CN1488646A

CN1488646A - SARS virus S protein and N protein fusion protein, and preparation and use thereof

Info

Publication number: CN1488646A
Application number: CNA031321623A
Authority: CN
Inventors: 李越希; 陶开华; 朱敏生
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-07-03
Filing date: 2003-07-03
Publication date: 2004-04-14
Anticipated expiration: 2023-07-03
Also published as: CN100386343C

Abstract

The invention is confluens protein of SARS virus S protein and N protein, and the manufacturing, application and the gene project technology, vaccine and diagnosing reductant. Through analyzing the cistine series of SARS s protein and N protein, sifts out SARS virus S protein segment with strong antigen epitope, namely the 162nd cistine to 460nd cistine, and the N protein segment of SARS virus with strong antigen epitope, anmely the first cistine to 258nd cistine. It synthesizes the new SARS virus S protein segment and N protein segment gene series, connects the two gene segments in series. Expresses the confluens protein of the two protein segments, the two are connected by glycine and serine, inserts a glycine adjoining to the frist cistine methionine of N protein segment, the confluens protein length is 560 cistines.

Description

Proteic fusion rotein of SARS virus S albumen and N and preparation thereof, application

Technical field

The present invention is connected the proteic brand-new gene fragment of the SARS virus S of chemosynthesis with the proteic gene fragment of N, utilize genetic engineering technique, preparation SARS virus S albumen and the proteic fusion rotein of N.By Computer Analysis, filter out the S protein fragments of the SARS virus that contains the strong antigen epi-position and the fusion rotein and the N protein fragments of N protein fragments, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, S gene order that chemosynthesis is brand-new and N gene order, utilize genetic engineering technique to express both fusion roteins, the fusion rotein of expressing can be used for vaccine and SARS virus antibody or detection of antigens etc., the present invention relates to genetic engineering technique, vaccine and diagnostic reagent field.

Background technology

SARS (Severe Acute Respiratory Syndrome) also claims SARS (Severe Acute Respiratory Syndrome), is a kind of potent virus sexually transmitted disease.On November 16th, 2,003 first routine patient SARS occurred in China Guangzhou, after this involved more than 30 countries in the world in the some months, and people more than 8000 infects, dead people more than 800.Mortality ratio according to WHO (WorldHealth Organization) statistics SARS can be up to about 15%, and the elderly's mortality ratio that infects this disease can reach 40% to 50%.The people has 10 to 14 days latent period after infecting SARS virus, occur high heat, cough, general malaise and headache, diarrhoea during morbidity, and respiratory distress syndrome can take place severe person in the week of one after the morbidity, so that the forfeiture respiratory function.

SARS virus is a kind of novel coronavirus, because this virus had not infected human in the past, so the crowd is to the general susceptible of this virus, SARS virus is a kind of sub-thread positive chain RNA virus, at present after measured to its complete genome sequence, announced about 10 total length virus gene sequence in the Genebank, laid a good foundation for utilizing genetic engineering technique research diagnostic reagent, vaccine and screening antiviral.

SARS virus can vitro culture with Vero-E6, set up immunofluorescence method and the enzyme linked immunosorbent detection method that detects SARS virus antibody so utilize the SARS virus of cultivating, but make Detection of antigen antibody with the SARS virus of cultivating, be difficult to mass production, also can produce false positive, developing special gene recombinant antigens is to set up the developing direction of SARS virus specific antibody detection reagent.The most desirable approach that prevention SARS infects is a vaccinate, but does not prevent the vaccine of SARS at present, both at home and abroad all actively developing the development of vaccine, especially Inactivated Vaccine is made fast progress, and recombinant vaccine is final developing direction.

Summary of the invention

SARS can stimulate body to produce multiple antibody after infecting human body, but sometimes in the serum only at the proteic antibody of part SARS virus, the time that may produce with various antibody or relevant with modulation on immune status, so when detecting intraserous SARS virus antibody, will use multiple antigen, with raising antibody recall rate.

The outermost S albumen of SARS virus is mediation virus and host cell bonded major protein, seals this proteic binding site and can stop virus infected cell, so the S albumen of SARS virus is the first-selected albumen that develops vaccine.The N albumen of SARS virus has higher conservative property, and it is very little to make a variation between each strain SARS virus, so be the ideal protein as antigen development SARS virus antibody test reagent.By Computer Analysis SARS virus S albumen and the proteic aminoacid sequence of N, we filter out the S protein fragments and the N protein fragments of the SARS virus that contains the strong antigen epi-position, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, the S protein fragments that chemosynthesis is brand-new and the gene order of N protein fragments, with two gene fragment series connection, utilize genetic engineering technique to express the fusion rotein of S protein fragments and N protein fragments, the fusion rotein of expression can be used for vaccine and SARS virus antibody or detection of antigens etc.

The present invention is that the SARS virus S albumen with chemosynthesis is connected with the proteic gene fragment of N, utilizes genetic engineering technique, the fusion rotein of preparation SARS virus S protein fragments and N protein fragments.By Computer Analysis SARS virus S albumen and the proteic aminoacid sequence of N, filter out the S protein fragments of the SARS virus that contains the strong antigen epi-position, i.e. 460 amino acid of the 162nd amino acid to the, totally 299 amino acid, with the N protein fragments of the SARS virus that contains the strong antigen epi-position, i.e. the 1st amino acid to 258 amino acid.The codon of selecting eucaryon and prokaryotic organism all to have a preference for, the SARS virus S protein fragments that chemosynthesis is brand-new and the gene order of N protein fragments, with two gene fragment series connection, utilize genetic engineering technique to express the fusion rotein of S protein fragments and N protein fragments, be connected with Serine with glycine between two protein fragments, behind first amino acid methionine of N protein fragments, insert a glycine, 560 amino acid of fusion rotein total length.The fusion rotein of expressing can be used for vaccine and SARS virus antibody or detection of antigens, and is used for immunity preparation anti-SARS virus monoclonal antibody and how anti-etc.

