CDNA SEQUENCE OF DENGUE VIRUS SEROTYPE 1 (SINGAPORE STRAIN)

Abstract: DEN1-S275/90 (ECACC V92042111) is a new strain of Dengue virus serotype 1. The complete cDNA sequence of this virus has been cloned and protein-coding fragments thereof have been used in the construction of expression plasmids. DEN1-S275/90 in inactivated form, DEN1-S275/90 polypeptides or fusion proteins thereof can be incorporated into vaccines for immunisation against DEN1-S275/90 and other DEN1 viruses. The invention further provides diagnostic reagents e.g. labelled antibodies to DEN1-S275/90 proteins, and kits to detect DEN1 virus.

Description: CDNA SEQUENCE OF DENGUE VIRUS SEROTYPE 1 (SINGAPORE STRAIN) The present invention relates to Dengue Virus Type 1. Dengue virus infection may lead to dengue fever (DF) or its more severe dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) . DHF is an important virus disease of global significance, especially in Southeast Asia. There are four serotypes of Dengue virus (DENl, DEN2, DEN3 and DEN4) belonging to the family Flaviviradae. The complete genomic seguence of DEN2 (Jamaica) has been published by Deubel et al; Virology 165, 234-244 (1988) . The complete genomic seguence of DEN3 (H87) has been published by Osatomi and Sumiyoshi; Virology 176. 643- 647 (1990) . The complete genomic seguence of DEN4 has been published by Zhao et al; Virology 155, 77-88. To date, only a partial seguence of any variant of DENl, DENl (Nauru Island) , has been determined; Mason et al, Virology 161, 262-267 (1987) . We have now identified a previously unknown strain of DENl and established its complete nucleotide seguence. The new strain, DEN1-S275/90, was deposited at the European Collection of Animal Cell Cultures (ECACC) Porton Down, GB under Budapest Treaty conditions on 21 April 1992 and given accession number V92042111. DEN1-S275/90 differs significantly from DEN2, DEN3 and DEN4 in terms of seguence homology. There are also a number of significant differences between DEN1-S275/90 and DENl (Nauru Island) . The present invention thus provides DEN1-S275/90 (ECACC V92042111) . The invention further provides DEN1- S275/90 (ECACC V92042111) for use as a diagnostic reagent. The invention also provides DEN1-S275/90 in inactivated form for use as a diagnostic reagent or a vaccine. The invention also provides the nucleic acid seguence of Seg. ID No. 1 and DNA seguences substantially corresponding to SEQ ID No. 1, e.g. degenerate variants thereof having one or more nucleotide changes but nevertheless capable of being translated to give the same protein seguence. The invention further provides fragments of such DNA polynucleotides, in particular the fragments encoding the C, C' ( PreH, M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 genes of the genome of the virus. The start and end points of these preferred fragments in the nucleic acid seguence of Seg I.D. No. 1 are shown below in Table 1. Table 1 also shows the start and end points of the proteins encoded by these genes, using the numbering of Seg. ID Nos. 1 and 2. TABLE 1 Start and end points of the nucleic acid (n) numbers encoding the genes of S275/90. The table also shows the start and end points of the corresponding proteins (p) within the polyprotein encoded by S275/90. The nucleic acid seguences of the invention may be used as probes in an assay to determine the presence or absence of DEN1-S275/90, or they may be incorporated into a vector, eg. an expression vector. Nucleic acid fragments according to the invention may be made by known methods of chemical synthesis or cloned from the virus itself using known recombinant technigues. Fragments according to the invention may also be produced by replication of DNA or RNA, by transcription from DNA to form RNA fragments or reverse transcription from RNA fragments to form DNA fragments. Such transcription may be in a cell free system or may be effected in cells for instance by cloning. Cell free systems include an appropriate replicase, transcriptase or reverse transcriptase, suitable nucleotide precursors and a nucleic acid template or appropriate seguence, together with buffers and any necessary or desirable cofactors. The present invention also provides a polyprotein as set forth in Seq. ID No. 1 and Seq. ID No. 2 and fragments thereof, eg. the C, C, PreM, M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 proteins as identified above in Table 1. The invention thus provides a polypeptide having an amino acid sequence substantially corresponding to the sequence shown in SEQ ID No. 2 or a fragment thereof. Fusion proteins which incorporate these peptides are also provided. The polyprotein and proteins according to the invention may be produced by synthetic peptide chemistry or by expressing vectors carrying DNA encoding the proteins in a suitable cell in order to produce expression of the DNA, followed by recovery of the expressed protein. Methods of expressing and recovering recombinant proteins, including fusion proteins, are well known in the art. For example, for expression of a polypeptide of the invention, an expression vector may be constructed. An expression vector is prepared which comprises a DNA seguence encoding a polypeptide of the invention and which is capable of expressing the polypeptide when provided with a suitable host, eucaryotic or procaryotic. Appropriate transcriptional and translational control elements are provided, including a promoter for the DNA sequence, a transcriptional termination site, and translational start and stop codons. The DNA seguence is provided in the correct frame such as to enable expression of the polypeptide to occur in a host compatible with the vector. The expression vector may be selected to be suitable to express the nucleic acid seguences of the invention in, for example, a bacterial e.g. i. coli, yeast, insect or mammalian cell. A baculovirus expression system may be used. The nucleic acid may be expressed in order that a protein or peptide encoded by the fragment alone is produced or alternatively it may be expressed to provide a fusion protein in which DEN1-S275/90 or a protein thereof, e.g. E, NSl, NS2, NS3 or NS5 as identified in Table 1 above is fused to a second amino acid sequence, e.g. a C-terminal seguence derived from glutathione S-transferase or maltose binding protein or a C-terminal or N-terminal signal seguence. Such a seguence may for example cause the fusion protein to be exported from the cell. The expression vector is then provided with an appropriate host. Cells harbouring the vector are grown so as to enable expression to occur. The vector may be a plasmid or a viral vector. Recovery and where desirable, further purification of the protein produced by an expression vector in a host cell may be by means known in the art. Such means are designed to separate the protein of the invention from the other proteins of the host cell. Suitable means include chromatographic separation of the recovered protein. The polyprotein and peptides of the invention may be used as immunogens for a vaccine against DEN1-S275/90 and other DENl viruses. Suitably, the proteins and peptides of the invention will be combined with a pharmaceutically acceptable carrier or diluent in order to prepare a sterile vaccine composition. The vaccine composition may then be used in a method of immunizing a human against DENl infections. Advantageously, a vaccine composition against DENl may comprise a mixture of two or more peptides. For example, it may comprise one non-structural (NS) peptide, eg. NSl or NS3, together with a capsid (C) , M or E peptide. A mixture of two or more NS peptides could also be used. The proteins and peptides of the invention may also be used as antigens in an immunoassay to detect the presence or absence of DENl, and especially DEN1-S272/90. The proteins and peptides are optionally labelled with a detectable label, eg a radioisotope, biotin or a fluorophore. The immunoassay may be conducted by bringing a known guantity of labelled protein (antigen) into contact with a sample suspected of containing antibody against DENl and detecting the presence or absence of antibody-antigen complex containing the labelled antigen. The invention also provides antibodies against the above-mentioned proteins and peptides of the invention. The antibodies may be monoclonal or polyclonal. Monoclonal antibodies -may be produced by hybridoma techniques known in the art or by recombinant means to provide hybrid antibodies such as humanized antibodies. The antibodies of the invention may be used in a method of treatment, eg passive immunisation, of DENl infections. The antibodies may also be used in a method of diagnosis, eg by immunoassay, to detect the presence or absence of DENl in a sample. The antibodies may be labelled as described above for the proteins and peptides of the invention. They may also be labelled with a toxin or isotope selected to kill virus-infected cells. Antibodies against NSl are particularly favoured since NSl is expressed on the surface of Dengue virus-infected cells. The antibodies of the invention may also be used in a method to detect the presence or absence of DENl protein in a sample. The method may comprise bringing the antibody into contact with a sample suspected to contain DENl proteins (antigens) and detecting the amount of antibody- antigen complex formed. Immunoassays according to the invention may be, for example, competitive (eg radioi mune assays - RIA) or non-competitive (eg enzyme linked immunosorbent assays - ELISA) . The following Examples illustrate the invention. In the accompanying drawings: Figure 1 is a diagrammatic representation of the cDNA of Dengue virus Type l (Singapore strain S275/90) and fragments of said DNA in expression vectors; Figure 2 shows gel results confirming serologic responses in mice after immunisations with fusion proteins prepared as in Examples 2 - 5 with or without complete Freund's adjuvant (CFA) . Gel Lanes: Lane 1: M, Lane 2: anti E, Lane 3: anti-E+ CFA, Lane 4: anti-NSl, Lane 5: anti-NS2, Lane 6: anti-NS2+ CFA, Lane 7: anti-NS3, Lane 8: anti-NS3+ CFA, Lane 9: anti-NS5, Lane 10: anti-NS5+ CFA, Lane 11: positive