Ala Gly Ser
1025               1030                1035                1040
Pro Glu Thr Leu Gly Val Leu Ser Ile Gly Thr Ala Gly Arg Glu Arg
1045                1050                1055
Lys Ile Val Asn Val Ala Ala Gly Asp Val Ser Gln Ala Ser Thr Glu
1060                1065                1070
Ala Ile Asn Gly Ser Gln Leu Tyr Ala Thr Asn Phe Met Leu Ser Lys
1075                1080                1085
Val Ala Gln Ser Val Lys Ser Asn Phe Gly Gly Asn Val Asn Leu Gly
1090                1095                1100
Thr Asp Gly Thr Ile Thr Phe Thr Asn Ile Gly Gly Thr Gly Gln Ala
1105               1110                1115                1120
Thr Ile His Asp Ala Ile Asn Asn Val Leu Thr Lys Gly Ile Tyr Leu
1125                1130                1135
Lys Ala Asp Gln Asn Asp Pro Thr Gly Asn Gln Gly Gln Lys Val Glu
1140                1145                1150
Leu Gly Asn Ala Ile Thr Leu Ser Ala Thr Asn Gln Trp Ala Asn Asn
1155                1160                1165
Gly Val Asn Tyr Lys Thr Asn Asn Leu Thr Thr Tyr Asn Ser Gln Asn
1170                1175                1180
Gly Thr Ile Leu Phe Gly Met Arg Glu Asp Pro Ser Val Lys Gln Ile
1185               1190                1195                1200
Thr Ala Gly Thr Tyr Asn Thr Thr Gly Asp Ala Asn Asn Lys Asn Gln
1205                1210                1215
Leu Asn Asn Thr Leu Gln Gln Thr Thr Leu Glu Ala Thr Gly Ile Thr
1220                1225                1230
Ser Ser Val Gly Ser Thr Asn Tyr Ala Gly Phe Ser Leu Gly Ala Asp
1235                1240                1245
Ser Val Thr Phe Ser Lys Gly Gly Ala Gly Thr Val Lys Leu Ser Gly
1250                1255                1260
Val Ser Asp Ala Thr Ala Asp Thr Asp Ala Ala Thr Leu Lys Gln Val
1265               1270                1275                1280
Lys Glu Tyr Arg Thr Thr Leu Val Gly Asp Asn Asp Ile Thr Ala Ala
1285                1290                1295
Asp Arg Ser Gly Gly Thr Ser Asn Gly Ile Thr Tyr Asn Leu Ser Leu
1300                1305                1310
Asn Lys Gly Thr Val Ser Ala Thr Glu Glu Lys Val Val Ser Gly Lys
1315                1320                1325
Thr Val Tyr Glu Ala Ile Arg Asn Ala Ile Thr Gly Asn Ile Phe Thr
1330                1335                1340
Ile Gly Leu Asp Asp Thr Thr Leu Asn Lys Ile Asn Asn Pro Ala Asp
1345               1350                1355                1360
Gln Asp Leu Ser Asn Leu Ser Glu Ser Gly Lys Asn Ala Ile Thr Gly
1365                1370                1375
Leu Val Asp Val Val Lys Lys Thr Asn Ser Pro Ile Thr Val Glu Pro
1380                1385                1390
Ser Thr Asp Ser Asn Lys Lys Lys Thr Phe Thr Val Gly Val Asp Phe
1395                1400                1405
Thr Asp Thr Ile Thr Glu Gly Asp Ala Thr Asp Asp Lys Lys Leu Thr
1410                1415                1420
Thr Ser Lys Ser Val Glu Ser Tyr Val Thr Asn Lys Leu Ala Asn Phe
1425               1430                1435                1440
Ser Thr Asp Ile Leu Leu Ser Asp Gly Arg Ser Gly Asn Ala Thr Thr
1445                1450                1455
Ala Asn Asp Gly Val Gly Lys Arg Arg Leu Ser Asp Gly Phe Thr Ile
1460                1465                1470
Lys Ser Glu Asn Phe Thr Leu Gly Ser Lys Gln Tyr Asn Gly Ser Asp
1475                1480                1485
Ser Leu Gly Val Met Tyr Asp Asp Gln Asn Gly Val Phe Lys Leu Ser
1490                1495                1500