Proteic fusion rotein of SARS virus S albumen and N and preparation thereof, application take following steps to implement:

1.SARS the screening of virus S protein and N proteantigen epi-position and the chemosynthesis of gene fragment thereof:

Utilize softwares such as ANTHEWIN, by Computer Analysis SARS virus S albumen and the proteic aminoacid sequence of N, find that the proteic N of S holds 460 amino acid of the 162nd amino acid to the to contain stronger antigenic determinant, the proteic N of N holds 258 amino acid of the 1st amino acid to the to contain stronger antigenic determinant.The codon of selecting eucaryon and prokaryotic organism all to have a preference for, chemosynthesis comprises S albumen and the proteic gene order of N of above-mentioned antigenic determinant respectively.NcoI restriction enzyme site (following setting-out part) and 8 protection bases (GCGAGTCACC) have been increased at the segmental 5 ' end of synthetic N protein gene; and after initiator codon ATG, inserted a codon GGA, increased BamHI restriction enzyme site (following setting-out part) and 3 protection bases (GCG) at 3 ' end.Increased BamHI restriction enzyme site (following setting-out part) and two protection bases (GT) at the segmental 5 ' end of synthetic S protein gene, increased terminator codon (TAA) and EcoRI restriction enzyme site (following setting-out part) and two protection bases (AC) at 3 ' end.Making two gene fragments of synthetic be easy to series connection is cloned in plasmid pET28a (+) the interior NcoI and EcoRI restriction enzyme site.

SARSN ( 1aa-258aa ) ：Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile Thr Phe GlyGly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly Arg Asn Gly Ala Arg ProLys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala LeuThr Gln His Gly Lys Glu Glu Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile AsnThr Asn Ser Gly Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg ValArg Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr LeuGly Thr Gly Pro Glu Ala Ser Leu Pro Tyr GlyAla Asn Lys Glu Gly Ile Val TrpVal Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn ProAsn Asn Asn Ala Ala Thr Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys GlyPhe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser ArgSer Arg Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro AlaArg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu Leu Asp Arg LeuAsn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr ValThr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

SARSS ( 162aa-460aa ) ：Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe LysHis Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys GlyTyr Gln Pro Ile Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys ProIle Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr AlaPhe Ser Pro Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Glv TyrLeu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp AlaVal Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe GluIle Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp ValVal Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala ThrLys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala AspTyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val SerAla Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp Ser Phe Val ValLys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp TyrAsn Tyr Lys Leu Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg AsnIle Asp Ala Thr Ser Thr Gly Asn TyrAsn Tyr Lys Tyr Arg Tyr Leu Arg His GlyLys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe

The dna sequence dna that contains SARS virus N proteantigen epitope gene (796 bp) of chemosynthesis: GCG AGT CA C CAT GGG ATC TGA TAA CGG TCC GCA GTC AAA CCA ACG TAG TGC CCC CCGCAT TAC ATT TGG TGG ACC CAC AGA TTC AAC TGA CAA TAA CCA GAA TGG AGG ACG CAATGG GGC AAG GCC AAA ACA GCG CCG ACC CCA AGG TTT ACC CAA TAA TAC TGC GTC TTGGTT CAC AGC TCT CAC TCA GCA TGG CAA GGA GGA ACT TAG ATT CCC TCG AGG CCA GGGCGT TCC AAT CAA CAC CAA TAG TGG TCC AGA TGA CCA AAT TGG CTA CTA CCG AAG AGCTAC CCG ACG AGT TCG TGG TGG TGA CGG CAA AAT GAA AGA GCT CAG CCC CAG ATG GTACTT CTA TTA CCT AGG AAC TGG CCC AGA AGC TTC ACT TCC CTA CGG CGC TAA CAA AGAAGG CAT CGT ATG GGT TGC AAC TGA GGG AGC CTT GAA TAC ACC CAA AGA CCA CAT TGGCAC CCG CAA TCC TAA TAA CAA TGC TGC CAC CGT GCT ACA ACT TCC TCA AGG AAC AACATT GCC AAA AGG CTT CTA CGC AGA GGG AAG CAG AGG CGG CAG TCA AGC CTC TTC TCGCTC CTC ATC ACG TAG TCG CGG TAA TTC AAG AAA TTC AAC TCC TGG CAG CAG TAG GGGAAA TTC TCC TGC TCG AAT GGC TAG CGG AGG TGG TGA AAC TGC CCT CGC GCT ATT GCTGCT AGA CAG ATT GAA CCA GCT TGA GAG CAA AGT TTC TGG TAA AGG CCA ACA ACA ACAAGG CCA AAC TGT CAC TAA GAA ATC TGC TGC TGA GGC ATC TAA AAA G GG ATC CGC G

The dna sequence dna that contains SARS virus S proteantigen epitope gene (916 bp) of chemosynthesis: GT GGATCCGAA TAC ATC TCT GAC GCA TTC TCT CTG GAC GTT TCC GAA AAG TCT GGT AACTTC AAA CAC CTG CGC GAG TTC GTG TTT AAA AAC AAA GAC GGT TTC CTG TAC GTT TACAAG GGC TAC CAG CCG ATC GAC GTA GTT CGT GAC CTG CCG TCT GGT TTT AAC ACT CTGAAA CCG ATC TTC AAG CTG CCG CTG GGT ATT AAC ATC ACT AAC TTC CGC GCT ATC CTGACT GCT TTC TCT CCG GCT CAG GAC ACT TGG GGC ACT TCT GCT GCA GCC TAC TTC GTTGGC TAC CTG AAG CCA ACT ACC TTT ATG CTG AAG TAC GAC GAA AAC GGT ACT ATC ACTGAT GCT GTT GAC TGC TCT CAG AAC CCA CTG GCT GAA CTG AAA TGC TCT GTT AAG AGCTTT GAG ATC GAC AAA GGT ATT TAC CAG ACC TCT AAC TTC CGT GTT GTT CCG TCT GGTGAC GTT GTG CGT TTC CCT AAC ATC ACT AAC CTG TGC CCG TTT GGT GAA GTT TTC AACGCT ACT AAA TTC CCT TCT GTC TAC GCA TGG GAG CGT AAA AAA ATT TCT AAC TGC GTTGCT GAT TAC TCT GTG CTG TAC AAC TCT ACC TTT TTC TCT ACC TTC AAG TGC TAC GGCGTT TCT GCT ACT AAG CTG AAC GAC CTG TGC TTC TCC AAC GTT TAC GCA GAT TCT TTCGTA GTT AAG GGT GAT GAC GTA CGT CAG ATC GCT CCA GGT CAG ACT GGT GTT ATC GCTGAC TAC AAC TAT AAA CTG CCG GAC GAT TTC ATG GGT TGC GTT CTG GCT TGG AAC ACTCGT AAC ATT GAC GCT ACT TCT ACT GGT AAC TAC AAC TAC AAA TAT CGT TAC CTG CGTCAC GGC AAA CTG CGT CCG TTC GAA CGT GAC ATC TCT AAC GTG CCG TTC TAA GAATTCAC

2. express the fusion rotein construction of recombinant plasmid of SARS virus S protein fragments and N protein fragments:

Extract plasmid pET28a (+) (U.S. Novagen company product),, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis, be dissolved in the deionized water with Nco I and EcoR I double digestion.With the SARS virus N protein gene fragment of Nco I and the chemosynthesis of BamH I double digestion,, after electrophoresis reclaims respectively, be dissolved in the deionized water with the SARS virus S protein gene fragment of BamH I and the chemosynthesis of EcoR I double digestion.

