Assembled viral particles and their use in a vaccine to rotaviral disease

TECHNICAL FIELD
The present invention relates generally to virus-like particles which are useful as vaccines and immunogens. In particular, the instant invention concerns assembled rotaviral structural proteins and the use of the assembly in preventing and ameliorating rotaviral infection.
BACKGROUND OF THE INVENTION
Rotaviruses cause gastrointestinal disorders and diarrhea in a wide variety of avian and mammalian species, including man. Several serotypes of rotavirus have been identified, four of which (serotypes 1 to 4) are found in humans and five of which (serotypes 3 to 7) are found in other animals. Recent studies indicate that cross protection among strains belonging to different serotypes may occur in animals including man. Ijaz et al., J Virol (1990) (In Press); Flores et al., J Clin Microbiol (1989) 27:512-518. The rotavirus genome is thought to consist of eleven segments of double-stranded RNA. The eleven genes encode the production of at least six structural proteins of the virus. In complete virus particles, these six proteins occur in a double-shelled arrangement. The outer shell or capsid is comprised of three proteins--virus protein 7 (VP7), virus protein 4 (VP4), and a third protein which has not yet been well characterized. There are three inner shell proteins designated virus protein 1 (VP1), virus protein 2 (VP2), and virus protein 6 (VP6).
VP7 is the major outer shell glycoprotein with an approximate molecular mass of 38 kD in its unreduced form (as determined by SDS-PAGE) and 42 kD in its reduced form (as determined by SDS-PAGE). VP7 has approximately 325 amino acids. The amino acid sequence of several rotavirus isolates has been determined and the sequences are approximately 75 to 86% homologous. Regions of conservation among human and animal species have been reviewed by Estes, M. K. et al., Microbiol Rev (1989) 53:410-449. This protein is known to bind to host cells (Sabara, M. et al., J Virol (1985) 53:58-66). Epitope mapping of VP7 using neutralizing monoclonal antibodies has localized a neutralizing-absorption domain to a component peptide with an approximate molecular mass of 14 kD (Sabara, M. et al., supra). Synthetic peptides derived from within this 14 kD fragment have also been shown to neutralize viral infectivity (Ijaz et al., supra).
A second outer capsid protein, VP4 (formerly designated VP3), is composed of 776 amino acids and has an approximate molecular mass of 82 kD in its unreduced form and 84 kD in its reduced form. The sequence of bovine VP4 has been determined (Potter, A. A. et al., Nucl Acid Res (1987) 15:4361) as has the partial amino acid sequence for simian VP4 (Mackow, et al., Proc Natl Acad Sci USA (1988) 85:645-649. VP4 possesses hemagglutinating activity, and induces the production of neutralizing antibodies which provide heterotypic passive protection in vivo (Offit, P. A. et al., J Virol (1986) 58:700-703). VP4 is responsible, in combination with VP7, in determining virus serotype.
Dimers of VP4 combine to form the surface spikes of rotavirus that extend distally from the rotavirus outer shell. VP4 is important in the penetration of the virus into the host cell and infectivity is increased by the cleavage of VP4 by trypsin. Trypsin enhanced infectivity is a common feature of all rotaviruses and the cleavage site for trypsin is also conserved as reviewed in Estes et al. (supra).
The inner capsid of rotavirus includes at least three proteins designated VP1, VP2 and VP6. Of interest herein is VP6 which is a 45 kD protein. Bovine VP6 appears to exist in trimeric units in both the virus particle and in infected cells, with the intersubunit linkage consisting of noncovalent interactions. These trimeric units complex further by virtue of disulfide bridges into larger units which likely represent the ring-like structures observed by several investigators using electron microscopy.
VP6 has been identified as the subgroup antigen and has also been described as the common rotavirus group antigen since some monoclonal antibodies raised against this protein react with all rotaviruses and polyclonal serum raised against a single rotavirus type can detect most other rotavirus strains. In addition to its antigenic properties, this nucleocapsid protein is extremely immunogenic and several investigators have found that the antibody raised to this protein has neutralizing ability. (See, e.g. Offit, et al., J Virol (1986) 58:700-703).
VP6 is an effective carrier protein (Redmond, M. J., et al. Mol Immunol (1990) (In Press) and VP4 is able to associate with VP6 monomeric and oligomeric protein units. (Redmond, M. J., et al. supra). The VP4-VP6 association has been shown to withstand harsh treatment such as boiling in SDS. Additionally, VP6 is capable of forming particles in vitro with VP2 and VP7 using a calcium dependent process (Ready, K. F. M., et al., Virology (1988) 167:269-273). The resulting assembly is immunoreactive with antibodies specific for the whole virus as well as for immunodominant sites on VP6 and VP7 and this immunoreactivity is equivalent to that of native bovine rotavirus (BRV). (Ready, K. F. M., et al., supra).
The present invention provides assembled viral particles including peptides or proteins corresponding to VP7 and/or VP4, or immunogenic regions thereof, in combination with VP6. These assembled particles are effective as vaccines and in eliciting the production of neutralizing antibodies. Such a vaccine provides an alternative to the use of a live attenuated virus vaccine.
DISCLOSURE OF THE INVENTION
The instant invention is based on the discovery that certain viral peptides, or epitopic regions thereof, when assembled into a viral particle, are able to elicit an immune response in a subject treated therewith. Vaccines including these assemblies are safer and more practical than those composed of attenuated virus.
Accordingly, in one aspect, the invention is directed to a viral particle assembly capable of eliciting an immunological response in a vertebrate subject. The viral particle assembly comprises:
(a) an inner capsid protein substantially homologous and functionally equivalent to VP6; and
(b) one or more outer capsid proteins selected from the group consisting of (i) a protein substantially homologous and functionally equivalent to VP4, or a functional fragment thereof, and (ii) a protein substantially homologous and functionally equivalent to VP7.
In another embodiment, the present invention is directed to a viral particle assembly capable of eliciting an immunological response in a vertebrate subject wherein the viral particle assembly comprises VP6 assembled with VP4 and VP7.
In yet further embodiments, the invention is directed to vaccine compositions including a pharmaceutically acceptable vehicle and the viral particle assemblies described above.
In other embodiments, the instant invention is directed to methods of treating and preventing rotaviral disease in a vertebrate subject using the above vaccine compositions.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-D show the nucleotide sequence and the predicted amino acid sequence of the VP6 protein of strain C486 (bovine).
FIGS. 2A-F compare the amino acid sequence of rotavirus VP6 derived from several strains: Bovine RF rotavirus (ROBMCP), human 1076 rotavirus (RO1HVP6), rotavirus segment 6 inner shell protein VP6 RNA (RO1S2VP6), equine H2 rotavirus (RO1VVP6H2), equine FI14 rotavirus (RO1VVP6F1), human Wa rotavirus (RO2SEG6), porcine Gottfried rotavirus (RO1PVP6), porcine group C rotavirus (PRVVP6), and simian SA11 rotavirus (ROTG6A).
FIG. 3 compares the amino acid sequence of rotavirus VP4 derived from several strains: K8, KU, DS1, M37, ST3, SA11, and RRV.
FIGS. 4A-F show the nucleotide sequence and corresponding amino acid sequence of the VP4 protein from strain C486 (bovine).
FIGS. 5A-C depict the nucleotide sequence and corresponding amino acid sequence of the VP7 protein from strain C486 (bovine)
FIGS. 6A-D compare the amino acid sequence of rotavirus VP7 derived from several strains: simian 11 rotavirus (ROTVP7), rhesus rotavirus (RORVP7), porcine OSU rotavirus (PRVOSUVP7), human rotavirus (ROHVP7A), porcine Gottfried rotavirus (PRVPRVP7G), bovine uk rotavirus (ROB7), and porcine major C rotavirus (PRVPRVVP7).
FIG. 7 is a graphic representation of the production of in vitro assembled rotavirus particles.

DETAILED DESCRIPTION
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, protein chemistry, molecular biology, microbiology and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications); Methods in Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual 2d Edition (Cold Spring Harbor Laboratory Press, 1989); Oligonucleotide Synthesis (M. J. Gait ed., 1984); and DNA Cloning, Volumes I and II (D. N. Glover, ed., 1985).
All patents, patent applications, and publications mentioned herein, whether supra or infra, are hereby incorporated by reference.
A. Definitions
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
By "VP6" is meant the art-recognized major viral protein of the inner capsid from any species or strain within the family Reoviridae. See, e.g., Kapikian, et al., in Virology (B. N. Fields et al., eds., 1988). Examples of rotavirus strains from which the VP6 protein can be isolated and employed in the present invention include, but are not limited to, Simian SA11, human D rotavirus, bovine UK rotavirus, human Wa or W rotavirus, human DS1 rotavirus, rhesus rotavirus, the "O" agent, bovine NCDV rotavirus, human S2 rotavirus, human KUN rotavirus, human 390 rotavirus, human P rotavirus, human M rotavirus, human Walk 57/14 rotavirus, human Mo rotavirus, human Ito rotavirus, human Nemoto rotavirus, human YO rotavirus, human McM2 rotavirus, rhesus monkey MMU18006 rotavirus, canine CU1 rotavirus, feline Taka rotavirus, equine H2 rotavirus, human St. Thomas No. 3 and No. 4 rotaviruses, human Hosokawa rotavirus, human Hochi rotavirus, porcine SB2 rotavirus, porcine Gottfried rotavirus, porcine SB1A rotavirus, porcine OSU rotavirus, equine H1 rotavirus, chicken Ch.2 rotavirus, turkey Ty.1 rotavirus, bovine C486 rotavirus, and strains derived therefrom. Thus VP6 for use in the present invention includes VP6 from any rotavirus strain, whether from subgroup I, subgroup II, or any as yet unidentified subgroup, as well as from any of the serotypes 1-7, as well as any as yet unidentified serotypes.
The VP6 protein comprises an amino acid sequence of rotavirus which is unique to the class, or any member of the class, of VP6 polypeptides. A representative nucleotide sequence and the deduced amino acid sequence of bovine recombinant (BR) VP6 strain C486 is shown in FIG. 1. FIG. 2 shows the amino acid sequence of several different strains of rotavirus. As can be seen, extensive homology is present between the depicted rotavirus strains. Other VP6 nucleotide and amino acid sequences will find use in the instant invention, the depicted sequences only being representative of already known VP6 sequences.
By "VP4" is meant an art-recognized viral protein of the outer capsid from any species or strain within the family Reoviridae. Examples of rotavirus strains from which the VP4 protein can be isolated and employed in the present invention include, but are not limited to, those described above with reference to VP6.
VP4 (formerly designated VP3), is composed of 776 amino acids. The amino acid sequence of VP4 derived from strains K8, KU, DS1, M37, ST3, SA11, and RRV is shown in FIG. 3. The nucleotide sequence and deduced amino acid sequence of VP4 from strain C486 (bovine) is shown in FIG. 4. Again, other sequences will find use herein.
By "VP7" is meant the art-recognized major viral protein of the outer capsid from any species or strain within the family Reoviridae. Examples of rotavirus strains from which the VP7 protein can be isolated and employed in the present invention include, but are not limited to, those described above with reference to VP6.
VP7 is composed of approximately 325 amino acids and the nucleotide sequence and deduced amino acid sequence of VP7 from strain C486 (bovine) is shown in FIG. 5. Arias et al., J Virol (1984) 50:657-661 describes the nucleotide sequence of VP7 from simian SA11. FIG. 6 depicts the amino acid sequences of several rotavirus strains. VP7 shows serotype restricted homology. The above sequences are meant to be representative and nonlimiting. Therefore, other functional sequences may also be employed with the present invention.
Two DNA or protein sequences are "substantially homologous" when at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra.
The term "functionally equivalent" refers to sequences of an analog of an outer or inner capsid rotavirus protein which define a chain that will produce a protein that elicits an immunological response equivalent to that elicited by the native sequence. Thus, the rotavirus proteins utilized herein need have the identical amino acid sequence of the native proteins.
A "functional fragment" of a rotavirus protein is a fragment with the capability of raising an immunological response equivalent to that elicited by the full sequence. It has been demonstrated that the distal end of VP4 is involved in the initial attachment of the virion to the cell, since infection may be blocked by monoclonal antibodies to this region. Furthermore, the enzyme trypsin enhances virus infectivity. This enhancement appears to act after adsorption, since trypsin does not affect the efficiency or rate of virus attachment to cells but does increase the levels of uncoated particles found in cells. The molecular mechanism for trypsin enhanced infectivity occurs via the cleavage of the VP4 protein into two fragments with approximate molecular weights of 28 kDa and 60 kDa, respectively. Therefore, the trypsin cleavage site of VP4 is important in rotavirus replication. Thus, fragments consisting of an amino acid sequence substantially homologous to at least the first 255 N-terminal amino acids, as depicted in FIG. 4, and which include the trypsin cleavage site, will also find use in the instant invention so long as these fragments are capable of raising an immunological response as defined above.
"A viral particle assembly" refers to an association between outer and inner capsid proteins of rotavirus, or proteins substantially homologous and functionally equivalent thereto, or functional fragments thereof. The particles can be assembled in vitro, as described more fully below, to resemble double shelled rotavirus.
The term "epitope" refers to the site on an antigen or hapten to which a specific antibody molecule binds. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site."
An "immunological response" to a composition or vaccine is the development in a vertebrate subject of a cellular and/or antibody mediated immune response to the composition or vaccine of interest. Usually, such a response consists of the subject producing antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.
An "immunogenic protein" is a protein which elicits an immunological response in a subject to which it is administered.
The terms "polypeptide" and "protein" are used interchangeably herein and are used in their broadest sense, i.e., to denote any polymer of amino acids (dipeptide or greater) linked through peptide bonds. Thus, the terms encompass oligopeptides, protein fragments, analogs, mutants, fusion proteins and the like.
"Native" proteins or polypeptides refer to proteins or polypeptides recovered from rotavirus or from rotavirus infected cells. Thus the term includes naturally occurring rotavirus proteins and fragments thereof. "Non-native" polypeptides refer to polypeptides that have been produced by recombinant DNA methods or by direct synthesis. "Recombinant" polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.
A "replicon" is any genetic element (e.g., a plasmid, a chromosome, a virus) that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control.
A "vector" is a replicon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment. An "expression vector" refers to a vector capable of autonomous replication or integration and contains control sequences which direct the transcription and translation of the desired DNA sequence in an appropriate host.
A "coding sequence" is a polynucleotide sequence which is transcribed and/or translated into a polypeptide.
A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (i.e., in the 3' direction) coding sequence.
A coding sequence is "under the control" of the promoter sequence in a cell when transcription of the coding sequence results from the binding of RNA polymerase to the promoter sequence; translation of the resulting mRNA then results in the polypeptide encoded within the coding sequence.
"Operably linked" refers to a juxtaposition wherein the components are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
"Control sequences" refers to those sequences which control the transcription and/or translation of the coding sequence(s); these may include, but are not limited to, promoter sequences, transcriptional initiation and termination sequences, and translational initiation and termination sequences. In addition, "control sequences" refers to sequences which control the processing of the polypeptide encoded within the coding sequence; these may include, but are not limited to sequences controlling secretion, protease cleavage, and glycosylation of the polypeptide.
A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell.
A "host cell" is a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence.
A cell has been "transformed" by exogenous DNA when such exogenuus DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. With respect to eucaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.
A "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heterologous region encodes a bacterial gene, the gene will usually be flanked by DNA that does not flank the bacterial gene in the genome of the source bacteria. Another example of the heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
A composition containing A is "substantially free of" B when at least about 85% by weight of the total of A+B in the composition is A. Preferably, A comprises at least about 90% by weight of the total of A+B in the composition, more preferably at least about 95%, or even 99% by weight.
A "vaccine composition," according to the present invention, is an otherwise conventional vaccine formulation employing either the viral particle assemblies alone or in combination with one or more unassembled purified viral proteins or with cell lysates having one or more of the individual outer and/or inner capsid proteins. Particularly useful is the addition of a crude cell lysate containing VP4 to the instant vaccine compositions. The preparation of vaccines containing the above active ingredients is well understood in the art. Typically, vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposomes. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccine. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Injectable vaccine formulations will contain an effective amount of the active ingredient, the exact amount being readily determined by one skilled in the art. The active ingredient can range from about 0.01% to about 95% (w/w) of the injectable composition, or even higher or lower if appropriate.
Additional vaccine formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulation. For suppositories, the vaccine composition will include traditional binders and carriers, such as polyalkaline glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like. These oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.
Furthermore, the viral particles may be formulated into vaccine compositions in either neutral or salt forms. If salts are used, the final preparation will typically contain less than 0.15M salt. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.
By "treating" is meant curing or ameliorating a subject that has contracted a rotaviral infection. "Preventing" rotaviral disease means preventing the occurrence of the infection, or tempering the severity of the infection if it is contracted subsequent to the administration of the instant compositions.
The vaccine composition of the present invention may be administered in a manner compatible with the dosage formulation, and in such amounts as will be therapeutically effective and immunogenic. A "therapeutically effective amount" of a vaccine composition is a dose sufficient to either prevent or treat rotaviral infection in a subject to which the composition is administered. The dosages of the viral particle assemblies which can treat or prevent rotaviral infection can be determined in view of this disclosure by one of ordinary skill in the art by running routine trials with appropriate controls. Comparison of the appropriate treatment groups to the controls will indicate whether a particular dosage is effective in preventing or treating a disease used in a controlled challenge. In general, effective dosage will vary depending on the mode of administration. For example, in the case of an intramuscular injection, generally from 0.001 .mu.g/kg to 10 .mu.g/kg will find use in the instant invention.
B. General Methods
Central to the instant invention is the discovery that assembled viral particles comprising rotavirus inner and outer capsid proteins, are able to elicit an immune response in a subject to which they are administered. The viral particles are assemblies of inner and outer capsid proteins of rotavirus. Particularly useful is a viral particle assembly including the inner capsid protein, VP6, with either or both of the outer capsid proteins, VP4 and VP7. VP6 appears to act as a "carrier" for the two outer capsid proteins, as described more fully below. These assemblies can be used alone, or in combination with unassembled, outer capsid proteins, provided as purified or partially purified proteins, or crude cell lysates, in a vaccine composition for the treatment and/or prevention of rotaviral infection.