Leu Asn Met Thr Ala Leu Thr Thr Ser Leu Ala Asn Thr Phe Ala Lys
1505               1510                1515                1520
Leu Asp Ala Ser Asn Leu Thr Asp Asp Ser Asn Lys Glu Lys Trp Arg
1525                1530                1535
Thr Ala Leu Asn Val Tyr Ser Lys Thr Glu Val Asp Ala Glu Ile Gln
1540                1545                1550
Lys Ser Lys Val Thr Leu Thr Pro Asp Ser Gly Leu Ile Phe Ala Thr
1555                1560                1565
Lys Gln Ala Gly Ser Gly Asn Asn Ala Gly Ile Asp Ala Gly Asn Lys
1570                1575                1580
Lys Ile Ser Asn Val Ala Asp Gly Asp Ile Ser Pro Thr Ser Gly Asp
1585               1590                1595                1600
Val Val Thr Gly Arg Gln Leu Tyr Ala Leu Met Gln Lys Gly Ile Arg
1605                1610                1615
Val Tyr Gly Asp Glu Val Ser Pro Thr Lys Thr Gln Thr Thr Ala Pro
1620                1625                1630
Thr Asn Ala Asn Pro Thr Ala Thr Thr Ala Pro Thr Ala Ser Ser Thr
1635                1640                1645
Gln Gly Trp Ala Thr Thr Ala Asn Thr Ala Gly Gly Val Ala Pro Ala
1650                1655                1660
Gly Asn Val Ala Thr Gly Asp Ile Ala Pro Thr Gln Pro Thr Leu Pro
1665               1670                1675                1680
Glu Met Asn Thr Ala Leu Val Asp Asp His Leu Ala Val Pro Leu Gly
1685                1690                1695
Gly Ser Leu Lys Ile His Gly Asp His Asn Val Lys Thr Thr Ile Ser
1700                1705                1710
Ala Asp Asn Gln Val Gly Ile Ser Leu Gln Pro Asn Ile Ser Ile Glu
1715                1720                1725
Asn Asn Leu Val Ile Gly Ser Asn Asp Pro Glu Lys Ala Lys Leu Ala
1730                1735                1740
Ala Gln Glu Gly Asn Ala Leu Val Ile Thr Asn Lys Asp Asp Gly Asn
1745               1750                1755                1760
Ala Ala Met Val Phe Asn Asn Glu Lys Asn Met Leu Val Leu Ser Asp
1765                1770                1775
Lys Glu Ala Lys Pro Arg Val Leu Leu Asp Gly Gln Asn Gly Ala Leu
1780                1785                1790
Thr Leu Val Gly Asn Asp Asp Ser Gln Val Thr Leu Ser Ser Lys Lys
1795                1800                1805
Gly Lys Asp Ile Asp Gly Asn Asp Leu Ser Arg Leu Ser Val Thr Thr
1810                1815                1820
Glu Arg Thr Asn Ala Asp Gly Gln Leu Glu Lys Val Glu Thr Ser Phe
1825               1830                1835                1840
Ala Thr Met Asp Asp Gly Leu Lys Phe Lys Ala Asp Gly Asp Lys Val
1845                1850                1855
Ile Asn Lys Lys Leu Asn Glu Thr Val Glu Ile Val Gly Asp Glu Asn
1860                1865                1870
Val Thr Thr Ser Ile Thr Asp Asp Asn Lys Val Lys Val Ser Leu Asn
1875                1880                1885
Lys Lys Ile Ala Ile Asp Glu Val Lys Ile Pro Asn Thr Asp Pro Asp
1890                1895                1900
Ala Gln Lys Gly Asp Ser Ile Val Ile Asn Asn Gly Gly Ile His Ala
1905               1910                1915                1920
Gly Asn Lys Val Ile Thr Gly Val Lys Ala Ser Asp Asp Pro Thr Ser
1925                1930                1935
Ala Val Asn Arg Gly Gln Leu Asn Thr Val Ile Asp Asn Val Gln Asn
1940                1945                1950
Asn Phe Asn Gln Val Asn Gln Arg Ile Gly Asp Leu Thr Arg Glu Ser
1955                1960                1965
Arg Ala Gly Ile Ala Gly Ala Met Ala Thr Ala Ser Leu Gln Asn Val