Above-mentioned three kinds of enzymes of volumetric molar concentration such as get and cut the back dna fragmentation, in same centrifuge tube, connect with the T4 dna ligase, make SARS virus N protein gene fragment and S protein gene fragment after BamH I enzyme is connected, be inserted between the Nco I and EcoR I site in the carrier pET28a (+), express the fusion rotein of a SARS virus S protein fragments and N protein fragments.

3. the screening of recombinant plasmid and evaluation:

With recombinant plasmid transformed e. coli bl21 (DE3), coating contains kantlex (60 μ g/ml) LB flat board, 37 ℃ of of of of of of and spends of puts the night.Next day, picking transformed a bacterium colony and a contrast bacterium (plasmid pET28a transformed bacteria) at random, extract plasmid respectively, plasmid with extraction is a template, right with the primer of primer of N protein gene fragment 5 ' end (5 '-GCGAGTCACCATGGGATCTGATAACGGTCCGCAGTCAAACCAACG-3 ') and S protein gene fragment 3 ' end (5 '-GTGAATTCTTAGAAAGGCACATTAGATATGT-3 ') composition primer, SARS virus N protein gene fragment that pcr amplification links together and S protein gene fragment, contain the positive recombinant plasmid of two gene fragments of connecting, the plasmid that should amplify the tandem gene fragment that is about 1701bp. will contain tandem gene carries out dna sequence analysis; SARSNS,：ATG GGA TCT GAT AAC GGT CCG CAG TCA AAC CAA CGT AGT GCC CCC CGC ATT ACA TTTGGT GGA CCC ACA GAT TCA ACT GAC AAT AAC CAG AAT GGA GGA CGC AAT GGG GCA AGGCCA AAA CAG CGC CGA CCC CAA GGT TTA CCC AAT AAT ACT GCG TCT TGG TTC ACA GCTCTC ACT CAG CAT GGC AAG GAG GAA CTT AGA TTC CCT CGA GGC CAG GGC GTT CCA ATCAAC ACC AAT AGT GGT CCA GAT GAC CAA ATT GGC TAC TAC CGA AGA GCT ACC CGA CGAGTT CGT GGT GGT GAC GGC AAA ATG AAA GAG CTC AGC CCC AGA TGG TAC TTC TAT TACCTA GGA ACT GGC CCA GAA GCT TCA CTT CCC TAC GGC GCT AAC AAA GAA GGC ATC GTATGG GTT GCA ACT GAG GGA GCC TTG AAT ACA CCC AAA GAC CAC ATT GGC ACC CGC AATCCT AAT AAC AAT GCT GCC ACC GTG CTA CAA CTT CCT CAA GGA ACA ACA TTG CCA AAAGGC TTC TAC GCA GAG GGA AGC AGA GGC GGC AGT CAA GCC TCT TCT CGC TCC TCA TCACGT AGT CGC GGT AAT TCA AGA AAT TCA ACT CCT GGC AGC AGT AGG GGA AAT TCT CCTGCT CGA ATG GCT AGC GGA GGT GGT GAA ACT GCC CTC GCG CTA TTG CTG CTA GAC AGATTG AAC CAG CTT GAG AGC AAA GTT TCT GGT AAA GGC CAA CAA CAA CAA GGC CAA ACTGTC ACT AAG AAA TCT GCT GCT GAG GCA TCT AAA AAGGGA TCCGAA TAC ATC TCT GACGCA TTC TCT CTG GAC GTT TCC GAA AAG TCT GGT AAC TTC AAA CAC CTG CGC GAG TTCGTG TTT AAA AAC AAA GAC GGT TTC CTG TAC GTT TAC AAG GGC TAC CAG CCG ATC GACGTA GTT CGT GAC CTG CCG TCT GGT TTT AAC ACT CTG AAA CCG ATC TTC AAG CTG CCGCTG GGT ATT AAC ATC ACT AAC TTC CGC GCT ATC CTG ACT GCT TTC TCT CCG GCT CAGGAC ACT TGG GGC ACT TCT GCT GCA GCC TAC TTC GTT GGC TAC CTG AAG CCA ACT ACCTTT ATG CTG AAG TAC GAC GAA AAC GGT ACT ATC ACT GAT GCT GTT GAC TGC TCT CAGAAC CCA CTG GCT GAA CTG AAA TGC TCT GTT AAG AGC TTT GAG ATC GAC AAA GGT ATTTAC CAG ACC TCT AAC TTC CGT GTT GTT CCG TCT GGT GAC GTT GTG CGT TTC CCT AACATC ACT AAC CTG TGC CCG TTT GGT GAA GTT TTC AAC GCT ACT AAA TTC CCT TCT GTCTAC GCA TGG GAG CGT AAA AAA ATT TCT AAC TGC GTT GCT GAT TAC TCT GTG CTG TACAAC TCT ACC TTT TTC TCT ACC TTC AAG TGC TAC GGC GTT TCT GCT ACT AAG CTG AACGAC CTG TGC TTC TCC AAC GTT TAC GCA GAT TCT TTC GTA GTT AAG GGT GAT GAC GTACGT CAG ATC GCT CCA GGT CAG ACT GGT GTT ATC GCT GAC TAC AAC TAT AAA CTG CCGGAC GAT TTC ATG GGT TGC GTT CTG GCT TGG AAC ACT CGT AAC ATT GAC GCT ACT TCTACT GGT AAC TAC AAC TAC AAA TAT CGT TAC CTG CGT CAC GGC AAA CTG CGT CCG TTCGAA CGT GAC ATC TCT AAC GTG CCG TTC TAA

The virus S protein fragment of the expression of recombinant plasmid SARS that makes up and the fusion of N protein fragments; 560 amino acid of total length; Be the N protein fragments at its N end; Long 259 amino acid; The C end is the S protein fragments; Long 299 amino acid; ,：Met Gly Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile Thr PheGly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly Arg Asn Gly Ala ArgPro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr AlaLeu Thr Gln His Gly Lys Glu Glu Leu Arg Phe Pro Arg Gly Gln Gly Val Pro IleAsn Thr Asn Ser Gly Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg ArgVal Arg Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr TyrLeu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys Glu Gly Ile ValTrp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg AsnPro Asn Asn Asn Ala Ala Thr Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro LysGly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser SerArg Ser Arg Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser ProAla Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu Leu Asp ArgLeu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln ThrVal Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Gly Ser Glu Tyr Ile Ser AspAla Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe Lys His Leu Arg Glu PheVal Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile AspVal Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu ProLeu Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro Ala GlnAsp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr Leu Lys Pro Thr ThrPhe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser GlnAsn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly IleTyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro AsnIle Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser ValTyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu TyrAsn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu AsnAsp Leu Cys Phe Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp ValArg Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu ProAsp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr SerThr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg Pro PheGlu Arg Asp Ile Ser Asn Val Pro Phe