The inner and outer capsid proteins for use in the viral particles of the instant invention can be prepared by any of several methods. First, the individual proteins can be isolated by successive degradation of purified virus with EDTA and either calcium chloride (CaCl.sub.2) or lithium chloride (LiCl) treatment by standard techniques. See, e.g., Almeida et al., J Med Virol (1979) 4:269-277; Bican et al., J Virol (1982) 43:1113-1117; Gorziglia et al., J Gen Virol (1985) 66:1889-1900; Ready et al., Virology (1987) 157:189-198. Alternatively, the viral proteins can be produced by recombinant DNA techniques, which are fully explained in the literature. See, e.g., Sambrook et al., supra; and DNA Cloning, supra.
DNA coding sequences encoding the viral polypeptides can be derived from the particular mRNA. See, e.g., Estes et al., supra; Both et al., J Virol (1984) 51:97-101; Cohen et al., Virology (1984) 138:178-182. Alternatively, a DNA sequence encoding the particular viral protein can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the viral protein amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; Jay et al., J Biol Chem (1984) 259:6311.
Once a coding sequence for the viral protein has been prepared or isolated, it can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform (in parenthesis) include the bacteriophage lambda (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1 106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells). See generally, DNA Cloning: Vols. I & II. supra; and Sambrook et al., supra.
The coding sequence for the viral protein can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the viral protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. In bacteria, for example, the viral protein is preferably made by the expression of a coding sequence containing a leader sequence which is removed by the bacterial host in post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.
An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). The control sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
A number of procaryotic expression vectors are known in the art. See, e.g., U.S. Pat. Nos. 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; see also UK Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and European Patent Application 103,395. Yeast expression vectors are also known in the art. See, e.g., U.S. Pat. Nos. 4,446,235; 4,443,539; 4,430,428; see also European Patent Applications 103,409; 100,561; and 96,491.
Particularly useful for expression of the rotaviral genes of the instant invention are insect cells and vectors suitable for use in these cells. Such systems are known in the art, and include, for example, insect expression transfer vectors constructed from the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Such expression vectors typically use the strong viral polyhedrin gene promoter to control expression of heterologous genes. Methods for the introduction of heterologous DNA into the desired site in the baculovirus are known in the art. (See, e.g. Smith, et al., Mol and Cell Biol (1983) 3:2156-2165; and Luckow and Sumners Virology (1989) 17:31. Insertion can be, for example, into a gene such as the polyhedrin gene, by homologous recombination. Insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Sequences encoding signal peptides can also be used in these expression systems since these peptides are recognized by insect cells and will cause the secretion of the expressed product into the expression medium.
Depending on the expression system and host selected, the viral protein is produced by growing host cells transformed by an expression vector described above under conditions whereby the protein is expressed. The protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the protein can be purified directly from cell-free media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
Purified VP6 protein exhibits structural polymorphism. Specifically, hexamers and small hexagonal lattices are present in many of the samples. Tubular particles form between about pH 5.0 and about pH 9.0, and are moderately stable to changes in temperature and ionic strength. The formation of these particles is fully reversible. Spherical particles reassembling single-shelled virus can be formed at about pH 4.0. A novel structure, in the form of sheets, composed of small-hole lattice, is formed in samples shifted from about pH 6.0 to about pH 4.0. These results demonstrate the importance of VP6 and of protein-protein interactions for rotavirus assembly.
Such protein-protein interactions are likely involved in the observed phenomenon that certain peptides can bind to VP6 in its monomeric form or to various oligomeric structures formed from VP6 monomers, such as in vitro assembled tubes and spheres. The attachment is mediated by a specific binding site(s) within VP6.
After the individual outer and inner capsid proteins have been either isolated, synthesized, or recombinantly produced, these proteins are assembled into viral particles using a calcium dependent process described in the examples and in Ready, K. F. M., et al., Virology (1988) 167:269-273. FIG. 7 depicts a representative scheme for the production of in vitro assembled rotavirus particles.
As explained above, the viral particles can be administered in vaccine compositions in combination with purified, unassembled outer capsid proteins, or with cell extracts including one or more of the individual outer capsid proteins. These cell extracts are prepared by techniques well known in the art. For example, cell extracts can be prepared by cultivating rotavirus in African Green Monkey MA104 cells, harvesting the cells and media together and removing the cells by centrifugation. The resulting supernatant includes rotavirus proteins. The proteins can be further purified using density gradient ultracentrifugation. The virus pellet is then subjected to treatment with CaCl.sub.2 and LiCl.sub.2, as described in Ready, K. F. M. and Sabara, M. Virology (1987) 157:189-198, to obtain the individual viral particles. VP4 is present in very small amounts in such preparations. Furthermore, small amounts of VP4 are often present in the VP7 fractions.
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Deposits of Strains Useful in Practicing the Invention
A deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. The accession number indicated was assigned after successful viability testing, and the requisite fees were paid. Access to said cultures will be available during pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR .sctn.1.14 and 35 USC .sctn.122. All restriction on availability of said cultures to the public will be irrevocably removed upon the granting of a patent based upon the application. Moreover, the designated deposits will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit; or for the enforceable life of the U.S. patent, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plasmid-containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description.
______________________________________Strain Deposit Date ATCC No.______________________________________pAC373BRV6 31 August 1987 40362(in E. coli)BVLVP4 24 October 1990(in A. californica)BYVP7 24 October 1990(in A. californica)______________________________________
C. Experimental
Materials and Methods
Enzymes were purchased from commercial sources, and used according to the manufacturers' directions. Radionucleotides and nitrocellulose filters were also purchased from commercial sources.
In the cloning of DNA fragments, except where noted, all DNA manipulations were done according to standard procedures. See, Sambrook et al., supra. Restriction enzymes, T.sub.4 DNA ligase, E. coli, DNA polymerase I, Klenow fragment, and other biological reagents were purchased from commercial suppliers and used according to the manufacturers' directions. Double-stranded DNA fragments were separated on agarose gels.
Animals
Rotavirus-free mice (CD1) were purchased from Harlan Sprague Dawley Inc., (Indianapolis, Ind.). The age of the mice at the time of purchase was approximately 6 weeks. The mice weighed between 25 and 30 grams. All mice were found to be seronegative for antibodies to rotavirus using an ELISA assay (Ijaz, M. K. et al. Exp Mol Path (1989) 51:186-204). The animals were housed in isolation units throughout the experiment.
Cells and Virus
MA104 cells (African green monkey) were cultured in Eagle's minimal essential media (MEM) supplemented with 10% fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, N.Y.). Bovine rotavirus isolate C486 was cultured from the feces of diarrheic calves by a method described previously (Babiuk, L. A. et al., J Clin Microbiol (1977) 6:610-617). Rotavirus C486 is publicly available from the ATCC, Rockville, Md. (accession no. VR-917). Simian rotavirus strain SA11 (serotype 3) was obtained from Dr. H. Malherbe (San Antonio, Tex.) and human rotavirus strain DS1 (serotype 2) and human rotavirus strain Wa (serotype 1) were obtained from Dr. H. Greenberg (Stanford University, Calif.).
These viruses were propagated in confluent MA104 cells in the presence of 1 .mu.g of trypsin (Difco Laboratories, Detroit, Mich.) per ml in the absence of FBS. Cells and supernatant were harvested together and cells were removed by centrifugation at 500g for 20 min. Virus was concentrated from the clarified supernatant fluids by pelleting through a 40% sucrose-cushion at 100,000 g for 21/2 hr at 15.degree. C. The virus pellet was resuspended in double distilled water and the amount of virus protein was estimated spectrophotometrically as described previously (Ijaz, M. K. et al., Antiviral Res (1987) 8:283-298). The resuspended virus was stored at -70.degree. C. Individual inner and outer capsid proteins were isolated from the preparation as described in the examples below.
Plaque Assay
A plaque assay for the quantitation of infectious rotavirus was performed according to the method described previously (Aha, P. M. and Sabara, M. I. J Virol (1990) 28:25-32). Briefly, 12 well tissue culture plates (NUNC) containing confluent monolayers of MA104 cells, were washed twice with MEM (without FBS). Serial tenfold dilutions of each rotavirus isolate were prepared in MEM containing trypsin (Difco Laboratories, Detroit, Mich.) to a final concentration of 10 .mu.g/ml. Following adsorption of the virus at 37.degree. C. for 1 hr, the inoculum was aspirated, cells were washed with MEM and overlaid with Dulbecco's Modified Eagle Medium (DMEM), containing 4% Sephadex G-75 beads (Pharmacia). The plates were incubated for 2 days at 37.degree. C., the overlay was aspirated and plates were stained with 0.5% crystal violet/80% methanol/PBS, washed and plaques enumerated.
ELISA Procedure
The ELISA was a modification of a previously described procedure (Sabara, M. et al., J Virol (1985) 53:58-66). All incubations were performed at room temperature (20.degree. C.) for 1 hr unless stated otherwise. Polystyrene, 96-well Immunolon 2 plates (Dynatech Labs Inc., Alexandria, Va.) were sensitized for each of the assay systems and used as follows.
For the detection of protein specific antibody, plates were coated overnight with the respective protein (5 picomoles/well) diluted in 0.05M carbonate bicarbonate buffer at pH 9.6. Unabsorbed protein was removed by extensive washing with distilled water. The uncoated sites on the plate were blocked by overnight treatment with 3% horse serum in 0.01M PAS and then washed with double distilled H.sub.2 O. The plated antigen was overlaid with 75 .mu.l of mouse antiserum/well in 0.01M PAS containing 1% horse serum and 0.05% Tween 20. Incubation was carried out for 2 hr at room temperature after which time the unbound antibody was removed by washing with 0.01M PAS containing 0.05% Tween 20 (PBST). A 1/5000 dilution (in PBST plus 1% horse serum) of biotinylated-goat anti-mouse serum (Zymed Laboratories Inc., San Francisco, Calif.) was then added per well and incubated for one hr at room temperature. After washing in PBST, plates were incubated for one hr with 75 .mu.l of streptavidin horseradish peroxidase conjugate diluted in PBST and 1% horse serum. After washing with PBST, the substrate (2.2'-Azino-di-[3 ethyl-benzthiazoline sulfonate], ABTS, Boehringer-Mannheim) was added. The color development was stopped after 10 min by the addition of 10% SDS. The optical density of the wells was determined at 405 nm by an ELISA reader (BioRad Laboratories, Richmond, Calif.). Titers were expressed as a reciprocal of the highest dilution with an OD of >2 SD over mean background levels.
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE):
Viral proteins were separated by SDS-PAGE under both reducing and non-reducing conditions according to the procedure described by Laemmli (Laemmli, U.K. Nature (1970) 227:680-685). Virus samples were resuspended in electrophoresis sample buffer (0.337M Tris pH 6.8, 6% SDS, 30% glycerol, 0.03% bromophenol blue) for running under nonreducing conditions and included 3.75% mercaptoethanol (BME) for reducing conditions. The samples were boiled for 5 to 10 min and analyzed following electrophoresis on a 10% polyacrylamide resolving gel with a 3% stacking gel.
Western Blotting of Rotavirus Proteins
Protein-specific antibodies were detected by the Western blotting technique described by Towbin et al. (Towbin, H. et al., Proc Natl Acad Sci USA (1979) 76:4350-4354). Viral proteins separated on a 10% polyacrylamide gel, were transferred to nitrocellulose paper (0.45 .mu.m) (BioRad Laboratories) by electroblotting at 100 volts for one hr in a buffer containing 20 mM Tris-190 mM glycine-20% methanol. Replica nitrocellulose strips were stained with amido black to determine the efficiency of protein transfer.
After transfer, reaction of viral protein with serum samples was determined as described previously (Braun, D. K. et al., J Virol (1983) 46:103-112). Non-specific reactions were blocked with 3% bovine serum albumin (BSA) in 0.01M TBS. After washing with TBST, the reaction was developed with protein A gold (BioRad Laboratories) for one hr. Following development, the protein bands were intensified by silver enhancement (Janssen Biotech, N.V., Belgium).
EXAMPLE 1
Isolation of Viral Proteins
A. Isolation of Native VP6
The VP6 viral protein was isolated from the purified virus suspension (described above) by successive degradation of purified virus with EDTA and either CaCl.sub.2 or LiCl, as follows. Outer capsid proteins were removed by incubating virus (3 mg/ml) in 50 mM EDTA, 0.01M Tris-HCl, pH 7.4 at 4.degree. C. for 30 min. Subviral particles were recovered by ultracentrifugation (100,000.times.g, 2-3 hr, 4.degree. C.) and resuspended in 0.01M Tris-HCl, pH 7.4 or 0.01M sodium borate, pH 9.0. They were then treated with either 1.5M CaCl.sub.2 with 0.01M Tris-HCl, pH 7.4 at 20.degree. C. for 20-30 min, or were frozen in 2M LiCl, 0.01M sodium borate, pH 9.0 at -70.degree. C. for 4 days. Cores and undegraded particles were separated from solubilized protein by ultracentrifugation. EDTA and salts were removed by extensive dialysis at 4.degree. C. against 0.01M Tris-HCl, pH 7.4, unless otherwise indicated. The purity of the samples was examined by polyacrylamide gel electrophoresis (PAGE) as described above.
B. Isolation of Native VP4
The limited number of copies of VP4 protein per virus particle makes the purification of large amounts of this protein difficult. However, VP4 is found in the supernatant obtained after ultracentrifugation following treatment of the subviral particles with 1.5M CaCl.sub.2 or 2M LiCl treatment of intact virus particles, as described in the isolation of native VP6. VP4 can also be purified from this pellet by e.g. HPLC, affinity chromatography, ion-exchange chromatography, etc.
C. Isolation of Native VP7
As with VP4, VP7 is also found in the supernatants described above but in larger amounts. See, Ready, K. F. M., et al. Virology (1988) 167:269-273. VP7 can be further purified using HPLC, affinity chromatography, ion-exchange chromatography, etc.
EXAMPLE 2
Production of Recombinant Viral Protein
A. Production of Recombinant VP6
The construction of recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) containing gene 6 from bovine rotavirus (BRV) and assembly of VP6 particles following infection of Spodoptera frugiperda (SF9) cells has been described previously (Redmond, M. J. et al., Mol Immunol, In Press. Briefly, genomic RNA extracted from purified bovine rotavirus strain C486 was used to produce cDNA. The cDNA was ligated into the Pst I site of pBR322 and used to transform E. coli strain DH1. The resulting colonies were probed with radiolabeled cDNA prepared from purified genomic RNA segment 6 as template.
Clone pR6-42 which contained a complete copy of the gene 6 RNA, was partially digested with Aha III which removed seven 5' noncoding nucleotides as well as the oligo-dC tails added during cDNA cloning. A Bam HI linker was then added.
The 3' oligo-dC tail and noncoding region were removed by digestion with Acc I which removes 56 noncoding nucleotides from the VP6 gene. A Bam HI linker was then added. The gene 6 cDNA was then ligated into the Bam HI site of the baculovirus transfer vector pAc373. This vector was designated pAC373BRV6 (ATCC no. 40362). Integration of the rotavirus gene into the genome of A. californica was then carried out by homologous recombination in S. frugidperda (SF9) cells as outlined by Summers, M. D. and Smith, G. E., Texas Agricultural Station Bulletin 1555:26-27. Recombinants were identified by plaque hybridization, as described above, using radiolabeled cDNA prepared from purified genomic RNA segment 6. Recombinants were plaque purified and analyzed for expression of recombinant gene 6 produced proteins by SDS-PAGE analysis and Western blotting using the methods described above.
The recombinant virus containing gene 6 was used to infect SF9 cells. Following incubation for 72 hr at 27C, the cells were lysed in a 2 ml NaHCO.sup.3 buffer (pH 7.5) containing 0.05% triton X-100 and 0.2 trypsin inhibitor units per ml. Cellular debris was removed by centrifugation at 1500 g. The supernatant was dialyzed against 0.1M glycine buffer (pH 3.0) for 24 hr. This dissociates VP6 aggregates into monomers. The dialysis solution was exchanged for 0.01M citrate buffer (pH 4.0) and dialysis continued for 24 hr, during which time spheres of VP6 formed. This dialysis buffer was exchanged for 0.01M Tris-HCl (pH 7.4)+1 mM sodium azide. Dialysis was then continued overnight at 4.degree. C. Nonaggregated material was removed by ultracentrifugation using 300,000 dalton molecular weight cutoff filters. The quality of the VP6 spheres produced by this method was determined by electromicroscopy and purity confirmed by SDS-PAGE.
B. Production of Recombinant VP4
VP4 was produced recombinantly as described for VP6 except that site directed mutagenesis was used to insert a Bam HI restriction site immediately upstream of the translational initiation codon of the cDNA including gene 4 (the gene encoding VP4), resulting in the deletion of nine nucleotides normally present preceding the VP4 initiation codon. The 3' end of the gene was left unmodified.
The modified gene 4 was ligated into transfer vector pVL941. Integration of gene 4 into the A. californica genome was carried out as above to render the expression vector BVLVP4.
Crude detergent lysates of the VP4-baculovirus infected SF9 cells were used as starting material in the production of viral particles as described below. The interaction between VP4 and VP6 is analogous to an affinity purification step. Thus, the VP6-VP4 complex can be separated from unbound VP4 and other cellular proteins by ultracentrifugation over sucrose gradients.
C. Production of Recombinant VP7
cDNA including a full length copy of the gene for VP7 (gene 8) was cloned and identified as described above. A clone (clone pr8-G) which contained the full length cDNA copy of the VP7 gene was digested with Aha III and Sph I which removed 7 and 27 nucleotides, respectively, from the 5' and 3' ends of the gene. Bam HI linkers were then added to both ends of the DNA.
Gene 8 was ligated into the Bam HI site of pACYM1 and integration of gene B into the genome of A. californica proceeded as described for VP6 to yield expression vector BYVP7. Recombinants were identified, plaque purified and assayed for expression as described above.