1970                1975                1980
Ala Leu Pro Gly Lys Thr Thr Ile Ser Val Gly Thr Ala Thr Phe Lys
1985               1990                1995                2000
Gly Glu Asn Ala Val Ala Ile Gly Met Ser Arg Leu Ser Asp Asn Gly
2005                2010                2015
Lys Val Gly Ile Arg Leu Ser Gly Met Ser Thr Ser Asn Gly Asp Lys
2020                2025                2030
Gly Ala Ala Met Ser Val Gly Phe Ser Phe
2035                2040
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2039 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Met Asn Lys Val Phe Lys Ile Lys Tyr Ser Val Val Lys Gln Glu Met
1               5                  10                  15
Ile Val Val Ser Glu Leu Ala Asn Asn Lys Asp Lys Thr Ala Ser Gln
20                  25                  30
Lys Asn Thr His Asn Thr Ala Phe Phe Gln Pro Leu Phe Thr Lys Cys
35                  40                  45
Thr Tyr Leu Ala Leu Leu Ile Asn Ile Ala Leu Gly Thr Ser Leu Phe
50                  55                  60
Pro Gln Leu Ala Asn Ala Lys Phe Leu Glu Val Tyr Asn Ser Ser Val
65                  70                  75                  80
Lys Leu Gln His Val Asn Ser Gly Val Pro Ser Asp Ser Val Asn Leu
85                  90                  95
Asn Pro Ser Gly Gly Glu Asn Val Gly Met Asn Ser Asn Gln Gly Val
100                 105                 110
Ala Ile Gly Arg Gly Ala Val Asn Asn Tyr Ser Ala Thr Gly Ser Ile
115                 120                 125
Ala Ile Gly Gln Gly Ala Lys Asn Asp Asn Trp Ala Thr Arg Ser Ile
130                 135                 140
Ala Ile Gly Gln Gly Ala Lys Asn Glu Ser Ile Ala Ser Asp Ser Val
145                 150                 155                 160
Ala Ile Ser Asn Ala Ile Asn Arg Phe Lys Lys Ser Ile Val Ile Gly
165                 170                 175
Leu Asn Thr Tyr Thr Gln Leu Asp Pro Arg Arg Ala Pro Glu Ser Arg
180                 185                 190
Gln Gly Ser Val Val Ile Gly Glu Asn Ala Lys Ser Ala Gly Asn Gln
195                 200                 205
Ser Val Ser Leu Gly Gln Asn Ala Trp Ser Lys Thr Asn Ser Ile Ser
210                 215                 220
Ile Gly Ala Gly Thr Phe Ala Glu Gly Lys Ser Thr Ile Ala Ile Gly
225                 230                 235                 240
Thr Asp Lys Ile Leu Gly Thr Asn Tyr Asn Asp Lys Leu Pro Ala Pro
245                 250                 255
Ser Trp Asp Gly Arg Thr Gly Lys Ala Pro Thr Asn Ser Ile Trp Asp
260                 265                 270
Ile Phe Ser Glu Leu Tyr Met Gly Lys Lys Thr Asn Gly Thr Asp Tyr
275                 280                 285
Asp Ala Lys Lys Asn Asp Arg Asp Pro Asn Lys Pro Glu Ala Phe Tyr
290                 295                 300
Thr Tyr Ser Asp Phe Lys Ser Arg Tyr Val Asn Asn Pro Ser Thr Ser
305                 310                 315                 320
Pro Thr Tyr Ala Ala Lys Leu Gly Ala Ile Ala Leu Gly Ser Arg Thr
325                 330                 335
Ile Ala Ala Gly Glu Met Ser Thr Ala Val Gly Ser Leu Ala Phe Ala
340                 345                 350
Leu Ala Asp Lys Ser Thr Ala Met Gly Leu Arg Ser Phe Val Ala Lys
355                 360                 365
Asp Ala Val Gly Gly Thr Ala Ile Gly Glu Glu Ser Arg Thr Phe Ala
370                 375                 380
Lys Asp Ser Val Ala Ile Gly Asn Lys Thr Glu Ala Ser Asn Ala Gly
