WO1993022440A1 - Cdna sequence of dengue virus serotype 1 (singapore strain) - Google Patents

Cdna sequence of dengue virus serotype 1 (singapore strain) Download PDF

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Publication number
WO1993022440A1
WO1993022440A1 PCT/CA1993/000182 CA9300182W WO9322440A1 WO 1993022440 A1 WO1993022440 A1 WO 1993022440A1 CA 9300182 W CA9300182 W CA 9300182W WO 9322440 A1 WO9322440 A1 WO 9322440A1
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leu
gly
thr
ala
val
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French (fr)
Inventor
Yin-Hwee Tan
Jianlin Fu
Boon-Huan Tan
Eu-Hian Yap
Yow-Cheong Chan
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National University of Singapore
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National University of Singapore
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Priority to EP93911702A priority Critical patent/EP0638122B1/en
Priority to US08/325,426 priority patent/US6017535A/en
Priority to AU42575/93A priority patent/AU679179B2/en
Priority to KR1019940703865A priority patent/KR950701684A/en
Priority to DE69315689T priority patent/DE69315689T2/en
Publication of WO1993022440A1 publication Critical patent/WO1993022440A1/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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 dengue fever
  • DHF dengue shock syndrome
  • DEN1, DEN2, DEN3 and DEN4 are serotypes of Dengue virus belonging to the family Flaviviradae.
  • 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
  • the present invention thus provides DEN1-S275/90
  • 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
  • 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.
  • 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
  • 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
  • inventions 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.
  • an expression vector for expression of a polypeptide of the invention, an expression vector may be constructed.
  • An expression vector is prepared which comprises a DNA
  • telomere 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
  • 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. E. coli, yeast, insect or
  • 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
  • a fusion protein in which DEN1-S275/90 or a protein thereof, e.g. E, NS1, 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.
  • 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
  • the polyprotein and peptides of the invention may be used as immunogens for a vaccine against DEN1-S275/90 and other DEN1 viruses.
  • 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 DEN1
  • a vaccine composition against DEN1 may comprise a mixture of two or more peptides.
  • it may comprise one non-structural (NS) peptide, eg. NS1 or NS3, together with a capsid (C), M or E peptide.
  • NS non-structural
  • C capsid
  • E capsid
  • 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 DEN1, and especially DEN1-S272/90.
  • the proteins and peptides are optionally labelled with a detectable label, eg a radioisotope, biotin or a
  • the immunoassay may be conducted by bringing a known guantity of labelled protein (antigen) into contact with a sample suspected of containing antibody against DEN1 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 DEN1 in a sample.
  • the antibodies may be
  • proteins and peptides of the invention may also be labelled with a toxin or isotope selected to kill virus-infected cells.
  • Antibodies against NS1 are particularly favoured since NS1 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 DEN1 protein in a sample.
  • the method may comprise bringing the antibody into contact with a sample suspected to contain DEN1 proteins (antigens) and detecting the amount of antibody- antigen complex formed.
  • Immunoassays according to the invention may be, for example, competitive (eg radioimmune assays - RIA) or non-competitive (eg enzyme linked
  • Figure 1 is a diagrammatic representation of the cDNA of Dengue virus Type l (Singapore strain S275/90) and
  • 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).
  • 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.
  • Lane 4 (+) anti-NS1
  • Lane 5 (-)
  • Lane 6 (+) anti-NS2
  • Lane 7 (-)
  • Lane 8 (+) anti-NS3
  • Lane 9 (-)
  • Lane 10 (+) anti- NS5
  • Lane 11 positive Dengue
  • Lane 12
  • Figure 4 shows fluorescence microscopy of C6/36 cells infected with Dengue Type 1 DI-275 and probed with
  • A control antiserum
  • B anti-E
  • C anti-NS1
  • D anti-NS2
  • E anti-NS3
  • F anti-NS5.
  • DEN1 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
  • 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
  • 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
  • primers 796 and 10090/B 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 EcoRI adaptors before insertion into the EcoRI sites of the pUC18 vector.
  • 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',
  • 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.
  • 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.
  • primer 10090/B which binds to the minus strand of the template at nucleotide positions 10,086 to 10,099 of strain S275/90
  • the PCR amplified cDNAs at the 5' noncoding region were double digested with XbaI and EcoRl, while those at the 3' noncoding region were digested with BamHI and EcoIR before cloning into the appropriate sites of the pUC18 vector.
  • the clones were screened and subjected to double-strand seguencing as described above.
  • 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. ? ⁇ he reading frame begins with the first AUG start codon, corresponding to
  • nucleotides 81 to 83 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).
  • 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.
  • the same purine-rich composition was
  • 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, NS1, 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
  • nonstructural regions (5' noncoding end to NS1, about 2400 nucleotides in length) of Nauru Island strain of DENl
  • nucleotide homology is 93.1% and when translated, the amino acid homology is 97.6%.
  • strain S275/90 shows a higher homology with DEN3 then with DEN2 and DEN4.
  • the nucleotide divergence of each gene is less than the
  • the cDNA regions for E, NS1, NS2, NS3 and NS5 of clone DI-275, a DENl cDNA clone derived from DEN1 virus Singapore Strain S275/90 as in Example 1, were amplified by the polymerase chain reaction (PCR) and digested with restriction enzymes.
  • PCR polymerase chain reaction
  • oligonucleotide primers used in the PCR as set out in Table 3 and Seq ID Nos. 3-12.
  • restriction enzymes were ligated to the pGEX-KG vector (Guan and Dixon, Anal. Biochem. 192, 262-267, 1991).
  • NS1 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 two parts of NS5 were ligated together, then ligated into the pGEX-KG vector.
  • Recombinant plasmids were transformed into E . coli DH5 ⁇ or c600 HF1 strains. All plasmids encoded Dengue virus proteins fused to the C-terminus of glutathione S-transferase or Maltose Binding Protein (MBP) .
  • 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 HPO 4 , 0.005 M NaH 2 PO 4 ) with 0.1 mg/ml lysozyme, 1% triton X-100, 0.5 ⁇ g/ml aprotinin, 0.05 ⁇ g/ml Leupeptin, 0.25 ⁇ g/ml pepstatin, 5mM DTT and 0.175 ⁇ g/ml PMSF, and kept on ice for 10 min.