VP7-baculovirus infected SF9 cells secrete VP7 directly into the growth medium. VP7 is essentially affinity purified therefrom by the formation of double shelled (VP6-VP7) particles, in the presence of 50 mM CaCl.sub.2. This separates the VP7 from the other components present in the growth medium. The final purification step is ultracentrifugation over sucrose gradients.
EXAMPLE 3
Assembly of Viral Particles
VP7 and VP4 protein concentrations in growth media and infected cell lysates were estimated on SDS-PGE gels stained with Coomassie Blue, and western blots probed with VP7 and VP4 specific antisera. A standard of native BRV was used at a predetermined concentration. The concentrations of proteins used give the following ratios to VP6.
VP6-VP4. 1:10 w/w
VP6-VP7. 1:10 w/w
The starting material for all particle preparations was 10 .mu.g of VP6. To prepare VP6-VP4 particles, 100 .mu.g of VP4 protein in a detergent lysate of SF9 cells (described in Example 2B) dialyzed in 10 mM Tris+50 mM CaCl.sub.2 pH 8 overnight at 4.degree. C. was mixed for 2 hr. at 37.degree. with VP6. If used as a vaccine, the preparation was centrifuged at 100,000 g. for 2 hr. through a 40% sucrose gradient.
To prepare VP6-4-7 particles, the Vp6-4 particles prepared above were added to 100 .mu.g of VP7. The VP7 preparation consisted of media from cell culture that had been dialyzed overnight into 0.1M Tris-HCl pH 8.0 containing 50 mM CaCl.sub.2.
The VP6-4+7 mixture was then dialyzed for 3-4 days against 0.1M Tris-HCl pH 8.0 containing 50 mM CaCl.sub.2 The particles were purified over sucrose as described previously.
To prepare VP6-VP7, 10 .mu.g of VP6 spheres were mixed with 100 .mu.g of VP7 contained in a preparation obtained by dialyzing tissue culture growth medium, from VP7-baculovirus infected SF9 cells, against 0.1m Tris-HCl pH 8.0 containing 50 mM CaCl.sub.2. Dialysis was continued for 3-4 days against the same buffer after the addition of VP6. VP6-7 particles, when required, were purified by ultracentrifugation. In order to produce VP6-7-4 particles, cell lysates containing VP4 equivalent to 100 .mu.g of purified virus were mixed for 2 hrs. at 37.degree. C., with VP6-7, then ultracentrifuged through 40% Sucrose for 2 hrs. at 100,000 G. After this time, particles were purified by centrifugation as described above.
EXAMPLE 4
Protective Capacity of the Assembled Viral Proteins
Vaccine compositions used in this example were as follows: For the primary immunizing dose, each immunogen (Table 1) was emulsified with Freund's complete adjuvant. For the second and third immunizations, each immunogen was emulsified with Freund's incomplete adjuvant. Equal volumes of the immunogen and the adjuvant were used.
Thirteen groups of mice were immunized intramuscularly with the preparations outlined in Table 1. Each mouse was immunized three times before and after breeding. The first immunization was given when the mice were seven weeks old and was followed by the second and third vaccinations at two week intervals. Litters were born when the mice were 12 to 14 weeks old.
Following birth, the mouse pups were allowed to suckle their dams and were challenged at 7 days of age with one of four rotavirus isolates. These isolates were bovine rotavirus strain C486 (serotype 6), simian rotavirus strain SA11 (serotype 3), human rotavirus strain DS1 (serotype 1) and Wa (serotype 2). The SA11 isolate was obtained from Dr. H. Malherbe (San Antonio, Tex.) and the strain Wa and DS1 isolates were obtained from Dr. H. Greenberg (Stanford University, Calif.). The strain C486 which was a local isolate adapted to grow in MA104 cells (Babiuk, L. A. et al., J Clin Microbiol (1977) 6:610-617). These viruses were grown in MA104 cells, harvested and concentrated for challenge by the method described previously (Ijaz et al., Antiviral Res (1987) 8:283-298. The challenge dose for each isolate was approximately 10.sup.4 PFU/mouse suspended in MEM in 100 .mu.l volume. For challenge, the virus preparations were administered by intubation of the stomach with a soft flexible plastic feeding tube. Trypan blue dye (GIBCO) was used as a marker to assess the accuracy of intubation.
The appearance of diarrhea was scored clinically up to 72 hr post-challenge using clinical scores as follows:
(-) no sign of diarrhea in live mice, or on necropsy;
(+) no external signs of diarrhea but semi-liquid colon contents at autopsy;
(++) fluid was apparent on palpation of the abdomen and the colon was filled with liquid feces and gas;
(+++) the external anal region was soiled with feces and intestinal fluid was present on palpation and;
(++++) liquid feces present around the anal region and on palpation of the abdomen intestinal fluid was present and oozed from the anus, severe dehydration, internal liquid content in colon and caecum and distention due to accumulation of gas.
TABLE 1__________________________________________________________________________HOMOLOGOUS PROTECTION OF NEONATAL MICE SUCKLING ON DAMSIMMUNIZED WITH RECOMBINANT ROTAVIRUS PROTEINS Clinical ScoreGroup Antigen Serum Titre Wa DS-1 SA-11 BRV PRN__________________________________________________________________________ 1 Sentinel -- ++++ ++++ ++++ ++++ 2 Placebo -- ++++ ++++ ++++ ++++ <40 3 BRV 1,540,830 0 0 0 0 >1,280 4 VP6 5,235 ++ ++ ++ ++ <40 5 VP7 6,105 ND ND ND + <40 6 VP4 53,850 0 0 + 0 780 7 VP6-VP4 31,150 0 0 + 0 1,000 8 VP6-VP7 252,520 0 0 ++ 0 <40 9 VP6-VP4 64,805 0 0 + 0 740 plus VP6-VP7 (mixture)10 VP6-VP4-VP7 35,235 ND ND ND 0 22511 VP6-VP7-VP4 69,685 0 0 + 0 23012 VP6 (100) 53,625 ND ND ND ++ <4013 VP6 (10) 29,960 ND ND ND ++ <4014 VP7 (100) 4,110 ND ND ND + <4015 VP7 (10) 27,610 ND ND ND ++ <4016 VP4 (100) 22,550 ND ND ND 0 60017 VP4 (10) 35,150 0 0 + 0 54018 VP6 + VP4 59,080 ND ND ND 0 >128019 VP6 + VP7 145,820 ND ND ND + <4020 VP6 + VP7 + VP4 44,620 ND ND ND 0 46021 VPS-VP7 plus 544,447 ND ND ND + <40 insect cell proteins__________________________________________________________________________ Group 3 is bovine rotavirus antigen and was given at a dose of 50 .mu.g/mouse. Groups 4-6 are partially purified viral proteins. 10 .mu.g/mouse of these proteins were administered. Groups 7-11 and 21 are assembled particles. Approximately 10 .mu.g of VP6 and VP7 were administered per mouse and approximately 3.2 .mu.g of VP4 administered per mouse. Groups 12-17 are partially purified viral proteins and the .mu.g/mouse administered is in parentheses. Groups 18-20 are mixed, crude lysates. Approximately 10 .mu.g/mouse of th mixed lysate was administered and 90 .mu.g/mouse of the insect cell protein (Group 21). ND = Not Done PRN = 50% plaque reduction nutralization titers.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 30(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1356 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 24..1214(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GGCTTTTAAACGAAGTCTTCAACATGGATGTCCTGTACTCCTTGTCAAAA50MetAspValLeuTyrSerLeuSerLys 15ACTCTTAAAGATGCTAGAGACAAAATTGTCGAAGGCACATTATACTCC98ThrLeuLysAspAlaArgAspLysIleValGluGlyThrLeuTyrSer1015 2025AATGTAAGTGATCTAATTCAACAATTTAATCAAATGATAATTACTATG146AsnValSerAspLeuIleGlnGlnPheAsnGlnMetIleIleThrMet30 3540AATGGAAATGAGTTCCAAACTGGAGGAATTGGTAATCTACCGATTAGA194AsnGlyAsnGluPheGlnThrGlyGlyIleGlyAsnLeuProIleArg45 5055AATTGGAATTTTGATTTTGGATTACTTGGAACAACTCTACTAAATTTA242AsnTrpAsnPheAspPheGlyLeuLeuGlyThrThrLeuLeuAsnLeu6065 70GATGCTAACTACGTCGAAACGGCCCGCAATACAAATGATTATTTTGTA290AspAlaAsnTyrValGluThrAlaArgAsnThrAsnAspTyrPheVal7580 85GATTTTGTAGATAATGTATGTATGGACGAAATGGTTAGAGAATCACAA338AspPheValAspAsnValCysMetAspGluMetValArgGluSerGln9095100 105AGAAATGGAATTGCACCACAATCAGATTCACTTATAAAGTTATCAGGC386ArgAsnGlyIleAlaProGlnSerAspSerLeuIleLysLeuSerGly110115 120ATTAAATTTAAAAGAATAAATTTTGACAATTCATCAGAATACATAGAG434IleLysPheLysArgIleAsnPheAspAsnSerSerGluTyrIleGlu125130 135AACTGGAATTTGCCAAATAGAAGACAAAGAACGGGTTTTACATTTCAT482AsnTrpAsnLeuProAsnArgArgGlnArgThrGlyPheThrPheHis1401451 50AAACCAAACATTTTCCCTTATTCAGCTTCATTCACGTTGAACAGATCA530LysProAsnIlePheProTyrSerAlaSerPheThrLeuAsnArgSer155160165CAACC TTCTCATGATAACTTGATGGGTACGATGTGGCTCAATGCGGGA578GlnProSerHisAspAsnLeuMetGlyThrMetTrpLeuAsnAlaGly170175180185T CAGAAATTCAGGTCGCTGGATTCGACTACTCATGTGCAATAAACGCG626SerGluIleGlnValAlaGlyPheAspTyrSerCysAlaIleAsnAla190195200 CCAGCTAATACGCAACAATTTGAGCATATTGTACAGCTTCGAAGGGTG674ProAlaAsnThrGlnGlnPheGluHisIleValGlnLeuArgArgVal205210215TTG ACTACAGCTACAATAACTCTTTTACCAGATGCAGAAAGATTTAGT722LeuThrThrAlaThrIleThrLeuLeuProAspAlaGluArgPheSer220225230TTTCCAAG AGTGATTACTTCAGCTGACGGAGCGACTACATGGTACTTC770PheProArgValIleThrSerAlaAspGlyAlaThrThrTrpTyrPhe235240245AATCCAGTGATTCTTA GACCAAATAACGTTGAAATAGAGTTTCTACTA818AsnProValIleLeuArgProAsnAsnValGluIleGluPheLeuLeu250255260265AACGGGCAGATA ATAAATACTTACCAAGCAAGATTTGGAACCATCATA866AsnGlyGlnIleIleAsnThrTyrGlnAlaArgPheGlyThrIleIle270275280GCTAGAAATTTT GATACAATTAGATTGTCATTTCAGTTGATGAGACCA914AlaArgAsnPheAspThrIleArgLeuSerPheGlnLeuMetArgPro285290295CCAAATATGACACC AGCGGTAGCGGCGTTATTTCCAAATGCGCAGCCA962ProAsnMetThrProAlaValAlaAlaLeuPheProAsnAlaGlnPro300305310TTTGAACATCACGCAACAG TAGGACTCACGCTTAGAATTGAATCTGCA1010PheGluHisHisAlaThrValGlyLeuThrLeuArgIleGluSerAla315320325GTTTGTGAATCAGTACTTGCCGACGCA AGCGAAACAATGCTAGCAAAT1058ValCysGluSerValLeuAlaAspAlaSerGluThrMetLeuAlaAsn330335340345GTGACATCTGTTAGACAAGAATAC GCGATACCAGTTGGACCAGTTTTT1106ValThrSerValArgGlnGluTyrAlaIleProValGlyProValPhe350355360CCACCAGGTATGAATTGGACTGA TTTGATCACTAACTATTCACCATCT1154ProProGlyMetAsnTrpThrAspLeuIleThrAsnTyrSerProSer365370375AGAGAGGATAACTTGCAGCGTGTAT TTACAGTGGCTTCCATTAGAAGC1202ArgGluAspAsnLeuGlnArgValPheThrValAlaSerIleArgSer380385390ATGCTTGTCAAATGAGGACCAAGCTAACCACTTG GTATCCGACTTTGGTGAG1254MetLeuValLys395TATGTAGCTACGTCAAGCTGTTTGAACTCTGTAAGTAAGGATGCGTCTACGTATTCGCTA1314CACAGAGTAATCACTCAGATGGCGTAGTGAGAGGATGTGACC 1356(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 397 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetAspValLeuTyrSerLeuSerLysThrLeuLysAspAlaArgAsp1 51015LysIleValGluGlyThrLeuTyrSerAsnValSerAspLeuIleGln202530GlnPheAsnGlnMetIleIleTh rMetAsnGlyAsnGluPheGlnThr354045GlyGlyIleGlyAsnLeuProIleArgAsnTrpAsnPheAspPheGly50556 0LeuLeuGlyThrThrLeuLeuAsnLeuAspAlaAsnTyrValGluThr65707580AlaArgAsnThrAsnAspTyrPheValAspPheValAspAsnValCys 859095MetAspGluMetValArgGluSerGlnArgAsnGlyIleAlaProGln100105110SerAspSerLeu IleLysLeuSerGlyIleLysPheLysArgIleAsn115120125PheAspAsnSerSerGluTyrIleGluAsnTrpAsnLeuProAsnArg130135 140ArgGlnArgThrGlyPheThrPheHisLysProAsnIlePheProTyr145150155160SerAlaSerPheThrLeuAsnArgSerGlnProSerHi sAspAsnLeu165170175MetGlyThrMetTrpLeuAsnAlaGlySerGluIleGlnValAlaGly180185190 PheAspTyrSerCysAlaIleAsnAlaProAlaAsnThrGlnGlnPhe195200205GluHisIleValGlnLeuArgArgValLeuThrThrAlaThrIleThr210 215220LeuLeuProAspAlaGluArgPheSerPheProArgValIleThrSer225230235240AlaAspGlyAlaThrThrTrpTyrPhe AsnProValIleLeuArgPro245250255AsnAsnValGluIleGluPheLeuLeuAsnGlyGlnIleIleAsnThr260265 270TyrGlnAlaArgPheGlyThrIleIleAlaArgAsnPheAspThrIle275280285ArgLeuSerPheGlnLeuMetArgProProAsnMetThrProAlaVal 290295300AlaAlaLeuPheProAsnAlaGlnProPheGluHisHisAlaThrVal305310315320GlyLeuThrLeuArg IleGluSerAlaValCysGluSerValLeuAla325330335AspAlaSerGluThrMetLeuAlaAsnValThrSerValArgGlnGlu340 345350TyrAlaIleProValGlyProValPheProProGlyMetAsnTrpThr355360365AspLeuIleThrAsnTyrSerProSerArgGluAspAsn LeuGlnArg370375380ValPheThrValAlaSerIleArgSerMetLeuValLys385390395(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 397 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:MetAspValLeuTyrSerLeuSerLysThrLeuLysAspAlaArgAsp15 1015LysIleValGluGlyThrLeuTyrSerAsnValSerAspLeuIleGln202530GlnPheAsnGlnMetIleIleThrMetAs nGlyAsnGluPheGlnThr354045GlyGlyIleGlyAsnLeuProIleArgAsnTrpAsnPheAspPheGly5055 