rabbit sera, Lane 12: negative rabbit sera. Lane 13: M; Figure 3 shows gel results confirming serologic response in rabbits after immunisations with fusion proteins prepared as in Examples 2 to 5. (-) , serum before immunisation; (+) serum after immunisation. Gel Lanes: Lane 1: (-) , Lane 2: (+) anti-E, Lane 3: (-) , Lane 4: (+) anti-NSl, Lane 5: (-) , Lane 6: (+) anti-NS2, Lane 7: (-) , Lane 8: (+) anti-NS3, Lane 9: (-) , Lane 10: (+) anti- NS5, Lane 11: positive Dengue, Lane 12: patient sera; Figure 4 shows fluorescence microscopy of C6/36 cells infected with Dengue Type 1 DI-275 and probed with antibodies against recombinant fusion proteins. A, control antiserum; B, anti-E; C, anti-NSl; D, anti-NS2; E, anti- NS3; F anti-NS5. EXAMPLE 1 DENl virus, strain S275/90, was isolated in 1990 from the serum of a DHF patient in Singapore by 3 passages in AP61 (Aedes psuedoscutellaris ) cells followed by 3 passages in C6/36 (Aedes albopictus) cells, and identified by immunofluorescence using type-specific monoclonal antibodies. After a further 8 to 13 passages in C6/36 cells, the virus-infected culture fluid was partially purified by precipitation with polyethylene glycol and ultracentrifugation on a 30% sucrose cushion (6) . The viral RNA was extracted from the purified virus by treatment with phenol in the presence of sodium dodecyl sulphate. Following cDNA synthesis (cDNA Synthesis System Plus, Amersham) using random primers, the assorted cDNAs were cloned into .EcoRI sites of pUC18 vector via EcoRI adaptors (Promega) . The Esherichia coli transformants containing Dengue-specific sequences were screened by colony hybridisation with 32 P-labelled cDNA probes prepared by reverse transcription of strain S275/90 RNA. The cloning procedure yielded overlapping cDNA clones containing inserts ranging in size from 0.5kb to 2.7kb. The ends of these primary clones and their subclσnes obtained by nested deletional analysis (Erase-a-Base System, Promega) were subjected to double-strand sequencing (Sequenase Version 2.0, United States Biochemical). The sequence data generated covers about 90% of the genomic sequence of S275/90. Potential secondary structures have been postulated for the 5' and 3' ends of flaviviruses (4, 7, 8), posing a problem in obtaining clones with intact ends. A different stragegy for sequencing the 5' and 3' noncoding regions was used to increase the chances of obtaining clones which contain these sequences as well as the terminal end sequences of the genome. cDNAs of strain S275/90 were obtained by random priming and oligo(dT) priming (after poly(A) tailing of the virus RNA]; these were amplified by polymerase chain reaction (PCR) in the presence of specific primers, 796 and 10090/B, respectively. The cDNAs of interest were then religated into pUC18 vector. The nucleotide seguences of the primers are as follows: primer 796, 5' CCG TGA ATC CTG GGT GTC 3'; primer 10090/B, 5' GGG AAT TCC AGT GGT GTG GATC 3' with a BamHI site at its 5' end. The seguences of the primers were selected from that of the initial clones of strain S275/90. To obtain the seguences at the 5' noncoding region, random cDNA clones were first generated as described above, followed by ligation to EcoRl adaptors before insertion into the _7σoRI sites of the pUC18 vector. These ligated products of assorted cDNA inserts were flanked by the reverse and forward sequencing primers of M13 in the pUC18 vector. The forward seguencing primer was thus used as one of the primers for PCR. The ligated cDNA clones were used as templates for PCR in the presence of primer 796 (which binds to the plus strand of the template at nucleotide position 808 to 825 of strain S275/90) and the commercial M13 single-strand primer (5'GTA AAA CGA CGG CCAGT 3 r , Pharmacia) . The amplified cDNAs thus contained the polylinker from the pUC18 vector at one end and an Xbal site (at nucleotide position 728) at the other end. For the 3' noncoding region, an additional step was included before cDNA synthesis. After extraction, the purified Dengue viral RNA was tailed by poly A polymerase (Bethesda Research Laboratories) with ATP. This was followed by cDNA synthesis using oligo(dT) as primer for the first strand cDNA synthesis. The same procedures of EcoRl adaptors ligation and insertion into JJcoRI sites of the pUC18 vector were repeated. The ligated products were again subjected to PCR amplification using the primer 10090/B (which binds to the minus strand of the template at nucleotide positions 10,086 to 10,099 of strain S275/90) and the commercial M13 single-strand primer. All samples were amplified by 30 cycles of PCR with melting, annealing and polymerisation conditions of 1 minute at 94°C, 2 minutes at 55°C and 3 minutes at 72°C, respectively. The amplified DNA was purified by electroelution in agarose gel followed by appropriate restriction enzyme digestions. The PCR amplified cDNAs at the 5' noncoding region were double digested with XJbal and .EcoRl, while those at the 3' noncoding region were digested with BamUI and EcoIR before cloning into the appropriate sites of the pUClδ vector. The clones were screened and subjected to double-strand seguencing as described above. The sequence data obtained from the overlapping cDNA clones was ordered by homology alignment with the published sequences of the four Dengue serotypes DENl, DEN2, DEN3 and DEN4 using the computer program of Wilbur and Lip an (9) . Seq ID No. 1 shows the complete nucleotide seguence of strain S275/90, which is 10,718 nucleotides in length, and its deduced amino acid sequence. ?ιhe reading frame begins with the first AUG start codon, corresponding to nucleotides 81 to 83, and contains an open reading frame of 10,188 nucleotides encoding a polyprotein of 3396 amino acids; there are 80 nucleotides in the 5' noncoding region and 450 nucleotides in the 3' noncoding region. The seguence in the 5' noncoding region preceding the first AUG codon of the open reading frame appears to be conserved for all Dengue virus types (1-4). The length of the 3 ' noncoding region of strain S275/90 is longer than that of DEN2 (412 nucleotides) , DEN3 (433 nucleotides) and DEN4 (384 nucleotides) . The nucleotide composition of strain S275/90 is 31.9% A, 25.9% G, 21.5% T and 20.7% C. As reported for the other flaviviruses, the same purine-rich composition was observed, and there is an absence of poly(A) tract at the 3 ' end. The individual protein coding segments are based on comparison with protein sequence data for all the proteins determined from the four Dengue serotypes. These cleavage sites may reveal the involvement of viral or cellular proteases involved in protein processing. The C, preM, M, E, NSl, NS2A, NS2B, NS3, NS4A, NS4B and NS5 proteins are cleaved at the sites M/MNQRKK, A/FXL, RXKR/SV, X/MRCXG, VQA/DXGCV, VXA/GXG, X/SWPLN, KXQR/XG, GRX/S, VXA/NE and R/G, respectively, where X refers to any residue. The cleavage sites of NS2A, NS3 and NS4B conform to the reported consensus seguences (4, 5) , which were originally established by Rice et al (10) . The nucleotide seguences of the structural and nonstructural regions 5' noncoding end to NSl, about 2400 nucleotides in length) of Nauru Island strain of DENl (isolated in 1974) and strain S275/90 were compared. Nucleotide variation shows that transitions are about 85.0% [transitions/(transitions + transversions) x 100%] in the structural region and 92.1% in the NSl region; 15% of these base changes are transversions in the structural region and 7.9% in the NSl region. The overall 236 nucleotide differences have given rise to 27 amino acid substitutions. As shown in Table 2, the nucleotide homology is 93.1% and when translated, the amino acid homology is 97.6%. Although both strains were isolated from different geographic regions with an interval of 16 years, a higher homology was still observed between the two strains. It can also be seen in Table 2 that strain S275/90 shows a higher homology with DEN3 then with DEN2 and DEN4. The nucleotide divergence of each gene is less than the translated amino acid divergence. The greatest nucleotide and amino acid changes, and hence the greatest evolution, lie in the nonstructural gene NS2A in all the four Dengue serotypes. A high homology is found in NS3 and NS5, which contain conserved sequences. Chu et al (11) compared three topotypes of DENl strains (Thailand, Philippines and Caribbean) genetically at the envelope region. They found nucleotide changes to be less than 5% but translational differences of 2% at the amino acid level. Our strain S275/90 shows nucleotide changes of 7.7% and amino acid changes of 2.6% in the envelope region. Rico-Hesse (6) compared nucleotide seguences within a chosen E/NS1 region to estimate evolutionary relationships among 40 DENl strains of different geographic range and time period. TABLE 2 HOMOLOGY (%) COMPARISON OF ALIGNED NUCLEOTIDE SEQUENCES OF THE FOUR DENGUE SEROTYPES WITH STRAIN S275/90 (AMINO ACID ALIGNMENT WITHIN BRACKETS) . REFERENCES 1. WESTAWAY, E.G., BRINTON, M.A. , GAIDAMOVICH,- S. Ya, HORZINEK, M.C. , IGARASHI, A., AARIAINEN, L. , LVOV, D.K. , PORTERFIELD, J.S., RUSSEL, P. . , and TRENT, D.W., Intervirology 24, 183-192 (1985) . 2. ROSEN, L. , Supplement to S. Afr. J. Med. 11, 40-42 (1986) . 3. HALSTEAD, S., Science 239, 476-481 (1988). 4. DEUBEL, V., KINNEY, R.M. , and TRENT, D.W. , Virology 165, 234-244 (1988). 5. OSATOMI, K. and SUMIYOSHI, H. , Virology 176, 643-647 (1990) . 6. RICO-HESSE, R. , PALLANSCH, M.A. , NOTTAY, B.K., AND EW, O.M., Virology 174, 479-493 (1990) 7. BRINTON, M.A., and DISPOTO, J.H., Virology 162, 290- 299 (1988) . 8. IRIE, K. , MOHAN, P.M., SASAGURI, Y. , PUTNAK, R. and PADMANABHAN, R. , Gene 75, 197-211 (1989) . 9. WILBUR, W.J., and LIPMAN, D.J. , Proc. Natl. Acad. Sci. USA 80, 726-730 (1983). 10. RICE, C ., LENCHES, E.M. , EDDY, S.R. , SHIN, S.J., SHEETS, R.L., and STRAUSS, J.H. , Science 229, 726-733 (1985) . 11. CHU, M.C, O'ROURKE, E.J. and TRENT, D.W. , J. Gen. Virol . 