4. the Screening and Identification of expressed fusion protein engineering bacteria:

The positive transformant that will contain recombinant plasmid, be seeded to and contain 3ml LB substratum (containing kanamycin 60 μ g/ml) in vitro, 37 ℃ of shaking culture 3h, add IPTG to final concentration 0.5mmol/L, continue shaking culture and induce 6h, centrifugal collection thalline carries out SDS-PAGE and detects, and recon is expressed SARS virus S albumen and the proteic fusion rotein of N that relative molecular weight is about 60kD, and contrast bacterium BL21 (DE3) does not have this protein band.

5. express the purifying of SARS virus S albumen and the proteic fusion rotein of N:

1) ultrasonic degradation of expression SARS virus S albumen and the proteic fusion rotein engineering bacteria of N

Centrifugal (8000rpm, 10min, 4 ℃) receives bacterium with the engineering bacteria of abduction delivering fusion rotein, thalline is resuspended in the bacterial lysate (20mmol/L PB pH7.4,10mmol/L EDTA, 1mmol/LDTT, 5% glycerine) of original fluid l/10 volume, ice-bath ultrasonic is broken bacterium, centrifugal collection supernatant.

2) the sulfuric acid amine fractionation precipitation of expression SARS virus S albumen and the proteic fusion rotein of N

Accurately measure the supernatant volume, add an amount of saturated sulfuric acid amine aqueous solution in supernatant, the limit edged stirs, and making the final concentration of sulfuric acid amine is 30%, puts in the ice bath and spends the night.Centrifugal collection supernatant adds an amount of solid sulfur acid amide powder, and the limit edged stirs, and making the final concentration of sulfuric acid amine is 50%, puts in the ice bath and spends the night.Next day centrifugal collecting precipitation.With l00ml balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2mmol/L DTT) suspension precipitation, in the dialysis tubing of packing into,, change liquid 1 time to 500ml balance liquid dialysed overnight.Centrifugal collection supernatant is directly gone up S-Sepearse FF cation seperation column purifying purifying

3) S-Sepearse FF cation seperation column purifying

With balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2mmol/L DTT) flushing balance S-Sepharose FF cation seperation column, will go up the good direct upper prop of supernatant of step dialysis then, last sample flow velocity 1.5ml/min.Fully wash post with balance liquid liquid behind the end of the sample, again successively with contain 50,100,200, the balance liquid eluted protein of 1000mmol/L NaCl, collect each elution peak albumen, with each peak albumen of 12%SDS-PAGE electrophoresis detection, determine which elution peak contains the SARS virus S albumen and the proteic fusion rotein of N of expression.100mmol/L NaCl eluted protein peak is SARS virus S albumen and the proteic fusion rotein of N.

6. the SARS virus S albumen of purifying and the proteic fusion rotein of N are used as Detection of antigen SARS virus antibody:

With the SARS virus S albumen of renaturation and the proteic fusion rotein of N with bag behind carbonate (pH9.6) doubling dilution of 50mmol/L by elisa plate, the indirect enzyme-linked immunosorbent method detects known anti-SARS virus positive serum and normal human serum, the proteic fusion rotein of SARS virus S albumen and N can react with the anti-SARS virus positive serum, not with the normal human serum reaction, illustrate that the SARS virus S albumen and the proteic fusion rotein of N of expressing have better antigenicity and specificity.

7. with horseradish peroxidase (HRP) mark SARS virus S albumen and the proteic fusion rotein of N, detect anti-SARS virus IgM or IgG antibody with pouncing on the method for obtaining or sandwich assay.The proteic fusion egg of SARS virus S albumen and N detects anti-SARS virus antibody as antigen with golden mark method.

8. with the SARS virus S albumen and the proteic fusion rotein of N of expressing, be used for vaccine, SARS virus antibody or detection of antigens and be used for immunity preparation anti-SARS virus monoclonal antibody and how anti-etc.

9. SARS virus S protein gene fragment is connected with the N gene fragment, expresses, prepare with the form of fusion rotein.

10. by gene recombination technology, utilize bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation SARS virus S albumen and the proteic fusion rotein of N.English abbreviation explanation: SARS (Severe Acute Respiratory Syndrome): SARS (Severe Acute Respiratory Syndrome); EDTA: Tetramethyl Ethylene Diamine; IPTG: isopropylthiogalactoside; DTT: dithiothreitol (DTT); SDS: dodecyl sodium sulfonate is received; PAGE: polyacrylamide gel electrophoresis; SKL: t-inosinic acid is received; PB: phosphate buffered saline buffer; DNA: thymus nucleic acid; RNA: Yeast Nucleic Acid; KD: kilodalton; Triton: Triton.

The advantage that the present invention compared with prior art has

The S protein fragments of the SARS virus that we express and the fusion rotein of N protein fragments have more advantage:

1. the enzyme-linked immunologic detecting kit of the SARS virus antibody of using now, the antigen of its use is totivirus antigen, produce dangerous, cost is high, with other coronavirus shortcomings such as cross reaction are arranged.The S protein fragments of the SARS virus of expressing and the fusion rotein of N protein fragments can overcome above-mentioned shortcoming as antigen.

2. the fusion rotein of the S protein fragments of the SARS virus of Biao Daing and N protein fragments exists with soluble form, is easy to purifying, and does not need renaturation to handle.

3. with the S protein fragments and the N protein fragments amalgamation and expression of SARS virus, two independent protein fragments are more convenient, economical than using respectively.When making the Detection of antigen anti-SARS virus antibody, no longer need to prepare respectively this two kinds of albumen, also do not need to adjust two antigenic usage ratios, so application is convenient and cost is lower with these two albumen.When detecting anti-SARS virus antibody, only need this a kind of fusion rotein of enzyme labelling, no longer need two albumen of mark respectively with prize law or sandwich assay.

4. there is not the SARS virus vaccine at present.Inactivated Vaccine has obtained bigger progress, but production cost height, dangerous big.The outermost S albumen of SARS virus is mediation virus and host cell bonded major protein, seals this proteic binding site and can stop virus infected cell, so the S albumen of SARS virus is the first-selected albumen that develops vaccine.N albumen can stimulate body to produce stronger cellular immunization.We utilize genetic engineering technique to express the S protein fragments of preparation SARS virus and the fusion rotein of N protein fragments, for the development recombinant vaccine lays the foundation.Recombinant vaccine has safety, advantage that cost is low.