  • the cells were sonicated at maximum power for 3 ⁇ 1 min while chilled.
  • the lysate was centrifuged at 12,000 ⁇ 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.
  • the beads were centrifuged and washed with PBS buffer (by centrifugation) at least 6 times, or until the wash solution read zero at A 280 in a spectrophotometer.
  • the beads were resuspended in thrombin 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.
  • E. coli containing the NS1 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 ⁇ 15 sec. After centrifugation at 1000 ⁇ g the supernatant was again centrifuged (25,000 ⁇ g). The pellet was resuspended in 2 ml H 2 O , adding a final concentration of 0.5% Triton X-100, 10 mM EDTA, and 100 mM NaCl, then centrifuged at 20,000 ⁇ 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 O, 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
  • 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.
  • 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 ⁇ g of fusion protein per injected dose.
  • mice 12-day old female Swiss mice were immunised with the soluble proteins of E, NS1, NS2, NS3 and NS5 fusion proteins with or without Freund's adjuvant.
  • the injections were intraperitoneal or
  • 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
  • cell culture medium was replaced with methionine-free medium containing 3 ⁇ g/ml actinomycin D for 3 h, followed by the addition of fresh medium with [ 35 S] methionine at 20 ⁇ Ci/ml and 3 ⁇ g/ml actinomycin D for a further 3 h.
  • the cells were washed with cold PBS,
  • RIPA buffer 100 mM Tris-HCl pH7.5, 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.1% NP 40, 1% sodium
  • 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, NS1, NS2, NS3 and NS5 had been generated in mice (Fig. 2) and in rabbits (Fig. 3). These antibodies reacted with the native E, NS1, NS2, NS3 and NS5 proteins synthesised in infected C6/36 cells.
  • 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
  • Fig 4 shows the antisera to E, NS1, NS2, NS3 and NS5 reacted specifically with the Dengue virus S275/90 infected cells, but control antiserum did no react.
  • MOLECULE TYPE RNA (genomic)
  • GCC CTT TTT CTT GCA CAT GCC ATA GGA ACA TCC
  • ATC ACT CAG AAA GGG 878 Ala Leu Phe Leu Ala His Ala Ile Gly Thr Ser Ile Thr Gln Lys Gly
  • 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 Ile Gln Thr Ser Gly
  • 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
  • AAA AGG AAA AGA AAC TTA ACA ATA ATG GAC CTA CAT CCA GGA TCG GGG AAA 5102 Arg Lys Arg Asn Leu Thr Ile Met Asp Leii His Pro Gly Ser Gly Lys
  • CAG CGC ACT CCA CAG GAC AAC CAG TTA GCA TAT GTG GTG ATA GGT TTG 6782 Gln Arg Thr Pro Gln Asp Asn Gln Leu Ala Tyr Val Val Ile Gly Leu
  • AGA GGA ACC GCC AAA TTG 7742 Arg Gly Glu Thr Thr Lys His Ala Val Ser Arg Gly Thr Ala Lys Leu
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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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 (DEN1, 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 DEN1, DEN1 (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 DEN1 (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.
Gene Start (n) End(n) Start (p) End (p)
C 81 422 1 114
C' 123 422 15 114
PreM 423 695 115 205
M 696 920 206 280
E 921 2402 281 774
NS1 2403 3464 775 1128
NS2A 3465 4112 1129 1344
NS2B 4113 4499 1345 1474
NS3 4500 6359 1475 2093
NS4A 6360 6809 2094 2242
NS4B 6810 7556 2243 2492
NS5 7557 10268 2493 3396
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. E. 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, NS1, 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 DEN1 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 DEN1
infections.
Advantageously, a vaccine composition against DEN1 may comprise a mixture of two or more peptides. For example, it may comprise one non-structural (NS) peptide, eg. NS1 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 DEN1, 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 DEN1 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 DEN1 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 NS1 are particularly favoured since NS1 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 DEN1 protein in a sample. The method may comprise bringing the antibody into contact with a sample suspected to contain DEN1 proteins (antigens) and detecting the amount of antibody- antigen complex formed. Immunoassays according to the invention may be, for example, competitive (eg radioimmune 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-NS1, 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-NS1, 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-NS1; D, anti-NS2; E, anti-NS3; F anti-NS5. EXAMPLE 1
DEN1 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 32P-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 EcoRI adaptors before insertion into the EcoRI 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',
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 XbaI and EcoRl, while those at the 3' noncoding region were digested with BamHI and EcoIR before cloning into the appropriate sites of the pUC18 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 DEN1, DEN2, DEN3 and DEN4 using the computer program of Wilbur and Lipman (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, NS1, 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 NS1, 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) × 100%] in the structural region and 92.1% in the NS1 region; 15% of these base changes are transversions in the structural region and 7.9% in the NS1 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 DEN1 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 DEN1 strains of different geographic range and time period.
Figure imgf000013_0001
REFERENCES
1. WESTAWAY, E.G., BRINTON, M.A., GAIDAMOVICH, S. Ya, HORZINEK, M.C., IGARASHI, A., KAARIAINEN, L. , LVOV, D.K., PORTERFIELD, J.S., RUSSEL, P.K., 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 KEW, 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, CM., 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, NS1, NS2, NS3 and NS5 of clone DI-275, a DENl cDNA clone derived from DEN1 virus Singapore Strain S275/90 as in Example 1, 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 NS1 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 SalI and ClaI; 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 A600 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 Na2HPO4, 0.005 M NaH2PO4) 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 min. The cells were sonicated at maximum power for 3 × 1 min while chilled. The lysate was centrifuged at 12,000 × 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 A280 in a spectrophotometer. The beads were resuspended in thrombin 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 NS1
FROM INCLUSION BODIES
E. coli containing the NS1 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 × 15 sec. After centrifugation at 1000 × g the supernatant was again centrifuged (25,000 × g). The pellet was resuspended in 2 ml H2O , adding a final concentration of 0.5% Triton X-100, 10 mM EDTA, and 100 mM NaCl, then centrifuged at 20,000 × 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 H2O, 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 A280 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/NS2-1, 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 NS1 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, NS1, 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 [35S] 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 × 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, NS1, NS2, NS3 and NS5 had been generated in mice (Fig. 2) and in rabbits (Fig. 3). These antibodies reacted with the native E, NS1, 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, NS1, 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, NS1, NS2 , NS3 and NS5) occurred in both mice and rabbits.