70 , 1701-1712 (1989 ) . EXAMPLE 2 CONSTRUCTION OF EXPRESSION PLASMIDS Standard recombinant DNA techniques were used for construction of the expression plasmids described below and summarised in Fig. 1 (Sambrook et al . , Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, N.Y.) . For construction of plasmids, the cDNA regions for E, NSl, NS2, NS3 and NS5 of clone DI-275, a DENl cDNA clone derived from DENl virus Singapore Strain S275/90 as in Example l, were amplified by the polymerase chain reaction (PCR) and digested with restriction enzymes. The restriction enzyme sites were built into the oligonucleotide primers used in the PCR as set out in Table 3 and Seq ID Nos. 3-12. Fragments of E, NS3 and NS5 cDNA digested with restriction enzymes were ligated to the pGEX-KG vector (Guan and Dixon, Anal. Biochem. 192, 262-267, 1991). Fragments of NSl and NS2 cDNA were ligated to pMAL-c and pMAL-cRI vectors (New England Biolabs) , respectively (Ford et al . , Prot. Exp. Pur. 2, 96-107, 1991; Maina et al . , Gene 74, 365-373, 1988; di Guan et al . , Gene, 67, 21-30, 1991). The construction of NS5 cDNA was done in two stages. The 5'-region, the cDNA fragment from nucleotide 7544-8365 of NS5, was made by PCR, digested with Sail and Clal; and the 3'-region, the fragment from nucleotide 8275 (Clal) to the 3'-end of NS5, was isolated directly from the cDNA of clone DI-275 (D-275 cDNA) by Clal and SacI double digestion. The two parts of NS5 were ligated together, then ligated into the pGEX-KG vector. Recombinant plasmids were transformed into E . coli DH5α or c600 HF1 strains. All plasmids encoded Dengue virus proteins fused to the C-terminus of glutathione S-transferase or Maltose Binding Protein (MBP) . EXAMPLE 3 PURIFICATION OF E. NS3 AND NS5 PROTEINS FROM RECOMBINANT E. COLI E. coli , harbouring E, NS3 and NS5 genes (separately) were grown in LB medium A 600 of 0.5 at 37°C, then induced with IPTG at 0.2mM for 2 h at 30°C The bacteria were harvested and resuspended on ice in MTPBS buffer (0.15 M NaCl, 0.016 M Na 2 HP0 4 , 0.005 M NaH 2 P0 4 ) with 0.1 mg/ml lysozyme, 1% triton X-100, 0.5 μg/ml aprotinin, 0.05 μg/ml Leupeptin, 0.25 μg/ml pepstatin, 5mM DTT and 0.175 μg/ml PMSF, and kept on ice for 10 in. The cells were sonicated at maximum power for 3 x 1 min while chilled. The lysate was centrifuged at 12,000 x g. The supernatant was added to 1 ml Glutathione-Sepharose 4B beads (Pharmacia) , and incubated at 4°C on a rotator for 1 h to absorb the fusion proteins. Then the beads were centrifuged and washed with PBS buffer (by centrifugation) at least 6 times, or until the wash solution read zero at A 2g0 in a spectrophotometer. The beads were resuspended in thro bin cleavage buffer,- and the Dengue virus proteins were cleaved off the beads with thrombin at 4°C for 1 hr. The supernatant, containing Dengue virus proteins, was recovered by centrifugation, and the proteins were stored at -80°C. EXAMPLE 4 SOLUBILISATION AND PURIFICATION OF A FUSION PROTEIN OF NSl FROM INCLUSION BODIES E. coli containing the NSl fusion protein was grown as above, except the tac promoters were induced with 0.3mM IPTG for 16 h. The bacteria were harvested, 1 gram wet weight of E. coli was resuspended in 5 ml lysis buffer with lysozyme at 1.6 mg/ml and was sonicated for 2 x 15 sec. After centrifugation at 1000 x g the supernatant was again centrifuged (25,000 x g) . The pellet was resuspended in 2 ml H,0 , adding a final concentration of 0.5% Triton X-100, 10 mM EDTA, and 100 mM NaCl, then centrifuged at 20,000 x g twice. The pellet was washed with 1 ml 2 M urea twice and dissolved in 8 M urea in 0.1 M Tris-HCl pH 8.8, 0.14 M 2- mercaptoethanol. The urea concentration was reduced to 1 M by adding H 2 0, and amylose resin (New England Biolabs) was added to adsorb the solubilised fusion protein at 22°C for 1 h. The amylose resin was washed with buffer (New England Biolabs) five times until the A 280 of the clarified supernatant was near zero. A final concentration of 50 mM maltose was then added to elute the fusion protein, which was recovered by removing the beads by centrifugation. EXAMPLE 5 PURIFICATION OF A SOLUBLE FUSION PROTEIN OF NS2 After growth of E . coli transformed with pMAL-cRI/NS2-l, lysis and sonicatibn as in Example 3 above, the clarified extract containing fehe soluble NS2 fusion protein was adsorbed onto amylose resin, followed by washing and elution of the NS2 fusion protein as in Example 4 above. EXAMPLE 6 IMMUNISATION OF RABBITS AND MICE The soluble fusion proteins of E, NS2, NS3 and NS5 purified from recombinant E. coli , as in Examples 3 and 5 above, and inclusion bodies containing the NSl fusion protein which had been purified up to the 2M urea wash stage as in Example 4, were placed directly in SDS loading buffer for preparative SDS-PAGE in 10% SDS-polyacrylamide gels. The proteins were visualised by staining with 0.05% Coomassie Blue for 10 min. The gel segments were cut and homogenized in sterile PBS, mixed with Freund's adjuvant and injected directly into white rabbits intramuscularly and subcutaneously on the first, sixth and twenty first days with about 200-500 μg of fusion protein per injected dose. The rabbits were bled 14 days after the last booster dose. For immunisation of mice, 12-day old female Swiss mice were immunised with the soluble proteins of E, NSl, NS2, NS3 and NS5 fusion proteins with or without Freund's adjuvant. The injections were intraperitoneal or subcutaneous on the first, fourth, and fourteenth day, using about 20 μg fusion protein per dose. The mice were bled 14 days after the last dose. The sera of rabbits and mice were used for IFA and immunoprecipitation assays. EXAMPLE 7 RADIOIMMUNOPRECIPITATIONS Radioimmunoprecipitations were done with rabbit and mouse antibodies against the structural and non-structural Dengue virus recombinant fusion proteins of D-275. At 36- 40 h post-infection of C6/36 cells with Dengue virus S275/90 strain, cell culture medium was replaced with methionine-free medium containing 3 μg/ml actinomycin D for 3 h, followed by the addition of fresh medium with [ 35 S] methionine at 20 μCi/ml and 3 μg/ml actinomycin D for a further 3 h. The cells were washed with cold PBS, dissolved in RIPA buffer [100 mM Tris-HCl pH7.5, 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.1% NP 40, 1% sodium dexoycholate, 100 μg/ml PMSF] on ice for 1 h, then clarified at 1000 x g for 10 min. The lysates were precleared with normal serum and protein A Sepharose. For immunoprecipitation, rabbit and mouse sera that had been preabsorbed with normal, uninfected C6/36 cell extract fixed by cold acetone were incubated with labeled antigen overnight at 4°C. The virus protein-antibody complexes were precipitated with protein A-Sepharose and were washed with immunoprecipitation buffer [10 mM Tris-HCl, pH7.4, 0.05% aprotinin, 1% NP40, 2 mM EDTA, 0.15 M NaCl], 6 times then 2X SDS-PAGE buffer was added, boiled for 2 min, and the supernatant was loaded on a 12% SDS-polyacrylamide gel.I After fixing enhancing and drying, the gel was exposed to X-ray film. The results confirmed that antibodies to recombinant E, NSl, NS2, NS3 and NS5 had been generated in mice (Fig. 2) and in rabbits (Fig. 3) . These antibodies reacted with the native E, NSl, NS2, NS3 and NS5 proteins synthesised in infected C6/36 cells. EXAMPLE 8 INDIRECT IMMUNOFLUORESCENCE ASSAY The C6/36 cells infected with Dengue virus S275/90 for 2 days were fixed on glass plates with cold acetone for immunofluorescence. 2-fold dilutions of the sera of rabbits or mice were incubated with the fixed cells for 1 h at 37°C, then washed with PBS. Secondary antibodies were linked to fluorescein and incubated for 1 h, followed by washing with PBS for observation using fluorescence microscopy. Fig 4 shows the antisera to E, NSl, NS2, NS3 and NS5 reacted specifically with the Dengue virus S275/90 infected cells, but control antiserum did no react. Quantitation of the result (as set out in Table 4) showed that an immune response to all recombinant Dengue virus proteins (E, NSl, NS2, NS3 and NS5) occurred in both mice and rabbits. TABLE 3 Oligonucleotides used to prepare cDNA fragments corresponding to Dengue virus proteins (by PCR) 1. pGEX-KG/EX-20 DIF920E EcoRl • E 5'CCA TGA ATT CCC^ATG CGA TGC GTG GGA DIF2400X Xhol £ 5'CAC ATC^TCG AGT CCG CTT GAA CCA TGA DIR2400S P maτ [ NS1 5' TGG TTC CCG GGG ACT CGG GAT GTG TA DIF3458H .Hindlll NSl 5'ACT A*AG CTT GAT CAT GCA GAG ACC ATT GA 3. pMAL-cRI/NS2-l DIR-NS2PM EcoRl NS2 5'AAT CAG^AAT TCT CTG CAG GGT CAG GGG AA DIF-NS2H Hindlll _NS2 5'ATA ACA AAG CTT ATC TTT GTT TCT TTT TCT 4. pGEX-KG/NS3 BHC6001 DIR-NS3B BamHI _ S3 5'GAA AGG ATC CTC TGG AGT GTT ATG GGA CAC A DIF-6360H Hindlll JTS3 5'ACC CAA GCT TCA TCT TCT TCC TGC TGC 5. pGEX-KG/NS5(C600 HF1) TAC GGG AGC C DIF-8365 5'CAA TGA TAT CTA GGT TGG CT TABLE 4 IMMUNE RESPONSES OF MICE AND RABBITS: INDIRECT IMMUNOFLUORESCENCE ASSAYS SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: National University of Singapore (B) STREET: 10 Kent Ridge Crescent (C) CITY;: Singapore (E) COUNTRY: Singapore (F) POSTAL CODE (ZIP): 0511 (ii) TITLE OF INVENTION: Dengue Virus (iii) NUMBER OF SEQUENCES: 12 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER: (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10718 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Dengue Fever Virus Type 1 (B) STRAIN: S275/90 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 81..10268 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GTGGACCGCA AAGAACAGTT TCGAATCGGA AGCTTGCTTA ACGTAGTTCT AACAGTTTTT 60 TATTAGAGAG CAGATCTCTG ATG AAC AAC CAA CGA AAA AAG ACG GCT CGA 110 Met Asn Asn Gin Arg Lys Lys Thr Ala Arg 1 5 10 CCG TCT TTC AAT ATG CTG AAA CGC GCG AGA AAC CGC GTG TCA ACT GGT 158 Pro Ser Phe Asn Met Leu Lys Arg Ala Arg Asn Arg Val Ser Thr Gly 15 20 25 TCA CAG TTG GCG AAG AGA TTC TCA AAA GGA TTG CTT TCA GGC CAA GGA 206 Ser Gin Leu Ala Lys Arg Phe Ser Lys Gly Leu Leu Ser Gly Gin Gly 30 35 40 CCC ATG AAA TTG GTG ATG GCT TTC ATA GCA TTC CTA AGA TTT CTA GCC 254 Pro Met Lys Leu Val Met Ala Phe lie Ala Phe Leu Arg Phe Leu Ala 45 50 55 ATA CCC CCA ACA GCA GGA ATT TTG GCT AGA TGG GGC TCA TTC AAG AAG 302 He Pro Pro Thr Ala Gly He Leu Ala Arg Trp Gly Ser Phe Lys Lys 60 65 70 AAT GGA GCG ATC AAA GTG CTA CGG GGT TTC AAG AAA GAA ATC TCA AAC 350 Asn Gly Ala He Lys Val Leu Arg Gly Phe Lys Lys Glu He Ser Asn 75 80 85 90 ATG TTG AAC ATA ATG AAT AGA AGG AAA AGA TCT GTG ACC ATG CTC CTC 398 Met Leu Asn He Met Asn Arg Arg Lys Arg Ser Val Thr Met Leu Leu 95 100 105 ATG CTG CTG CCC ACA GCC TTG GCG TTC CAT TTG ACT ACA CGA GGG GGA 446 Met Leu Leu Pro Thr Ala Leu Ala Phe His Leu Thr Thr Arg Gly Gly 110 115 120 GAG CCA CAC ATG ATA GTT AGC AAG CAG GAA AGA GAA AAG TCA CTC TTG 494 Glu Pro His Met He Val Ser Lys Gin Glu Arg Glu Lys Ser Leu Leu 125 130 135 TTT AAG ACC TCT GTA GGT GTC AAC ATG TGC ACC CTT ATA GCG ATG GAT 542 Phe Lys Thr Ser Val Gly Val Asn Met Cys Thr Leu He Ala Met Asp 140 145 150 TTG GGA GAG TTA TGT GAG GAC ACA ATG ACT TAC ;kλA TGC CCT CGA ATT 590 Leu Gly Glu Leu Cys Glu Asp Thr Met Thr Tyr."'