60LeuLeuGlyThrThrLeuLeuAsnLeuAspAlaAsnTyrValGluThr65707580AlaArgAsnThrAsnAspTyrPheValAspPheVa lAspAsnValCys859095MetAspGluMetValArgGluSerGlnArgAsnGlyIleAlaProGln100105 110SerAspSerLeuIleLysLeuSerGlyIleLysPheLysArgIleAsn115120125PheAspAsnSerSerGluTyrIleGluAsnTrpAsn LeuProAsnArg130135140ArgGlnArgThrGlyPheThrPheHisLysProAsnIlePheProTyr145150155 160SerAlaSerPheThrLeuAsnArgSerGlnProSerHisAspAsnLeu165170175MetGlyThrMetTrpLeuAsnAlaGlySerGluI leGlnValAlaGly180185190PheAspTyrSerCysAlaIleAsnAlaProAlaAsnThrGlnGlnPhe195200 205GluHisIleValGlnLeuArgArgValLeuThrThrAlaThrIleThr210215220LeuLeuProAspAlaGluArgPheSerPheProArgValIleTh rSer225230235240AlaAspGlyAlaThrThrTrpTyrPheAsnProValIleLeuArgPro245250 255AsnAsnValGluIleGluPheLeuLeuAsnGlyGlnIleIleAsnThr260265270TyrGlnAlaArgPheGlyThrIleIleAlaArgAsn PheAspThrIle275280285ArgLeuSerPheGlnLeuMetArgProProAsnMetThrProAlaVal290295300AlaAlaLeuPheProAsnAlaGlnProPheGluHisHisAlaThrVal305310315320GlyLeuThrLeuArgIleGluSerAlaValCysGluSer ValLeuAla325330335AspAlaSerGluThrMetLeuAlaAsnValThrSerValArgGlnGlu340345 350TyrAlaIleProValGlyProValPheProProGlyMetAsnTrpThr355360365AspLeuIleThrAsnTyrSerProSerArgGluAspAsnL euGlnArg370375380ValPheThrValAlaSerIleArgSerMetLeuValLys385390395(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 397 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:MetAspValLeuTyrSerLeuSerLysThrLeuLysAspAlaArgAsp15 1015LysIleValGluGlyThrLeuTyrSerAsnValSerAspLeuIleGln202530GlnPheAsnGlnMetIleI leThrMetAsnGlyAsnGluPheGlnThr354045GlyGlyIleGlyAsnLeuProIleArgAsnTrpAsnPheAspPheGly5055 60LeuLeuGlyThrThrLeuLeuAsnLeuAspAlaAsnTyrValGluThr65707580AlaArgAsnThrIleAspTyrPheV alAspPheValAspAsnValCys859095MetAspGluMetValArgGluSerGlnArgAsnGlyIleAlaProGln100 105110SerAspSerLeuIleLysLeuSerGlyIleLysPheLysArgIleAsn115120125PheAspAsnSerSerGluTyrIleGlu AsnTrpAsnLeuProAsnArg130135140ArgGlnArgThrGlyPheThrPheHisLysProAsnIlePheProTyr145150 155160SerAlaSerPheThrLeuAsnArgSerGlnProAlaHisAspAsnLeu165170175MetGlyThrMetTrpLeuAsnAla GlySerGluIleGlnValAlaGly180185190PheAspTyrSerCysAlaIleAsnAlaProAlaAsnThrGlnGlnPhe1952 00205GluHisIleValGlnLeuArgArgValLeuThrThrAlaThrIleThr210215220LeuLeuProAspAlaGluArgPheSerPheProA rgValIleThrSer225230235240AlaAspGlyAlaThrThrTrpTyrPheAsnProValIleLeuArgPro245 250255AsnAsnValGluIleGluPheLeuLeuAsnGlyGlnIleIleAsnThr260265270TyrGlnAlaArgPheGlyThrIleIl eAlaArgAsnPheAspThrIle275280285ArgLeuSerPheGlnLeuMetArgProProAsnMetThrProAlaVal290295 300AlaAlaLeuPheProAsnAlaGlnProPheGluHisHisAlaThrVal305310315320GlyLeuThrLeuArgIleGluSerAlaVal CysGluSerValLeuAla325330335AspAlaSerGluThrMetLeuAlaAsnValThrSerValArgGlnGlu340 345350TyrAlaIleProValGlyProValPheProProGlyMetAsnTrpThr355360365AspLeuIleThrAsnTyrSerProSerArg GluAspAsnLeuGlnArg370375380ValPheThrValAlaSerIleArgSerMetLeuValLys385390395(2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 397 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:MetAspValLeuTyrSerLeuSerLysThrLeuLysAspAlaArgAsp1 51015LysIleValGluGlyThrLeuTyrSerAsnValSerAspLeuIleGln202530GlnPheAsn GlnMetIleIleThrMetAsnGlyAsnGluPheGlnThr354045GlyGlyIleGlyAsnLeuProIleArgAsnTrpAsnPheAspPheGly50 5560LeuLeuGlyThrThrLeuLeuAsnLeuAspAlaAsnTyrValGluThr65707580AlaArgAsnThrIle AspTyrPheValAspPheValAspAsnValCys859095MetAspGluMetValArgGluSerGlnArgAsnGlyIleAlaProGln10 0105110SerAspSerLeuArgLysLeuSerGlyIleLysPheLysArgIleAsn115120125PheAspAsnSerSerGl uTyrIleGluAsnTrpAsnLeuGlnAsnArg130135140ArgGlnArgThrGlyPheThrPheHisLysProAsnIlePheProTyr145150 155160SerAlaSerPheThrLeuAsnArgSerGlnProAlaHisAspAsnLeu165170175MetGlyThrMetTrp LeuAsnAlaGlySerGluIleGlnValAlaGly180185190PheAspTyrSerCysAlaIleAsnAlaProAlaAsnThrGlnGlnPhe195 200205GluHisIleValGlnLeuArgArgValLeuThrThrAlaThrIleThr210215220LeuLeuProAspAlaGluArgPhe SerPheProArgValIleAsnSer225230235240AlaAspGlyAlaThrThrTrpTyrPheAsnProValIleLeuArgPro245 250255AsnAsnValGluValGluPheLeuLeuAsnGlyGlnIleIleAsnThr260265270TyrGlnAlaArgPheG lyThrIleIleAlaArgAsnPheAspThrIle275280285ArgLeuSerPheGlnLeuMetArgProProAsnMetThrProThrVal290 295300AlaAlaLeuPheProAsnAlaGlnProPheGluHisHisAlaThrVal305310315320GlyLeuThrLeuArgIleGl uSerAlaValCysGluSerValLeuAla325330335AspAlaSerGluThrMetLeuAlaAsnValThrSerValArgGlnGlu340 345350TyrAlaIleProValGlyProValPheProProGlyMetAsnTrpThr355360365AspLeuIleThrAsnTyrSer ProSerArgGluAspAsnLeuGlnArg370375380ValPheThrValAlaSerIleArgSerMetLeuIleLys385390395(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 397 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:MetAspValLeuTyrSerLeuSerLysThrLeuLysAspAlaArgAsp 151015LysIleValGluGlyThrLeuTyrSerAsnValSerAspLeuIleGln202530 GlnPheAsnGlnMetIleIleThrMetAsnGlyAsnGluPheGlnThr354045GlyGlyIleGlyAsnLeuProThrArgAsnTrpSerPheAspPheGly 505560LeuLeuGlyThrThrLeuLeuAsnLeuAspAlaAsnTyrValGluThr65707580AlaArg AsnThrIleAspTyrPheValAspPheValAspAsnValCys859095MetAspGluMetValArgGluSerGlnArgAsnGlyIleAlaProGln 100105110SerGluSerLeuArgLysLeuSerGlyIleLysPheLysArgIleAsn115120125PheAspA snSerSerGluTyrIleGluAsnTrpAsnLeuGlnAsnArg130135140ArgGlnArgThrGlyPheThrPheHisLysProAsnIlePheProTyr145 150155160SerAlaSerPheThrLeuAsnArgSerGlnProAlaHisAspAsnLeu165170175MetGl yThrMetTrpLeuAsnAlaGlySerGluIleHisValAlaGly180185190PheAspTyrSerCysAlaIleAsnAlaProAlaAsnIleGlnGlnPhe 195200205GluHisIleValGlnLeuArgArgValLeuThrThrAlaThrIleThr210215220LeuLeuProAspAla GluArgPheSerPheProArgValIleAsnSer225230235240AlaAspGlyAlaThrThrTrpTyrPheAsnProValIleLeuArgPro 245250255AsnAsnValGluValGluPheLeuLeuAsnGlyGlnIleIleAsnThr260265270TyrGln AlaArgPheGlyThrIleValAlaArgAsnPheAspThrIle275280285ArgLeuSerPheGlnLeuMetArgProProAsnMetThrProSerVal290 295300AlaAlaLeuPheProAsnAlaGlnProPheGluHisHisAlaThrVal305310315320GlyLeuThrL euArgIleGluSerAlaIleCysGluSerValLeuAla325330335AspAlaSerGluThrMetLeuAlaAsnValThrSerValArgGlnGlu 340345350TyrAlaIleProValGlyProValPheProProGlyMetAsnTrpThr355360365AspLeuIleTh rAsnTyrSerProSerArgGluAspAsnLeuHisArg370375380ValPheThrValAlaSerIleArgSerMetLeuValLys385390 395(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 399 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:MetAspValLeuTyrSerLeuSerLysThrLeuLysAspAl aArgAsp151015LysIleValGluGlyThrLeuTyrSerAsnValSerAspLeuIleGln2025 30GlnPheAsnGlnMetValIleThrMetAsnGlyAsnGluPheGlnThr354045GlyGlyIleGlyAsnLeuProIleArgAsnTrpAsnPheAspPh eGly505560LeuLeuGlyThrThrLeuLeuAsnLeuAspAlaAsnTyrValGluThr65707580 AlaArgAsnThrIleAspTyrPheValAspPheValAspAsnValCys859095MetAspGluMetValArgGluSerGlnArgAsnGlyIleAlaPr oGln100105110SerAspSerLeuArgLysLeuSerGlyIleLysPheLysArgIleAsn115120125 PheAspAsnSerSerGluTyrIleGluAsnTrpAsnLeuGlnAsnArg130135140ArgGlnArgLysGlyPheThrPheHisLysProAsnIlePheProTyr1 45150155160SerAlaSerPheThrLeuAsnArgSerGlnProAlaHisAspAsnLeu165170175MetGlyThrMetTrpLeuAsnAlaGlySerGluIleGlnValAlaGly180185190PheAspTyrSerCysAlaIleAsnAlaProAlaAsnThrGlnGln Phe195200205GluHisIleValGlnLeuArgArgValLeuThrThrAlaThrIleThr210215220LeuLe uProAspAlaGluArgPheSerPheProArgValIleAsnSer225230235240AlaAspGlyThrThrThrTrpTyrPheAsnProValIlePheArgPro 245250255AsnAsnValGluIleGluPheLeuLeuAsnGlyGlnIleIleAsnAsn260265270 TyrGlnAlaArgPheGlyThrIleIleAlaArgAsnPheAspThrIle275280285ArgLeuSerPheGlnLeuMetArgProProProGlnAsnMetThrPro 290295300AlaValAlaAlaLeuPheProAsnAlaProProPheGluHisHisAla305310315320 ThrValGlyLeuThrLeuArgIleGluSerAlaIleCysGluSerVal325330335LeuAlaAspAlaSerGluThrMetLeuAlaAsnValThrSerValArg340345350GlnGluTyrAlaValProValGlyProValPheProProGlyMetAsn355360365T rpThrAspLeuIleThrAsnTyrSerProSerArgGluAspAsnLeu370375380GlnArgValPheThrValAlaSerIleArgSerMetLeuIleLys385 390395(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 397 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:MetGluValLeuTyrSerIleSerL ysThrLeuLysAspAlaArgAsp151015LysIleValGluGlyThrLeuTyrSerAsnValSerAspIleIleGln20 2530GlnPheAsnGlnIleIleValThrMetAsnGlyAsnGluPheGlnThr354045GlyGlyIleGlyThrLeuProIleArgA snTrpThrPheAspPheGly505560LeuLeuGlyThrThrLeuLeuAsnLeuAspAlaAsnTyrValGluThr657075 80AlaArgThrThrIleGluTyrPheIleAspPheIleAspAsnValCys859095MetAspGluMetThrArgGluSerGlnA rgAsnGlyIleAlaProGln100105110SerAspAlaLeuArgLysLeuSerGlyIleLysPheLysArgIleAsn115120 125PheAspAsnSerSerGluTyrIleGluAsnTrpAsnLeuGlnAsnArg130135140ArgGlnArgThrGlyPheValPheHisLysProAsnIl ePheProTyr145150155160SerAlaSerPheThrLeuAsnArgSerGlnProLeuHisAsnAspLeu165170 175MetGlyThrMetTrpLeuAsnAlaGlySerGluIleGlnValAlaGly180185190PheAspTyrSerCysAlaIleAsnAlaPro AlaAsnThrGlnGlnPhe195200205GluHisIleValGlnLeuArgArgAlaLeuThrThrAlaThrIleThr210215 220IleLeuProAspAlaGluArgPheSerPheProArgValIleAsnSer225230235240AlaAspGlyAlaThrThrTrpPhePheAsnPro ValIleLeuArgPro245250255AsnAsnValGluValGluPheLeuLeuAsnGlyGlnIleIleAsnThr260265 270TyrGlnAlaArgPheGlyThrIleIleAlaArgAsnPheAspThrIle275280285ArgLeuSerPheGlnLeuMetArgProProAsnM etThrProAlaVal290295300AsnAlaLeuPheProGlnAlaGlnProPheGlnHisHisAlaThrVal305310315 320GlyLeuThrLeuArgIleAspSerAlaValCysGluSerValLeuAla325330335AspSerAsnGluThrMetLeuAlaAsnValTh rAlaValArgGlnGlu340345350TyrAlaValProValGlyProValPheProProGlyMetAsnTrpThr355360 365GluLeuIleThrAsnTyrSerProSerArgGluAspAsnLeuGlnArg370375380ValPheThrValAlaSerIleArgSerMetLeuIleLys 385390395(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 397 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:MetGluValLeuTyr SerLeuSerLysThrLeuLysAspAlaArgAsp151015LysIleValGluGlyThrLeuTyrSerAsnValSerAspLeuIleGln20 2530GlnPheAsnGlnMetIleValThrMetAsnGlyAsnAspPheGlnThr354045GlyGlyIleGlyAsnLeu ProValArgAsnTrpThrPheAspPheGly505560LeuLeuGlyThrThrLeuLeuAsnLeuAspAlaAsnTyrValGluThr6570 7580AlaArgThrThrIleGluTyrPheIleAspPheIleAspAsnValCys859095MetAspGluMetAlaArg GluSerGlnArgAsnGlyIleAlaProGln100105110SerGluAlaPheArgLysLeuAlaGlyIleLysPheLysArgIleAsn115 120125PheAspAsnSerSerGluTyrIleGluAsnTrpAsnLeuGlnAsnArg130135140ArgGlnArgThrGlyPheIlePheHisL ysProAsnIlePheProTyr145150155160SerAlaSerPheThrLeuAsnArgSerGlnProMetHisAspAsnLeu165 170175MetGlyThrMetTrpLeuAsnAlaGlySerGluIleGlnValAlaGly180185190PheAspTyrSerCysAlaIl eAsnAlaProAlaAsnIleGlnGlnPhe195200205GluHisIleValGlnLeuArgArgAlaLeuThrThrAlaThrIleThr210215 220LeuLeuProAspAlaGluArgPheSerPheProArgValIleAsnSer225230235240AlaAspGlyAlaThrThrTrpPhe PheAsnProValIleLeuArgPro245250255AsnAsnValGluValGluPheLeuLeuAsnGlyGlnIleIleAsnThr260 265270TyrGlnAlaArgPheGlyThrIleValAlaArgAsnPheAspThrIle275280285ArgLeuSerPheGlnLeuMetArg ProProAsnMetThrProAlaVal290295300AspAlaLeuPheProGlnAlaGlnProPheGlnHisHisAlaThrVal305310 315320GlyLeuThrLeuArgIleGluSerAlaValCysGluSerValLeuAla325330335AspAlaAsnGluThrLeuLeuA laAsnValThrAlaValArgGlnGlu340345350TyrAlaIleProValGlyProValPheProProGlyMetAsnTrpThr355 360365GluLeuIleThrAsnTyrSerProSerArgGluAspAsnLeuGlnArg370375380ValPheThrValAlaSerIleArgSerMetLe uIleLys385390395(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 397 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:MetGlu ValLeuTyrSerLeuSerLysThrLeuLysAspAlaArgAsp151015LysIleValGluGlyThrLeuTyrSerAsnValSerAspLeuIleGln 202530GlnPheAsnGlnMetIleValThrMetAsnGlyAsnAspPheGlnThr354045GlyGlyIle GlyAsnLeuProValArgAsnTrpThrPheAspPheGly505560LeuLeuGlyThrThrLeuLeuAsnLeuAspAlaAsnTyrValGluAsn65 707580AlaArgThrIleIleGluTyrPheIleAspPheIleAspAsnValCys859095MetAspGlu MetAlaArgGluSerGlnArgAsnGlyValAlaProGln100105110SerGluAlaLeuArgLysLeuAlaGlyIleLysPheLysArgIleAsn 115120125PheAspAsnSerSerGluTyrIleGluAsnTrpAsnLeuGlnAsnArg130135140ArgGlnArgThrGlyPhe ValPheHisLysProAsnIlePheProTyr145150155160SerAlaSerPheThrLeuAsnArgSerGlnProMetHisAspAsnLeu 165170175MetGlyThrMetTrpLeuAsnAlaGlySerGluIleGlnValAlaGly180185190PheAspTyrS erCysAlaIleAsnAlaProAlaAsnIleGlnGlnPhe195200205GluHisIleValGlnLeuArgArgAlaLeuThrThrAlaThrIleThr210 215220LeuLeuProAspAlaGluArgPheSerPheProArgValIleAsnSer225230235240AlaAspGlyAlaTh rThrTrpPhePheAsnProValIleLeuArgPro245250255AsnAsnValGluValGluPheLeuLeuAsnGlyGlnIleIleAsnThr 260265270TyrGlnAlaArgPheGlyThrIleIleAlaArgAsnPheAspAlaIle275280285ArgLeuLeuPheGln LeuMetArgProProAsnMetThrProAlaVal290295300AsnAlaLeuPheProGlnAlaGlnProPheGlnHisHisAlaThrVal305310 