Figure imgf000020_0001
Figure imgf000021_0001
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:1:
(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:1:
GTGGACCGCA AAGAACAGTT TCGAATCGGA AGCTTGCTTA ACGTAGTTCT AACAGTTTTT 60
TATTAGAGAG CAGATCTCTG ATG AAC AAC CAA CGA AAA AAG ACG GCT CGA 110
Met Asn Asn Gln 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 Gln Leu Ala Lys Arg Phe Ser Lys Gly Leu Leu Ser Gly Gln 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 Ile Pro Pro Thr Ala Gly Ile 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 Ile Lys Val Leu Arg Gly Phe Lys Lys Glu Ile 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 Ile 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 Ile Val Ser Lys Gln 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 Ile Ala Met Asp
140 145 150
TTG GGA GAG TTA TGT GAG GAC ACA ATG ACT TAC AAA TGC CCT CGA ATT 590 Leu Gly Glu Leu Cys Glu Asp Thr Met Thr Tyr Lys Cys Pro Arg Ile
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 Gln 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 Gln Ile
220 225 230
CAA AGA GTG GAG ACT TGG GCT TTG CGA CAC CCA GGA TTC ACG GTG ATA 830 Gln Arg Val Glu Thr Trp Ala Leu Arg His Pro Gly Phe Thr Val Ile
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 Ile Gly Thr Ser Ile Thr Gln Lys Gly
255 260 265
ATT ATT TTC ATT TTG TTA ATG CTA GTA ACA CCA TCC ATG GCC ATG CGA 926 Ile Ile Phe Ile 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 Ile 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 Ile 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 Ile Glu Ala Lys Ile 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 Gln 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 Gln 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 Ile Val
395 400 405 410
CAA TAT GAA AAC TTA AAA TAT TCA GTG ATA GTC ACT GTC CAC ACT GGG 1358 Gln Tyr Glu Asn Leu Lys Tyr Ser Val Ile 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 Gln His Gln Val Gly Asn Glu Thr Thr Glu His Gly Thr Ile Ala
430 435 440
ACC ATA ACA CCT CAA GCT CCT ACG TCG GAA ATA CAG CTG ACC GAC TAC 1454 Thr Ile Thr Pro Gln Ala Pro Thr Ser Glu Ile Gln 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 Gln 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 Gln Glu Thr Trp Asn Arg Gln 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 Gln Glu Val Val Val Leu Gly Ser Gln 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 Ile Gln 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 Ile 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 Gln 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 Gln Val Lys Tyr Glu Gly Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser
605 610 615
ACC CAA GAT GAG AAA GGA GTG ACC CAG AAT AGA TTG ATA ACA GCC AAT 1982 Thr Gln Asp Glu Lys Gly Val Thr Gln Asn Arg Leu Ile Thr Ala Asn
620 625 630
CCT ATA GTT ACT GAC AAA GAA AAA CCA GTC AAC ATT GAG ACA GAA CCA 2030 Pro Ile Val Thr Asp Lys Glu Lys Pro Val Asn Ile 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 Ile 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 Gln Cys Trp Phe Lys Lys Gly Ser Ser Ile 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 Ile 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 Ile 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 Gln 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 Ile Gly Ile Gly Ile 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 Ile 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 Gln 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 Ile Asn Trp Lys Gly Arg Glu Leu Lys Cys Gly Ser Gly Ile 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 Gln Tyr Lys Phe Gln 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 Ile 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 Ile Arg Ser Ala Thr Arg Leu Glu Asn Ile Met Trp
830 835 840
AAG CAA ATA TCA AAT GAA CTG AAC CAC ATC TTA CTT GAA AAT GAC ATG 2654 Lys Gln Ile Ser Asn Glu Leu Asn His Ile 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 Ile Leu Ala Gln Gly
860 865 870
AAA AAA ATG ATT AGA CCA CAA CCC ATG GAA CAC AAA TAC TCA TGG AAA 2750 Lys Lys Met Ile Arg Pro Gln 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 Ile Ile Gly Ala Asp Ile Gln Asn Thr Thr
895 900 905
TTC ATC ATT GAC GGC CCA GAT ACT CCA GAA TGT CCT GAT GAC CAA AGA 2846 Phe Ile Ile Asp Gly Pro Asp Thr Pro Glu Cys Pro Asp Asp Gln Arg
910 915 920
GCA TGG AAC ATT TGG GAA GTT GAG GAC TAT GGG TTC GGA ATT TTC ACG 2894 Ala Trp Asn Ile Trp Glu Val Glu Asp Tyr Gly Phe Gly Ile Phe Thr
925 930 935
ACA AAC ATA TGG TTG AAA TTG CGT GAC TCC TAC ACC CAA ATG TGT GAC 2942 Thr Asn Ile Trp Leu Lys Leu Arg Asp Ser Tyr Thr Gln 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 Ile 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 Ile 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 Ile 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 Ile Ile Pro
1005 1010 1015
AAG ATC TAT GGA GGA CCA ATA TCT CAG CAC AAC TAC AGA CCA GGA TAT 3182 Lys Ile Tyr Gly Gly Pro Ile Ser Gln 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 Gln 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 Ile Ile
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 Ile 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 Ile Ser Ile Met Ile 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 Ile Met Gly Gln 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 Ile Arg Leu Cys Ile 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 Ile 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 Ile Met
1245 1250 1255