^ys Cys Pro Arg He 155 160 165 "' 170 ACT GAG GCG GAA CCA GAT GAC GTT GAT TGT TGG TGC AAT GCT ACA GAC 638 Thr Glu Ala Glu Pro Asp Asp Val Asp Cys Trp Cys Asn Ala Thr Asp 175 180 185 ACA TGG GTG ACC TAT GGA ACA TGT TCC CAA ACT GGC GAG CAC CGA CGG 686 Thr Trp Val Thr Tyr Gly Thr Cys Ser Gin Thr Gly Glu His Arg Arg 190 195 200 GAC AAA CGT TCC GTC GCA CTG GCC CCA CAC GTG GGA CTT GGT CTA GAA 734 Asp Lys Arg Ser Val Ala Leu Ala Pro His Val Gly Leu Gly Leu Glu 205 210 215 ACA AGA ACC GAA ACG TGG ATG TCC TCT GAA GGC GCT TGG AAA CAA ATA 782 Thr Arg Thr Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys Gin He 220 225 230 CAA AGA GTG GAG ACT TGG GCT TTG CGA CAC CCA GGA TTC ACG GTG ATA 830 Gin Arg Val Glu Thr Trp Ala Leu Arg His Pro Gly Phe Thr Val He 235 240 245 250 GCC CTT TTT CTT GCA CAT GCC ATA GGA ACA TCC ATC ACT CAG AAA GGG 878 Ala Leu Phe Leu Ala His Ala He Gly Thr Ser He Thr Gin Lys Gly 255 260 265 ATT ATT TTC ATT TTG TTA ATG CTA GTA ACA CCA TCC ATG GCC ATG CGA 926 He He Phe He Leu Leu Met Leu Val Thr Pro Ser Met Ala Met Arg 270 275 280 TGC GTG GGA ATA GGC AGC AGG GAC TTC * GTG GAA GGA CTA TCA GGA GCA 974 Cys Val Gly He Gly Ser Arg Asp Phe Val Glu Gly Leu Ser Gly Ala 285 290 295 ACT TGG GTA GAC GTG GTA CTG GAA CAT GGA AGT TGC GTC ACC ACC ATG 1022 Thr Trp Val Asp Val Val Leu Glu His Gly Ser Cys Val Thr Thr Met 300 305 310 GCA AAA GAC AAA CCA ACA TTG GAC ATT GAA CTC CTG AAA ACG GAG GTC 1070 Ala Lys Asp Lys Pro Thr Leu Asp He Glu Leu Leu Lys Thr Glu Val 315 320 325 330 ACG AAC CCT GCC GTC CTG CGC AAA CTG TGC ATT GAA GCT AAA ATA TCA 1118 Thr Asn Pro Ala Val Leu Arg Lys Leu Cys He Glu Ala Lys He Ser 335 340 345 AAC ACC ACC ACC GAT TCA AGA TGT CCA ACA CAA GGA GAA GCT ACA CTG 1166 Asn Thr Thr Thr Asp Ser Arg Cys Pro Thr Gin Gly Glu Ala Thr Leu 350 355 360 GTG GAA GAA CAA GAC GCG AAC TTT GTG TGT CGA CGA ACG TTC GTG GAC 1214 Val Glu Glu Gin Asp Ala Asn Phe Val Cys Arg Arg Thr Phe Val Asp 365 370 375 AGA GGC TGG GGT AAT GGC TGC GGA CTA TTT GGA AAA GGA AGC CTA CTG 1262 Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Leu Leu 380 385 390 ACG TGT GCT AAG TTC AAG TGT GTG ACA AAA CTA GAA GGA AAG ATA GTT 1310 Thr Cys Ala Lys Phe Lys Cys Val Thr Lys Leu Glu Gly Lys He Val 395 400 405 410 CAA TAT GAA AAC TTA AAA TAT TCA GTG ATA GTC ACT GTC CAC ACT GGG 1358 Gin Tyr Glu Asn Leu Lys Tyr Ser Val He Val Thr Val His Thr Gly 415 420 425 GAC CAG CAC CAG GTG GGA AAC GAG ACT ACA GAA CAT GGA ACA ATT GCA 1406 Asp Gin His Gin Val Gly Asn Glu Thr Thr Glu His Gly Thr He Ala 430 435 440 ACC ATA ACA CCT CAA GCT CCT ACG TCG GAA ATA CAG CTG ACC GAC TAC 1454 Thr He Thr Pro Gin Ala Pro Thr Ser Glu He Gin Leu Thr Asp Tyr 445 450 455 GGA GCC CTC ACA TTG GAC TGC TCA CCT AGA ACT GGG CTG GAC TTT AAT 1502 Gly Ala Leu Thr Leu Asp Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn 460 465 470 GAG ATG GTG CTA TTG ACA ATG AAA GAA AAA TCA TGG CTT GTT CAC AAA 1550 Glu Met Val Leu Leu Thr Met Lys Glu Lys Ser Trp Leu Val His Lys 475 480 485 490 CAA TGG TTT CTA GAC TTA CCA CTG CCT TGG ACT TCG GGG GCT TCA ACA 1598 Gin Trp Phe Leu Asp Leu Pro Leu Pro Trp Thr Ser Gly Ala Ser Thr 495 500 505 TCC CAA GAG ACT TGG AAC AGA CAA GAT TTG CTG GTC ACA TTC AAG ACA 1646 Ser Gin Glu Thr Trp Asn Arg Gin Asp Leu Leu Val Thr Phe Lys Thr 510 515 520 GCT CAT GCA AAG AAG CAG GAA GTA GTC GTA CTG GGA TCA CAG GAA GGA 1694 Ala His Ala Lys Lys Gin Glu Val Val Val Leu Gly Ser Gin Glu Gly 525 530 535 GCA ATG CAC ACT GCG TTG ACT GGG GCG ACA GAA ATC CAA ACG TCT GGA 1742 Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu He Gin Thr Ser Gly 540 545 550 ACG ACA ACA ATT TTT GCA GGA CAC CTG AAA TGT AGA CTA AAA ATG GAC 1790 Thr Thr Thr He Phe Ala Gly His Leu Lys Cys Arg Leu Lys Met Asp 555 560 565 570 AAA CTG ACT CTA AAA GGG ATG TCA TAT GTG ATG TGC ACA GGC TCA TTT 1838 Lys Leu Thr Leu Lys Gly Met Ser Tyr Val Met Cys Thr Gly Ser Phe 575 580 585 AAG CTA GAG AAG GAA GTG GCT GAG ACC CAG CAT GGA ACT GTT TTA GTG 1886 Lys Leu Glu Lys Glu Val Ala Glu Thr Gin His Gly Thr Val Leu Val 590 595 600 CAG GTT AAA TAC GAA GGA ACA GAT GCA CCA TGC AAG ATC CCC TTT TCG 1934 Gin Val Lys Tyr Glu Gly Thr Asp Ala Pro Cys Lys He Pro Phe Ser 605 610 615 ACC CAA GAT GAG AAA GGA GTG ACC CAG AAT AGA TTG ATA ACA GCC AAT 1982 Thr Gin Asp Glu Lys Gly Val Thr Gin Asn Arg Leu He Thr Ala Asn 620 625 630 CCT ATA GTT ACT GAC AAA GAA AAA CCA GTC AAC ATT GAG ACA GAA CCA 2030 Pro He Val Thr Asp Lys Glu Lys Pro Val Asn He Glu Thr Glu Pro 635 640 645 650 CCT TTT GGT GAG AGC TAC ATC GTG GTA GGG GCA GGT GAA AAA GCT TTG 2078 Pro Phe Gly Glu Ser Tyr He Val Val Gly Ala Gly Glu Lys Ala Leu 655 660 665 AAA CAA TGC TGG TTC AAG AAA GGA AGC AGC ATA GGG AAA ATG TTC GAA 2126 Lys Gin Cys Trp Phe Lys Lys Gly Ser Ser He Gly Lys Met Phe Glu 670 675 680 GCA ACC GCC CGA GGA GCA CGA AGG ATG GCT ATC CTG GGA GAC ACC GCA 2174 Ala Thr Ala Arg Gly Ala Arg Arg Met Ala He Leu Gly Asp Thr Ala 685 690 695 TGG GAC TTC GGT TCT ATA GGA GGA GTG TTC ACG TCT GTG GGA AAA TTA 2222 Trp Asp Phe Gly Ser He Gly Gly Val Phe Thr Ser Val Gly Lys Leu 700 705 710 GTG CAT CAG GTT TTT GGA ACC GCA TAT GGG GTT CTG TTC AGC GGT GTT 2270 Val His Gin Val Phe Gly Thr Ala Tyr Gly Val Leu Phe Ser Gly Val 715 720 725 730 TCT TGG ACC ATG AAA ATA GGA ATA GGG ATT CTG CTG ACA TGG TTG GGA 2318 Ser Trp Thr Met Lys He Gly He Gly He Leu Leu Thr Trp Leu Gly 735 740 745 TTA AAT TCA AGG AGC ACG TCA CTT TCG ATG ACG TGC ATT GCA GTT GGC 2366 Leu Asn Ser Arg Ser Thr Ser Leu Ser Met Thr Cys He Ala Val Gly 750 755 760 ATG GTC ACA CTG TAC CTA GGA GTC ATG GTT CAA GCG GAC TCG GGA TGT 2414 Met Val Thr Leu Tyr Leu Gly Val Met Val Gin Ala Asp Ser Gly Cys 765 770 775 GTA ATC AAC TGG AAG GGC AGA GAA CTC AAA TGT GGA AGT GGC ATT TTT 2462 Val He Asn Trp Lys Gly Arg Glu Leu Lys Cys Gly Ser Gly He Phe 780 785 790 GTC ACT AAT GAA GTC CAC ACT TGG ACA GAG CAA TAC AAA TTT CAA GCT 2510 Val Thr Asn Glu Val His Thr Trp Thr Glu Gin Tyr Lys Phe Gin Ala 795 800 805 810 GAC TCC CCA AAA AGA CTA TCA GCA GCC ATC GGA AAG GCA TGG GAG GAG 2558 Asp Ser Pro Lys Arg Leu Ser Ala Ala He Gly Lys Ala Trp Glu Glu 815 820 825 GGT GTG TGT GGA ATT CGA TCA GCC ACT CGT CTC GAG AAC ATC ATG TGG 2606 Gly Val Cys Gly He Arg Ser Ala Thr Arg Leu Glu Asn He Met Trp 830 835 840 AAG CAA ATA TCA AAT GAA CTG AAC CAC ATC TTA CTT GAA AAT GAC ATG 2654 Lys Gin He Ser Asn Glu Leu Asn His He Leu Leu Glu Asn Asp Met 845 850 855 AAA TTC ACA GTG GTT GTA GGA GAT GTT GTT GGG ATC TTG GCC CAA GGG 2702 Lys Phe Thr Val Val Val Gly Asp Val Val Gly He Leu Ala Gin Gly 860 865 870 AAA AAA ATG ATT AGA CCA CAA CCC ATG GAA CAC AAA TAC TCA TGG AAA 2750 Lys Lys Met He Arg Pro Gin Pro Met Glu His Lys Tyr Ser Trp Lys 875 880 885 890 AGC TGG GGA AAA GCC AAA ATC ATA GGA GCA GAC ATA CAG AAC ACC ACC 2798 Ser Trp Gly Lys Ala Lys He He Gly Ala Asp He Gin Asn Thr Thr 895 900 905 TTC ATC ATT GAC GGC CCA GAT ACT CCA GAA TGT CCT GAT GAC CAA AGA 2846 Phe He He Asp Gly Pro Asp Thr Pro Glu Cys Pro Asp Asp Gin Arg 910 915 920 GCA TGG AAC ATT TGG GAA GTT GAG GAC TAT GGG TTC GGA ATT TTC ACG 2894 Ala Trp Asn He Trp Glu Val Glu Asp Tyr Gly Phe Gly He Phe Thr 925 930 935 ACA AAC ATA TGG TTG AAA TTG CGT GAC TCC TAC ACC CAA ATG TGT GAC 2942 Thr Asn He Trp Leu Lys Leu Arg Asp Ser Tyr Thr Gin Met Cys Asp 940 945 950 CAC CGG CTA ATG TCA GCT GCC ATC AAG GAC AGC AAG GCA GTC CAT GCT 2990 His Arg Leu Met Ser Ala Ala He Lys Asp Ser Lys Ala Val His Ala 955 960 965 970 GAT ATG GGG TAC TGG ATA GAA AGT GAA AAG AAC GAG ACC TGG AAG CTG 3038 Asp Met Gly Tyr Trp He Glu Ser Glu Lys Asn Glu Thr Trp Lys Leu 975 980 985 GCA AGA GCC TCT TTC ATA GAA GTT AAA ACA TGT GTC TGG CCA AAA TCC 3086 Ala Arg Ala Ser Phe He Glu-Val Lys Thr Cys Val Trp Pro Lys Ser 990 995 1000 CAC ACT CTA TGG AGC AAT GGA GTT CTG GAA AGT GAA ATG ATA ATT CCA 3134 His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met He He Pro 1005 1010 1015 AAG ATC TAT GGA GGA CCA ATA TCT CAG CAC AAC TAC AGA CCA GGA TAT 3182 Lys He Tyr Gly Gly Pro He Ser Gin His Asn Tyr Arg Pro Gly Tyr 1020 1025 1030 TTC ACA CAA ACG GCA GGG CCA TGG CAC CTA GGC AAG TTG GAA CTG GAT 3230 Phe Thr Gin Thr Ala Gly Pro Trp His Leu Gly Lys Leu Glu Leu Asp 1035 1040 1045 1050 TTT GAT TTG TGT GAG GGT ACC ACA GTT GTT GTG GAT GAA CAT TGT GGA 3278 Phe Asp Leu Cys Glu Gly Thr Thr Val Val Val Asp Glu His Cys Gly 1055 1060 1065 AAT CGA GGT CCA TCT CTT AGA ACC ACA ACA GTC ACA GGA AAG ATA ATT 3326 Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Val Thr Gly Lys He He 1070 1075 .1080 CAT GAA TGG TGT TGC AGA TCT TGT ACG CTA CCA CCC TTA CGT TTC AAA 3374 His Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Phe Lys 1085 1090 1095 GGA GAA GAT GGA TGT TGG TAC GGT ATG GAA ATC AGA CCA GTC AAG GAA 3422 Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu He Arg Pro Val Lys Glύ 1100 1105 1110 AAG GAA GAG AAT CTA GTC AAA TCA ATG GTC TCT GCA GGG TCA GGG GAA 3470 Lys Glu Glu Asn Leu Val Lys Ser Met Val Ser Ala Gly Ser Gly Glu 1115 1120 1125 1130 GTG GAC AGC TTT TCA CTA GGA CTG CTA TGC ATA TCA ATA ATG ATC GAA 3518 Val Asp Ser Phe Ser Leu Gly Leu Leu Cys He Ser He Met He Glu 1135 1140 1145 GAG GTG ATG AGA TCC AGA TGG AGC AGA AAA ATG CTG ATG ACT GGA ACA 3566 Glu Val Met Arg Ser Arg Trp Ser Arg Lys Met Leu Met Thr Gly Thr 1150 1155 1160 CTG GCT GTG TTC CTC CTT CTC ATA ATG GGA CAA TTG ACA TGG AAT GAT 3614 Leu Ala Val Phe Leu Leu Leu He Met Gly Gin Leu Thr Trp Asn Asp 1165 1170 1175 CTG ATC AGG TTA TGC ATC ATG GTT GGA GCC AAT GCT TCA GAC AGG ATG 3662 Leu He Arg Leu Cys He Met Val Gly Ala Asn Ala Ser Asp Arg Met 1180 1185 1190 GGG ATG GGA ACA ACG TAC CTA GCT CTG ATG GCC ACT TTT AAA ATG AGA 3710 Gly Met Gly Thr Thr Tyr Leu Ala Leu Met Ala Thr Phe Lys Met Arg 1195 1200 1205 1210 CCA ATG