Description of drawings

The invention will be further described below with reference to accompanying drawing:

Fig. 1 is that molecular biology software carries out analytical results to the proteic epitope of SARS virus N.The result shows, from 258 amino acid of the 1st amino acid to the, contains strong wetting ability epitope at the N of SARS virus albumen n end, i.e. the position that arrow indicates in the figure.

Fig. 2 is that molecular biology software carries out analytical results to the proteic epitope of SARS virus S.The result shows, from 460 amino acid of the 162nd amino acid to the, contains strong wetting ability epitope at the S of SARS virus albumen n end, i.e. the position that arrow indicates in the figure.

Fig. 3 is the construction of recombinant plasmid schema of expressing the fusion rotein of the S protein fragments of SARS virus and N protein fragments.

Fig. 4 is the pcr amplification product with 8 transformants of Agarose gel detection of 1.2%.M: the low-molecular-weight dna standard (TaKaRa, DL2000); 2,3,4,5,6: the 2,3,4,5, No. 65 transformants amplify the target gene fragment of 1701bp, i.e. the position that arrow indicates in the figure, 1,7,8: 1st, 7, No. 83 transformants do not amplify the target gene fragment of 1701bp.

Fig. 5 is the SDS-PAGE analytical results of expressing the engineering bacteria of the S protein fragments of SARS virus and N protein fragments fusion rotein.M: low molecular weight protein (LMWP) standard (Pharmacia); 2,4,5,6: 2nd, 4,5, No. 64 recons are all expressed relative molecular weight and are about 60000 purpose fusion rotein, i.e. the position that arrow indicates in the figure.1,3,7,8: 1st, 3,7, No. 84 transformants are not expressed target protein.

Fig. 6 is the SDS-PAGE analytical results of expressing before and after the fusion rotein purifying of the S protein fragments of SARS virus and N protein fragments.M: low molecular weight protein (LMWP) standard (Pharmacia); 1: contrast bacterium BL21 (DE3); 2: the engineering bacteria of the N protein fragments of expression SARS virus and the fusion rotein of S protein fragments; Behind the 3:S-Sepharose FF cation seperation column purifying the S protein fragments of SARS virus and the fusion rotein of N protein fragments.

Embodiment

The detailed description of embodiment of the present invention:

The analysis of the S albumen of SARS virus and N proteantigen epi-position, synthetic the reaching of gene are expressed

By Computer Analysis SARS virus N albumen and the proteic aminoacid sequence of S, filter out the N protein fragments of the SARS virus that contains the strong antigen epi-position, i.e. the 1st amino acid to 258 amino acid, S protein fragments with the SARS virus that contains the strong antigen epi-position, i.e. 460 amino acid of the 162nd amino acid to the, totally 299 amino acid.The codon of selecting eucaryon and prokaryotic organism all to have a preference for, the SARS virus N protein fragments that chemosynthesis is brand-new and the gene order of S protein fragments, utilize genetic engineering technique that two gene fragments are connected, be cloned into the NcoI/EcoRI site in the plasmid pET28a (+), express the fusion rotein of S protein fragments and N protein fragments.With recombinant plasmid transformed e. coli bl21 (DE3), screening has obtained the engineering bacteria of highly effectively expressing SARS virus S protein fragment and N protein fragments fusion rotein, and the fusion rotein of the SARS virus of expression accounts for about 30% of tropina total amount.

Materials and methods

1. bacterial classification and plasmid: host bacterium BL21 (DE3) and expression vector pET28a (+) are U.S. Novagen company product.

2. molecular biology reagent: restriction enzyme NcoI, BamHI, EcoRI, and the T4 dna ligase be TaKaRa company product.Plasmid purification test kit and the test kit that reclaims dna fragmentation in the sepharose are TaKaRa company product.DTT and IPTG are TaKaRa company product.Other reagent is import or homemade analytical reagent.

3. gene fragment is synthetic: helped synthetic by Dalian TaKaRa company.

The enzyme of gene clone method: DNA cut, connection, electrophoresis; The extraction of plasmid, conversion; General molecular cloning methods such as proteic SDS-PAGE analysis carry out according to a conventional method.Other test kit by specification is operated.

5.DNA sequential analysis: with TaKaRa company plasmid purification test kit plasmid purification, with the full-automatic sequenator order-checking of DNA.

The result

Utilize softwares such as ANTHEWIN, by Computer Analysis SARS virus S albumen and the proteic aminoacid sequence (GeneBank of N, closing number: AY278488), find that the proteic N of N holds 258 amino acid of the 1st amino acid to the to contain stronger antigenic determinant (Fig. 1), the proteic N of S holds 460 amino acid of the 162nd amino acid to the to contain stronger antigenic determinant (Fig. 2).The codon of selecting eucaryon and prokaryotic organism all to have a preference for, chemosynthesis comprises S albumen and the proteic gene order of N of above-mentioned antigenic determinant respectively.NcoI restriction enzyme site (following setting-out part) and 8 protection bases (GCGAGTCACC) have been increased at the segmental 5 ' end of synthetic N protein gene; and after initiator codon ATG, inserted a codon GGA, increased BamHI restriction enzyme site (following setting-out part) and 3 protection bases (GCG) at 3 ' end.Increased BamHI restriction enzyme site (following setting-out part) and two protection bases (GT) at the segmental 5 ' end of synthetic S protein gene, increased terminator codon (TAA) and EcoRI restriction enzyme site (following setting-out part) and two protection bases (AC) at 3 ' end.Making two gene fragments of synthetic be easy to series connection is cloned in plasmid pET28a (+) the interior NcoI and EcoRI restriction enzyme site.