ATT TTA AAA TTA TTG ACT GAC TTT CAG TCA CAT CAG CTG TGG GCT ACC 3902 Ile Leu Lys Leu Leu Thr Asp Phe Gln Ser His Gln 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 Ile 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 Gln Lys Thr Thr Trp Leu Pro Val Leu Leu
1310 1315 1320 GGA TCT CTT GGA TGC AAA CCA CTA ACC ATG TTT CTC ATA GCA GAA AAC 4094 Gly Ser Leu Gly Cys Lys Pro Leu Thr Met Phe Leu Ile Ala Glu Asn
1325 1330 1335
AAA ATC TGG GGA AGG AAA AGT TGG CCC CTC AAT GAA GGA ATC ATG GCT 4142 Lys Ile Trp Gly Arg Lys Ser Trp Pro Leu Asn Glu Gly Ile Met Ala
1340 1345 1350
GTT GGA ATA GTC AGC ATC CTA CTA AGT TCA CTC CTC AAA AAT GAT GTG 4190 Val Gly Ile Val Ser Ile 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 Ile Ala Gly Gly Met Leu Ile Ala Cys Tyr
1375 1380 1385
GTT ATA TCT GGA AGC TCA GCC GAC TTA TCA CTA GAG AAA GCG GCT GAG 4286 Val Ile 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 Ile
1405 1410 1415
TTA GTG GAG GTC CAA GAT GAT GGA ACC ATG AAG ATA AAA GAT GAA GAG 4382 Leu Val Glu Val Gln Asp Asp Gly Thr Met Lys Ile 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 Ile 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 Ile 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 Gln Lys Lys Lys Gln 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 Ile Tyr Arg
1485 1490 1495
ATT ATG CAG AGA GGA CTG TTG GGC AGG TCC CAA GTA GGA GTG GGA GTT 4622 Ile Met Gln Arg Gly Leu Leu Gly Arg Ser Gln 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 Gln 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 Gln 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 Ile Ser Tyr Gly Gly Gly Trp Arg Phe Gln 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 Gln Val Ile 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 Gln 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 Ile 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 Ile Val Asn Arg Glu Gly Lys Ile 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 Ile Ala Gln Ala
1630 1635 1640
AAA GCA TCA CAA GAA GGG CCC CTA CCA GAG ATT GAG GAC GAG GTG TTT 5054 Lys Ala Ser Gln Glu Gly Pro Leu Pro Glu Ile 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 Ile 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 Ile Val Arg Glu Ala Ile Arg Arg Asn
1675 1680 1685 1690
GTG CGC ACA CTA ATT TTG GCT CCC ACA AGG GTT GTC GCT TCC GAA ATG 5198 Val Arg Thr Leu Ile Leu Ala Pro Thr Arg Val Val 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 Ile Arg Tyr Gln 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 Ile 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 Ile Ile Met Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile 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 Ile 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 Ile Phe Met Thr Ala Thr Pro Pro Gly Ser Val Glu Ala Phe Pro Gln
1790 1795 1800
AGC AAT GCA GTT ATC CAA GAT GAG GAA AGA GAC ATT CCT GAG AGA TCA 5534 Ser Asn Ala Val Ile Gln Asp Glu Glu Arg Asp Ile 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 Ile 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 Ile Lys Ser Gly Asn Asp Ile 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 Ile Gln 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 Gln 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 Ile Ser Glu Met Gly Ala Asn Phe Arg Ala Asp Arg Val Ile
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 Ile 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 Ile 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 Gln Arg Arg Gly Arg Ile Gly Arg Asn Gln Asn Lys Glu Gly Asp Gln
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 Gln 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 Ile 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 Ile Ile Pro Ala Leu Phe Glu Pro Glu Arg Glu Lys Ser Ala Ala Ile
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 Gln 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 Gln 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 Ile Leu Glu Ile Gly Lys Leu
2095 2100 2105
CCA CAA CAC TTG ACG CAA AGG GCC CAG AAT GCC TTG GAC AAC CTG GTT 6446 Pro Gln His Leu Thr Gln Arg Ala Gln 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 Gln 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 Ile Glu Thr Leu Met Leu Leu Ala Leu Ile
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 Ile 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 Ile Ala Ala Ser Ile
2190 2195 2200
ATA CTA GAG TTT TTC CTG ATG GTG CTG CTT ATT CCA GAG CCA GAC AGA 6734 Ile Leu Glu Phe. Phe Leu Met Val Leu Leu Ile 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 Gln Arg Thr Pro Gln Asp Asn Gln Leu Ala Tyr Val Val Ile 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 Ile 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 Ile 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 Ile 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 Ile Glu Asn Thr Thr Ala Asn Ile Ser Leu Thr Ala Ile Ala Asn
2300 2305 2310
CAG GCA GCT ATA TTG ATG GGA CTT GAT AAA GGA TGG CCA ATA TCG AAG 7070 Gln Ala Ala Ile Leu Met Gly Leu Asp Lys Gly Trp Pro Ile 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 Ile Gly Val Pro Leu Leu Ala Leu Gly Cys Tyr Ser Gln 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 Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr Arg Glu Ala Gln
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 Ile Met Lys Asn Pro Thr Val Asp Gly Ile
2380 2385 2390
GTT GCA ATA GAT TTG GAC CCT GTG GTT TAT GAT GCA AAA TTT GAG AAA 7310 Val Ala Ile 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 Gln Leu Gly Gln Ile Met Leu Leu Ile Leu Cys Thr Ser Gln Ile 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 Ile 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 Ile Ala Val Ser Met Ala Asn Ile 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 Gln 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 Ile 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 Ile