TTT GCT GTC GGG CTG TTG TTC CGC AGA CTA ACA TCT AGA GAA 3758 Pro Met Phe Ala Val Gly Leu Leu Phe Arg Arg Leu Thr Ser Arg Glu 1215 1220 1225 GTT CTT CTT CTT ACA ATT GGA TTG AGT CTA GTG GCA TCT GTG GAG TTA 3806 Val Leu Leu Leu Thr He Gly Leu Ser Leu Val Ala Ser Val Glu Leu 1230 1235 1240 CCA AAT TCC CTG GAG GAG CTG GGG GAT GGA CTT GCA ATG GGC ATT ATG 3854 Pro Asn Ser Leu Glu Glu Leu Gly Asp Gly Leu Ala Met Gly He Met 1245 1250 1255 ATT TTA AAA TTA TTG ACT GAC TTT CAG TCA CAT CAG CTG TGG GCT ACC 3902 He Leu Lys Leu Leu Thr Asp Phe Gin Ser His Gin Leu Trp Ala Thr 1260 1265 1270 TTG CTG TCC TTG ACA TTT GTC AAA ACA ACG TTT TCC TTG CAC TAT GCA 3950 Leu Leu Ser Leu Thr Phe Val Lys Thr Thr Phe Ser Leu His Tyr Ala 1275 1280 1285 1290 TGG AAG ACA ATG GCT ATG GTA CTG TCA ATT GTA TCT CTC TTC CCC TTA 3998 Trp Lys Thr Met Ala Met Val Leu Ser He Val Ser Leu Phe Pro Leu 1295 1300 1305 TGC CTG TCC ACG ACC TCC CAA AAA ACA ACA TGG CTT CCG GTG CTA TTG 4046 Cys Leu Ser Thr Thr Ser Gin Lys Thr Thr Trp Leu Pro Val Leu Leu 1310 1315 1320 GGA TCT CTT GGA GC AAA CCA CTA ACC ATG TTT CTC ATA GCA GAA AAC 4094 Gly Ser Leu Gly Cys Lys Pro Leu Thr Met Phe Leu He Ala Glu Asn 1325 1330 1335 AAA ATC TGG GGA AGG AAA AGT TGG CCC CTC AAT GAA GGA ATC ATG GCT 4142 Lys He Trp Gly Arg Lys Ser Trp Pro Leu Asn Glu Gly He Met Ala 1340 1345 1350 GTT GGA ATA GTC AGC ATC CTA CTA AGT TCA CTC CTC AAA AAT GAT GTG 4190 Val Gly He Val Ser He Leu Leu Ser Ser Leu Leu Lys Asn Asp Val 1355 1360 1365 1370 - CCG CTA GCT GGG CCA CTA ATA GCT GGA GGC ATG CTA ATA -GCA TGT TAC 4238 Pro Leu Ala Gly Pro Leu He Ala Gly Gly Met Leu He Ala Cys Tyr 1375 1380 1385 GTT ATA TCT GGA AGC TCA GCC GAC TTA TCA CTA GAG AAA GCG GCT GAG 4286 Val He Ser Gly Ser Ser Ala Asp Leu Ser Leu Glu Lys Ala Ala Glu 1390 1395 1400 GTC TCC TGG GAA GAA GAA GCA GAA CAC TCT GGT GCC TCA CAC AAT ATA 4334 Val Ser Trp Glu Glu Glu Ala Glu His Ser Gly Ala Ser His Asn He 1405 1410 1415 TTA GTG GAG GTC CAA GAT GAT GGA ACC ATG AAG ATA AAA GAT GAA GAG 4382 Leu Val Glu Val Gin Asp Asp Gly Thr Met Lys He Lys Asp Glu Glu 1420 1425 1430 AGA GAT GAC ACG CTA ACC ATT CTC CTT AAA GCA ACC CTG CTA GCA GTT 4430 Arg Asp Asp Thr Leu Thr He Leu Leu Lys Ala Thr Leu Leu Ala Val 1435 1440 1445 1450 TCA GGG GTG TAC CCA TTA TCA ATA CCA GCA ACC CTT TTT GTG TGG TAC 4478 Ser Gly Val Tyr Pro Leu Ser He Pro Ala Thr Leu Phe Val Trp Tyr 1455 1460 1465 TTT TGG CAG AAA AAG AAA CAA AGA TCT GGA GTG TTA TGG GAC ACA CCT 4526 Phe Trp Gin Lys Lys Lys Gin Arg Ser Gly Val Leu Trp Asp Thr Pro 1470 1475 1480 AGC CCT CCA GAA GTG GAA AGA GCA GTC CTT GAT GAT GGT ATC TAT AGA 4574 Ser Pro Pro Glu Val Glu Arg Ala Val Leu Asp Asp Gly He Tyr Arg 1485 1490 1495 ATT ATG CAG AGA GGA CTG TTG GGC AGG TCC CAA GTA GGA GTG GGA GTT 4622 He Met Gin Arg Gly Leu Leu Gly Arg Ser Gin Val Gly Val Gly Val 1500 1505 1510 TTC CAA GAC GGC GTG TTC CAC ACA ATG TGG CAC GTC ACC AGG GGA GCT 4670 Phe Gin Asp Gly Val Phe His Thr Met Trp His Val Thr Arg Gly Ala 1515 1520 1525 1530 GTC CTT ATG TAC CAA GGG AAG AGG CTG GAA CCA AGC TGG GCC AGT GTC 4718 Val Leu Met Tyr Gin Gly Lys Arg Leu Glu Pro Ser Trp Ala Ser Val 1535 1540 1545 AAA AAA GAC TTG ATC TCA TAT GGA GGA GGT TGG AGG TTT CAA GGA TCC 4766 Lys Lys Asp Leu He Ser Tyr Gly Gly Gly Trp Arg Phe Gin Gly Ser 1550 1555 1560 TGG AAC ACG GGA GAA GAA GTG CAG GTG ATT GCT GTT GAA CCA GGA AAA 4814 Trp Asn Thr Gly Glu Glu Val Gin Val He Ala Val Glu Pro Gly Lys 1565 1570 1575 AAC CCC AAA AAT GTA CAG ACA GCG CCG GGT ACC TTC AAG ACC CCT GAA 4862 Asn Pro Lys Asn Val Gin Thr Ala Pro Gly Thr Phe Lys Thr Pro Glu 1580 1585 1590 GGT GAA GTT GGA GCT ATT GCC CTA GAT TTT AAA CCC GGC ACA TCT GGA 4910 Gly Glu Val Gly Ala He Ala Leu Asp Phe Lys Pro Gly Thr Ser Gly 1595 1600 1605 1610 TCT CCC ATC GTG AAC AGA GAA GGA AAA ATA GTA GGT CTT TAT GGA AAT 4958 Ser Pro He Val Asn Arg Glu Gly Lys He Val Gly Leu Tyr Gly Asn 1615 1620 1625 GGA GTA GTG ACA ACA AGT GGA ACC TAC GTC AGT GCC ATA GCC CAA GCC 5006 Gly Val Val Thr Thr Ser Gly Thr Tyr Val Ser Ala He Ala Gin Ala 1630 1635 1640 AAA GCA TCA CAA GAA GGG CCC CTA CCA GAG ATT GAG GAC GAG GTG TTT 5054 Lys Ala Ser Gin Glu Gly Pro Leu Pro Glu He Glu Asp Glu Val Phe 1645 1650 1655 AGG AAA AGA AAC TTA ACA ATA ATG GAC CTA CAT CCA GGA TCG GGG AAA 5102 Arg Lys Arg Asn Leu Thr He Met Asp Leii His Pro Gly Ser Gly Lys 1660 1665 1670 ACA AGA AGA TAT CTT CCA GCC ATA GTC CGT GAG GCC ATA AGA AGG AAC 5150 Thr Arg Arg Tyr Leu Pro Ala He Val Arg Glu Ala He Arg Arg Asn 1675 1680 1685 1690 GTG CGC ACA CTA ATT TTG GCT CCC ACA AGG GTT 'feTC GCT TCC GAA ATG 5198 Val Arg Thr Leu He Leu Ala Pro Thr Arg Val j'al Ala Ser Glu Met 1695 1700 '• -- 1705 GCA GAG GCG CTC AAG GGA ATG CCA ATA AGG TAC CAA ACA ACA GCA GTG 5246 Ala Glu Ala Leu Lys Gly Met Pro He Arg Tyr Gin Thr Thr Ala Val 1710 1715 1720 AAG AGT GAA CAC ACA GGA AAA GAG ATA GTT GAC CTC ATG TGT CAC GCC 5294 Lys Ser Glu His Thr Gly Lys Glu He Val Asp Leu Met Cys His Ala 1725 1730 1735 ACT TTC ACC ATG CGT CTC CTG TCT CCC GTG AGA GTT CCC AAT TAC AAC 5342 Thr Phe Thr Met Arg Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn 1740 1745 1750 ATG ATT ATC ATG GAT GAA GCA CAT TTT ACC GAT CCA GCC AGC ATA GCG 5390 Met He He Met Asp Glu Ala His Phe Thr Asp Pro Ala Ser He Ala 1755 1760 1765 1770 CGC AGA GGG TAC ATC TCA ACC CGA GTG GGC ATG GGT GAA GCA GCT GCG 5438 Arg Arg Gly Tyr He Ser Thr Arg Val Gly Met Gly Glu Ala Ala Ala 1775 1780 1785 ATC TTC ATG ACA GCC ACT CCC CCA GGA TCG GTG GAG GCC TTT CCA CAG 5486 He Phe Met Thr Ala Thr Pro Pro Gly Ser Val Glu Ala Phe Pro Gin 1790 1795 1800 AGC AAT GCA GTT ATC CAA GAT GAG GAA AGA GAC ATT CCT GAG AGA TCA 5534 Ser Asn Ala Val He Gin Asp Glu Glu Arg Asp He Pro Glu Arg Ser 1805 1810 1815 TGG AAC TCA GGC TAT GAG TGG ATC ACT- GAC TTC CCA GGT AAA ACA GTC 5582 Trp Asn Ser Gly Tyr Glu Trp He Thr Asp Phe Pro Gly Lys Thr Val 1820 1825 1830 TGG TTT GTT CCA AGC ATC AAA TCA GGA AAT GAC ATT GCC AAC TGC TTA 5630 Trp Phe Val Pro Ser He Lys Ser Gly Asn Asp He Ala Asn Cys Leu 1835 1840 1845 1850 AGA AAG AAT GGG AAA CGG GTG ATT CAA TTG AGC AGG AAA ACC TTT GAT 5678 Arg Lys Asn Gly Lys Arg Val He Gin Leu Ser Arg Lys Thr Phe Asp 1855 1860 1865 ACA GAG TAC CAA AAA ACA AAA AAC AAC GAC TGG GAC TAT GTC GTC ACA 5726 Thr Glu Tyr Gin Lys Thr Lys Asn Asn Asp Trp Asp Tyr Val Val Thr 1870 1875 1880 ACA -GAT ATC TCC GAA ATG GGA GCA AAC TTC CGA GCC GAC AGG GTG ATA 5774 Thr Asp He Ser Glu Met Gly Ala Asn Phe Arg Ala Asp Arg Val He 1885 1890 1895 GAC CCA AGA CGG TGT CTG AAA CCG GTA ATA CTA AAA GAT GGT CCA GAG 5822 Asp Pro Arg Arg Cys Leu Lys Pro Val He Leu Lys Asp Gly Pro Glu 1900 1905 1910 CGC GTC ATT CTA GCC GGA CCG ATG CCA GTG ACT GTG GCC AGT GCT GCC 5870 Arg Val He Leu Ala Gly Pro Met Pro Val Thr Val Ala Ser Ala Ala 1915 1920 1925 1930 CAG AGG AGA GGA AGA ATT GGA AGG AAC CAA AAC AAA GAA GGT GAT CAG 5918 Gin Arg Arg Gly Arg He Gly Arg Asn Gin Asn Lys Glu Gly Asp Gin 1935 1940 1945 TAC GTT TAC ATG GGA CAG CCT TTA AAT AAT GAT GAG GAT CAC GCT CAT 5966 Tyr Val Tyr Met Gly Gin Pro Leu Asn Asn Asp Glu Asp His Ala His 1950 1955 1960 TGG ACA GAA GCA AAA ATG CTC CTT GAC AAT ATA AAC ACA CCA GAA GGG 6014 Trp Thr Glu Ala Lys Met Leu Leu Asp Asn He Asn Thr Pro Glu Gly 1965 1970 1975 ATC ATC CCA GCC CTC TTT GAG CCA GAG AGA GAA AAG AGT GCA GCA ATA 6062 He He Pro Ala Leu Phe Glu Pro Glu Arg Glu Lys Ser Ala Ala He 1980 1985 1990 GAC GGG GAG TAC AGA CTG CGG GGA GAA GCA AGA AAA ACG TTT GTG GAG 6110 Asp Gly Glu Tyr Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Glu 1995 2000 2005 2010 CTC ATG AGA AGA GGA GAT CTA CCT GTC TGG CTA TCC TAC AAA GTT GCC 6158 Leu Met Arg Arg Gly Asp Leu Pro Val Trp Leu Ser Tyr Lys Val Ala 2015 2020 2025 TCA GAA GGC TTC CAG TAC TCT GAC AGA AGA TGG TGC TTT GAC GGG GAA 6206 Ser Glu Gly Phe Gin Tyr Ser Asp Arg Arg Trp Cys Phe Asp Gly Glu 2030 2035 2040 AGG AAC AAC CAG GTG TTG GAG GAG AAC ATG GAC GTG GAG ATG TGG ACA 6254 Arg Asn Asn Gin Val Leu Glu Glu Asn Met Asp Val Glu Met Trp Thr 2045 2050 2055 AAA GAA GGA GAA CGA AAG AAA CTA CGA CCC CGC TGG CTG GAT GCC AGA 6302 Lys Glu Gly Glu Arg Lys Lys Leu Arg Pro Arg Trp Leu Asp Ala Arg 2060 2065 2070 ACA TAC TCA GAC CCA CTG GCC CTG CGC GAG TTT AAA GAG TTT GCA GCA 6350 Thr Tyr Ser Asp Pro Leu Ala Leu Arg Glu Phe Lys Glu Phe Ala Ala 2075 2080 2085 2090 GGA AGA AGA AGT GTC TCA GGT GAT CTA ATA TTA ' GAA ATA GGG AAA CTT 6398 Gly Arg Arg Ser Val Ser Gly Asp Leu He Leu Glu He Gly Lys Leu 2095 2100 2105 CCA CAA CAC TTG ACG CAA AGG GCC CAG AAT GCC TTG GAC AAC CTG GTT 6446 Pro Gin His Leu Thr Gin Arg Ala Gin Asn Ala Leu Asp Asn Leu Val 2110 2115 2120 ATG TTG CAC AAC TCC GAA CAA GGA GGA AGA GCC TAC AGA CAT GCA ATG 6494 Met Leu His Asn Ser Glu Gin Gly Gly Arg Ala Tyr Arg His Ala Met 2125 2130 2135 GAA GAA CTT CCA GAC ACC ATA GAA ACG TTG ATG CTC CTA GCT TTG ATA 6542 Glu Glu Leu Pro Asp Thr He Glu Thr Leu Met Leu Leu Ala Leu He 2140 2145 2150 GCT GTG TTA ACT GGT GGA GTG ACG CTG TTC TTC CTA TCA GGA AAG GGC 6590 Ala Val Leu Thr Gly Gly Val Thr Leu Phe Phe Leu Ser Gly Lys Gly 2155 2160 2165 2170 CTA GGG AAA ACA TCT ATT GGC CTA CTC TGC GTG ATG GCT TCA AGC GTA 6638 Leu Gly Lys Thr Ser He Gly Leu Leu Cys Val Met Ala Ser Ser Val 2175 2180 2185 CTG CTA TGG ATG GCC AGC GTG GAG CCT CAT TGG ATA GCG GCC TCC ATC 6686 Leu Leu Trp Met Ala Ser Val Glu Pro His Trp He Ala Ala Ser He 2190 2195 2200 ATA CTA GAG TTT TTC CTG ATG GTG CTG CTT ATT CCA GAG CCA GAC AGA 6734 He Leu Glu Phe. Phe Leu Met Val Leu Leu He Pro Glu Pro Asp Arg 2205 2210 2215 CAG CGC ACT CCA CAG GAC AAC CAG TTA GCA TAT GTG GTG ATA GGT TTG 6782 Gin Arg Thr Pro Gin Asp Asn Gin Leu Ala Tyr Val Val He Gly Leu 2220 2225 2230 TTA TTC ATG ATA CTC ACA GTG GCA GCC AAT GAG ATG GGA TTA TTG GAA 6830 Leu Phe Met He Leu Thr Val Ala Ala Asn Glu Met Gly Leu Leu Glu 2235 2240 2245 2250 ACC ACA AAG AAA GAC TTA GGG ATT GGC CAT GTA GCC GCC GAA AAC CAC 6878 Thr Thr Lys Lys Asp Leu Gly He Gly His Val Ala Ala Glu Asn His 2255 2260 2265 CAC CAT GCT ACA ATG CTG GAC GTA GAC CTA CGT CCA GCT TCA GCC TGG 6926 His His Ala Thr Met Leu Asp Val Asp Leu Arg Pro Ala Ser Ala Trp 2270 2275 2280 ACC CTC TAT GCA GTA GCC ACA ACA GTT ATC ACC CCC ATG ATG AGA CAC 6974 Thr Leu Tyr Ala Val Ala Thr Thr Val He Thr Pro Met Met Arg His 2285 2290 2295 ACA ATT GAA AAT ACA ACG GCA AAT ATT TCC CTG ACA GCC ATT GCA AAC 7022 Thr He Glu Asn Thr Thr Ala Asn He Ser Leu Thr Ala He Ala Asn 2300 2305 