SARSN ( 1aa-258aa ) ：Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile Thr Phe GlyGly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly Arg Asn Gly Ala Arg ProLys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala LeuThr Gln His Gly Lys Glu Glu Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile AsnThr Asn Ser Gly Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg ValArg Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr LeuGly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys Glu Gly Ile Val TrpVal Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn ProAsn Asn Asn Ala Ala Thr Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys GlyPhe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser ArgSer Arg Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro AlaArg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu Leu Asp Arg LeuAsn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr ValThr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

SARSS ( 162aa-460aa ) ：Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe LysHis Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys GlyTyr Gln Pro Ile Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys ProIle Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr AlaPhe Ser Pro Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly TyrLeu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp AlaVal Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe GluIle Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp ValVal Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala ThrLys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala AspTyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val SerAla Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp Ser Phe Val ValLys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp TyrAsn Tyr Lys Leu Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg AsnIle Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His GlyLys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe

The dna sequence dna (916bp) that contains SARS virus S proteantigen epitope gene of chemosynthesis: GT GGATCCGAA TAC ATC TCT GAC GCA TTC TCT CTG GAC GTT TCC GAA AAG TCT GGT AACTTC AAA CAC CTG CGC GAG TTC GTG TTT AAA AAC AAA GAC GGT TTC CTG TAC GTT TACAAG GGC TAC CAG CCG ATC GAC GTA GTT CGT GAC CTG CCG TCT GGT TTT AAC ACT CTGAAA CCG ATC TTC AAG CTG CCG CTG GGT ATT AAC ATC ACT AAC TTC CGC GCT ATC CTGACT GCT TTC TCT CCG GCT CAG GAC ACT TGG GGC ACT TCT GCT GCA GCC TAC TTC GTTGGC TAC CTG AAG CCA ACT ACC TTT ATG CTG AAG TAC GAC GAA AAC GGT ACT ATC ACTGAT GCT GTT GAC TGC TCT CAG AAC CCA CTG GCT GAA CTG AAA TGC TCT GTT AAG AGCTTT GAG ATC GAC AAA GGT ATT TAC CAG ACC TCT AAC TTC CGT GTT GTT CCG TCT GGTGAC GTT GTG CGT TTC CCT AAC ATC ACT AAC CTG TGC CCG TTT GGT GAA GTT TTC AACGCT ACT AAA TTC CCT TCT GTC TAC GCA TGG GAG CGT AAA AAA ATT TCT AAC TGC GTTGCT GAT TAC TCT GTG CTG TAC AAC TCT ACC TTT TTC TCT ACC TTC AAG TGC TAC GGCGTT TCT GCT ACT AAG CTG AAC GAC CTG TGC TTC TCC AAC GTT TAC GCA GAT TCT TTCGTA GTT AAG GGT GAT GAC GTA CGT CAG ATC GCT CCA GGT CAG ACT GGT GTT ATC GCTGAC TAC AAC TAT AAA CTG CCG GAC GAT TTC ATG GGT TGC GTT CTG GCT TGG AAC ACTCGT AAC ATT GAC GCT ACT TCT ACT GGT AAC TAC AAC TAC AAA TAT CGT TAC CTG CGTCAC GGC AAA CTG CGT CCG TTC GAA CGT GAC ATC TCT AAC GTG CCG TTC TAA GAATTCAC

Above-mentioned three kinds of enzymes of volumetric molar concentration such as get and cut the back dna fragmentation, in same centrifuge tube, connect with the T4 dna ligase, make SARS virus N protein gene fragment and S protein gene fragment after BamH I enzyme is connected, be inserted between the Nco I and EcoR I site in the carrier pET28a (+), express the fusion rotein of a SARS virus S protein fragments and N protein fragments.Make up flow process and see Fig. 3.

3. the screening of recombinant plasmid and evaluation:

The recombinant plasmid transformed that the last step was connected arrives e. coli bl21 (DE3), and the converted product coating is contained on the solid LB substratum of kantlex (60 μ g/ml), puts 37 ℃ of overnight incubation.8 transformant bacterium colonies of random choose next day (being labeled as respectively 1-8 number) are inoculated into respectively and contain 4ml liquid LB substratum (containing kantlex 60 μ g/ml) and in vitro, put 37 ℃ of shaking culture 6h, get bacterium liquid 1ml, centrifugal receipts bacterium.Use 50 μ l deionized water suspension thalline respectively, boiling water boils 5min, centrifugal (4 ℃, 12000rpm) 5min, get supernatant (in plasmid is arranged) 1 μ l as pcr template, right with the primer of primer of N protein gene fragment 5 ' end (5 '-GCGAGTCACCATGGGATCTGATAACGGTCCGCAGTCAAACCAACG-3 ') and S protein gene fragment 3 ' end (5 '-GTGAATTCTTAGAAAGGCACATTAGATATGT-3 ') composition primer, SARS virus N protein gene fragment that pcr amplification links together and S protein gene fragment, contain the positive recombinant plasmid of two gene fragments of connecting, should amplify the tandem gene fragment that is about 1701bp.The PCR reaction density is: plasmid template 1 μ l, upstream and downstream primer each 1 μ l, 10x Buffer 5.0 μ l, 2.5 mmol/L dNTP, 4.0 μ l, Taq archaeal dna polymerase 0.5 μ l (2.5U), deionized water 37.5 μ l, cumulative volume 50 μ l.Amplification condition is: 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 60 seconds, 30 circulations; Last 72 ℃ were extended 7 minutes.Get pcr amplification product 5 μ l, Agarose gel detection with 1.2%, the result is in 8 transformants, 2nd, 3,4,5, No. 65 transformants amplify the target gene fragment (see figure 4) of 1701bp, and the 1st, 7, No. 83 transformants do not amplify this gene fragment.Tentative confirmation has 5 transformants to contain SARS virus N protein gene fragment and the segmental tandem gene of S protein gene.