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 Ile 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 Ile Gly Glu Ser Ser Pro Asn Pro Thr Ile 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 Gln Phe Cys Ile Lys Ile 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 Gln Met Gln 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 Ile 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 Ile Ile Gly Gln Arg Ile Glu Asn Ile 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 Ile
2815 2820 2825
CCC ATG GTC ACA CAA ATA GCC ATG ACT GAC ACC ACA CCC TTT GGA CAA 8606 Pro Met Val Thr Gln Ile Ala Met Thr Asp Thr Thr Pro Phe Gly Gln
2830 2835 2840
CAG AGG GTG TTT AAA GAG AAA GTT GAC ACG CGC ACA CCA AAA GCA AAA 8654 Gln 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 Gln Ile 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 Ile 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 Ile 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 Gln 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 Gln 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 Ile 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 Ile Leu Arg Asp Ile Ser Lys Ile 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 Ile 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 Gln Asn Glu Ala Lys Ile Thr Asp Ile 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 Ile Phe Lys Leu Thr Tyr Gln 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 Gln 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 Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln 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 Gln Leu Ile Arg Gln 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 Ile 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 Ile Ser Gly Asp Asp Cys Val Val Lys Pro Ile 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 Ile Pro Gln 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 Gln Gln Val Pro Phe Cys Ser His His Phe His Gln Leu Ile 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 Ile Val Val Pro Cys Arg Asn Gln 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 Gln Gly Ala Gly Trp Ver 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 Gln Met Trp Gln 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 Ile 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 Ile 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 Gln 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 Ile 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 Gln 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 Ile Gly Leu Thr Ala Arg Ala Thr Trp Ala Thr Asn Ile
3340 3345 3350
CAA GTG GCC ATA AAC CAA GTG AGA AGA -CTA ATC GGG AAT GAG AAT TAT 10190 Gln Val Ala Ile Asn Gln Val Arg Arg Leu Ile 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
AAATCAAACA AGGCAAGAAG TCAGGCCGGA TTAAGCCATA GTACGGTAAG AGCTATGCTG 10348
CCTGTGAGCC CCGTCCAAGG ACGTAAAATG AAGTCAGGCC GAAAGCCACG GTTTGAGCAA 10408
ACCGTGCTGC CTGTAGCTTC ATCGTGGGGA TGTAAAAACC TGGGAGGCTG CAACCCATGG 10468
AAGCTGTACG CATGGGGTAG CAGACTAGTG GTTAGAGGAG ACCCCTCCCA AAACATAACG 10528
CAGCAGCGGG GCCCAACACC AGGGGAAGCT GTATCCTGGT GGTAAGGACT AGAGGTTAGA 10588
GGAGACCCCC GGCATAACAA TAAACAGCAT ATTGACGCTG GGAGAGACCA GAGATCCTGC 10648
TGTCTCTACA GCATCATTCC AGGCACAGAA CGCCAGAAAA TGGAATGGTG CTGTTGAATC 10708
AACAGGTTCT 10718
(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 Gln 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 Gln Leu Ala Lys Arg
20 25 30
Phe Ser Lys Gly Leu Leu Ser Gly Gln Gly Pro Met Lys Leu Val Met
35 40 45
Ala Phe Ile Ala Phe Leu Arg Phe Leu Ala Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Ala Arg Trp Gly Ser Phe Lys Lys Asn Gly Ala Ile Lys Val
65 70 75 80
Leu Arg Gly Phe Lys Lys Glu Ile Ser Asn Met Leu Asn Ile 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 Ile Val
115 120 125
Ser Lys Gln Glu Arg Glu Lys Ser Leu Leu Phe Lys Thr Ser Val Gly
130 135 140 Val Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Leu Cys Glu 145 150 155 160
Asp Thr Met Thr Tyr Lys Cys Pro Arg Ile 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 Gln 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 Gln Ile Gln Arg Val Glu Thr Trp 225 230 235 240
Ala Leu Arg His Pro Gly Phe Thr Val Ile Ala Leu Phe Leu Ala His
245 250 255
Ala Ile Gly Thr Ser Ile Thr Gln Lys Gly Ile Ile Phe Ile Leu Leu
260 265 270
Met Leu Val Thr Pro Ser Met Ala Met Arg Cys Val Gly Ile 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 Ile Glu Leu Leu Lys Thr Glu Val Thr Asn Pro Ala Val Leu
325 330 335
Arg Lys Leu Cys Ile Glu Ala Lys Ile Ser Asn Thr Thr Thr Asp Ser
340 345 350
Arg Cys Pro Thr Gln Gly Glu Ala Thr Leu Val Glu Glu Gln 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 Ile Val Gln Tyr Glu Asn Leu Lys
405 410 415
Tyr Ser Val Ile Val Thr Val His Thr Gly Asp Gln His Gln Val Gly
420 425 430
Asn Glu Thr Thr Glu His Gly Thr Ile Ala Thr Ile Thr Pro Gln Ala
435 440 445
Pro Thr Ser Glu Ile Gln 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 Gln Trp Phe Leu Asp Leu 485 490 495
Pro Leu Pro Trp Thr Ser Gly Ala Ser Thr Ser Gln Glu Thr Trp Asn
500 505 510
Arg Gln Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala Lys Lys Gln
515 520 525
Glu Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu 530 535 540
Thr Gly Ala Thr Glu Ile Gln Thr Ser Gly Thr Thr Thr Ile 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 Gln His Gly Thr Val Leu Val Gln Val Lys Tyr Glu Gly
595 600 605
Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly 610 615 620
Val Thr Gln Asn Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp Lys 625 630 635 640
Glu Lys Pro Val Asn Ile Glu Thr Glu Pro Pro Phe Gly Glu Ser Tyr
645 650 655 Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Gln Cys Trp Phe Lys
660 665 670
Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala
675 680 685
Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Ile 690 695 700
Gly Gly Val Phe Thr Ser Val Gly Lys Leu Val His Gln Val Phe Gly 705 710 715 720
Thr Ala Tyr Gly Val Leu Phe Ser Gly Val Ser Trp Thr Met Lys Ile
725 730 735
Gly Ile Gly Ile Leu Leu Thr Trp Leu Gly Leu Asn Ser Arg Ser Thr
740 745 750
Ser Leu Ser Met Thr Cys Ile Ala Val Gly Met Val Thr Leu Tyr Leu
755 760 765
Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Ile Asn Trp Lys Gly 770 775 780
Arg Glu Leu Lys Cys Gly Ser Gly Ile Phe Val Thr Asn Glu Val His 785 790 795 800
Thr Trp Thr Glu Gln Tyr Lys Phe Gln Ala Asp Ser Pro Lys Arg Leu
805 810 815 Ser Ala Ala Ile Gly Lys Ala Trp Glu Glu Gly Val Cys Gly Ile Arg 820 825 830
Ser Ala Thr Arg Leu Glu Asn Ile Met Trp Lys Gln Ile Ser Asn Glu
835 840 845
Leu Asn His Ile Leu Leu Glu Asn Asp Met Lys Phe Thr Val Val Val 850 855 860
Gly Asp Val Val Gly Ile Leu Ala Gln Gly Lys Lys Met Ile Arg Pro 865 870 875 880 Gln Pro Met Glu His Lys Tyr Ser Trp Lys Ser Trp Gly Lys Ala Lys
885 890 895 Ile Ile Gly Ala Asp Ile Gln Asn Thr Thr Phe Ile Ile Asp Gly Pro
900 905 910
Asp Thr Pro Glu Cys Pro Asp Asp Gln Arg Ala Trp Asn Ile Trp Glu
915 920 925
Val Glu Asp Tyr Gly Phe Gly Ile Phe Thr Thr Asn Ile Trp Leu Lys 930 935 940
Leu Arg Asp Ser Tyr Thr Gln Met Cys Asp His Arg Leu Met Ser Ala 945 950 955 960
Ala Ile Lys Asp Ser Lys Ala Val His Ala Asp Met Gly Tyr Trp Ile
965 970 975
Glu Ser Glu Lys Asn Glu Thr Trp Lys Leu Ala Arg Ala Ser Phe Ile
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 Ile Ile Pro Lys Ile Tyr Gly Gly Pro 1010 1015 1020
Ile Ser Gln His Asn Tyr Arg Pro Gly Tyr Phe Thr Gln 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 Ile Ile 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 Ile 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 Ile Ser Ile Met Ile 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 Ile Met Gly Gln Leu Thr Trp Asn Asp Leu Ile Arg Leu Cys Ile 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 Ile
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 Ile Met Ile Leu Lys Leu Leu Thr 1250 1255 1260
Asp Phe Gln Ser His Gln 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 Ile Val Ser Leu Phe Pro Leu Cys Leu Ser Thr Thr Ser
1300 1305 1310
Gln Lys Thr Thr Trp Leu Pro Val Leu Leu Gly Ser Leu Gly Cys Lys
1315 1320 1325
Pro Leu Thr Met Phe Leu Ile Ala Glu Asn Lys Ile Trp Gly Arg Lys 1330 1335 1340
Ser Trp Pro Leu Asn Glu Gly Ile Met Ala Val Gly Ile Val Ser Ile 1345 1350 1355 1360
Leu Leu Ser Ser Leu Leu Lys Asn Asp Val Pro Leu Ala Gly Pro Leu
1365 1370 1375 Ile Ala Gly Gly Met Leu Ile Ala Cys Tyr Val Ile 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 Ile Leu Val Glu Val Gln Asp 1410 1415 1420
Asp Gly Thr Met Lys Ile Lys Asp Glu Glu Arg Asp Asp Thr Leu Thr 1425 1430 1435 1440 Ile Leu Leu Lys Ala Thr Leu Leu Ala Val Ser Gly Val Tyr Pro Leu
1445 1450 1455
Ser Ile Pro Ala Thr Leu Phe Val Trp Tyr Phe Trp Gln Lys Lys Lys
1460 1465 1470
Gln 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 Ile Tyr Arg Ile Met Gln Arg Gly Leu 1490 1495 1500
Leu Gly Arg Ser Gln Val Gly Val Gly Val Phe Gln Asp Gly Val Phe 1505 1510 1515 1520
His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met Tyr Gln Gly
1525 1530 1535
Lys Arg Leu Glu Pro Ser Trp Ala Ser Val Lys Lys Asp Leu Ile Ser
1540 1545 1550
Tyr Gly Gly Gly Trp Arg Phe Gln Gly Ser Trp Asn Thr Gly Glu Glu
1555 1560 1565
Val Gln Val Ile Ala Val Glu Pro Gly Lys Asn Pro Lys Asn Val Gln 1570 1575 1580
Thr Ala Pro Gly Thr Phe Lys Thr Pro Glu Gly Glu Val Gly Ala Ile 1585 1590 1595 1600
Ala Leu Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro Ile Val Asn Arg
1605 1610 1615
Glu Gly Lys Ile Val Gly Leu Tyr Gly Asn Gly Val Val Thr Thr Ser
1620 1625 1630
Gly Thr Tyr Val Ser Ala Ile Ala Gln Ala Lys Ala Ser Gln Glu Gly
1635 1640 1645
Pro Leu Pro Glu Ile Glu Asp Glu Val Phe Arg Lys Arg Asn Leu Thr 1650 1655 1660
Ile Met Asp Leu His Pro Gly Ser Gly Lys Thr Arg Arg Tyr Leu Pro 1665 1670 1675 1680
Ala Ile Val Arg Glu Ala Ile Arg Arg Asn Val Arg Thr Leu Ile 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 Ile Arg Tyr Gln Thr Thr Ala Val Lys Ser Glu His Thr Gly
1715 1720 1725
Lys Glu Ile 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 Ile Ile Met Asp Glu 1745 1750 1755 1760
Ala His Phe Thr Asp Pro Ala Ser Ile Ala Arg Arg Gly Tyr Ile Ser
1765 1770 1775
Thr Arg Val Gly Met Gly Glu Ala Ala Ala Ile Phe Met Thr Ala Thr
1780 1785 1790
Pro Pro Gly Ser Val Glu Ala Phe Pro Gln Ser Asn Ala Val Ile Gln
1795 1800 1805
Asp Glu Glu Arg Asp Ile Pro Glu Arg Ser Trp Asn Ser Gly Tyr Glu 1810 1815 1820 Trp Ile Thr Asp Phe Pro Gly Lys Thr Val Trp Phe Val Pro Ser Ile 1825 1830 1835 1840
Lys Ser Gly Asn Asp Ile Ala Asn Cys Leu Arg Lys Asn Gly Lys Arg
1845 1850 1855
Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Gln Lys Thr
1860 1865 1870
Lys Asn Asn Asp Trp Asp Tyr Val Val Thr Thr Asp Ile Ser Glu Met
1875 1880 1885
Gly Ala Asn Phe Arg Ala Asp Arg Val Ile Asp Pro Arg Arg Cys Leu 1890 1895 1900
Lys Pro Val Ile Leu Lys Asp Gly Pro Glu Arg Val Ile Leu Ala Gly 1905 1910 1915 1920
Pro Met Pro Val Thr Val Ala Ser Ala Ala Gln Arg Arg Gly Arg Ile
1925 1930 1935
Gly Arg Asn Gln Asn Lys Glu Gly Asp Gln Tyr Val Tyr Met Gly Gln
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 Ile Asn Thr Pro Glu Gly Ile Ile Pro Ala Leu Phe 1970 1975 1980