2310 CAG GCA GCT ATA TTG ATG GGA CTT GAT AAA GGA TGG CCA ATA TCG AAG 7070 Gin Ala Ala He Leu Met Gly Leu Asp Lys Gly Trp Pro He Ser Lys 2315 2320 2325 2330 ATG GAC ATA GGA GTT CCA CTT CTC GCC TTG GGG TGC TAT TCC CAG GTG 7118 Met Asp He Gly Val Pro Leu Leu- Ala Leu Gly Cys Tyr Ser Gin Val 2335 2340 2345 AAT CCA CTG ACG CTG ACA GCG GCG GTA TTG ATG CTA GTG GCT CAT TAC 7166 Asn Pro Leu Thr Leu Thr Ala Ala Val Leu Met Leu Val Ala His Tyr 2350 2355 2360 GCC ATA ATT GGA CCT GGA CTG CAA GCA AAA GCG ACT AGA GAA GCT CAA 7214 Ala He He Gly Pro Gly Leu Gin Ala Lys Ala Thr Arg Glu Ala Gin 2365 2370 2375 AAA AGG ACA GCG GCC GGA ATA ATG AAA AAT CCA ACC GTT GAT GGA ATT 7262 Lys Arg Thr Ala Ala Gly He Met Lys Asn Pro Thr Val Asp Gly He 2380 2385 2390 GTT GCA ATA GAT TTG GAC CCT GTG GTT TAT GAT GCA AAA TTT GAG AAA 7310 Val Ala He Asp Leu Asp Pro Val Val Tyr Asp Ala Lys Phe Glu Lys 2395 2400 2405 2410 CAA CTA GGC CAA ATA ATG TTG TTG ATA CTA TGC ACA TCA CAG ATC CTC 7358 Gin Leu Gly Gin He Met Leu Leu He Leu Cys Thr Ser Gin He Leu 2415 2420 2425 TTG ATG CGG ACT ACA TGG GCC TTG TGT GAA TCC ATC ACA CTG GCC ACT 7406 Leu Met Arg Thr Thr Trp Ala Leu Cys Glu Ser He Thr Leu Ala Thr 2430 2435 2440 GGA CCT CTG ACC ACG CTT TGG GAG GGA TCT CCA GGA AAA TTT TGG AAC 7454 Gly Pro Leu Thr Thr Leu Trp Glu Gly Ser Pro Gly Lys Phe Trp Asn 2445 2450 2455 ACC ACG ATA GCG GTT TCC ATG GCA AAC ATT TTC AGG GGA AGT TAT CTA 7502 Thr Thr He Ala Val Ser Met Ala Asn He Phe Arg Gly Ser Tyr Leu 2460 2465 2470 GCA GGA GCA GGC CTG GCC TTC TCA TTA ATG AAA TCT CTA GGA GGA GGT 7550 Ala Gly Ala Gly Leu Ala Phe Ser Leu Met Lys Ser Leu Gly Gly Gly 2475 2480 2485 2490 AGG AGA GGT ACG GGA GCC AAG GGG AAA CAC TGG GAG AGA AAT GGA AAA 7598 Arg Arg Gly Thr Gly Ala Lys Gly Lys His Trp Glu Arg Asn Gly Lys 2495 2500 2505 GAC AGA CTG AAC CAA CTG AGC AAG TCA GAA TTC AAC ACT TAC AAA AGG 7646 Asp Arg Leu Asn Gin Leu Ser Lys Ser Glu Phe Asn Thr Tyr Lys Arg 2510 2515 2520 AGT GGG ATT ATG GAA GTG GAC AGA TCC GAA GCC AAA GAG GGA CTG AAA 7694 Ser Gly He Met Glu Val Asp .Arg Ser Glu Ala Lys Glu Gly Leu Lys 2525 2530 2535 AGA GGA GAA ACA ACC AAA CAT GCA GTG TCG AGA GGA ACC GCC AAA TTG 7742 Arg Gly Glu Thr Thr Lys His Ala Val Ser Arg Gly Thr Ala Lys Leu 2540 2545 2550 AGG TGG TTC GTG GAG AGG AAC CTT GTG AAA CCA GAA GGG AAA GTC ATA 7790 Arg Trp Phe Val Glu Arg Asn Leu Val Lys Pro Glu Gly Lys Val He 2555 2560 2565 2570 GAC CTC GGT TGT GGA AGA GGT GGC TGG TCA TAC TAT TGC GCT GGG CTG 7838 Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Ala Gly Leu 2575 2580 2585 AAG AAA GTC ACA GAA GTG AAG GGA TAC ACA AAA GGA GGA CCT GGA CAT 7886 Lys Lys Val Thr Glu Val Lys Gly Tyr Thr Lys Gly Gly Pro Gly His 2590 2595 2600 GAG GAA CCA ATC CCA ATG GCG ACC TAT GGA TGG AAC CTA GTA AAG CTA 7934 Glu Glu Pro He Pro Met Ala Thr Tyr Gly Trp Asn Leu Val Lys Leu 2605 2610 2615 TAC TCC GGG AAA GAC GTA TTC TTT ACA CCA CCT GAG AAG TGT GAC ACC 7982 Tyr Ser Gly Lys Asp Val Phe Phe Thr Pro Pro Glu Lys Cys Asp Thr 2620 2625 2630 CTT TTG TGT GAT ATT GGT GAG TCC TCT CCA AAC CCA ACT ATA GAA GAA 8030 Leu Leu Cys Asp He Gly Glu Ser Ser Pro Asn Pro Thr He Glu Glu 2635 2640 2645 2650 GGA AGA ACG TTA CGC GTC CTA AAG ATG GTG GAA CCA TGG CTC AGA GGG 8078 Gly Arg Thr Leu Arg Val Leu Lys Met Val Glu Pro Trp Leu Arg Gly 2655 2660 2665 AAC CAA TTT TGC ATA AAA ATT CTA AAT CCC TAC ATG CCA AGT GTG GTG 8126 Asn Gin Phe Cys He Lys He Leu Asn Pro Tyr Met Pro Ser Val Val 2670 2675 2680 GAA ACT CTG GAG CAA ATG CAA AGA AAA CAT GGA GGA ATG CTA GTG CGG 8174 Glu Thr Leu Glu Gin Met Gin Arg Lys His Gly Gly Met Leu Val Arg 2685 2690 2695 AAT CCA CTT TCA AGA AAT TCT ACT CAT GAA ATG TAT TGG GTT TCA TGT 8222 Asn Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Val Ser Cys 2700 2705 2710 GGA ACA GGA AAC ATT GTG TCA GCA GTA AAC ATG ACA TCT AGA ATG TTG 8270 Gly Thr Gly Asn He Val Ser Ala Val Asn Met Thr Ser Arg Met Leu 2715 2720 2725 2730 CTA AAT CGA TTC ACA ATG GCT CAC AGG AAA CCA ACA TAT GAA AGA GAC 8318 Leu Asn Arg Phe Thr Met Ala His Arg Lys Pro Thr Tyr Glu Arg Asp 2735 2740 2745 GTG GAC TTA GGC GCT GGA ACA AGA CAT GTG GCA GTG GAA CCA GAG GTA 8366 Val Asp Leu Gly Ala Gly Thr Arg His Val Ala Val Glu Pro Glu Val 2750 2755 2760 GCC AAC CTA GAT ATC ATT GGC CAG AGG ATA GAG AAC ATA AAA CAT GAA 8414 Ala Asn Leu Asp He He Gly Gin Arg He Glu Asn He Lys His Glu 2765 2770 2775 CAT AAG TCA ACA TGG CAT TAT GAT GAG GAC AAT CCA TAT AAA ACA TGG 8462 His Lys Ser Thr Trp His Tyr Asp Glu Asp Asn Pro Tyr Lys Thr Trp 2780 2785 2790 GCC TAT CAT GGA TCA TAT GAG GTC AAG CCA TCA GGA TCA GCC TCA TCC 8510 Ala Tyr His Gly Ser Tyr Glu Val Lys Pro Ser Gly Ser Ala Ser Ser 2795 2800 2805 2810 ATG GTC AAT GGC GTG GTG AAA CTG CTC ACC AAA CCA TGG GAT GCC ATC 8558 Met Val Asn Gly Val Val Lys Leu Leu Thr Lys Pro Trp Asp Ala He 2815 2820 2825 CCC ATG GTC ACA CAA ATA GCC ATG ACT GAC ACC ACA CCC TTT GGA CAA 8606 Pro Met Val Thr Gin He Ala Met Thr Asp Thr Thr Pro Phe Gly Gin 2830 2835 2840 CAG AGG GTG TTT AAA GAG AAA GTT GAC ACG CGC ACA CCA AAA GCA AAA 8654 Gin Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Pro Lys Ala Lys 2845 2850 2855 CGA GGC ACA GCA GAA ATC ATG GAG GTG ACA GCC AGG TGG TTA TGG GGT 8702 Arg Gly Thr Ala Gin He Met Glu Val Thr Ala Arg Trp Leu Trp Gly 2860 2865 2870 TTT CTC TCT AGA AAC AAA AAA CCA AGA ATT TGT ACA AGA GAG GAG TTC 8750 Phe Leu Ser Arg Asn Lys Lys Pro Arg He Cys Thr Arg Glu Glu Phe 2875 2880 2885 2890 ACA AGA AAA GTT AGG TCA AAC GCA GCC ATT GGA GCA GTG TTC GTT GAT 8798 Thr Arg Lys Val Arg Ser Asn Ala Ala He Gly Ala Val Phe Val Asp 2895 2900 2905 GAA AAT CAA TGG AAC TCA GCA AAA GAA GCA GTG GAA GAT GAG CGG TTC 8846 Glu Asn Gin Trp Asn Ser Ala Lys Glu Ala Val Glu Asp Glu Arg Phe 2910 2915 2920 TGG GAC CTT GTG CAC AGA GAG AGG GAG CTT CAC AAA CAG GGA AAA TGT 8894 Trp Asp Leu Val His Arg Glu Arg Glu Leu His Lys Gin Gly Lys Cys 2925 2930 2935 GCC ACG TGT GTT TAC AAC ATG ATG GGG AAG AGA GAG AAA AAA CTA GGA 8942 Ala Thr Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly 2940 2945 2950 GAG TTC GGA * AAG GCA AAA GGA AGT CGT GCA ATA TGG TAC ATG TGG TTG 8990 Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala He Trp Tyr Met Trp Leu 2955 2960 2965 ' 2970 GGA GCA CGC TTT CTA GAG TTC GAA GCT CTT GGT TTC ATG AAC GAA GAT 9038 Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Met Asn Glu Asp 2975 2980 2985 CAC TGG TTC AGT AGA GAG AAT TCA CTC AGT GGA GTG GAA GGA GAA GGA 9086 His Trp Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly 2990 2995 3000 CTC CAC AAA CTC GGA TAT ATA CTC AGA GAC ATA TCA AAG ATT CCA GGG 9134 Leu His Lys Leu Gly Tyr He Leu Arg Asp He Ser Lys He Pro Gly 3005 3010 3015 GGA AAT ATG TAT GCA GAT GAC ACA GCC GGA TGG GAT ACA AGG ATA ACA 9182 Gly Asn Met Tyr Ala. Asp Asp Thr Ala Gly Trp Asp Thr Arg He Thr 3020 3025 3030 GAG GAT GAT CTT CAG AAT GAG GCC AAA ATT ACT GAC ATC ATG GAG CCC 9230 Glu Asp Asp Leu Gin Asn Glu Ala Lys He Thr Asp He Met Glu Pro 3035 3040 3045 3050 GAA CAT GCC CTA CTG GCT ACG TCA ATC TTC AAG CTG ACC TAC CAA AAT 9278 Glu His Ala Leu Leu Ala Thr Ser He Phe Lys Leu Thr Tyr Gin Asn 3055 3060 3065 AAG GTG GTA AGG GTA CAG AGA CCA GCG AAA AAT GGA ACC GTG ATG GAT 9326 Lys Val Val Arg Val Gin Arg Pro Ala Lys Asn Gly Thr Val Met Asp 3070 3075 3080 GTC ATA TCC AGA CGT GAC CAG AGA GGA AGT GGC CAG GTC GGA ACT TAT 9374 Val He Ser Arg Arg Asp Gin Arg Gly Ser Gly Gin Val Gly Thr Tyr 3085 3090 3095 GGC TTA AAC ACT TTC ACT AAC ATG GAA GCC CAG CTA ATA AGA CAA ATG 9422 Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gin Leu He Arg Gin Met 3100 3105 3110 GAG TCT GAG GGA ATC TTT TCA CCC AGC GAA TTG GAG ACC CCA AAT TTA 9470 Glu Ser Glu Gly He Phe Ser Pro Ser Glu Leu Glu Thr Pro Asn Leu 3115 3120 3125 3130 GCC GAG AGA GTT CTC GAC TGG CTG GAA AAA TAT GGC GTC GAA AGG CTG 9518 Ala Glu Arg Val Leu Asp Trp Leu Glu Lys Tyr Gly Val Glu Arg Leu 3135 3140 3145 AAA AGA ATG GCA ATC AGC GGA GAT GAC TGC GTG GTG AAA CCA ATT GAT 9566 Lys Arg Met Ala He Ser Gly Asp Asp Cys Val Val Lys Pro He Asp 3150 3155 3160 GAC AGG TTC GCA ACA GCC TTA ACA GCT CTG AAT GAT ATG GGA AAA GTA 9614 Asp Arg Phe Ala Thr Ala Leu Thr Ala Leu Asn Asp Met Gly Lys Val 3165 3170 3175 AGA AAA GAT ATA CCA CAA TGG GAA CCC TCA AAA GGA TGG AAT GAT TGG 9662 Arg Lys Asp He Pro Gin Trp Glu Pro Ser Lys Gly Trp Asn Asp Trp 3180 3185 3190 CAA CAG GTG CCT TTT TGT TCA CAC CAT TTC CAC CAG CTG ATT ATG AAG 9710 Gin Gin Val Pro Phe Cys Ser His His Phe His Gin Leu He Met Lys 3195 3200 3205 3210 GAT GGG AGG GAA ATA GTG GTG CCA TGC CGC AAC CAA GAT GAA CTT GTG 9758 Asp Gly Arg Glu He Val Val Pro Cys Arg Asn Gin Asp Glu Leu Val 3215 3220 3225 GGT AGG GCT AGA GTA TCA CAA GGT GCT GGA TGG i'JGC CTG AGA GAA ACT 9806 Gly Arg Ala Arg Val Ser Gin Gly Ala Gly Trp • ■ er Leu Arg Glu Thr 3230 3235 " - 3240 GCA TGC CTA GGC AAG TCA TAT GCA CAA ATG TGG CAG CTG ATG TAC TTC 9854 Ala Cys Leu Gly Lys Ser Tyr Ala Gin Met Trp Gin Leu Met Tyr Phe 3245 3250 3255 CAC AGG AGA GAC CTG AGA CTA GCT GCT AAT GCT ATC TGT TCA GCC GTT 9902 His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala He Cys Ser Ala Val 3260 3265 3270 CCA GTT GAT TGG GTC CCA ACC AGC CGC ACC ACT TGG TCG ATC CAT GCC 9950 Pro Val Asp Trp Val Pro Thr Ser Arg Thr Thr Trp Ser He His Ala 3275 3280 3285 3290 CAT CAC CAA TGG ATG ACA ACA GAA GAC ATG TTG TCA GTG TGG AAT AGG 9998 His His Gin Trp Met Thr Thr Glu Asp Met Leu Ser Val Trp Asn Arg 3295 3300 3305 GTT TGG ATA GAG GAA AAC CCA TGG ATG GAG GAC AAA ACC CAT GTA TCC 10046 Val Trp He Glu Glu Asn Pro Trp Met Glu Asp Lys Thr His Val Ser 3310 3315 3320 AGT TGG GAA GAT GTT CCA TAT TTA GGA AAA AGG GAA GAT CAG TGG TGT 10094 Ser Trp Glu Asp Val Pro Tyr Leu Gly Lys Arg Glu Asp Gin Trp Cys 3325 3330 3335 GGA TCC CTG ATA GGC TTA ACA GCA AGG GCT ACC TGG GCC ACC AAC ATA 10142 Gly Ser Leu He Gly Leu Thr Ala Arg Ala Thr Trp Ala Thr Asn He 3340 3345 3350 CAA GTG GCC ATA AAC CAA GTG AGA AGA-CTA ATC GGG AAT GAG AAT TAT 10190 Gin Val Ala He Asn Gin Val Arg Arg Leu He Gly Asn Glu Asn Tyr 3355 3360 3365 3370 CTA GAT TAC ATG ACA TCA ATG AAG AGA TTC AAG AAC GAG AGT GAT CCG 10238 Leu Asp Tyr Met Thr Ser Met Lys Arg Phe Lys Asn Glu Ser Asp Pro 3375 3380 3385 AAG GGG CAC TCT GGT GAG TCA ACA CAC TTA TGAAAATAAA GGAAAATAAG 10288 Lys Gly His Ser Gly Glu Ser Thr His Leu 3390 3395 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3396 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Asn