315320GlyLeuThrLeuArgIleGluSerAlaValCysGluSerValLeuAla325330335AspAlaAsnGlu ThrLeuLeuAlaAsnValThrAlaValArgGlnGlu340345350TyrAlaIleProValGlyProValPheProProGlyMetAsnTrpThr355 360365GluLeuIleThrAsnTyrSerProSerArgGluAspAsnLeuGlnArg370375380ValPheThrValAlaSerIleA rgSerMetLeuIleLys385390395(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 394 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: MetAspValLeuPheSerIleAlaLysThrValSerAspLeuLysLys151015LysValValValGlyThrIleTyrThrAsnValGluAspIleIl eGln202530GlnThrAsnGluLeuIleArgThrLeuAsnGlyAsnThrPheHisThr354045 GlyGlyIleGlyThrGlnProGlnLysGluTrpAsnPheGlnLeuPro505560GlnLeuGlyThrThrLeuLeuAsnLeuAspAspAsnTyrValGlnAla65 707580ThrArgSerValIleAspTyrLeuAlaSerPheIleGluAlaValCys859095 AspAspGluIleValArgGluAlaSerArgAsnGlyMetGlnProGln100105110SerProThrLeuIleAlaLeuAlaSerSerLysPheLysThrIleAsn 115120125PheAsnAsnSerSerGlnSerIleLysAsnTrpSerAlaGlnSerGly130135140ValArgIle GlnPheMetAsnIleAsnProMetValPheGluTyrArg145150155160AsnSerTyrIleLeuGlnArgAlaAsnProGlnTyrGlyAsnValMet 165170175GlyLeuArgTyrTyrThrAlaSerAsnThrCysGlnLeuAlaAlaPhe180185190 AspSerThrLeuAlaGluAsnAlaProAsnAsnThrGlnArgPheIle195200205TyrAsnGlyArgLeuLysArgProIleSerAsnValLeuMetLysIle 210215220GluAlaGlyAlaProAsnIleAsnAsnLeuThrIleLeuProAspPro225230235240ThrA snGlnThrThrTrpLeuTyrAsnProAspGlnLeuMetAsnGly245250255ThrPheThrIleGluPheTyrAsnAsnGlyGlnLeuValAspMetVal 260265270ArgAsnMetGlyValValThrValArgThrPheAspSerTyrArgIle275280285ThrIl eAspMetIleArgProAlaAlaMetThrGlnTyrValGlnArg290295300LeuPheProGlnGlyGlyProTyrProTyrGlnAlaAlaTyrMetLeu305 310315320ThrLeuSerIleLeuAspAlaThrThrGluSerValLeuCysAspSer325330335His SerValAspTyrSerIleValAlaAsnTrpArgArgAspSerAla340345350MetProAlaGlyThrValPheGlnProGlyPheProTrpGluGlnThr 355360365LeuSerAsnTyrThrValAlaGlnGluAspAsnLeuGluArgLeuLeu370375380LeuValAlaSer ValLysArgMetValMet385390(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 775 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:MetAlaSerL euIleTyrArgGlnLeuLeuSerAsnSerTyrValThr151015AsnIleSerAspGluValAsnGluIleGlyThrLysLysThrThrAsn 202530ValThrValAsnProGlyProPheAlaGlnThrGlyTyrAlaProVal354045AspTrpGlyHisG lyGluLeuProAspSerThrLeuValGlnProThr505560LeuAspGlyProTyrGlnProThrLeuAsnLeuProValThrAspTyr6570 7580TrpMetLeuIleAlaProThrArgGluGlyLysValAlaGluGlyThr859095AsnThrThrAspA rgTrpPheAlaCysValLeuValGluProAsnVal100105110GlnAsnThrGlnArgGlnTyrValLeuAspGlyGlnAsnValGlnLeu115 120125HisValSerAsnAspSerSerThrSerTrpLysPheIleLeuPheIle130135140LysLeuThrProTyrGlyThrTy rThrGlnTyrSerThrLeuSerThr145150155160ProHisLysLeuCysAlaTrpMetLysArgAspAsnArgValTyrTrp165 170175TyrGlnGlyAlaThrProAsnAlaSerGluSerTyrTyrLeuThrIle180185190AsnAsnAspAsnSer AsnValSerSerAspAlaGluPheTyrLeuIle195200205ProGlnSerGlnThrAlaMetCysThrGlnTyrIleAsnAsnGlyLeu210 215220ProProIleGlnAsnThrArgAsnIleValProValAsnIleThrSer225230235240ArgGlnIleLysAspVal ArgAlaGlnMetAsnGluAspIleValIle245250255SerLysThrSerLeuTrpLysGluMetGlnTyrAsnArgAspIleIle260 265270IleArgPheLysPheAlaAsnSerIleIleLysSerGlyGlyLeuGly275280285TyrLysTrpSerGluIleS erPheLysProMetAsnTyrGlnTyrThr290295300TyrThrArgAspGluGluGluValThrAlaHisThrThrCysSerVal305310 315320AsnGlyValAsnAspPheAsnTyrAsnGlyGlyThrLeuProThrAsp325330335PheAlaIleSerArgPh eGluValIleLysGluAsnSerTyrValTyr340345350ValAspTyrTrpAspAspSerGlnAlaPheArgAsnMetValTyrVal355 360365ArgSerLeuAlaAlaAsnLeuAsnAspValValCysThrGlyGlySer370375380TyrSerPheAlaLeuProValGlyAsn HisProValMetSerGlyGly385390395400AlaValThrLeuThrSerAlaGlyValThrLeuSerThrGlnTyrThr405 410415AspTyrValSerLeuAsnSerLeuGlnPheArgPheArgLeuAlaVal420425430SerGluProSerPheSer IleSerArgThrArgMetSerGlyIleTyr435440445GlyLeuProAlaValAsnProAsnAsnSerAlaGluTyrTyrGluIle4504 55460AlaGlyArgPheSerLeuIleSerLeuValProThrAsnAspAspTyr465470475480GlnThrProIleAlaAsnSerV alThrValArgGlnAspLeuGluArg485490495GlnLeuGlyGluLeuArgGluGluPheAsnSerLeuSerGlnGluIle500 505510AlaValSerGlnLeuIleAspLeuAlaThrLeuProLeuAspMetPhe515520525SerMetPheSerGlyIleLysSe rThrValGluAlaValLysSerMet530535540ThrThrAsnValMetLysArgPheLysThrSerSerLeuAlaAsnAla545550 555560IleSerAspLeuThrSerAsnMetSerGluAlaAlaSerSerValArg565570575LeuThrSerValArgSerVal GlyThrIleThrLeuProArgAlaArg580585590ValSerLeuGlnValGlyAspAspLeuArgSerMetGlnAspValSer595 600605ThrGlnValSerAsnValSerArgAsnLeuArgLeuLysGluPheThr610615620ThrGlnThrAspThrLeuSerPheAspAsp IleSerAlaAlaValLeu625630635640LysThrLysLeuAspLysSerThrGlnIleSerGlnGlnThrMetPro645 650655AspIleIleAlaGluSerSerGluLysPheIleProLysArgSerTyr660665670ArgIleValAspGluAspIleA rgPheGluThrGlyIleAspGlyThr675680685PheTyrAlaTyrLysValAspThrPheAsnGluIleProPheAspMet690695 700GluArgPheAsnLysLeuIleThrAspSerProValLeuSerAlaIle705710715720IleAspPheLysThrLeuLysAsnLe uAsnAspAsnTyrGlyIleThr725730735LysLysGlnAlaMetGluLeuLeuHisSerAsnProLysThrLeuLys740 745750GluPheIleAsnAsnAsnAsnProIleIleArgAsnArgIleGluAsn755760765LeuIleSerGlnCysArgLeu 770775(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 775 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:MetAlaSerLeuIleTyrArgGlnLeuLeu ThrAsnSerTyrSerVal151015AspLeuHisAspGluIleGluGlnIleGlySerGluLysThrGlnAsn2025 30ValThrValAsnProGlyProPheAlaGlnThrArgTyrAlaProVal354045AsnTrpGlyHisGlyGluIleAsnAspSerThr ThrValGluProIle505560LeuAspGlyProTyrGlnProThrThrPheLysProLeuThrAspTyr657075 80TrpIleLeuIleAsnSerAsnThrAsnGlyValValTyrGluSerThr859095AsnAsnSerAspPheTrpThrAlaValValAla IleGluProHisVal100105110IleGlnValAspArgGlnTyrThrValPheGlyGluAsnLysGlnPhe115120 125AsnValArgAsnAspSerAspLysTrpLysPheLeuGluMetPheArg130135140GlySerSerGlnAsnGluPheTyrAsnArgArgThrLeuThrS erAsp145150155160ThrLysLeuValGlyIleLeuLysTyrGlyGlyArgIleTrpThrPhe165170 175HisGlyGluThrProArgAlaThrThrAspSerSerAsnThrAlaAsn180185190LeuAsnAspIleSerIleIleIleHisSerGluPh eTyrIleIlePro195200205ArgSerGlnGluSerLysCysAsnGluTyrIleAsnAsnGlyLeuPro210215220ProIleGlnAsnThrArgAsnValValProLeuSerLeuSerSerArg225230235240SerIleGlnTyrLysArgAlaGlnValAsnGluAspIle ThrIleSer245250255LysThrSerLeuTrpLysGluMetGlnCysAsnArgAspIleIleIle260265 270ArgPheLysPheGlyAsnSerIleValLysLeuGlyGlyLeuGlyTyr275280285LysTrpSerGluIleSerTyrLysAlaAlaAsnTyrGln TyrAsnTyr290295300LeuArgAspGlyGluGlnValThrAlaHisThrThrCysSerValAsn305310315 320GlyValAsnAsnPheSerTyrAsnGlyGlySerLeuProThrAspPhe325330335SerValSerArgTyrGluValIleLysGluAsnSerT yrValTyrVal340345350AspTyrTrpAspAspSerLysAlaPheArgAsnMetValTyrValArg355360 365SerLeuAlaAlaAsnLeuAsnSerValLysCysThrGlyGlySerTyr370375380AspPheSerIleProValGlyAlaTrpProValMetAsnGlyGlyAl a385390395400ValSerLeuHisPheAlaGlyValThrLeuSerThrGlnPheThrAsp405410 415PheValSerLeuAsnSerLeuArgPheArgPheSerLeuThrValAsp420425430GluProSerPheSerIleLeuArgThrArgThrValAsn LeuTyrGly435440445LeuProAlaAlaAsnProAsnAsnGlyAsnGluTyrTyrGluIleSer450455460 GlyArgPheSerLeuIleSerLeuValProThrAsnAspAspTyrGln465470475480ThrProIleMetAsnSerValThrValArgGlnAspLeuGlu ArgGln485490495LeuThrAspLeuArgGluGluPheAsnSerLeuSerGlnGluIleAla500505 510MetSerGlnLeuIleAspLeuAlaLeuLeuProLeuAspMetPheSer515520525MetPheSerGluLeuLysSerThrIleAspLeuThrLysSerM etAla530535540ThrSerValMetLysLysPheArgLysSerLysLeuAlaThrSerIle54555055556 0SerGluMetThrHisSerLeuSerAspAlaAlaSerSerAlaSerArg565570575SerValSerIleArgSerAsnIleSerThrIleSerAsnTr pThrAsn580585590ValSerAsnAspValSerAsnValThrAsnSerLeuSerAspIleSer595600605ThrGlnThrSerThrIleSerLysAsnLeuArgLeuLysGluMetIle610615620ThrGlnThrGluGlyMetSerPheAspAspIleSerAlaAlaValLeu 625630635640LysThrLysIleAspMetSerThrGlnIleGlyLysAsnThrLeuPro645650 655AspIleValThrGluAlaSerGluLysPheIleProLysArgSerTyr660665670ArgIleLeuLysAspAspGluValMetGluIleAsnThrGlu GlyLys675680685ValPheAlaTyrLysIleAspThrLeuAsnGluValProPheAspVal690695700As nLysPheAlaGluLeuValThrAsnSerProValIleSerAlaIle705710715720IleAspPheLysThrLeuLysAsnLeuAsnAspAsnTyrGlyIleT hr725730735ArgIleGluAlaLeuAsnLeuIleLysSerAsnProAsnValLeuArg74074575 0AsnPheIleAsnGlnAsnAsnProIleIleArgAsnArgIleGluGln755760765LeuIleLeuGlnCysLysLeu770775(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 775 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:MetAlaSerLeuIleTyrArgGlnLeuLeuThrAsnSerTyrSerVal 151015AspLeuHisAspGluIleGluGlnIleGlySerGluLysThrGlnSer202530 ValThrValAsnProGlyProPheAlaGlnThrArgTyrAlaProVal354045AsnTrpGlyHisGlyGluIleAsnAspSerThrThrValGluProVal 505560LeuAspGlyProTyrGlnProThrThrPheLysProProThrAspTyr65707580TrpLeu LeuIleSerSerAsnThrAsnGlyValValTyrGluSerThr859095AsnAsnAsnAspPheTrpThrAlaValIleAlaIleGluProHisVal 100105110SerGlnValAsnArgGlnTyrThrLeuPheGlyGluAsnLysGlnPhe115120125AsnValG luAsnAsnSerAspLysTrpLysPhePheGluMetPheLys130135140GlySerSerGlnGlyAsnPheSerAsnArgArgThrLeuThrSerSer145 150155160AsnArgLeuValGlyMetLeuLysTyrGlyGlyArgValTrpThrPhe165170175HisGl yGluThrProArgAlaThrThrAspSerSerAsnThrAlaAsp180185190LeuAsnAsnIleSerIleIleIleHisSerGluPheTyrIleIlePro 195200205ArgSerGlnGluSerLysCysAsnGluTyrIleAsnAsnGlyLeuPro210215220ProIleGlnAsnThr ArgAsnValValProLeuSerLeuSerSerArg225230235240SerIleGlnTyrArgArgAlaGlnValAsnGluAspIleThrIleSer 245250255LysThrSerLeuTrpLysGluMetGlnTyrAsnArgAspIleIleIle260265270ArgPhe LysPheGlyAsnSerValIleLysLeuGlyGlyLeuGlyTyr275280285LysTrpSerGluIleSerTyrLysAlaAlaAsnTyrGlnTyrSerTyr290 295300SerArgAspGlyGluGlnValThrAlaHisThrThrCysSerValAsn305310315320GlyValAsnA snPheSerTyrAsnGlyGlySerLeuProThrAspPhe325330335SerIleSerArgTyrGluValIleLysGluAsnSerTyrValTyrIle 340345350AspTyrTrpAspAspSerLysAlaPheArgAsnMetValTyrValArg355360365SerLeuAlaAl aAsnLeuAsnSerValLysCysThrGlyGlySerTyr370375380AsnPheArgLeuProValGlyLysTrpProIleMetAsnGlyGlyAla385 390395400ValSerLeuHisPheAlaGlyValThrLeuSerThrGlnPheThrAsp405410415PheValSer LeuAsnSerLeuArgPheArgPheSerLeuThrValAsp420425430GluProSerPheSerIleLeuArgThrArgThrIleAsnLeuTyrGly 435440445LeuProAlaAlaAsnProAsnAsnGlyAsnGluTyrTyrGluMetSer450455460GlyArgPheSerLeuIle SerLeuValGlnThrAsnAspAspTyrGln465470475480ThrProIleMetAsnSerValThrValArgGlnAspLeuGluArgGln 485490495LeuAsnAspLeuArgGluGluPheAsnSerLeuSerGlnGluIleAla500505510MetSerGlnL euIleAspLeuAlaLeuLeuProLeuAspMetPheSer515520525MetPheSerGlyIleLysSerThrIleAspLeuThrLysSerMetAla530 535540ThrSerValMetLysLysPheArgLysSerLysLeuAlaThrSerIle545550555560SerGluMetThrAs nSerLeuSerAspAlaAlaSerSerAlaSerArg565570575SerAlaSerIleArgSerAsnIleSerThrIleSerAsnTrpThrAsn 580585590ThrSerLysSerValSerAsnValThrAspSerValAsnAspIleSer595600605ThrGlnThrSerThr IleSerLysLysLeuArgLeuArgGluMetIle610615620ThrGlnThrGluGlyLeuSerPheAspAspIleSerAlaAlaValLeu625630 635640LysThrLysIleAspMetSerThrGlnIleGlyLysAsnThrLeuPro645650655AspIleValThr GluAlaSerGluLysPheIleProLysArgSerTyr660665670ArgValLeuLysAspAspGluValMetGluIleAsnThrGluGlyLys675 680685PhePheAlaTyrLysValAspThrLeuAsnGluIleProPheAspIle690695700AsnLysPheAlaGluLeuValT hrAspSerProValIleSerAlaIle705710715720IleAspPheLysThrLeuLysAsnLeuAsnAspAsnTyrGlyIleThr72 5730735ArgIleGluAlaPheAsnLeuIleLysSerAsnProAsnValLeuArg740745750AsnPheIleAsnGl nAsnAsnProIleIleArgAsnArgIleGluGln755760765LeuIleLeuGlnCysLysLeu770775(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 775 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:MetAlaSerLeuIleTyrArgGlnLeuLeuThrAsnSerTyrSerVal15 1015GluLeuSerAspGluIleAsnThrIleGlySerGluLysThrGlnAsn202530ValThrIleAsnProGlyPr oPheAlaGlnThrAsnTyrAlaProVal354045ValLeuGluSerTrpGluValAsnAspSerThrThrIleGluProVal5055 60LeuAspGlyProTyrGlnProThrPheLysProProSerThrAspTyr65707580TrpIleLeuLeuAsnProThrAspGl nGlnValValLeuGluGlyThr859095AsnLysThrAspIleTrpIleAlaLeuLeuLeuValGluProAsnVal100 105110ThrAsnGlnSerArgGlnTyrThrLeuPheGlyGluThrLysGlnIle115120125ThrValGluAsnAsnThrAsnLysTrp LysPhePheGluMetPheArg130135140LysAsnValSerAlaGluPheGlnHisLysArgThrLeuThrSerAsp1451501 55160ThrLysLeuAlaGlyPheLeuLysHisTyrAsnSerValTrpThrPhe165170175HisGlyGluThrProHisAlaThrT hrAspTyrSerSerThrSerAsn180185190LeuSerGluValGluThrValIleHisValGluPheTyrIleIlePro19520 0205ArgSerGlnGluSerLysCysValGluTyrIleAsnThrGlyLeuPro210215220ProMetGlnAsnThrArgAsnIleValProValAl aLeuSerSerArg225230235240SerValThrTyrGlnArgAlaGlnValAsnGluAspIleIleIleSer245 250255LysThrSerLeuTrpLysGluMetGlnCysAsnArgAspIleIleIle260265270ArgPheLysPheAsnAsnSerIleVal