Glu Pro Glu Arg Glu Lys Ser Ala Ala Ile 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 Gln Tyr
2020 2025 2030
Ser Asp Arg Arg Trp Cys Phe Asp Gly Glu Arg Asn Asn Gln 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 Ile Leu Glu Ile Gly Lys Leu Pro Gln His Leu Thr Gln
2100 2105 2110
Arg Ala Gln Asn Ala Leu Asp Asn Leu Val Met Leu His Asn Ser Glu
2115 2120 2125
Gln Gly Gly Arg Ala Tyr Arg His Ala Met Glu Glu Leu Pro Asp Thr 2130 2135 2140
Ile Glu Thr Leu Met Leu Leu Ala Leu Ile 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 Ile 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 Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu
2195 2200 2205
Met Val Leu Leu Ile Pro Glu Pro Asp Arg Gln Arg Thr Pro Gln Asp 2210 2215 2220
Asn Gln Leu Ala Tyr Val Val Ile Gly Leu Leu Phe Met Ile 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 Ile 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 Ile Thr Pro Met Met Arg His Thr Ile Glu Asn Thr Thr 2290 2295 2300
Ala Asn Ile Ser Leu Thr Ala Ile Ala Asn Gln Ala Ala Ile Leu Met 2305 2310 2315 2320
Gly Leu Asp Lys Gly Trp Pro Ile Ser Lys Met Asp Ile Gly Val Pro
2325 2330 2335
Leu Leu Ala Leu Gly Cys Tyr Ser Gln Val Asn Pro Leu Thr Leu Thr
2340 2345 2350
Ala Ala Val Leu Met Leu Val Ala His Tyr Ala Ile Ile Gly Pro Gly
2355 2360 2365
Leu Gln Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Thr Ala Ala Gly 2370 2375 2380
Ile Met Lys Asn Pro Thr Val Asp Gly Ile Val Ala Ile Asp Leu Asp 2385 2390 2395 2400
Pro Val Val Tyr Asp Ala Lys Phe Glu Lys Gln Leu Gly Gln Ile Met
2405 2410 2415
Leu Leu Ile Leu Cys Thr Ser Gln Ile Leu Leu Met Arg Thr Thr Trp
2420 2425 2430
Ala Leu Cys Glu Ser Ile 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 Ile Ala Val Ser 2450 2455 2460
Met Ala Asn Ile 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 Gln Leu 2500 2505 2510
Ser Lys Ser Glu Phe Asn Thr Tyr Lys Arg Ser Gly Ile 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 Ile 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 Ile 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 Ile Gly 2625 2630 2635 2640
Glu Ser Ser Pro Asn Pro Thr Ile Glu Glu Gly Arg Thr Leu Arg Val
2645 2650 2655
Leu Lys Met Val Glu Pro Trp Leu Arg Gly Asn Gln Phe Cys Ile Lys
2660 2665 2670
Ile Leu Asn Pro Tyr Met Pro Ser Val Val Glu Thr Leu Glu Gln Met
2675 2680 2685
Gln 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 Ile 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 Ile Ile
2755 2760 2765
Gly Gln Arg Ile Glu Asn Ile 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 Ile Pro Met Val Thr Gln Ile
2820 2825 2830 Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu 2835 2840 2845
Lys Val Asp Thr Arg Thr Pro Lys Ala Lys Arg Gly Thr Ala Gln Ile 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 Ile Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser
2885 2890 2895
Asn Ala Ala Ile Gly Ala Val Phe Val Asp Glu Asn Gln 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 Gln 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 Ile 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
Ile Leu Arg Asp Ile Ser Lys Ile Pro Gly Gly Asn Met Tyr Ala Asp 3010 3015 3020
Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Asp Asp Leu Gln Asn 3025 3030 3035 3040
Glu Ala Lys Ile Thr Asp Ile Met Glu Pro Glu His Ala Leu Leu Ala
3045 3050 3055
Thr Ser Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val Val Arg Val Gln
3060 3065 3070
Arg Pro Ala Lys Asn Gly Thr Val Met Asp Val Ile Ser Arg Arg Asp
3075 3080 3085
Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr 3090 3095 3100
Asn Met Glu Ala Gln Leu Ile Arg Gln Met Glu Ser Glu Gly Ile 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 Ile Ser
3140 3145 3150
Gly Asp Asp Cys Val Val Lys Pro Ile Asp Asp Arg Phe Ala Thr Ala
3155 3160 3165 Leu Thr Ala Leu Asn Asp Met Gly Lys Val Arg Lys Asp Ile Pro Gln
3170 3175 3180
Trp Glu Pro Ser Lys Gly Trp Asn Asp Trp Gln Gln Val Pro Phe Cys
3185 3190 3195 3200
Ser His His Phe His Gln Leu Ile Met Lys Asp Gly Arg Glu Ile Val
3205 3210 3215
Val Pro Cys Arg Asn Gln Asp Glu Leu Val Gly Arg Ala Arg Val Ser
3220 3225 3230
Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser
3235 3240 3245
Tyr Ala Gln Met Trp Gln Leu Met Tyr Phe His Arg Arg Asp Leu Arg
3250 3255 3260
Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro Val Asp Trp Val Pro
3265 3270 3275 3280
Thr Ser Arg Thr Thr Trp Ser Ile His Ala His His Gln Trp Met Thr
3285 3290 3295
Thr Glu Asp Met Leu Ser Val Trp Asn Arg Val Trp Ile 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 Gln Trp Cys Gly Ser Leu Ile Gly Leu
3330 3335 3340
Thr Ala Arg Ala Thr Trp Ala Thr Asn Ile Gln Val Ala Ile Asn Gln
3345 3350 3355 3360
Val Arg Arg Leu Ile 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)
(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 NO:5:
TGGTTCCCGG GGACTCGGGA TGTGTA 26
(2) INFORMATION FOR SEQ ID NO: 6:
(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: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

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, NS1,
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/NS1-104, 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, NS1, NS2, NS3, NS5 and fusion proteins thereof capable of eliciting antibodies to a DEN1 viral protein.
16. An antibody against a polypeptide as claimed in any one of claims 9 to 11 capable of binding a DEN1 viral protein, optionally carrying a revealing label.
17. A test kit for the detection of the presence or absence of DEN1 virus comprising the antibody of claim 16 or the polypeptide of claim 9 or 13 fixed to a solid support.