Asn Gin Arg Lys Lys Thr Ala Arg Pro Ser Phe Asn Met Leu 1 5 10 15 Lys Arg Ala Arg Asn Arg Val Ser Thr Gly Ser Gin Leu Ala Lys Arg 20 25 30 Phe Ser Lys Gly Leu Leu Ser Gly Gin Gly Pro Met Lys Leu Val Met 35 40 45 Ala Phe He Ala Phe Leu Arg Phe Leu Ala He Pro Pro Thr Ala Gly 50 55 60 He Leu Ala Arg Trp Gly Ser Phe Lys Lys Asn Gly Ala He Lys Val 65 70 75 80 Leu Arg Gly Phe Lys Lys Glu He Ser Asn Met Leu Asn He Met Asn 85 90 95 Arg Arg Lys Arg Ser Val Thr Met Leu Leu Met Leu Leu Pro Thr Ala 100 105 110 Leu Ala Phe His Leu Thr Thr Arg Gly Gly Glu Pro His Met He Val 115 120 125 Ser Lys Gin Glu Arg Glu Lys Ser Leu Leu Phe Lys Thr Ser Val Gly 130 135 140 Val Asn Met Cys Thr Leu He Ala Met Asp Leu Gly Glu Leu Cys Glu 145 150 155 160 Asp Thr Met Thr Tyr Lys Cys Pro Arg He Thr Glu Ala Glu Pro Asp 165 170 175 Asp Val Asp Cys Trp Cys Asn Ala Thr Asp Thr Trp Val Thr Tyr Gly 180 185 190 Thr Cys Ser Gin Thr Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala 195 200 205 Leu Ala Pro His Val Gly Leu Gly Leu Glu Thr Arg Thr Glu Thr Trp 210 215 220 Met Ser Ser Glu Gly Ala Trp Lys Gin He Gin Arg Val Glu Thr Trp 225 230 235 240 Ala Leu Arg His Pro Gly Phe Thr Val He Ala Leu Phe Leu Ala His 245 250 255 Ala He Gly Thr Ser He Thr Gin Lys Gly He He Phe He Leu Leu 260 265 270 Met Leu Val Thr Pro Ser Met Ala Met Arg Cys Val Gly He Gly Ser 275 280 285 Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val 290 295 300 Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asp Lys Pro Thr 305 310 315 320 Leu Asp He Glu Leu Leu Lys Thr Glu Val Thr Asn Pro Ala Val Leu 325 330 335 Arg Lys Leu Cys He Glu Ala Lys He Ser Asn Thr Thr Thr Asp Ser 340 345 350 Arg Cys Pro Thr Gin Gly Glu Ala Thr Leu Val Glu Glu Gin Asp Ala 355 360 365 Asn Phe Val Cys Arg Arg Thr Phe Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly Leu Phe Gly Lys Gly Ser Leu Leu Thr Cys Ala Lys Phe Lys 385 390 395 400 Cys Val Thr Lys Leu Glu Gly Lys He Val Gin Tyr Glu Asn Leu Lys 405 410 415 Tyr Ser Val He Val Thr Val His Thr Gly Asp Gin His Gin Val Gly 420 425 430 Asn Glu Thr Thr Glu His Gly Thr He Ala Thr He Thr Pro Gin Ala 435 440 445 Pro Thr Ser Glu He Gin Leu Thr Asp Tyr Gly Ala Leu Thr Leu Asp 450 455 460 Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Thr 465 470 475 480 Met Lys Glu Lys Ser Trp Leu Val His Lys Gin Trp Phe Leu Asp Leu 485 490 495 Pro Leu Pro Trp Thr Ser Gly Ala Ser Thr Ser Gin Glu Thr Trp Asn 500 505 510 Arg Gin Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala Lys Lys Gin 515 520 525 Glu Val Val Val Leu Gly Ser Gin Glu Gly Ala Met His Thr Ala Leu 530 535 540 Thr Gly Ala Thr Glu He Gin Thr Ser Gly Thr Thr Thr He Phe Ala 545 550 555 560 Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Thr Leu Lys Gly 565 570 575 Met Ser Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys Glu Val ' 580 585 590 Ala Glu Thr Gin His Gly Thr Val Leu Val Gin Val Lys Tyr Glu Gly 595 600 605 Thr Asp Ala Pro Cys Lys He Pro Phe Ser Thr Gin Asp Glu Lys Gly 610 615 620 Val Thr Gin Asn Arg Leu He Thr Ala Asn Pro He Val Thr Asp Lys 625 630 635 640 Glu Lys Pro Val Asn He Glu Thr Glu Pro Pro Phe Gly Glu Ser Tyr 645 650 655 He Val Val Gly Ala Gly Glu Lys Ala Leu Lys Gin Cys Trp Phe Lys 660 665 670 Lys Gly Ser Ser He Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala 675 680 685 Arg Arg Met Ala He Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser He 690 695 700 Gly Gly Val Phe Thr Ser Val Gly Lys Leu Val His Gin Val Phe Gly 705 710 715 720 Thr Ala Tyr Gly Val Leu Phe Ser Gly Val Ser Trp Thr Met Lys He 725 730 735 Gly He Gly He Leu Leu Thr Trp Leu Gly Leu Asn Ser Arg Ser Thr 740 745 750 Ser Leu Ser Met Thr Cys He Ala Val Gly Met Val Thr Leu Tyr Leu 755 760 765 Gly Val Met Val Gin Ala Asp Ser Gly Cys Val He Asn Trp Lys Gly 770 775 780 Arg Glu Leu Lys Cys Gly Ser Gly He Phe Val Thr Asn Glu Val His 785 790 795 800 Thr Trp Thr Glu Gin Tyr Lys Phe Gin Ala Asp Ser Pro Lys Arg Leu 805 810 815 Ser Ala Ala He Gly Lys Ala Trp Glu Glu Gly Val Cys Gly He Arg 820 825 830 Ser Ala Thr Arg Leu Glu Asn He Met Trp Lys Gin He Ser Asn Glu 835 840 845 Leu Asn His He Leu Leu Glu Asn Asp Met Lys Phe Thr Val Val Val 850 855 860 Gly Asp Val Val Gly He Leu Ala Gin Gly Lys Lys Met He Arg Pro 865 870 875 880 Gin Pro Met Glu His Lys Tyr Ser Trp Lys Ser Trp Gly Lys Ala Lys 885 890 895 He He Gly Ala Asp He.Gin Asn Thr Thr Phe He He Asp Gly Pro 900 905 910 Asp Thr Pro Glu Cys Pro Asp Asp Gin Arg Ala Trp Asn He Trp Glu 915 920 925 Val Glu Asp Tyr Gly Phe Gly He Phe Thr Thr Asn He Trp Leu Lys 930 935 940 Leu Arg Asp Ser Tyr Thr Gin Met Cys Asp His Arg Leu Met Ser Ala 945 950 955 960 Ala He Lys Asp Ser Lys Ala Val His Ala Asp Met Gly Tyr Trp He 965 970 975 Glu Ser Glu Lys Asn Glu Thr Trp Lys Leu Ala Arg Ala Ser Phe He 980 985 990 Glu Val Lys Thr Cys Val Trp Pro Lys Ser His Thr Leu Trp Ser Asn 995 1000 1005 Gly Val Leu Glu Ser Glu Met He He Pro Lys He Tyr Gly Gly Pro 1010 1015 1020 He Ser Gin His Asn Tyr Arg Pro Gly Tyr Phe Thr Gin Thr Ala Gly 1025 1030 1035 1040 Pro Trp His Leu Gly Lys Leu Glu Leu Asp Phe Asp Leu Cys Glu Gly 1045 1050 1055 Thr Thr Val Val Val Asp Glu His Cys Gly Asn Arg Gly Pro Ser Leu 1060 1065 1070 Arg Thr Thr Thr Val Thr Gly Lys He He His Glu Trp Cys Cys Arg 1075 1080 1085 Ser Cys Thr Leu Pro Pro Leu Arg Phe Lys Gly Glu Asp Gly Cys Trp 1090 1095 1100 Tyr Gly Met Glu He Arg Pro Val Lys Glu Lys Glu Glu Asn Leu Val 1105 1110 1115 1120 Lys Ser Met Val Ser Ala Gly Ser Gly Glu Val Asp Ser Phe Ser Leu 1125 1130 1135 Gly Leu Leu Cys He Ser He Met He Glu Glu Val Met Arg Ser Arg 1140 1145 1150 Trp Ser Arg Lys Met Leu Met Thr Gly Thr Leu Ala Val Phe Leu Leu 1155 1160 1165 Leu He Met Gly Gin Leu Thr Trp Asn Asp Leu He Arg Leu Cys He 1170 1175 1180 Met Val Gly Ala Asn Ala Ser Asp Arg Met Gly Met Gly Thr Thr Tyr 1185 1190 1195 1200 Leu Ala Leu Met Ala Thr Phe Lys Met Arg Pro Met Phe Ala Val Gly 1205 1210 1215 Leu Leu Phe Arg Arg Leu Thr Ser Arg Glu Val Leu Leu Leu Thr He 1220 1225 1230 Gly Leu Ser Leu Val Ala Ser Val Glu Leu Pro Asn Ser Leu Glu Glu 1235 1240 1245 Leu Gly Asp Gly Leu Ala Met Gly He Met He Leu Lys Leu Leu Thr 1250 1255 1260 Asp Phe Gin Ser His Gin Leu Trp Ala Thr Leu Leu Ser Leu Thr Phe 1265 1270 1275 1280 Val Lys Thr Thr Phe Ser Leu His Tyr Ala Trp Lys Thr Met Ala Met 1285 1290 1295 Val Leu Ser He Val Ser Leu Phe Pro Leu Cys Leu Ser Thr Thr Ser 1300 1305 1310 Gin Lys Thr Thr Trp Leu Pro Val Leu Leu Gly Ser Leu Gly Cys Lys 1315 1320 1325 Pro Leu Thr Met Phe Leu He Ala Glu Asn Lys He Trp Gly Arg Lys 1330 1335 1340 Ser Trp Pro Leu Asn Glu Gly He Met Ala Val Gly He Val Ser He 1345 1350 1355 1360 Leu Leu Ser Ser Leu Leu Lys Asn Asp Val Pro Leu Ala Gly Pro Leu 1365 1370 1375 He Ala Gly Gly Met Leu He Ala Cys Tyr Val He Ser Gly Ser Ser 1380 1385 1390 Ala Asp Leu Ser Leu Glu Lys Ala Ala Glu Val Ser Trp Glu Glu Glu 1395 1400 1405 Ala Glu His Ser Gly Ala Ser His Asn He Leu Val Glu Val Gin Asp 1410 1415 1420 Asp Gly Thr Met Lys He Lys Asp Glu Glu Arg Asp Asp Thr Leu Thr 1425 1430 1435 1440 He Leu Leu Lys Ala Thr Leu Leu Ala Val Ser Gly Val Tyr Pro Leu 1445 1450 1455 Ser He Pro Ala Thr Leu Phe Val Trp Tyr Phe Trp Gin Lys Lys Lys 1460 1465 1470 Gin Arg Ser Gly Val Leu Trp Asp Thr Pro Ser Pro Pro Glu Val Glu 1475 1480 1485 Arg Ala Val Leu Asp Asp Gly He Tyr Arg He Met Gin Arg Gly Leu 1490 1495 1500 Leu Gly Arg Ser Gin Val Gly Val Gly Val Phe Gin Asp Gly Val Phe 1505 1510 1515 1520 His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met Tyr Gin Gly 1525 1530 1535 Lys Arg Leu Glu Pro Ser Trp Ala Ser Val Lys Lys Asp Leu He Ser 1540 1545 1550 Tyr Gly Gly Gly Trp Arg Phe Gin Gly Ser Trp Asn Thr Gly Glu Glu 1555 1560 1565 Val Gin Val He Ala Val Glu Pro Gly Lys Asn Pro Lys Asn Val Gin 1570 1575 1580 Thr Ala Pro Gly Thr Phe Lys Thr Pro Glu Gly Glu Val Gly Ala He 1585 1590 1595 1600 Ala Leu Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro He Val Asn Arg 1605 1610 1615 Glu Gly Lys He Val Gly Leu Tyr Gly Asn Gly Val Val Thr Thr Ser 1620 1625 1630 Gly Thr Tyr Val Ser Ala He Ala Gin Ala Lys : ' jla Ser Gin Glu Gly 1635 1640 , ;;J 1645 Pro Leu Pro Glu He Glu Asp Glu Val Phe Arg Lys Arg Asn Leu Thr 1650 1655 1660 He Met Asp Leu His Pro Gly Ser Gly Lys Thr Arg Arg Tyr Leu Pro 1665 1670 1675 1680 Ala He Val Arg Glu Ala He Arg Arg Asn Val Arg Thr Leu He Leu 1685 1690 1695 Ala Pro Thr Arg Val Val Ala Ser Glu Met Ala Glu Ala Leu Lys Gly 1700 1705 1710 Met Pro He Arg Tyr Gin Thr Thr Ala Val Lys Ser Glu His Thr Gly 1715 1720 1725 Lys Glu He Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu 1730 1735 1740 Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Met He He Met Asp Glu 1745 1750 1755 1760 Ala His Phe Thr Asp Pro Ala Ser He Ala Arg Arg Gly Tyr He Ser 1765 1770 1775 Thr Arg Val Gly Met Gly Glu Ala Ala Ala He Phe Met Thr Ala Thr 1780 1785 1790 Pro Pro Gly Ser Val Glu Ala Phe Pro Gin Ser Asn Ala Val He Gin 1795 1800 1805 Asp Glu Glu Arg Asp He Pro Glu Arg Ser Trp Asn Ser Gly Tyr Glu 1810 1815 ' 1820 Trp He Thr Asp Phe Pro Gly Lys Thr Val Trp Phe Val Pro Ser He 1825 1830 1835 1840 Lys Ser Gly Asn Asp He Ala Asn Cys Leu Arg Lys Asn Gly Lys Arg 1845 1850 1855 Val He Gin Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Gin Lys Thr 1860 1865 1870 Lys Asn Asn Asp Trp Asp Tyr Val Val Thr Thr Asp He Ser Glu Met 1875 1880 1885 Gly Ala Asn Phe Arg Ala Asp Arg Val He Asp Pro Arg Arg Cys Leu Ϊ890 1895 1900 Lys Pro Val He Leu Lys Asp Gly Pro Glu Arg Val He Leu Ala Gly 1905 1910 " 1915 1920 Pro Met Pro Val Thr Val Ala Ser Ala Ala Gin Arg Arg Gly Arg He 1925 1930 1935 Gly Arg Asn Gin Asn Lys Glu Gly Asp Gin Tyr Val Tyr Met Gly Gin 1940 1945 1950 Pro Leu Asn Asn Asp Glu Asp His Ala His Trp Thr Glu Ala Lys Met 1955 1960 1965 Leu Leu Asp Asn He Asn Thr Pro Glu Gly He He Pro Ala Leu Phe 1970 1975 1980 Glu Pro Glu Arg Glu Lys Ser Ala Ala He Asp Gly Glu Tyr Arg Leu 1985 1990 1995 2000 Arg Gly Glu Ala Arg Lys Thr Phe Val Glu Leu Met Arg Arg Gly Asp 2005 2010 2015 Leu Pro Val Trp Leu Ser Tyr Lys Val Ala Ser Glu Gly Phe Gin Tyr 2020 2025 2030 Ser Asp Arg Arg Trp Cys Phe Asp Gly Glu Arg Asn Asn Gin Val Leu 2035 2040 2045 Glu Glu Asn Met Asp Val Glu Met Trp Thr Lys Glu Gly Glu Arg Lys 2050 2055 2060 Lys Leu Arg Pro Arg Trp Leu Asp Ala Arg Thr Tyr Ser Asp Pro Leu 2065 2070 2075 2080 Ala Leu Arg Glu Phe Lys Glu Phe