385                 390                 395                 400
Ser Met Ala Tyr Gly Tyr Lys Ala Lys Ala Val Gly Ala Gly Ala Ile
405                 410                 415
Ala Ile Gly Ala Glu Val Ala Ala Gly Ala Glu Phe Asp Ser Ser Gln
420                 425                 430
Ala Gly Asn Leu Leu Leu Asn Arg Gly Ala Tyr Ala Thr Leu Lys Ser
435                 440                 445
Ala Asp Lys Ser Asp Asp Ile Lys Ala Gly Asp Ala Ile Asn Val Phe
450                 455                 460
Thr Gln Phe Phe Asp Asn Met Leu Thr Gln Gly Ser His Leu Thr Tyr
465                 470                 475                 480
Glu Asn Thr Thr Tyr Leu Thr Thr Ser Ala Gly Asp Ile Lys Lys Thr
485                 490                 495
Leu Ala Ala Val Gly Asp Gly Gly Lys Asn Ala Ile Ala Ile Gly Asn
500                 505                 510
Lys Thr Phe Ala Ser Lys Ala Asn Ser Val Ala Leu Gly Ser Tyr Ala
515                 520                 525
Leu Ala Ser Ala Gln Asn Ala Phe Ala Leu Gly Ser Tyr Ser Leu Val
530                 535                 540
Ser Pro Leu Ala Ala Asn Thr Ile Val Ile Gly Val Gly Gly Tyr Ala
545                 550                 555                 560
Thr Gly Ser Asn Ser Phe Val Gly Gly Ser Trp Val Ser Thr Leu Ser
565                 570                 575
Ala Arg Thr Val Val Leu Gly Tyr Ser Ala Ser Ile Ser Ser Asp Ser
580                 585                 590
His Asp Ser Leu Ala Met Gly Val Asn Ala Phe Ile Gly Asn Gly Ser
595                 600                 605
Asn Ser Ser Leu Ala Leu Gly Thr Gly Ser Thr Ile Ala Lys Asn Ala
610                 615                 620
Lys Ser Pro Asp Ser Leu Ala Ile Gly Lys Asp Ser Arg Ile Asp Ala
625                 630                 635                 640
Lys Asp Thr Asp Asn Gly Val Leu Tyr Thr Pro Gln Val Tyr Asp Glu
645                 650                 655
Thr Thr Arg Ala Phe Arg Thr Phe Asp Glu Asn Lys Asp Tyr Met Arg
660                 665                 670
Gln Ala Met Ala Leu Gly Phe Asn Ala Lys Val Ser Arg Gly Lys Gly
675                 680                 685
Lys Met Glu Thr Gly Ile Asn Ser Met Ala Ile Gly Ala Arg Ser Gln
690                 695                 700
Ala Thr Leu Gln Asn Ser Thr Ala Leu Gly Val Asn Ala Lys Thr Asp
705                 710                 715                 720
Tyr Thr Trp Glu Gln Leu Glu Ala Asp Pro Trp Val Ser Lys Gly Ala
725                 730                 735
Ile Ser Ile Pro Thr Ser Gly Lys Ile Gly Val Ile Ser Val Gly Ser
740                 745                 750
Lys Gly Ser Glu Arg Arg Ile Val Asn Val Ala Ser Gly Ser Leu Asp
755                 760                 765
Thr Asp Ala Val Asn Val Ala Gln Leu Lys Thr Ile Glu Glu Arg Phe
770                 775                 780
Gln Ser Glu Ile Asp Leu Leu Gln Asn Gly Gly Gly Val Gln Tyr Leu
785                 790                 795                 800
Ser Val Glu Lys Thr Asn Ile Asn Gly Glu Ala Gly Arg Val Ala Ser
805                 810                 815
Gln Ile Arg Lys Gly Glu Ser Tyr Lys Arg Tyr Val Lys Leu Lys Thr
820                 825                 830
Gln Leu Leu Tyr Leu Asp Ala Arg Lys Lys Leu Asn Gly Glu Lys Phe
835                 840                 845
Asp Gln Thr Ser Leu Asp Lys Ile Ser Lys Ala Val Gln Glu Leu Glu
850                 855                 860
Ala Glu Tyr Ser Gly Glu Leu Lys Thr Thr Ala Ser Glu Leu Asn Arg