PCT/CA1993/000182 1992-04-29 1993-04-28 Cdna sequence of dengue virus serotype 1 (singapore strain) Ceased WO1993022440A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP93911702A EP0638122B1 (en) 1992-04-29 1993-04-28 Cdna sequence of dengue virus serotype 1 (singapore strain)
US08/325,426 US6017535A (en) 1992-04-29 1993-04-28 cDNA sequence of Dengue virus serotype 1 (Singapore strain)
AU42575/93A AU679179B2 (en) 1992-04-29 1993-04-28 CDNA sequence of dengue virus serotype 1 (singapore strain)
KR1019940703865A KR950701684A (en) 1992-04-29 1993-04-28 CDNA sequence of dengue virus serotype 1 (Singapore strain) of dengue virus serotype 1 (Singapore strain)
DE69315689T DE69315689T2 (en) 1992-04-29 1993-04-28 CDNS SEQUENCE OF DENGUE VIRUS SEROTYPE 1 (SINGAPORE STEM)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9209243.6 1992-04-29
GB929209243A GB9209243D0 (en) 1992-04-29 1992-04-29 Dengue virus

Publications (1)

Publication Number Publication Date
WO1993022440A1 true WO1993022440A1 (en) 1993-11-11

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US (1) US6017535A (en)
EP (1) EP0638122B1 (en)
KR (1) KR950701684A (en)
AT (1) ATE161048T1 (en)
AU (1) AU679179B2 (en)
CA (1) CA2134666A1 (en)
DE (1) DE69315689T2 (en)
GB (1) GB9209243D0 (en)
MY (1) MY110899A (en)
SG (1) SG47384A1 (en)
WO (1) WO1993022440A1 (en)

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EP0800084A1 (en) * 1996-04-02 1997-10-08 Immuno GmbH Technique for detecting infections with TBE-virus and other flaviviruses
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WO2004092412A3 (en) * 2003-03-31 2005-03-03 Roche Diagnostics Gmbh Compositions and methods for detecting certain flaviviruses, including members of the japanese encephalitis virus serogroup
US7052878B1 (en) * 1999-12-01 2006-05-30 The United States Of America As Represented By The Secretary Of The Navy Serotype and dengue group specific flurogenic probe based PCR (TaqMan) assays against the respective C and NS5 genomic and 3′ non-coding regions of dengue virus
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Cited By (19)

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Publication number Priority date Publication date Assignee Title
US6676936B1 (en) 1988-07-14 2004-01-13 The United States Of America As Represented By The Department Of Health And Human Services. Chimeric and/or growth-restricted flaviviruses
WO1996040933A1 (en) * 1995-06-07 1996-12-19 The Government Of The United States Of America, Represented By The Secretary Department Of Health And Human Services Infectious dengue 2 virus pdk-53 as quadravalent vaccine
EP0800084A1 (en) * 1996-04-02 1997-10-08 Immuno GmbH Technique for detecting infections with TBE-virus and other flaviviruses
DE19613253A1 (en) * 1996-04-02 1997-10-09 Immuno Gmbh Technique for the detection of infections with the TBE virus and other flaviviruses
FR2767324A1 (en) * 1997-08-14 1999-02-19 Pasteur Institut Diagnostic kit for detecting antibodies to dengue virus serotypes
WO1999009414A1 (en) * 1997-08-14 1999-02-25 Institut Pasteur Use of recombinant envelope proteins for diagnosing the dengue virus
WO2000075665A1 (en) * 1999-06-09 2000-12-14 Institut Pasteur Early detection of flaviviruses using the ns1 glycoprotein
FR2794865A1 (en) * 1999-06-09 2000-12-15 Pasteur Institut METHOD OF EARLY DETECTION OF FLAVIVIRUSES AND ITS APPLICATIONS
US8709427B2 (en) 1999-06-09 2014-04-29 Institut Pasteur Assay for the diagnosis of flaviviral infection using antibodies with high affinity for NS1 protein of flavivirusi in hexameric form
US6870032B1 (en) 1999-06-09 2005-03-22 Institut Pasteur Assay for the diagnosis of flaviviral infection using antibodies with high affinity for NS1 protein of flavivirus in hexameric form
US7282341B2 (en) 1999-06-09 2007-10-16 Institut Pasteur Assay for the diagnosis of flaviviral infection using antibodies with high affinity for NS1 protein of flavivirus in hexameric form
US7052878B1 (en) * 1999-12-01 2006-05-30 The United States Of America As Represented By The Secretary Of The Navy Serotype and dengue group specific flurogenic probe based PCR (TaqMan) assays against the respective C and NS5 genomic and 3′ non-coding regions of dengue virus
US7348138B2 (en) * 2000-10-04 2008-03-25 The Trustees Of The University Of Pennsylvania Method of inducing cell death using West Nile virus capsid protein
US7510827B2 (en) 2003-03-31 2009-03-31 Roche Molecular Systems, Inc. Compositions and methods for detecting certain Flaviviruses, including members of the Japanese encephalitis virus serogroup
CN100557027C (en) * 2003-03-31 2009-11-04 霍夫曼-拉罗奇有限公司 Compositions and methods for detecting certain flaviviruses including members of Japanese encephalitis virus serogroups
WO2004092412A3 (en) * 2003-03-31 2005-03-03 Roche Diagnostics Gmbh Compositions and methods for detecting certain flaviviruses, including members of the japanese encephalitis virus serogroup
US8846312B2 (en) 2003-03-31 2014-09-30 Roche Molecular Systems, Inc. Compositions and methods for detecting certain flaviviruses, including members of the Japanese encephalitis virus serogroup
US8999675B2 (en) 2009-08-31 2015-04-07 Gen-Probe Incorporated Dengue virus assay
WO2012153153A1 (en) 2011-05-11 2012-11-15 Diagon Kft. Procedure for rapid determination of viruses using nucleic acid-based molecular diagnostics, and a kit for this purpose

Also Published As

Publication number Publication date
EP0638122B1 (en) 1997-12-10
MY110899A (en) 1999-06-30
KR950701684A (en) 1995-04-28
CA2134666A1 (en) 1993-11-11
SG47384A1 (en) 1998-04-17
AU4257593A (en) 1993-11-29
ATE161048T1 (en) 1997-12-15
DE69315689D1 (en) 1998-01-22
AU679179B2 (en) 1997-06-26
US6017535A (en) 2000-01-25
GB9209243D0 (en) 1992-06-17
DE69315689T2 (en) 1998-04-02
EP0638122A1 (en) 1995-02-15

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