LysLeuGlyGlyLeuGlyTyr275280285LysTrpSerGluIleSerPheLysAlaAlaAsnTyrGlnTyrAsnTyr290295 300LeuArgAspGlyGluGlnValThrAlaHisThrThrCysSerValAsn305310315320GlyValAsnAsnPheSerTyrAsnGlyGly SerLeuProThrAspPhe325330335SerValSerArgTyrGluValIleLysGluAsnSerTyrValTyrVal3403 45350AspTyrTrpAspAspSerGlnAlaPheArgAsnMetValTyrValArg355360365SerLeuAlaAlaAsnLeuAsnSerValLysC ysThrGlyGlyAsnTyr370375380AsnPheGlnLeuProValGlyAlaTrpProValMetSerGlyGlyAla385390395 400ValSerLeuHisPheAlaGlyValThrLeuSerThrGluPheThrAsp405410415PheValSerLeuAsnSerLeuArgPheAr gPheSerLeuThrValGlu420425430GluProProPheSerIleLeuArgThrArgValSerGlyLeuTyrGly435440 445LeuProAlaPheAsnProAsnSerGlyHisGluTyrTyrGluIleAla450455460GlyArgPheSerPheIleLeuLeuValProSerAsnAsp AspTyrGln465470475480ThrProIleMetAsnSerValThrValArgGlnAspLeuGluArgGln485490 495LeuGlyAspLeuArgGluGluPheAsnSerLeuSerGlnGluIleAla500505510MetThrGlnLeuIleAspLeuAlaLeuLeu ProLeuAspMetPheSer515520525MetPheSerGlyIleLysSerThrIleAspAlaAlaLysSerMetAla530535 540ThrLysValMetLysLysPheLysArgSerGlyLeuAlaThrSerIle545550555560SerGluLeuThrGlySerLeuSerAsnAlaAlaS erSerIleSerArg565570575SerSerSerIleArgSerAsnIleSerSerIleSerValTrpThrAsp580585 590ValSerGluGlnIleAlaGlySerSerAspSerValSerAsnIleSer595600605ThrGlnMetSerAlaIleSerArgArgLeuArgLe uArgGluIleThr610615620ThrGlnThrGluGlyMetAsnPheAspAspIleSerAlaAlaValLeu625630635 640LysThrLysIleAspArgSerThrHisIleSerProAspThrLeuPro645650655AspIleMetThrGluSerSerLysLysPheIle ProLysArgAlaTyr660665670ArgValLeuLysAspAspGluValMetGluAlaAspValAspGlyLys675680 685PhePheAlaTyrLysValAspThrPheGluGluValProPheAspVal690695700AspLysPheValAspLeuValThrAspSerProValIleSer AlaIle705710715720IleAspPheLysThrLeuLysAsnLeuAsnAspAsnTyrGlyIleThr725730 735ArgSerGlnAlaLeuAspLeuIleArgSerAspProArgValLeuArg740745750AspPheIleAsnGlnAsnAsnProIleIleLysA snArgIleGluGln755760765LeuIleLeuGlnCysArgLeu770775(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 775 amino acids (B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:MetAlaSerLeuIleTyrArgGlnLeuLeuThrAsnSerTyrThrVal1510 15GluLeuSerAspGluIleAsnThrIleGlySerGluLysSerGlnAsn202530IleThrIleAsnProGlyProPheAlaGlnThrAsnTyrA laProVal354045ValLeuGluSerTrpGluValAsnAspSerThrThrIleGluProVal505560L euAspGlyProTyrGlnProThrPheLysProProSerThrAspTyr65707580TrpIleLeuLeuAsnProThrAsnGlnGlnValValLeuGluGlyT hr859095AsnLysThrAspIleTrpIleAlaLeuLeuLeuValGluProAsnVal100105110ThrAsnGlnSerArgGlnTyrThrLeuPheGlyGluThrLysGlnIle115120125ThrValGluAsnAsnThrAsnLysTrpLysPhePheGluMetPheArg130135140SerSerValSerSerGluPheGlnHisLysArgThrLeuThrSerAsp145150155160 ThrLysLeuAlaGlyPheLeuLysHisTyrAsnSerValTrpSerPhe165170175HisGlyGluThrProHisAlaThrThrAspTyrSerSerThrSer Asn180185190LeuSerGluValGluThrValIleHisValGluPheTyrIleIleSer195200205 ArgSerGlnGluSerLysCysValGluTyrIleAsnThrGlyLeuPro210215220ProMetGlnAsnThrArgAsnIleValProValAlaLeuSerSerArg225 230235240SerValThrTyrGlnArgAlaGlnValAsnGluAspIleIleIleSer245250255 LysThrSerLeuTrpLysGluMetGlnTyrAsnArgAspIleIleIle260265270ArgPheLysPheAsnAsnSerIleIleLysLeuGlyGlyLeuGlyTy r275280285LysTrpSerGluIleSerPheLysAlaAlaAsnTyrGlnTyrAsnTyr290295300LeuArg AspGlyGluGlnValThrAlaHisThrThrCysSerValAsn305310315320GlyValAsnAsnPheSerTyrAsnGlyGlyLeuLeuProThrHisPhe 325330335SerValSerArgTyrGluValIleLysGluAsnSerTyrValTyrVal340345350 AsnTyrTrpAspAspSerGlnAlaLeuArgAsnMetValTyrValArg355360365SerLeuAlaAlaAsnLeuAsnSerValLysCysThrGlyGlyAsnTyr 370375380AsnPheGlnLeuProValGlyAlaTrpProValMetSerGlyGlyAla385390395400Va lSerLeuHisPheAlaGlyValThrLeuSerThrLysPheThrAsp405410415PheValSerLeuAsnSerLeuArgPheArgPheSerLeuThrValGlu 420425430GluProProPheSerIleLeuArgThrArgValSerGlyLeuTyrGly435440445Leu ProAlaSerAsnProAsnSerGlyHisGluTyrTyrGluIleAla450455460GlyArgPheSerLeuIleSerLeuValProSerAsnAspAspTyrGln465 470475480ThrProIleMetAsnSerIleThrValArgGlnAspLeuGluArgGln485490495 LeuGlyAspLeuArgGluGluPheAsnSerLeuSerGlnGluIleAla500505510IleThrGlnLeuIleAspLeuAlaLeuLeuProLeuAspMetPheSer 515520525MetPheSerGlyIleLysSerThrIleAspAlaAlaLysSerMetAla530535540ThrLysValM etLysLysPheLysArgSerGlyLeuAlaThrSerIle545550555560SerGluLeuThrArgSerLeuSerAsnAlaAlaSerSerIleSerArg 565570575SerSerSerIleArgSerAsnIleSerSerValSerGluTrpThrAsp580585590Va lSerGluGlnIleAlaGlySerSerAspSerValArgAsnIleSer595600605ThrGlnIleSerAlaIleSerArgArgLeuArgLeuArgGluIleThr 610615620ThrGlnThrGluGlyMetAsnPheIleAspIleSerAlaAlaValLeu625630635640LysThr LysIleAspArgSerThrHisIleArgProAspThrLeuPro645650655AspIleIleThrGluSerSerGluLysPheIleProLysArgAlaTyr 660665670ArgValLeuLysAspAspGluValMetGluAlaAspValAspGlyLys675680685PhePhe AlaTyrLysValAspThrPheGluGluValProPheAspVal690695700AspLysPheValAspLeuValThrAspSerProValIleSerAlaIle705 710715720IleAspPheLysThrLeuLysAsnLeuAsnAspAsnTyrGlyIleThr725730735ArgS erGlnAlaLeuAspLeuIleArgSerAspProArgValLeuArg740745750AspPheIleAsnGlnAsnAsnProIleIleLysAsnArgIleGluGln 755760765LeuIleLeuGlnCysArgLeu770775(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 776 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:MetAlaAlaLeuIleTyrArgGlnLeuLeuThrAsnSerTyrThrVal151015GluLeuSer AspGluIleGlnGluIleGlySerThrLysThrGlnAsn202530ValThrValAsnProGlyProPheAlaGlnThrAsnTyrAlaProVal35 4045AsnTrpGlyProGlyGluThrAsnAspSerThrThrValGluProVal505560LeuAspGlyProTyrGlnPro ThrThrPheAsnProProValSerTyr65707580TrpMetLeuLeuAlaProThrAsnAlaGlyValValValGluGlyThr85 9095AsnAsnThrAsnArgTrpLeuAlaThrIleLeuIleGluProAsnVal100105110GlnGlnValGluArg ThrTyrThrLeuPheGlyGlnGlnValGlnVal115120125ThrValSerAsnAspSerGlnThrLysTrpLysPheValAspLeuSer130 135140LysGlnThrGlnAspGlyAsnTyrSerGlnHisGlySerLeuLeuSer145150155160ThrProLysLeuTyrGly ValMetLysHisGlyGlyLysIleTyrThr165170175TyrAsnGlyGluThrProAsnAlaAsnThrGlyTyrTyrSerThrThr180 185190AsnPheAspThrValAsnMetThrAlaTyrCysAspPheTyrIleIle195200205ProLeuAlaGlnGluAlaL ysCysThrGluTyrIleAsnAsnGlyLeu210215220ProProIleGlnAsnThrArgAsnIleValProValSerIleValSer225230 235240ArgAsnIleValTyrThrArgAlaGlnProAsnGlnAspIleValVal245250255SerLysThrSerLeuTr pLysGluMetGlnTyrAsnArgAspIleVal260265270IleArgPheLysPheAlaAsnSerIleIleLysSerGlyGlyLeuGly275 280285TyrLysTrpSerGluValSerPheLysProAlaPheTyrGlnTyrThr290295300TyrThrArgAspGlyGluGluValThr AlaHisThrThrCysSerVal305310315320AsnGlyValAsnAspPheAsnTyrAsnGlyGlySerLeuProThrAsp325 330335PheValIleSerLysTyrGluValIleLysGluAsnSerPheValTyr340345350IleAspTyrTrpAspAsp SerGlnAlaPheArgAsnMetValTyrVal355360365ArgSerLeuAlaAlaAspLeuAsnSerValMetCysThrGlyGlyAsp3703 75380TyrSerPheAlaLeuProValGlyAsnTyrProValMetThrGlyGly385390395400AlaValSerLeuHisSerAlaG lyValThrLeuSerThrGlnPheThr405410415AspPheValSerLeuAsnSerLeuArgPheArgPheArgLeuSerVal420 425430GluGluProProPheSerIleLeuArgThrArgValSerGlyLeuTyr435440445GlyLeuProAlaAlaLysProAs nAsnSerGlnGluTyrTyrGluIle450455460AlaGlyArgPheSerLeuIleSerLeuValProLeuAsnAspAspTyr465470 475480GlnThrProIleMetAsnSerValThrValArgGlnAspLeuGluArg485490495GlnLeuGlyGluLeuArgAsp GluPheAsnAsnLeuSerGlnGlnIle500505510AlaMetSerGlnLeuIleAspLeuAlaLeuLeuProLeuAspMetPhe515 520525SerMetPheSerGlyIleLysSerThrIleAspAlaAlaLysSerMet530535540AlaThrAsnValMetLysArgPheLysLys SerSerLeuAlaAsnSer545550555560ValSerThrLeuThrAspSerLeuSerAspAlaAlaSerSerIleSer565 570575ArgSerAlaSerValArgSerValSerSerThrAlaSerAlaTrpThr580585590GluValSerAsnIleAlaSerA spIleAsnValThrThrSerSerIle595600605SerThrGlnThrSerThrIleSerArgArgLeuArgLeuLysGluMet610615 620AlaThrGlnThrAspGlyMetAsnPheAspAspIleSerAlaAlaVal625630635640LeuLysThrLysIleAspLysSerTh rGlnLeuAsnThrAsnThrLeu645650655ProGluIleValThrGluAlaSerGluLysPheIleProAsnArgAla660 665670TyrArgValIleLysAspAspGluValLeuGluAlaSerIleAspGly675680685LysTyrPheAlaTyrLysValGluThr PheGluGluIleProPheAsp690695700ValGlnLysPheAlaAspLeuValThrAspSerProValIleSerAla705710 715720IleIleAspPheLysThrLeuLysAsnLeuAsnAspAsnTyrGlyIle725730735SerArgGlnGlnAlaLeuAsnLeu LeuArgSerAspProArgValLeu740745750ArgGluPheIleAsnGlnAspAsnProIleIleArgAsnArgIleGlu7557 60765SerLeuIleMetGlnCysArgLeu770775(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 776 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(i i) MOLECULE TYPE: cDNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:MetAlaSerLeuIleTyrArgGlnLeuLeuThrAsnSerTyrThrVal151015AspLeuSerAspGluIleGlnGluIle GlySerThrLysThrGlnAsn202530ValThrIleAsnLeuGlyProPheAlaGlnThrGlyTyrAlaProVal3540 45AsnTrpGlyProGlyGluThrAsnAspSerThrThrValGluProVal505560LeuAspGlyProTyrGlnProThrSerPheAsnProPro ValAspTyr65707580TrpMetLeuLeuAlaProThrAlaAlaGlyValValValGluGlyThr8590 95AsnAsnThrAspArgTrpLeuAlaThrIleLeuValGluProAsnVal100105110ThrSerGluThrArgSerTyrThrLeuPheGl yThrGlnGluGlnIle115120125ThrIleAlaTyrAlaSerGlnThrGlnTrpLysPheIleAspValVal130135 140LysThrThrGlnAsnGlySerTyrSerGlnTyrGlyProLeuGlnSer145150155160ThrProLysLeuTyrAlaValMetLysHisAsnGly LysIleTyrThr165170175TyrAsnGlyGluThrProAsnValThrThrLysTyrTyrSerThrThr180185 190AsnTyrAspSerValAsnMetThrAlaPheCysAspPheTyrIleIle195200205ProArgGluGluGluSerThrCysThrGluTyrIle AsnAsnGlyLeu210215220ProProIleGlnAsnThrArgAsnIleValProLeuAlaLeuSerAla225230235 240ArgAsnIleIleSerHisArgAlaGlnAlaAsnGluAspIleValVal245250255SerLysThrSerLeuTrpLysGluMetGlnTyrA snArgAspIleThr260265270IleArgPheLysPheAlaSerSerIleValLysSerGlyGlyLeuGly275280 285TyrLysTrpSerGluIleSerPheLysProAlaAsnTyrGlnTyrThr290295300TyrThrArgAspGlyGluAspValThrAlaHisThrThrCysSe rVal305310315320AsnGlyMetAsnAspPheAsnPheAsnGlyGlySerLeuProThrAsp325330 335PheIleIleSerArgTyrGluValIleLysGluAsnSerTyrValTyr340345350ValAspTyrTrpAspAspSerGlnAlaPheArgAsn MetValTyrVal355360365ArgSerLeuAlaAlaAsnLeuAsnSerValIleCysThrGlyGlyAsp370375380TyrSerPheAlaLeuProValGlyGlnTrpProValMetThrGlyGly385390395400AlaValSerLeuHisSerAlaGlyValThrLeuSerThr GlnPheThr405410415AspPheValSerPheAsnSerLeuArgPheArgPheArgLeuThrVal420425 430GluGluProSerPheSerIleThrArgThrArgValGlyGlyLeuTyr435440445GlyLeuProAlaAlaTyrProAsnAsnGlyLysGluTyrT yrGluVal450455460AlaGlyArgLeuSerLeuIleSerLeuValProSerAsnAspAspTyr465470475 480GlnThrProIleThrAsnSerValThrValArgGlnAspLeuGluArg485490495GlnLeuGlyGluLeuArgGluGluPheAsnAlaLeuSe rGlnGluIle500505510AlaMetSerGlnLeuIleTyrLeuAlaLeuLeuProLeuAspMetPhe515520 525SerMetPheSerGlyIleLysSerThrIleAspAlaAlaLysSerMet530535540AlaThrSerValMetLysLysPheLysLysSerGlyLeuAlaAsnSer545550555560ValSerThrLeuThrAspSerLeuSerAspAlaAlaSerSerIleSer565570 575ArgGlyAlaSerIleArgSerValGlySerSerAlaSerAlaTrpThr580585590AspValSerThrGlnIleThrAspValSerSerSerVal SerSerIle595600605SerThrGlnThrSerThrIleSerArgArgLeuArgLeuLysGluMet610615620 AlaThrGlnThrGluGlyMetAsnPheAspAspIleSerAlaAlaVal625630635640LeuLysThrLysIleAspArgSerThrGlnIleSerProAsnT hrLeu645650655ProAspIleValThrGluAlaSerGluLysPheIleProAsnArgAla660665 670TyrArgValIleAsnAsnAspGluValPheGluAlaGlyThrAspGly675680685ArgTyrPheAlaTyrArgValGluThrPheAspGluIleProPh eAsp690695700ValGlnLysPheAlaAspLeuValThrAspSerProValIleSerAla705710715720IleIleAspPheLysThrLeuLysAsnLeuAsnAspAsnTyrGlyIle725730735SerArgGlnGlnAlaPheAsnLeuLeuArgSerAspProArg ValLeu740745750ArgGluPheIleAsnGlnAspAsnProIleIleArgAsnArgIleGlu755760765GlnLeuIleMetGlnCysArgLeu770775(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2363 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE: (A) NAME/KEY: CDS(B) LOCATION: 10..2337(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:GGCTATAAAATGGCTTCACTCATTTATAGACAGTTGCTTACTAATTCA48MetAlaSerLeuIleTyrArgGlnLeuLeuThrAsnSer1 510TACACAGTAGAACTTTCAGATGAAATCCAAGAAATTGGATCGACTAAG96TyrThrValGluLeuSerAspGluIleGlnGluIleGlySerThrLys15 2025ACTCAAAACGTTACCGTTAATCCAGGACCGTTCGCGCAAACAAATTAC144ThrGlnAsnValThrValAsnProGlyProPheAlaGlnThrAsnTyr3035 4045GCTTCAGTTAATTGGGGACCTGGTGAAACGAATGACTCAACTACAGTT192AlaSerValAsnTrpGlyProGlyGluThrAsnAspSerThrThrVal50 5560GAACCAGTGCTTGATGGACCATATCAACCAACGACTTTTAATCCACCT240GluProValLeuAspGlyProTyrGlnProThrThrPheAsnProPro65 7075GTAAGTTATTGGATGTTGTTAGCACCAACGAACGCGGGGGTGGTAGAT288ValSerTyrTrpMetLeuLeuAlaProThrAsnAlaGlyValValAsp8085 90CAAGGTACGAACAATACAAACAGATGGTTAGCGACAATATTAATTAAA336GlnGlyThrAsnAsnThrAsnArgTrpLeuAlaThrIleLeuIleLys95100 105CCAAATGTACAGCAAGTTGAGCGAACATATACATTATTTGGGCAACAA384ProAsnValGlnGlnValGluArgThrTyrThrLeuPheGlyGlnGln110115120 