865                 870                 875                 880
Val Ala Met Gln Leu Asn Ala Glu Thr Thr Val Asn Asp Phe Gly Lys
885                 890                 895
Phe Asn Gln Tyr Lys Thr Gln Ile Glu Asn Ala Thr Asn Ala Asp Ser
900                 905                 910
Glu Lys Asn Val Gly Gly Leu Ser Pro Gln Val Ile Ala Gln Leu Lys
915                 920                 925
Ala Asn Asn Asn Tyr Leu Asn Asp Gly Ala Lys Gly Gln Asp Ser Ile
930                 935                 940
Ala Phe Gly Trp Gln Ala Lys Thr Ser Glu Ala Asn Asn Gly Leu Ala
945                 950                 955                 960
Gly Lys Gln Ala Ile Ala Ile Gly Phe Gln Ala Asn Ser Ser Ala Glu
965                 970                 975
Asn Ala Ile Ser Ile Gly Thr Asn Ser Asp Thr Ser Met Thr Gly Ala
980                 985                 990
Val Ala Ile Gly Lys Gly Ala Thr Val Thr Ala Gly Gly Lys Pro Ser
995                1000                1005
Ile Ala Leu Gly Gln Asp Ser Thr Val Ala Asn Ser Ala Ile Ser Arg
1010                1015                1020
Thr Ser Ser Val Met Ile Asn Gly Leu Thr Phe Asn Asn Phe Ala Gly
1025               1030                1035                1040
Ser Pro Glu Thr Leu Gly Val Leu Ser Ile Gly Thr Ala Gly Lys Glu
1045                1050                1055
Arg Lys Ile Val Asn Val Ala Ala Gly Asp Ile Ser Gln Thr Ser Thr
1060                1065                1070
Glu Ala Ile Asn Gly Ser Gln Leu Tyr Ala Thr Asn Phe Met Leu Asn
1075                1080                1085
Lys Leu Ala Gln Ser Val Lys Thr Asn Phe Gly Gly Asn Ala Asn Leu
1090                1095                1100
Ala Thr Asp Gly Thr Ile Thr Phe Thr Asn Ile Gly Gly Thr Gly Gln
1105               1110                1115                1120
Asp Thr Ile His Asp Ala Ile Asn Asn Val Leu Thr Lys Leu Ile Ser
1125                1130                1135
Leu Ser Ala Thr Glu Glu Glu Glu Val Val Ser Gly Glu Ala Val Tyr
1140                1145                1150
Asp Ala Leu Lys Gly Ala Lys Pro Thr Val Ser Ala Glu Ala Asn Lys
1155                1160                1165
Gly Ile Thr Gly Leu Val Asp Val Val Lys Lys Ala Asn Ser Pro Ile
1170                1175                1180
Thr Val Glu Pro Ser Thr Asp Asn Asn Lys Lys Lys Thr Phe Thr Val
1185               1190                1195                1200
Gly Leu Met Lys Asp Ile Glu Gly Val Asn Ser Ile Thr Phe Asp Lys
1205                1210                1215
Ser Gly Gln Asp Leu Asn Gln Val Thr Gly Arg Met Ser Ser Ala Gly
1220                1225                1230
Leu Thr Phe Lys Lys Gly Asp Thr Thr Asn Gly Ser Thr Thr Thr Phe
1235                1240                1245
Ala Glu Asp Gly Leu Thr Ile Asp Ser Thr Thr Asn Ser Ala Gln Thr
1250                1255                1260
Asn Leu Val Lys Val Ser Arg Asp Gly Phe Ser Val Lys Asn Gly Ser
1265               1270                1275                1280
Asp Glu Ser Lys Leu Ala Ser Thr Lys Leu Ser Ile Gly Ala Glu Asn
1285                1290                1295
Ala Glu His Val Glu Val Thr Lys Ser Gly Ile Ala Leu Lys Ala Asp
1300                1305                1310
Asn Thr Ser Asp Lys Ser Ser Ile Thr Leu Ala Gln Asp Ala Ile Thr
1315                1320                1325
Leu Ala Gly Asn Ala Thr Gly Thr Ala Ile Lys Leu Thr Gly Val Ala
1330                1335                1340