Ala Ala Gly Arg Arg Ser Val Ser 2085 2090 2095 Gly Asp Leu He Leu Glu He Gly Lys Leu Pro Gin His Leu Thr Gin 2100 2105 2110 Arg Ala Gin Asn Ala Leu Asp Asn Leu Val Met Leu His Asn Ser Glu 2115 2120 2125 Gin Gly Gly Arg Ala Tyr Arg His Ala Met Glu Glu Leu Pro Asp Thr 2130 2135 2140 He Glu Thr Leu Met Leu Leu Ala Leu He Ala Val Leu Thr Gly Gly 2145 2150 2155 2160 Val Thr Leu Phe Phe Leu Ser Gly Lys Gly Leu Gly Lys Thr Ser He 2165 2170 2175 Gly Leu Leu Cys Val Met Ala Ser Ser Val Leu Leu Trp Met Ala Ser 2180 2185 2190 Val Glu Pro His Trp He Ala Ala Ser He He Leu Glu Phe Phe Leu 2195 2200 2205 Met Val Leu Leu He Pro Glu Pro Asp Arg Gin Arg Thr Pro Gin Asp 2210 2215 2220 Asn Gin Leu Ala Tyr Val Val He Gly Leu Leu Phe Met He Leu Thr 2225 2230 2235 2240 Val Ala Ala Asn Glu Met Gly Leu Leu Glu Thr Thr Lys Lys Asp Leu 2245 2250 2255 Gly He Gly His Val Ala Ala Glu Asn His His His Ala Thr Met Leu 2260 2265 2270 Asp Val Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala 2275 2280 2285 Thr Thr Val He Thr Pro Met Met Arg His Thr He Glu Asn Thr Thr 2290 2295 2300 Ala Asn He Ser Leu Thr Ala He Ala Asn Gin Ala Ala He Leu Met 2305 2310 2315 2320 Gly Leu Asp Lys Gly Trp Pro He Ser Lys Met Asp He Gly Val Pro 2325 2330 2335 Leu Leu Ala Leu Gly Cys Tyr Ser Gin Val Asn Pro Leu Thr Leu Thr 2340 2345 2350 Ala Ala Val Leu Met Leu Val Ala His Tyr Ala He He Gly Pro Gly 2355 2360 2365 Leu Gin Ala Lys Ala Thr Arg Glu Ala Gin Lys Arg Thr Ala Ala Gly 2370 2375 2380 He Met Lys Asn Pro Thr Val Asp Gly He Val Ala He Asp Leu Asp 2385 2390 2395 2400 Pro Val Val Tyr Asp Ala Lys Phe Glu Lys Gin Leu Gly Gin He Met 2405 2410 2415 Leu Leu He Leu Cys Thr Ser Gin He Leu Leu Met Arg Thr Thr Trp 2420 2425 2430 Ala Leu Cys Glu Ser He Thr Leu Ala Thr Gly Pro Leu Thr Thr Leu 2435 2440 2445 Trp Glu Gly Ser Pro Gly Lys Phe Trp Asn Thr Thr He Ala Val Ser 2450 2455 2460 Met Ala Asn He Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Ala 2465 2470 2475 2480 Phe Ser Leu Met Lys Ser Leu Gly Gly Gly Arg Arg Gly Thr Gly Ala 2485 2490 2495 Lys Gly Lys His Trp Glu Arg Asn Gly Lys Asp Arg Leu Asn Gin Leu 2500 2505 2510 Ser Lys Ser Glu Phe Asn Thr Tyr Lys Arg Ser Gly He Met Glu Val 2515 2520 2525 Asp Arg Ser Glu Ala Lys Glu Gly Leu Lys Arg Gly Glu Thr Thr Lys 2530 2535 2540 His Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe Val Glu Arg 2545 .2550 2555 2560 Asn Leu Val Lys Pro Glu Gly Lys Val He Asp Leu Gly Cys Gly Arg 2565 2570 2575 Gly Gly Trp Ser Tyr Tyr Cys Ala Gly Leu Lys Lys Val Thr Glu Val 2580 2585 2590 Lys Gly Tyr Thr Lys Gly Gly Pro Gly His Glu Glu Pro He Pro Met 2595 2600 2605 Ala Thr Tyr Gly Trp Asn Leu Val Lys Leu Tyr Ser Gly Lys Asp Val 2610 2615 2620 Phe Phe Thr Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp He Gly 2625 2630 2635 2640 Glu Ser Ser Pro Asn Pro Thr He Glu Glu Gly Arg Thr Leu Arg Val 2645 2650 2655 Leu Lys Met Val Glu Pro Trp Leu Arg Gly Asn Gin Phe Cys He Lys 2660 2665 2670 He Leu Asn Pro Tyr Met Pro Ser Val Val Glu Thr Leu Glu Gin Met 2675 2680 2685 Gin Arg Lys His Gly Gly Met Leu Val Arg Asn Pro Leu Ser Arg Asn 2690 2695 2700 Ser Thr His Glu Met Tyr Trp Val Ser Cys Gly Thr Gly Asn He Val 2705 2710 2715 2720 Ser Ala Val Asn Met Thr Ser Arg Met Leu Leu Asn Arg Phe Thr Met 2725 2730 2735 Ala His Arg Lys Pro Thr Tyr Glu Arg Asp Val Asp Leu Gly Ala Gly 2740 2745 2750 Thr Arg His Val Ala Val Glu Pro Glu Val Ala Asn Leu Asp He He 2755 2760 2765 Gly Gin Arg He Glu Asn He Lys His Glu His Lys Ser Thr Trp His 2770 2775 2780 Tyr Asp Glu Asp Asn Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr 2785 2790 2795 2800 Glu Val Lys Pro Ser Gly Ser Ala Ser Ser Met Val Asn Gly Val Val 2805 2810 2815 Lys Leu Leu Thr Lys Pro Trp Asp Ala He Pro Met Val Thr Gin He 2820 2825 2830 Ala Met Thr Asp Thr Thr Pro Phe Gly Gin Gin Arg Val Phe Lys Glu 2835 2840 2845 Lys Val Asp Thr Arg Thr Pro Lys Ala Lys Arg Gly Thr Ala Gin He 2850 2855 2860 Met Glu Val Thr Ala Arg Trp Leu Trp Gly Phe Leu Ser Arg Asn Lys 2865 2870 2875 2880 Lys Pro Arg He Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser 2885 2890 2895 Asn Ala Ala He Gly Ala Val Phe Val Asp Glu Asn Gin Trp Asn Ser 2900 2905 2910 Ala Lys Glu Ala Val Glu Asp Glu Arg Phe Trp Asp Leu Val His Arg 2915 2920 2925 Glu Arg Glu Leu His Lys Gin Gly Lys Cys Ala Thr Cys Val Tyr Asn 2930 2935 2940 Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys 2945 2950 2955 2960 Gly Ser Arg Ala He Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu 2965 2970 2975 Phe Glu Ala Leu Gly Phe Met Asn Glu Asp His Trp Phe Ser Arg Glu 2980 2985 2990 Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr 2995 3000 3005 He Leu Arg Asp He Ser Lys He Pro Gly Gly Asn Met Tyr Ala Asp 3010 3015 3020 Asp Thr Ala Gly Trp Asp Thr Arg He Thr Glu Asp Asp Leu Gin Asn 3025 3030 3035 3040 Glu Ala Lys He Thr Asp He Met Glu Pro Glu His Ala Leu Leu Ala 3045 3050 3055 Thr Ser He Phe Lys Leu Thr Tyr Gin Asn Lys Val Val Arg Val Gin 3060 3065 3070 Arg Pro Ala Lys Asn Gly Thr Val Met Asp Val He Ser Arg Arg Asp 3075 3080 3085 Gin Arg Gly Ser Gly Gin Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr 3090 3095 3100 Asn Met Glu Ala Gin Leu He Arg Gin Met Glu Ser Glu Gly He Phe 3105 3110 3115 3120 Ser Pro Ser Glu Leu Glu Thr Pro Asn Leu Ala Glu Arg Val Leu Asp 3125 3130 3135 Trp Leu Glu Lys Tyr Gly Val Glu Arg Leu Lys Arg Met Ala He Ser 3140 3145 3150 Gly Asp Asp Cys Val Val Lys Pro He Asp Asp Arg Phe Ala Thr Ala 3155 3160 3165 Leu Thr Ala Leu Asn Asp Met Gly Lys Val Arg Lys Asp He Pro Gin 3170 3175 3180 Trp Glu Pro Ser Lys Gly Trp Asn Asp Trp Gin Gin Val Pro Phe Cys 3185 3190 3195 3200- Ser His His Phe His Gin Leu He Met Lys Asp Gly Arg Glu He Val 3205 3210 3215 Val Pro Cys Arg Asn Gin Asp Glu Leu Val Gly Arg Ala Arg Val Ser 3220 3225 3230 Gin Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser 3235 3240 3245 Tyr Ala Gin Met Trp Gin Leu Met Tyr Phe His Arg Arg Asp Leu Arg 3250 3255 3260 Leu Ala Ala Asn Ala He Cys Ser Ala Val Pro Val Asp Trp Val Pro 3265 3270 3275 3280 Thr Ser Arg Thr Thr Trp Ser He His Ala His His Gin Trp Met Thr 3285 3290 3295 Thr Glu Asp Met Leu Ser Val Trp Asn Arg Val Trp He Glu Glu Asn 3300 3305 3310 - Pro Trp Met Glu Asp Lys Thr His Val Ser Ser Trp Glu Asp Val Pro 3315 3320 3325 Tyr Leu Gly Lys Arg Glu Asp Gin Trp Cys Gly Ser Leu He Gly Leu 3330 3335 3340 Thr Ala Arg Ala Thr Trp Ala Thr Asn He Gin Val Ala He Asn Gin 3345 3350 3355 3360 Val Arg Arg Leu He Gly Asn Glu Asn Tyr Leu Asp Tyr Met Thr Ser * 3365 3370 3375 Met Lys Arg Phe Lys Asn Glu Ser Asp Pro Lys Gly His Ser Gly Glu 3380 3385 3390 Ser Thr His Leu 3395 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPEt nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCATGAATTC CCATGCGATG CGTGGGA 27 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CACATCTCGA GTCCGCTTGA ACCATGA 27 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) . (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:5: TGGTTCCCGG GGACTCGGGA TGTGTA 26 (2) INFORMATION FOR SEQ ID NO:6: t (i) SEQUENCE CHARACTERISTICS: .. J . (A) LENGTH: 29 base pairs "*" ' (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:6: ACTAAGCTTG ATCATGCAGA GACCATTGA 29 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AATCAGAATT CTCTGCAGGG TCAGGGGAA 29 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs ' (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATAACAAAGC TTATCTTTGT TTCTTTTTCT 30 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GAAAGGATCC TCTGGAGTGT TATGGGACAC A 31 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ACCCAAGCTT CATCTTCTTC CTGCTGC 27 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AGGAGGTCGA CGAGGTACGG GAGCC 25 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CAATGATATC TAGGTTGGCT 20

Claims: CLAIMS 1. DEN1-S275/90 (ECACC V92042111) 2. DEN1-S275/90 (ECACC V92042111) in inactivated form. 3. A DNA polynucleotide encoding DEN1-S275/90 (ECACC V92042111) whose seguence is substantially as shown in Seq. ID No. 1 4. A fragment of a DNA polynucleotide as claimed in claim 3, said fragment encoding the C, C , PreM, M, E, NSl, NS2A, NS2B, NS3, NS4A, NS4B or NS5 gene of DEN1-S275/90 (ECACC V92042111) . 5. A DNA polynucleotide or a fragment thereof according to claim 3 or claim 4 in an expression vector. 6. An expression vector as claimed in claim 5 selected from pGEX-KG/EX-20, pMAL-c/NSl-104, pMAL-cRI/NS2- 1, pGEX-KG/NS3 BH C600-1 and pGEX-KG/NS5 C600 HF1. 7. A cell harbouring an expression vector as claimed in claim 5 or claim 6. 8. A cell as claimed in claim 7 which is E.coli or an insect cell. 9. A polypeptide in substantially isolated form Which is the C, C, PreM, M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5 polypeptide of DEN1-S275/90 (ECACC V92042111) . 10. A polypeptide as claimed in claim 9 which is in the form of a fusion protein. 11. A fusion protein as claimed in claim 10 which is coded by an expression vector selected from the expression vectors of claim 6. 12. A method of preparing a polypeptide as claimed in any one of claims 9 to 11 which comprises culturing a cell line according to claim 7 or claim 8 and recovering the polypeptide. 13. A polypeptide as claimed in claim 9 carrying a label. 14. A vaccine comprising one or more polypeptides as claimed in any one of claims 9 to 11 or the inactivated virus as claimed in claim 2 in combination with a pharmaceutically acceptable carrier or diluent. 15. The vaccine of claim 14 wherein one polypeptide is selected from E, NSl, NS2, NS3, NS5 and fusion proteins thereof capable of eliciting antibodies to a DENl viral protein. 16. An antibody against a polypeptide as claimed in any one of claims 9 to 11 capable of binding a DENl viral protein, optionally carrying a revealing label. 17. A test kit for the detection of the presence or absence of DENl virus comprising the antibody of claim 16 or the polypeptide of claim 9 or 13 fixed to a solid support.

Inventors: CHAN YOW-CHEONG, FU JIANLIN, TAN BOON-HUAN, TAN YIN-HWEE, YAP EU-HIAN, CHAN, YOW-CHEONG, FU, JIANLIN, TAN, BOON-HUAN, TAN, YIN-HWEE, YAP, EU-HIAN
Applicants: CHAN YOW CHEONG, FU JIANLIN, TAN BOON HUAN, TAN YIN HWEE, UNIV SINGAPORE, YAP EU HIAN, NATIONAL UNIVERSITY OF SINGAPORE, TAN, YIN-HWEE
Application year: 1993
Main classification: C12N15
IPC codes: A61K39, C07K14, C12N7, C12N15