125GTTCAAGTAACAGTATCAAATGATTCACAGACAAAGTGGAAGTTTGTG432ValGlnValThrValSerAsnAspSerGlnThrLysTrpLysPheVal130135 140GATCTAAGTAAGCAGACACAAGATGGTAATTATTCACAACACGGTCCT480AspLeuSerLysGlnThrGlnAspGlyAsnTyrSerGlnHisGlyPro145150 155CTACTGTCAACACCGAAACTGTATGGAGTGATGAAACATGGAGGTAAA528LeuLeuSerThrProLysLeuTyrGlyValMetLysHisGlyGlyLys160165 170ATTTACACTTATAATGGAGAGACACCGAACGCAACTACTGGTTACTAC576IleTyrThrTyrAsnGlyGluThrProAsnAlaThrThrGlyTyrTyr175180185TCT ACAACTAACTTTGACACTGTAAACATGACAGCATATTGTGATTTT624SerThrThrAsnPheAspThrValAsnMetThrAlaTyrCysAspPhe190195200205 TATATAATTCCATTAGCACAAGAAGCAAAATGCACTGAATACATAAAT672TyrIleIleProLeuAlaGlnGluAlaLysCysThrGluTyrIleAsn210215220AATGGATTACCACCAATACAAAATACGAGAAATATCGTACCAGTTTCG720AsnGlyLeuProProIleGlnAsnThrArgAsnIleValProValSer225230235A TAGTATCAAGGAATATTGTATATACAAGAGCACAACCTAATCAAGAC768IleValSerArgAsnIleValTyrThrArgAlaGlnProAsnGlnAsp240245250ATAGTG GTATCAAAAACTTCATTATGGAAAGAGATGCAATATAATAGA816IleValValSerLysThrSerLeuTrpLysGluMetGlnTyrAsnArg255260265GATATAGTGATAAGA TTTAAATTTGCTAACTCAATCATAAAATCAGGG864AspIleValIleArgPheLysPheAlaAsnSerIleIleLysSerGly270275280285GGATTGGGATA TAAATGGTCAGAAGTGTCATTTAAACCAGCTAATTAT912GlyLeuGlyTyrLysTrpSerGluValSerPheLysProAlaAsnTyr290295300CAGTACACAT ATACCAGAGATGGTGAAGAAGTTACTGCACATACTACG960GlnTyrThrTyrThrArgAspGlyGluGluValThrAlaHisThrThr305310315TGTTCAGTAAAT GGAATAAATGATTTTAATTATAATGGTGGATCATTA1008CysSerValAsnGlyIleAsnAspPheAsnTyrAsnGlyGlySerLeu320325330CCGACTGATTTCGTAATA TCAAAATATGAAGTGATTAAGGAAAATTCT1056ProThrAspPheValIleSerLysTyrGluValIleLysGluAsnSer335340345TTTGTGTATATAGACTACTGGGACGA TTCACAAGCATTTAGAAACATG1104PheValTyrIleAspTyrTrpAspAspSerGlnAlaPheArgAsnMet350355360365GTATATGTACGCTCGTTGGCAG CCGATTTAAATTCGGTAATGTGTACA1152ValTyrValArgSerLeuAlaAlaAspLeuAsnSerValMetCysThr370375380GGAGGTGACTATAGTTTTGCG ATTCCAGTTGGTAATTATCCAGTTATG1200GlyGlyAspTyrSerPheAlaIleProValGlyAsnTyrProValMet385390395ACTGGGGGTGCTGTGTCATTGCAT TCAGCAGGTGTAACTTTATCAACG1248ThrGlyGlyAlaValSerLeuHisSerAlaGlyValThrLeuSerThr400405410CAGTTTACAGATTTCGTATCATTAAATTC ACTGAGATTTAGATTTAGA1296GlnPheThrAspPheValSerLeuAsnSerLeuArgPheArgPheArg415420425TTATCAGTAGAAGAACCGCCGTTCTCAATTCTACGGA CCAGAGTTAGT1344LeuSerValGluGluProProPheSerIleLeuArgThrArgValSer430435440445GGATTGTATGGACTTCCAGCGGCAAAACCGAAT AATTCACAAGAATAT1392GlyLeuTyrGlyLeuProAlaAlaLysProAsnAsnSerGlnGluTyr450455460TATGAGATAGCTGGGAGATTTTCATTAATATCA CTCGTACCGTCAAAT1440TyrGluIleAlaGlyArgPheSerLeuIleSerLeuValProSerAsn465470475GATGATTATCAGACACCAATAATAAATTCAGTCAC TGTACGACAAGAT1488AspAspTyrGlnThrProIleIleAsnSerValThrValArgGlnAsp480485490TTAGAACGACAATTAGGAGAACTAAGAGATGAATTTAACA ATTTATCA1536LeuGluArgGlnLeuGlyGluLeuArgAspGluPheAsnAsnLeuSer495500505CAACAAATCGCTATGTCACAACTGATAGATCTTGCGTTACTACCGTTA 1584GlnGlnIleAlaMetSerGlnLeuIleAspLeuAlaLeuLeuProLeu510515520525GACATGTTCTCAATGTTTTCAGGGATTAAGAGTACAATTGACGCA GCG1632AspMetPheSerMetPheSerGlyIleLysSerThrIleAspAlaAla530535540AAGTCTATGGCGACGAATGTAATGAAGAGATTTAAAAAGTCAAG TCTC1680LysSerMetAlaThrAsnValMetLysArgPheLysLysSerSerLeu545550555GCTAACTCAGTGTCAACGCTCACTGATTCATTGTCTGATGCAGCAT CA1728AlaAsnSerValSerThrLeuThrAspSerLeuSerAspAlaAlaSer560565570TCAATTTCTAGAAGTGCATCGGTTAGATCAGTTAGTTCAACTGCATCA 1776SerIleSerArgSerAlaSerValArgSerValSerSerThrAlaSer575580585GCTTGGACGGAAGTATCTAACATTACATCAGATATTAATGTGACAACG1824AlaT rpThrGluValSerAsnIleThrSerAspIleAsnValThrThr590595600605AGCTCGATCTCTACACAGACATCAACAATAAGCAGAAGGTTAAGACTA1872 SerSerIleSerThrGlnThrSerThrIleSerArgArgLeuArgLeu610615620AAAGAAATGGCGACTCAAACGGACGGTATGAATTTTGATGATATATCA1920 LysGluMetAlaThrGlnThrAspGlyMetAsnPheAspAspIleSer625630635GCAGCAGTACTCAAGACTAAAATTGATAAATCAACCCAGTTAAATACA1968Al aAlaValLeuLysThrLysIleAspLysSerThrGlnLeuAsnThr640645650AATACATTGCCGGAAATAGTAACTGAGGCTTCAGAAAAGTTTATACCA2016AsnThrL euProGluIleValThrGluAlaSerGluLysPheIlePro655660665AATAGAGCGTACCGTGTAATTAAAGATGATGAAGTGCTAGAGGCTAGT2064AsnArgAlaTyrArg ValIleLysAspAspGluValLeuGluAlaSer670675680685ACTGATGGTAAATATTTCGCTTACAAAGTTGAAACCATTTTGAAGAGA2112ThrAspGlyLys TyrPheAlaTyrLysValGluThrIleLeuLysArg690695700TTCCATTCGATGTACAAATTCGCTGACTTAGTGACTGACTCACCAGTT2160PheHisSerMe tTyrLysPheAlaAspLeuValThrAspSerProVal705710715ATATCGGCAATAATTGACTTTAAAACTCTTAAGAATCTAAATGATAAT2208IleSerAlaIleI leAspPheLysThrLeuLysAsnLeuAsnAspAsn720725730TACGGAATAAGCAGACAACAAGCACTAAATCTTCTAAGATCTGATCCG2256TyrGlyIleSerArgGln GlnAlaLeuAsnLeuLeuArgSerAspPro735740745CGAGTATTACGTGAATTTATTAATCAGGATAATCCAATAATACGAAAT2304ArgValLeuArgGluPheIleAsnGln AspAsnProIleIleArgAsn750755760765AGAATAGAAAGTTTGATAATGCAATGTCGCTTGTAAGCAACTGAACAAGAGGA2357ArgIleGluSerLeuIleMetGln CysArgLeu770775TGTGAC2363(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 776 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:MetAlaSerLeuIleTyrArgGlnLeuLeuThrAsnSerTyrThrVal151015GluLeuSerAspGluIleGlnGluI leGlySerThrLysThrGlnAsn202530ValThrValAsnProGlyProPheAlaGlnThrAsnTyrAlaSerVal3540 45AsnTrpGlyProGlyGluThrAsnAspSerThrThrValGluProVal505560LeuAspGlyProTyrGlnProThrThrPheAsnProProValSerTyr65 707580TrpMetLeuLeuAlaProThrAsnAlaGlyValValAspGlnGlyThr859095AsnAsnThrAsnAr gTrpLeuAlaThrIleLeuIleLysProAsnVal100105110GlnGlnValGluArgThrTyrThrLeuPheGlyGlnGlnValGlnVal11512 0125ThrValSerAsnAspSerGlnThrLysTrpLysPheValAspLeuSer130135140LysGlnThrGlnAspGlyAsnTyrSerGlnHisGlyProLeuLeuS er145150155160ThrProLysLeuTyrGlyValMetLysHisGlyGlyLysIleTyrThr165170175Tyr AsnGlyGluThrProAsnAlaThrThrGlyTyrTyrSerThrThr180185190AsnPheAspThrValAsnMetThrAlaTyrCysAspPheTyrIleIle195 200205ProLeuAlaGlnGluAlaLysCysThrGluTyrIleAsnAsnGlyLeu210215220ProProIleGlnAsnThrArgAsnIleValProVa lSerIleValSer225230235240ArgAsnIleValTyrThrArgAlaGlnProAsnGlnAspIleValVal245250 255SerLysThrSerLeuTrpLysGluMetGlnTyrAsnArgAspIleVal260265270IleArgPheLysPheAlaAsnSerIleIleLysSerGlyGlyLeuGly 275280285TyrLysTrpSerGluValSerPheLysProAlaAsnTyrGlnTyrThr290295300TyrThrArgAspGlyGluGluVal ThrAlaHisThrThrCysSerVal305310315320AsnGlyIleAsnAspPheAsnTyrAsnGlyGlySerLeuProThrAsp325 330335PheValIleSerLysTyrGluValIleLysGluAsnSerPheValTyr340345350IleAspTyrTrpAspAspSerGlnAlaPheArgAsnMe tValTyrVal355360365ArgSerLeuAlaAlaAspLeuAsnSerValMetCysThrGlyGlyAsp370375380TyrSerPheAla IleProValGlyAsnTyrProValMetThrGlyGly385390395400AlaValSerLeuHisSerAlaGlyValThrLeuSerThrGlnPheThr405 410415AspPheValSerLeuAsnSerLeuArgPheArgPheArgLeuSerVal420425430GluGluProProPheSerIleLeuArg ThrArgValSerGlyLeuTyr435440445GlyLeuProAlaAlaLysProAsnAsnSerGlnGluTyrTyrGluIle450455460A laGlyArgPheSerLeuIleSerLeuValProSerAsnAspAspTyr465470475480GlnThrProIleIleAsnSerValThrValArgGlnAspLeuGluArg 485490495GlnLeuGlyGluLeuArgAspGluPheAsnAsnLeuSerGlnGlnIle500505510AlaMetSerGlnLeu IleAspLeuAlaLeuLeuProLeuAspMetPhe515520525SerMetPheSerGlyIleLysSerThrIleAspAlaAlaLysSerMet530535 540AlaThrAsnValMetLysArgPheLysLysSerSerLeuAlaAsnSer545550555560ValSerThrLeuThrAspSerLeuSerAspAlaAlaSerSer IleSer565570575ArgSerAlaSerValArgSerValSerSerThrAlaSerAlaTrpThr580585590GluV alSerAsnIleThrSerAspIleAsnValThrThrSerSerIle595600605SerThrGlnThrSerThrIleSerArgArgLeuArgLeuLysGluMet610 615620AlaThrGlnThrAspGlyMetAsnPheAspAspIleSerAlaAlaVal625630635640LeuLysThrLysIleAspLysSerThrGln LeuAsnThrAsnThrLeu645650655ProGluIleValThrGluAlaSerGluLysPheIleProAsnArgAla660665 670TyrArgValIleLysAspAspGluValLeuGluAlaSerThrAspGly675680685LysTyrPheAlaTyrLysValGluThrIleLeuLysArgPheHisSer69 0695700MetTyrLysPheAlaAspLeuValThrAspSerProValIleSerAla705710715720IleIleAspPheLysThrL euLysAsnLeuAsnAspAsnTyrGlyIle725730735SerArgGlnGlnAlaLeuAsnLeuLeuArgSerAspProArgValLeu7407 45750ArgGluPheIleAsnGlnAspAsnProIleIleArgAsnArgIleGlu755760765SerLeuIleMetGlnCysArgLeu770 775(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1062 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 49..1026(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:GGCTTTAAAAGCGAGAATTT CCGTTTGGCTAGCGGTTAGCTCCTTTTAATGTATGGT57MetTyrGlyATTGAATATACCACAA TTCTAATCTTCTTGACATCGATTACATTATTG105IleGluTyrThrThrIleLeuIlePheLeuThrSerIleThrLeuLeu51015AATTATATCTTAAAATCAATAACG AGAATAATGGACTATATAATTTAC153AsnTyrIleLeuLysSerIleThrArgIleMetAspTyrIleIleTyr20253035AGATTTCTGCTTATAGTAGTG ATCTTGGCCACCATAATAAATGCGCAA201ArgPheLeuLeuIleValValIleLeuAlaThrIleIleAsnAlaGln404550AACTATGGAGTAAATTTGCC AATTACAGGTTCAATGGATACTGCGTAT249AsnTyrGlyValAsnLeuProIleThrGlySerMetAspThrAlaTyr556065GCAGACTCTACACAAAGTGAGC CATTTTTGACATCAACCCTTTGTTTG297AlaAspSerThrGlnSerGluProPheLeuThrSerThrLeuCysLeu707580TATTATCCTGTTGAGGCATCAAACGAA ATAGCTGATACCGAATGGAAA345TyrTyrProValGluAlaSerAsnGluIleAlaAspThrGluTrpLys859095GATACCTTATCACAATTGTTCTTGACAAAAGGATGG CCAACAGGATCA393AspThrLeuSerGlnLeuPheLeuThrLysGlyTrpProThrGlySer100105110115GTGTACCTTAAAGAATATGCTGATATAGCGGC CTTTTCAGTGGAACCA441ValTyrLeuLysGluTyrAlaAspIleAlaAlaPheSerValGluPro120125130CAGTTATACTGCGATTATAATTTAGTTTTAA TGAAATATGACTCTACA489GlnLeuTyrCysAspTyrAsnLeuValLeuMetLysTyrAspSerThr135140145CAAGAACTAGATATGTCTGAATTGGCCGATCTT ATATTGAACGAATGG537GlnGluLeuAspMetSerGluLeuAlaAspLeuIleLeuAsnGluTrp150155160CTGTGCAATCCAATGGACATAACGCTATATTATTATCAG CAGACTGAT585LeuCysAsnProMetAspIleThrLeuTyrTyrTyrGlnGlnThrAsp165170175GAAGCAAATAAATCGATATGGACGGGCTCTTCTTGCACGGTTAAAGT G633GluAlaAsnLysSerIleTrpThrGlySerSerCysThrValLysVal180185190195TGTCCATTAAATACACAAACACTTGGTATTGGATGTCTAATAA CTAAT681CysProLeuAsnThrGlnThrLeuGlyIleGlyCysLeuIleThrAsn200205210CCAGACACGTTTGAAACAGTTGCGACAATGGAGAAGTTAGTG ATTACA729ProAspThrPheGluThrValAlaThrMetGluLysLeuValIleThr215220225GATGTTGTAGATGGTGTCAATCACAAATTAAACGTCACAACGGCA ACG777AspValValAspGlyValAsnHisLysLeuAsnValThrThrAlaThr230235240TGCACCATACGCAACTGTAAAAAGTTAGGACCAAGGGAGAACGTAGCA 825CysThrIleArgAsnCysLysLysLeuGlyProArgGluAsnValAla245250255GTCATACAGGTAGGCGGCGCGAACATTTTAGACATCACAGCTGATCCA873Val IleGlnValGlyGlyAlaAsnIleLeuAspIleThrAlaAspPro260265270275ACAACTACACCACAGACAGAGACAATGATGCGAATAAATTGGAAAAAA921ThrThrThrProGlnThrGluThrMetMetArgIleAsnTrpLysLys280285290TGGTGGCAAGTCTTTTACACGGTAGTGGATTACGTCAATCAGATAATT96 9TrpTrpGlnValPheTyrThrValValAspTyrValAsnGlnIleIle295300305CAGACAATGTCCAAAAGATCTACATCGCTTAATTCGTCGGCGTTCTAC1017 GlnThrMetSerLysArgSerThrSerLeuAsnSerSerAlaPheTyr310315320TATAGAGTGTAGGTGCATGCTAGATTAGAGTTGTATGATGTGACC1062TyrArg Val325(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 326 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:MetTyrGlyIleGluTyrThrThrIleLeuIlePheLeuThrSerIle1 51015ThrLeuLeuAsnTyrIleLeuLysSerIleThrArgIleMetAspTyr202530IleIleTyrArgPheLeuL euIleValValIleLeuAlaThrIleIle354045AsnAlaGlnAsnTyrGlyValAsnLeuProIleThrGlySerMetAsp5055 60ThrAlaTyrAlaAspSerThrGlnSerGluProPheLeuThrSerThr65707580LeuCysLeuTyrTyrProValGluAlaSerAsnGluIleAlaAsp Thr859095GluTrpLysAspThrLeuSerGlnLeuPheLeuThrLysGlyTrpPro100105110ThrGlySe rValTyrLeuLysGluTyrAlaAspIleAlaAlaPheSer115120125ValGluProGlnLeuTyrCysAspTyrAsnLeuValLeuMetLysTyr13013 5140AspSerThrGlnGluLeuAspMetSerGluLeuAlaAspLeuIleLeu145150155160AsnGluTrpLeuCysAsnProMetAspIleThrL euTyrTyrTyrGln165170175GlnThrAspGluAlaAsnLysSerIleTrpThrGlySerSerCysThr1801851 90ValLysValCysProLeuAsnThrGlnThrLeuGlyIleGlyCysLeu195200205IleThrAsnProAspThrPheGluThrValAlaThrMetGluLysLeu210 215220ValIleThrAspValValAspGlyValAsnHisLysLeuAsnValThr225230235240ThrAlaThrCysThrIleArgAs nCysLysLysLeuGlyProArgGlu245250255AsnValAlaValIleGlnValGlyGlyAlaAsnIleLeuAspIleThr260265 270AlaAspProThrThrThrProGlnThrGluThrMetMetArgIleAsn275280285TrpLysLysTrpTrpGlnValPheTyrThrValValAspTyrValA sn290295300GlnIleIleGlnThrMetSerLysArgSerThrSerLeuAsnSerSer305310315320AlaPheTyrTyr ArgVal325(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 326 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:MetTyrGlyIleGluTyrThrThrIleL euIlePheLeuThrSerIle151015ThrLeuLeuAsnTyrIleLeuLysSerIleThrArgIleMetAspTyr20 2530IleIleTyrArgPheLeuLeuIleValValIleLeuAlaThrIleIle354045AsnAlaGlnAsnTyrGlyValAsnLeuProI leThrGlySerMetAsp505560ThrAlaTyrAlaAspSerThrGlnSerGluProPheLeuThrSerThr657075 80LeuCysLeuTyrTyrProValGluAlaSerAsnGluIleAlaAspThr859095GluTrpLysAspThrLeuSerGlnLeuPheL