Asp Gly Asn Ile Thr Val Asn Ser Lys Asp Ala Val Asn Gly Gly Gln
1345               1350                1355                1360
Leu Arg Thr Leu Leu Gly Val Asp Ser Gly Ala Lys Ile Gly Gly Thr
1365                1370                1375
Glu Lys Thr Thr Ile Ser Glu Ala Ile Ser Asp Val Lys Gln Ala Leu
1380                1385                1390
Thr Asp Ala Thr Leu Ala Tyr Lys Ala Asp Asn Lys Asn Gly Lys Thr
1395                1400                1405
Val Lys Leu Thr Asp Gly Leu Asn Phe Thr Ser Thr Thr Asn Ile Asp
1410                1415                1420
Ala Ser Val Glu Asp Asn Gly Val Val Lys Phe Thr Leu Lys Asp Lys
1425               1430                1435                1440
Leu Thr Gly Leu Lys Thr Ile Ala Thr Glu Ser Leu Asn Ala Ser Gln
1445                1450                1455
Asn Ile Ile Ala Gly Gly Thr Val Thr Val Gly Gly Glu Thr Glu Gly
1460                1465                1470
Ile Val Leu Thr Lys Ser Gly Ser Gly Asn Asp Arg Thr Leu Ser Leu
1475                1480                1485
Ser Gly Ala Gly Asn Ala Ala Thr Asp Gly Ile Lys Val Ser Gly Val
1490                1495                1500
Lys Ala Gly Thr Ala Asp Thr Asp Ala Val Asn Lys Gly Gln Leu Asp
1505               1510                1515                1520
Lys Leu Phe Lys Ala Ile Asn Asp Ala Leu Gly Thr Thr Asp Leu Ala
1525                1530                1535
Val Thr Lys Asn Pro Asn Gln Thr Ser Ile Phe Asn Pro Ile Asn Gly
1540                1545                1550
Thr Ala Pro Thr Thr Phe Lys Asp Ala Val Asp Lys Leu Thr Thr Ala
1555                1560                1565
Val Asn Thr Gly Trp Gly Ser Lys Val Gly Ile Leu Ala Thr Gly Ile
1570                1575                1580
Asp Gly Ile Asp Ala Gly Asn Lys Lys Ile Ser Asn Val Ala Asp Gly
1585               1590                1595                1600
Asp Ile Ser Pro Thr Ser Gly Asp Val Val Thr Gly Arg Gln Leu Tyr
1605                1610                1615
Ala Leu Met Gln Lys Gly Ile Arg Val Tyr Gly Asp Glu Val Ser Pro
1620                1625                1630
Thr Lys Thr Gln Thr Thr Ala Pro Thr Ala Ser Ser Thr Gln Gly Gly
1635                1640                1645
Ala Thr Thr Ala Asn Thr Ala Gly Gly Val Ala Pro Ala Gly Asn Val
1650                1655                1660
Ala Thr Gly Asp Ile Ala Pro Thr Gln Pro Ala Leu Pro Glu Met Lys
1665               1670                1675                1680
Thr Ala Leu Val Gly Asp His Leu Ala Val Pro Leu Gly Gly Ser Leu
1685                1690                1695
Lys Ile His Gly Asp His Asn Val Lys Thr Thr Ile Ser Ala Gly Asn
1700                1705                1710
Gln Val Gly Ile Ser Leu Gln Pro Asn Ile Ser Ile Glu Asn Asn Leu
1715                1720                1725
Val Ile Gly Ser Asn Lys Pro Glu Lys Ala Lys Leu Ala Ala Gln Glu
1730                1735                1740
Gly Asn Ala Leu Val Ile Thr Asn Lys Asp Asp Gly Asn Ala Ala Met
1745               1750                1755                1760
Val Phe Asn Asn Glu Lys Asn Met Leu Val Leu Ser Asp Lys Lys Ala
1765                1770                1775
Lys Pro Arg Ala Val Leu Asp Gly Gln Asn Gly Ala Leu Thr Leu Val
1780                1785                1790
Gly Asn Asp Asp Ser Gln Val Thr Leu Ser Ser Lys Lys Gly Lys Asp
1795                1800                1805
Ile Asp Gly Asn Asp Leu Ser Arg Leu Ser Val Thr Thr Glu Arg Thr