Extract the plasmid of No. 5 recons; Measure the interior SARS Nucleocapsid genetic fragment of plasmid and the tandem gene sequence of S GFP fragment; Dna sequence analysis confirms; SARSSN,：ATG GGA TCT GAT AAC GGT CCG CAG TCA AAC CA ACG TAG TGC CCC CCG CAT TAC ATTTGG TGG ACC CAC AGA TTC AAC TGA CAA TAA CCA GAA TGG AGG ACG CAA TGG GGC AAGGCC AAA ACA GCG CCG ACC CCA AGG TTT ACC CAA TAA TAC TGC GTC TTG GTT CAC AGCTCT CAC TCA GCA TGG CAA GGA GGA ACT TAG ATT CCC TCG AGG CCA GGG CGT TCC AATCAA CAC CAA TAG TGG TCC AGA TGA CCA AAT TGG CTA CTA CCG AAG AGC TAC CCG ACGAGT TCG TGG TGG TGA CGG CAA AAT GAA AGA GCT CAG CCC CAG ATG GTA CTT CTA TTACCT AGG AAC TGG CCC AGA AGC TTC ACT TCC CTA CGG CGC TAA CAA AGA AGG CAT CGTATG GGT TGC AAC TGA GGG AGC CTT GAA TAC ACC CAA AGA CCA CAT TGG CAC CCG CAATCC TAA TAA CAA TGC TGC CAC CGT GCT ACA ACT TCC TCA AGG AAC AAC ATT GCC AAAAGG CTT CTA CGC AGA GGG AAG CAG AGG CGG CAG TCA AGC CTC TTC TCG CTC CTC ATCACG TAG TCG CGG TAA TTC AAG AAA TTC AAC TCC TGG CAG CAG TAG GGG AAA TTC TCCTGC TCG AAT GGC TAG CGG AGG TGG TGA AAC TGC CCT CGC GCT ATT GCT GCT AGA CAGATT GAA CCA GCT TGA GAG CAA AGT TTC TGG TAA AGG CCA ACA A CAA CAA GGC CAA ACTGTC ACT AAG AAA TCT GCT GCT GAG GCA TCT AAA AAGGGA TCCGAA TAC ATC TCT GACGCA TTC TCT CTG GAC GTT TCC GAA AAG TCT GGT AAC TTC AAA CAC CTG CGC GAG TTCGTG TTT AAA AAC AAA GAC GGT TTC CTG TAC GTT TAC AAG GGC TAC CAG CCG ATC GACGTA GTT CGT GAC CTG CCG TCT GGT TTT AAC ACT CTG AAA CCG ATC TTC AAG CTG CCGCTG GGT ATT AAC ATC ACT AAC TTC CGC GCT ATC CTG ACT GCT TTC TCT CCG GCT CAGGAC ACT TGG GGC ACT TCT GCT GCA GCC TAC TTC GTT GGC TAC CTG AAG CCA ACT ACCTTT ATG CTG AAG TAC GAC GAA AAC GGT ACT ATC ACT GAT GCT GTT GAC TGC TCT CAGAAC CCA CTG GCT GAA CTG AAA TGC TCT GTT AAG AGC TTT GAG ATC GAC AAA GGT ATTTAC CAG ACC TCT AAC TTC CGT GTT GTT CCG TCT GGT GAC GTT GTG CGT TTC CCT AACATC ACT AAC CTG TGC CCG TTT GGT GAA GTT TTC AAC GCT ACT AAA TTC CCT TCT GTCTAC GCA TGG GAG CGT AAA AAA ATT TCT AAC TGC GTT GCT GAT TAC TCT GTG CTG TACAAC TCT ACC TTT TTC TCT ACC TTC AAG TGC TAC GGC GTT TCT GCT ACT AAG CTG AACGAC CTG TGC TTC TCC AAC GTT TAC GCA GAT TCT TTC GTA GTT AAG GGT GAT GAC GTACGT GAG ATC GCT CCA GGT CAG ACT GGT GTT ATC GCT GAC TAC AAC TAT AAA CTG CCGGAC GAT TTC ATG GGT TGC GTT CTG GCT TGG AAC ACT CGT AAC ATT GAC GCT ACT TCTACT GGT AAC TAC AAC TAC AAA TAT CGT TAC CTG CGT CAC GGC AAA CTG CGT CCG TTCGAA CGT GAC ATC TCT AAC GTG CCG TTC TAA

Above-mentioned 8 transformants are seeded to contain 3ml LB substratum (containing kanamycin 60 μ g/ml) in vitro, 37 ℃ of shaking culture 3h, add IPTG to final concentration 0.5mmol/L, continue shaking culture and induce 6h, centrifugal collection thalline carries out SDS-PAGE and detects, 2nd, 4,5, No. 64 transformants are expressed SARS virus S albumen and the proteic fusion rotein of N that relative molecular weight is about 60kD, and the 1st, 3,7, No. 84 transformants do not have this protein band (Fig. 5).

Express the purifying of SARS virus S albumen and the proteic fusion rotein of N

According to the aminoacid sequence of expressing SARS virus S albumen and the proteic fusion rotein of N, analyze its physicochemical property, determine suitable purification process.The isoelectric pH 9.7 of SARS virus S albumen and the proteic fusion rotein of N is expressed in machine analysis as calculated, so our decision is in pH is 7.4 phosphate buffered saline buffer, with S-SepharoseFF positively charged ion purifying.Concrete steps are as follows:

Material and method

1. main agents:

S-SepharoseFF positively charged ion gel is a Pharmacia company product, and IPTG, DTT are TaTaRa company product.Other reagent is homemade or the import analytical reagent.

2. express the abduction delivering and the ultrasonic degradation of SARS virus S albumen and the proteic fusion rotein engineering bacteria of N

Have from inoculation on the LB flat board of No. 5 engineering bacterias, choose single bacterium colony, be inoculated in the Erlenmeyer flask that contains 200ml LB liquid nutrient medium, add kantlex, put overnight incubation in 37 ℃ of shaking tables to final concentration 60 μ g/ml with toothpick.Be inoculated into 4 Erlenmeyer flasks that respectively contain 200ml LB liquid nutrient medium with bacterium liquid next day, and every bottle graft kind bacterium liquid 50ml put the interior shaking culture of 37 ℃ of shaking tables 1 hour, added IPTG then to final concentration 0.1mmol/L, abduction delivering 4 hours.

Centrifugal (8000rpm, 10min, 4 ℃) receives bacterium with the 1000ml engineering bacteria of abduction delivering fusion rotein, thalline is resuspended in the bacterial lysate (20mmol/L PB pH7.4,10mmol/L EDTA, 1mmol/L DTT, 5% glycerine) of l00ml, ice-bath ultrasonic is broken bacterium 10min, centrifugal (12000rpm, 30min, 4 ℃) collects supernatant.

3. express the sulfuric acid amine fractionation precipitation of SARS virus S albumen and the proteic fusion rotein of N

Accurately measure the supernatant volume, add an amount of saturated sulfuric acid amine aqueous solution in supernatant, the limit edged stirs, and making the final concentration of sulfuric acid amine is 30%, puts in the ice bath and spends the night.Next day, centrifugal (12000rpm, 20min, 4 ℃) collected supernatant, added an amount of solid sulfur acid amide powder, and the limit edged stirs, and making the final concentration of sulfuric acid amine is 50%, puts in the ice bath and spends the night.Next day centrifugal (12000rpm, 20min, 4 ℃) collecting precipitation.With 100ml balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2mmol/L DTT) suspension precipitation, in the dialysis tubing of packing into,, change liquid 1 time to 500ml balance liquid dialysed overnight.Centrifugal (12000rpm, 20min, 4 ℃) collects supernatant, directly goes up S-Sepearse FF cation seperation column purifying purifying

4.S-Sepearse FF cation seperation column purifying

With balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2mmol/L DTT) flushing balance S-Sepharose FF cation seperation column, will go up the good direct upper prop of supernatant of step dialysis then, last sample flow velocity 1.5ml/min.Fully wash post with balance liquid behind the end of the sample, again successively with contain 50,100,200, the balance liquid eluted protein of 1000mmol/L NaCl, collect each elution peak albumen, with each peak albumen of 12%SDS-PAGE electrophoresis detection, determine which elution peak contains the SARS virus S albumen and the proteic fusion rotein of N of expression.