euThrLysGlyTrpPro100105110ThrGlySerValTyrLeuLysGluTyrAlaAspIleAlaAlaPheSer115120 125ValGluProGlnLeuTyrCysAspTyrAsnLeuValLeuMetLysTyr130135140AspSerThrGlnGluLeuAspMetSerGluLeuAlaAspLe uIleLeu145150155160AsnGluTrpLeuCysAsnProMetAspIleThrLeuTyrTyrTyrGln165170 175GlnThrAspGluAlaAsnLysSerIleTrpThrGlySerSerCysThr180185190ValLysValCysProLeuAsnThrGlnThrLeu GlyIleGlyCysLeu195200205IleThrAsnProAspThrPheGluThrValAlaThrMetGluLysLeu210215 220ValIleThrAspValValAspGlyValAsnHisLysLeuAsnValThr225230235240ThrAlaThrCysThrIleArgAsnCysLysLysLeu GlyProArgGlu245250255AsnValAlaValIleGlnValGlyGlyAlaAsnIleLeuAspIleThr260265 270AlaAspProThrThrThrProGlnThrGluThrMetMetArgIleAsn275280285TrpLysLysTrpTrpGlnValPheTyrThrValValA spTyrValAsn290295300GlnIleIleGlnThrMetSerLysArgSerThrSerLeuAsnSerSer305310315 320AlaPheTyrTyrArgVal325(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 326 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: MetTyrGlyIleGluTyrThrThrValLeuThrPheLeuIleSerThr151015IleLeuLeuAsnTyrIleLeuLysSerLeuThrArgIleMetAspPhe 202530IleIleTyrArgPheLeuPheIleIleValIleLeuSerProPheLeu354045Arg AlaGlnAsnTyrGlyIleAsnLeuProIleAlaGlySerMetAsp505560ThrAlaTyrAlaAsnSerThrGlnGluGluProPheLeuThrSerThr65 707580LeuCysLeuTyrTyrProThrGluAlaAlaThrGluIleAsnAspAsn859095Ser TrpLysAspThrLeuSerGlnLeuPheLeuThrLysGlyTrpPro100105110ThrGluSerValTyrPheLysGluTyrThrAsnIleAlaSerPheSer 115120125ValAspProGlnLeuTyrCysAspTyrAsnValValLeuMetLysTyr130135140AspAlaThrLeuG lnLeuAspMetSerGluLeuAlaAspLeuIleLeu145150155160AsnGluTrpLeuCysAsnProMetAspIleThrLeuTyrTyrTyrGln 165170175GlnThrAspGluAlaAsnLysTrpIleSerMetGlySerSerCysThr180185190IleLy sValCysProLeuAsnThrGlnThrLeuGlyIleGlyCysLeu195200205ThrThrAspAlaThrThrPheGluGluValProThrAlaGluLysLeu210 215220ValIleThrAspValValAspGlyValAsnHisLysLeuAspValThr225230235240ThrAlaThr CysThrIleArgAsnCysLysLysLeuGlyProArgGlu245250255AsnValAlaValIleGlnValGlyGlySerAspIleLeuAspIleThr 260265270AlaAspProThrThrAlaProGlnThrGluArgMetMetArgIleAsn275280285TrpLysLys TrpTrpGlnValPheTyrThrValValAspTyrValAsp290295300GlnIleIleGlnValMetSerLysArgSerArgSerLeuAsnSerAla305 310315320AlaPheTyrTyrArgVal325(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 326 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:MetTyrGlyIleGluTyrThrThrValLeuThrPheLeuIleSerLeu151015IleLeuLeuAsnTyrIleLeu LysSerLeuThrArgMetMetAspCys202530IleIleTyrArgPheLeuPheIleValValIleLeuSerProLeuLeu35 4045LysAlaGlnAsnTyrGlyIleAsnLeuProIleThrGlySerMetAsp505560ThrAlaTyrAlaAsnSerThrGlnGluGluThr PheLeuThrSerThr65707580LeuCysLeuTyrTyrProThrGluAlaAlaThrGluIleAsnAspAsn85 9095SerTrpLysAspThrLeuSerGlnLeuPheLeuThrLysGlyTrpPro100105110ThrGlySerValTyrPheLysGluTy rThrAspIleAlaSerPheSer115120125ValAspProGlnLeuTyrCysAspTyrAsnValValLeuMetLysTyr130135 140AspAlaThrLeuGlnLeuAspMetSerGluLeuAlaAspLeuIleLeu145150155160AsnGluTrpLeuCysAsnProMetAspIle AlaLeuTyrTyrTyrGln165170175GlnThrAspGluAlaAsnLysTrpIleSerMetGlySerSerCysThr180 185190IleLysValCysProLeuAsnThrGlnThrLeuGlyIleGlyCysLeu195200205ThrThrAspThrAlaThrPheGluGluVal AlaThrAlaGluLysLeu210215220ValIleThrAspValValAspGlyValAsnHisLysLeuAspValThr225230235 240ThrAlaThrCysThrIleArgAsnCysLysLysLeuGlyProArgGlu245250255AsnValAlaValIleGlnValGlyGlyS erAspValLeuAspIleThr260265270AlaAspProThrThrAlaProGlnThrGluArgMetMetArgIleAsn275280 285TrpLysLysTrpTrpGlnValPheTyrThrValValAspTyrValAsn290295300GlnIleIleGlnAlaMetSerLysArgSerArgSerLe uAsnSerAla305310315320AlaPheTyrAsnArgIle325(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 326 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:MetTyrGlyIleGluTyrThrThrValLeuThrPheLeuIleSerLeu1510 15ValPheValAsnTyrIleLeuLysSerValThrArgThrMetAspPhe202530IleIleTyrArgPheLeuLeuValIleValValLeuAlaProLe uIle354045LysAlaGlnAsnTyrGlyIleAsnLeuProIleThrGlySerMetAsp505560ThrPr oTyrMetAsnSerThrThrSerGluThrPheLeuThrSerThr65707580LeuCysLeuTyrTyrProAsnGluAlaAlaThrGluIleAlaAspThr 859095LysTrpThrGluThrLeuSerGlnLeuPheLeuThrLysGlyTrpPro100105110 ThrGlySerValTyrPheLysGlyTyrAlaAspIleAlaSerPheSer115120125ValGluProGlnLeuTyrCysAspTyrAsnIleValLeuMetLysTyr 130135140AspGlyAsnLeuGlnLeuAspMetSerGluLeuAlaGlyLeuIleLeu145150155160As nGluTrpLeuCysAsnProMetAspIleMetLeuTyrTyrTyrGln165170175GlnThrAspGluAlaAsnLysTrpIleSerMetGlyThrSerCysThr 180185190IleLysValCysProLeuAsnThrGlnThrLeuGlyIleGlyCysSer195200205Thr ThrAspIleAsnSerPheGluThrValAlaAsnAlaGluLysLeu210215220AlaIleThrAspValValAspGlyValAsnHisLysLeuAspValThr225 230235240ThrSerThrCysThrIleArgAsnCysLysLysLeuGlyProArgGlu245250255 AsnValAlaValIleGlnValGlyGlyProAsnIleLeuAspIleThr260265270AlaAspProThrThrAlaProGlnThrGluArgMetMetArgIleAsn 275280285TrpLysArgTrpTrpGlnValPheTyrThrIleValAspTyrValAsn290295300GlnIleValG lnValMetSerLysArgSerArgSerLeuAspSerAla305310315320AlaPheTyrTyrArgVal325(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 326 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:MetTyrGlyIleGluTyrThrThrIleLeuIlePheLeuIleSerIle15 1015IleLeuLeuAsnTyrIleLeuLysSerValThrArgIleMetAspTyr202530IleIleTyrArgPheL euLeuIleThrValAlaLeuPheAlaLeuThr354045ArgAlaGlnAsnTyrGlyLeuAsnLeuProIleThrGlySerMetAsp50 5560AlaValTyrThrAsnSerThrGlnGluGluValPheLeuThrSerThr65707580LeuCysLeuTyrTyrProThrG luAlaSerThrGlnIleAsnAspGly859095AspTrpLysAspSerLeuSerGlnMetPheLeuThrLysGlyTrpPro100 105110ThrGlySerValTyrPheLysGluTyrSerAsnIleValAspPheSer115120125ValAspProGlnLeuTyrCysAsp TyrAsnLeuValLeuMetLysTyr130135140AspGlnSerLeuLysLeuAspMetSerGluLeuAlaAspLeuIleLeu145150 155160AsnGluTrpLeuCysAsnProMetAspValThrLeuTyrTyrTyrGln165170175GlnSerGlyGluSerAsnLys TrpIleSerMetGlySerSerCysThr180185190ValLysValCysProLeuAsnThrGlnThrLeuGlyIleGlyCysGln195 200205ThrThrAsnValAspSerPheGluMetIleAlaGluAsnGluLysLeu210215220AlaIleValAspValValAspGlyIleAsnH isLysIleAsnLeuThr225230235240ThrThrThrCysThrIleArgAsnCysLysLysLeuGlyProArgGlu245 250255AsnValAlaValIleGlnValGlyGlySerAsnValLeuAspIleThr260265270AlaAspProThrThrAsnProGl nThrGluArgMetMetArgValAsn275280285TrpLysLysTrpTrpGlnValPheTyrThrIleValAspTyrIleAsn290295 300GlnIleValGlnValMetSerLysArgSerArgSerLeuAsnSerAla305310315320AlaPheTyrTyrArgVal 325(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 326 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:MetTyrGlyIleGluTyrThrThrValLeuLeuTyrLeu IleSerPhe151015ValLeuMetSerTyrIleLeuLysThrIleThrLysMetMetAspTyr2025 30IleIleTyrArgIleThrPheIleIleValValLeuSerValLeuSer354045AsnAlaGlnAsnTyrGlyIleAsnLeuProIleThrGlySer MetAsp505560ThrAlaTyrAlaAsnSerThrGlnAspAsnAsnPheLeuSerSerThr65707580LeuCysLeuTyrTyrProSerGluAlaProThrGlnIleAsnAspAsn859095GluTrpLysAspThrLeuSerGlnLeuPheLeuThrLysGly TrpPro100105110ThrGlySerValTyrPheAsnGluTyrSerAsnValLeuGluPheSer115120125 IleAspProLysLeuHisCysAspTyrAsnIleValLeuIleArgPhe130135140AlaSerGlyGluGluLeuAspIleSerGluLeuAlaAspLeuIleLeu 145150155160AsnGluTrpLeuCysAsnProMetAspIleThrLeuTyrTyrTyrGln16517017 5GlnThrGlyGluAlaAsnLysTrpIleSerMetGlySerSerCysThr180185190ValLysValCysProLeuAsnThrGlnThrLeuGlyIleGlyCy sGln195200205ThrThrAsnThrAlaThrPheGluThrValAlaAspSerGluLysLeu210215220Ala IleValAspValValAspSerValAsnHisLysLeuAspValThr225230235240SerThrThrCysThrIleArgAsnCysAsnLysLeuGlyProArgGlu245250255AsnValAlaIleIleGlnValGlyGlySerAsnIleLeuAspIleThr260265270AlaAsnProThrThrSerProGlnThrGluArgMetMetArgValAsn275280285TrpLysLysTrpTrpGlnValPheTyrThrValValAspTyrIleAsn 290295300GlnIleValGlnValMetSerLysArgSerArgSerLeuAspSerSer305310315320 SerPheTyrTyrArgVal325(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 317 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:MetAlaGluLeu AlaCysPheCysTyrProHisLeuGluAsnAspSer151015TyrLysPheIleProPheAsnAsnLeuAlaIleLysCysMetLeuThr 202530AlaLysValAspArgLysAspGlnAspLysPheTyrAsnSerIleIle354045TyrGlyIleAlaPro ProProGlnPheLysLysArgTyrAsnThrAsn505560AspAsnSerArgGlyMetAsnTyrGluThrSerMetPheAsnLysVal6570 7580AlaValLeuIleCysGluAlaLeuAsnSerIleLysValThrGlnSer859095AspValAlaAsnVal LeuSerArgValValSerValArgHisLeuGlu100105110AsnLeuValLeuArgArgGluAsnHisGlnAspValLeuPheHisSer115 120125LysGluLeuLeuLeuLysSerValLeuIleAlaIleGlyHisSerLys130135140GluIleGluThrThrAlaThrAla GluGlyGlyGluIleValPheGln145150155160AsnAlaAlaPheThrMetTrpLysLeuThrTyrLeuGluHisLysLeu165 170175MetProIleLeuAspGlnAsnPheIleGluTyrLysIleThrValAsn180185190GluAspLysProIleS erGluSerHisValLysGluLeuIleAlaGlu195200205LeuArgTrpGlnTyrAsnLysPheAlaValIleThrHisGlyLysGly210 215220HisTyrArgValValLysTyrSerSerValAlaAsnHisAlaAspArg225230235240ValTyrAlaThrPheLysSe rAsnAsnLysAsnGlyAsnValLeuGlu245250255PheAsnLeuLeuAspGlnArgIleIleTrpGlnAsnTrpTyrAlaPhe260 265270ThrSerSerMetLysGlnGlyAsnThrLeuAspIleCysLysLysLeu275280285LeuPheGlnLysMetLysArg GluSerAsnProPheLysGlyLeuSer290295300ThrAspArgLysMetAspGluValSerGlnIleGlyIle305310315(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 317 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:MetAlaGluLeuAlaCysPheCysTyrProHisLeuGluLysAspSer 151015TyrLysPheIleProPheAsnSerLeuAlaIleLysCysMetLeuThr202530 AlaLysValAspLysLysAspGlnAspLysPheTyrAsnSerIleVal354045TyrGlyIleAlaProProProGlnPheLysLysArgTyrAsnThrAsn 505560AspAsnSerArgGlyMetAsnPheGluThrSerMetPheAsnLysVal65707580AlaIle LeuIleCysGluAlaLeuAsnSerIleLysValThrGlnSer859095AspValAlaAsnValLeuSerArgValValSerValArgHisLeuGlu 100105110AsnLeuValLeuArgLysGluAsnHisGlnAspValLeuPheHisSer115120125LysGluL euLeuLeuLysAlaValLeuIleAlaIleGlyGlnSerLys130135140GluIleGluThrThrAlaThrAlaGluGlyGlyGluIleValPheGln145 150155160AsnAlaAlaPheThrMetTrpLysLeuThrTyrLeuAspHisLysLeu165170175MetPr oIleLeuAspGlnAsnPheIleGluTyrLysIleThrLeuAsn180185190GluAspLysProIleSerAspAlaCysValLysGluLeuValAlaGlu 195200205LeuArgTrpGlnTyrAsnArgPheAlaValIleThrHisGlyLysGly210215220HisTyrArgValVal LysTyrSerSerValAlaAsnHisAlaAspArg225230235240ValPheAlaThrTyrLysAsnAsnAlaLysSerGlyAsnValThrAsp 245250255PheAsnLeuLeuAspGlnArgIleIleTrpGlnAsnTrpTyrAlaPhe260265270ThrSer SerMetLysGlnGlyAsnThrLeuAspValCysLysLysLeu275280285LeuPheGlnLysMetLysGlnGluLysAsnProPheLysGlyLeuSer290 295300ThrAspArgLysMetAspGluValSerHisValGlyIle305310315
As can be seen, vaccination with the viral particles, VP4 alone, and mixed crude cell lysates, provided protection to challenged neonatal mice.
Thus, viral particle assemblies, vaccine compositions containing these assemblies, and methods of treating and preventing rotaviral disease in a vertebrate subject are disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the intention as defined by the appended claims.

Number	Date	Country
0259149	Mar 1988	EPX
0273366	Jul 1988	EPX

Assembled viral particles and their use in a vaccine to rotaviral disease

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Date Issued

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Examiners

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CPC

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Abstract

Description

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US Referenced Citations (1)

Foreign Referenced Citations (2)

Non-Patent Literature Citations (23)

Entry
Ijaz et al., J. Virol. (1990) Pre-publication copy.
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Chen et al., "Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP-4-specific epitope" Journal of Virology (1992) 66(1):432-439.
Ijaz et al., "Heterotypic passive protection induced by synthetic peptides corresponding to VP7 and VP4 of bovine rotavirus" Journal of Virology (1991) 65(6):3106-3113.
Redmond et al., "Rotavirus vaccinology" Kurstak, E., ed., Control of Virus Diseases (1992) Marcel Dekker, Inc., New York, Chapter 16, pp. 387-404.
Redmond et al., "Assembly of recombinant rotavirus proteins into virus-like particles and assessment of vaccine potential" Vaccine (1992) 11(2):273-281.
Redmond et al., "Rotavirus particles function as immunological carriers for the delivery of peptides from infectious agents and endogenous proteins" Molecular Immunology (1991) 28(3):269-278.
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