1810                1815                1820
Asn Ala Asp Gly Gln Leu Glu Lys Val Glu Thr Ser Phe Ala Thr Met
1825               1830                1835                1840
Asp Asp Gly Leu Lys Phe Lys Ala Asp Gly Asp Lys Val Ile Asn Lys
1845                1850                1855
Lys Leu Asn Glu Thr Val Glu Ile Val Gly Asp Glu Asn Val Thr Thr
1860                1865                1870
Ser Ile Thr Asp Asp Asn Lys Val Lys Val Ser Leu Asn Lys Lys Ile
1875                1880                1885
Ala Ile Asp Glu Val Lys Ile Pro Asn Thr Asp Pro Asp Ala Gln Lys
1890                1895                1900
Gly Asp Ser Ile Val Ile Asn Asn Gly Gly Ile His Ala Gly Asn Lys
1905               1910                1915                1920
Val Ile Thr Gly Val Lys Ala Ser Asp Asp Pro Thr Ser Ala Val Asn
1925                1930                1935
Arg Gly Gln Leu Asn Thr Val Ile Asp Asn Val Gln Asn Asn Phe Asn
1940                1945                1950
Gln Val Asn Gln Arg Ile Gly Asp Leu Thr Arg Glu Ser Arg Ala Gly
1955                1960                1965
Ile Ala Gly Ala Met Ala Thr Ala Ser Leu Gln Asn Val Ala Leu Pro
1970                1975                1980
Gly Lys Thr Thr Ile Ser Val Gly Thr Ala Thr Phe Lys Gly Glu Asn
1985               1990                1995                2000
Ala Val Ala Ile Gly Met Ser Arg Leu Ser Asp Asn Gly Lys Val Gly
2005                2010                2015
Ile Arg Leu Ser Gly Met Ser Thr Ser Asn Gly Asp Lys Gly Ala Ala
2020                2025                2030
Met Ser Val Gly Phe Thr Phe
2035
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Leu Phe Ser Lys Ile Ser Asp Lys Lys Asn Leu Phe Phe Phe Ile
1               5                  10                  15
Tyr Ser Ser Ile Lys Arg Lys Phe Ile Met Lys Lys Thr Leu Ile Ala
20                  25                  30
Leu Ala Val Ile Thr Met Phe Ser Ser Ala Ala Asn Ala Ala Val Ile
35                  40                  45
Tyr Glu Lys Glu Gly Thr Lys Ile Asp Ile Asp Gly Arg Met His Phe
50                  55                  60
Glu Leu Arg Asn Asp Ser Gly Lys Arg Ser Asp Leu Gln Asp Ala Gly
65                  70                  75                  80
Ser Arg Val Arg Val Arg Ala Phe Gln Glu Ile Gly Asn Gly Phe Ser
85                  90                  95
Thr Tyr Gly Ala Val Glu Phe Arg Phe Ser Thr Lys Lys Asp Gly Ser
100                 105                 110
Glu Gln Ser Ile Gly Ser Asp Leu Arg Ala His Arg Phe Phe Ala Gly
115                 120                 125
Ile Lys Gln Lys Asp Ile Gly Glu Leu Thr Phe Gly Lys Gln Leu His
130                 135                 140
Leu Gly Asp Leu Val Pro Lys Ala Asn Tyr Ser Tyr Asp Leu Gly Ala
145                 150                 155                 160
Asn Ser Phe Phe Gly Ala His Ser Lys Val Ala His Phe Ile Ser Val
165                 170                 175
Pro Phe Asn Gly Val Arg Val Ser Ala
180                 185

Claims

1. An isolated polypeptide having the amino acid sequence at the N-terminal end as shown in SEQ ID:2 from Haemophilus paragallinarum, said polypeptide having a molecular weight of about 130 KD and which polypeptide prevents infection and onset of avian infectious coryza.

2. The isolated polypeptide of claim 1 which is a recombinant polypeptide obtained by a genetic recombination technique.