The result

Different concns NaCl albumen of wash-out from the S-Sepharose FF post is carried out SDS-PAGE to be analyzed, the result shows (see figure 6), electrophoresis result shows, 100mmol/L NaCl eluted protein peak is SARS virus S albumen and the proteic fusion rotein of N, the S albumen and the proteic fusion rotein of N (about 60kD place) of a dense SARS virus of colour developing, no obvious SARS virus S albumen and the proteic fusion rotein band of N in other elution peak.

The evaluation and the application of purifying SARS virus S albumen and the proteic fusion rotein of N

The reorganization SARS virus S albumen and the proteic fusion rotein of N of purifying are used as antigen, by the indirect ELISA test method, detect anti-SARS virus antibody (IgM or IgG) positive and negative serum, with the SARS virus S albumen of evaluation expression and the antigenicity and the specificity of the proteic fusion rotein of N.Experimental result shows that this recombination fusion protein has good antigenicity and specificity, can be used as Detection of antigen anti-SARS virus antibody etc.

Material and method

1. anti-SARS virus antibody positive serum and normal human serum: the positive control in the anti-SARS virus antibody ELISA test kit that GBI company produces, and the anti-SARS virus antibody positive serum of 2 parts of deactivations.Normal human serum is preserved by this laboratory.

2. integrated enzyme reaction material: elisa plate is that 96 orifice plates are produced in Shenzhen, and the mouse-anti people μ chain monoclonal antibody of horseradish peroxidase (HRP) mark is available from Sigma company.Other material is the conventional material of integrated enzyme reaction.

3.ELISA test: adopt the anti-SARS virus antibody (IgG or IgM) in the indirect ELISA detection serum.Basic step is: with the S albumen and the proteic fusion rotein of N of 50mmol/L carbonate solution (pH9.6) dilution SARS virus, wraps by elisa plate, and every hole 100 μ l, 4 ℃ are spent the night.Inferior daily confining liquid (10mmol/L phosphate buffered saline buffer, pH7.4,10% lowlenthal serum, 20% calf serum, 0.05% sulphur sulphur mercury, 5% sucrose) sealing, every hole 130 μ l, 37 ℃ of 1h (or 4 ℃ spend the night).With anti-SARS virus antibody to be measured (IgG or IgM) the moon, positive serum, with sample diluent (10mmol/L phosphate buffered saline buffer, pH7.4,10% lowlenthal serum, 20% calf serum, 0.05% sulphur sulphur mercury, 0.5mmol/L NaCl) dilution back (IgG dilution in 1: 20, IgM dilution in 1: 100), add to respectively in the enzyme linked plate holes after the sealing, every hole 100 μ l, each sample adds 2 holes, 37 ℃ of reaction 30min, with PBST washing lotion (10mmol/L phosphate buffered saline buffer, pH7.4,0.5% tween 20) wash 5 times after, add the anti-human IgG or the anti-people μ chain monoclonal antibody of horseradish peroxidase-labeled of dilution in 1: 5000, every hole 100 μ l, 37 ℃ of reaction 30min, PBST washes 5 times, adds substrate tetramethyl biphenyl ammonia (TMB) solution 100 μ l, 37 ℃ of lucifuge colour developing 10min, add 50 μ l 4N sulfuric acid mixing termination reactions, measure the A450 value with enzyme connection instrument.

The result

1. indirect ELISA detects anti-SARS virus antibody (IgG or IgM) in the serum

The SARS virus S albumen of purifying and bovine serum albumin (the 5th part) co-electrophoresis, the colour developing back of proteic fusion rotein of N and known different concns are compared, determine that the S albumen and the proteic fusion rotein concentration of N of the SARS virus of purifying is about 0.5mg/ml.By elisa plate, detect known 2 parts of anti-SARS virus IgG antibody positive serum and 1 part of anti-SARS virus IgM antibody positive serum with 1: 500 dilution back of 50mmol/L carbonate solution (pH9.6) bag.The result shows that color reaction all appears in 2 parts of anti-SARS virus antibody positive serums, and the A450 value is respectively 1.335 and 1.098.Color reaction also appears in 1 part of anti-SARS virus antibody (IgM) positive serum, and the A450 value is 0.872.Detected 180 portions of normal human serums simultaneously, their A450 value illustrates that all less than 0.05 the S albumen of SARS virus and the proteic fusion rotein of N have antigenicity and specificity preferably.

SARS virus S protein fragments and N protein fragments fusion rotein sequence table＜110〉Li Yuexi＜120〉SARS virus S protein fragments and N protein fragments fusion rotein and preparation thereof, use＜160〉2＜210〉1＜211〉560＜212〉PRT＜213〉SARS virus S protein fragments and N protein fragments fusion rotein＜220〉＜fusion rotein of 223〉SARS virus S protein fragments and N protein fragments, it is the fusion rotein of proteic the 1st amino acid to 258 amino acid fragment of N of proteic 460 amino acid fragments of the 162nd amino acid to the of S of SARS virus and SARS virus, the N protein fragments is at the N of fusion rotein end, the S protein fragments is at the C of fusion rotein end, be connected with Serine with glycine between two protein fragments, behind first amino acid methionine of N protein fragments, insert a glycine, 560 amino acid of fusion rotein total length.<400>lMet?Gly?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg1 5 10 15Ile?Thr?Phe?Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly

20 25 30Gly?Arg?Asn?Gly?Ala?Arg?Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro

35 40 45Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln?His?Gly?Lys?Glu

50 55 60Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn?Thr?Asn?Ser65 70 75 80Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val

85 90 95Arg?Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe

100 105 110Tyr?Tyr?Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn

115 120 125Lys?Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?ASn?Thr?Pro

130 135 140Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val145 150 155 160Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala?Glu

165 170 175Gly?Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg?Ser

180 185 190Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser

195 200 205Pro?Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu

210 215 220Leu?Leu?Asp?Arg?Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly225 230 235 240Gln?Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala

245 250 255Ser?Lys?Lys?Gly?Ser?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val

260 265 270Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys

275 280 285Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp

290 295 300Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe305 310 315 320Lys?Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr

325 330 335Ala?Phe?Ser?Pro?Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr

340 345 350Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu

355 360 365Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala

370 375 380Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr385 390 395 400Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe

405 410 415Pro?Asn?Ile?Thr?Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr

420 425 430Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys

435 440 445Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe

450 455 460Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser465 470 475 480Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln

485 490 495Ile?Ala?Pro?Gly?Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu

500 505 510Pro?Asp?Asp?Phe?Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile

515 520 525Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg

530 535 540His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe545 550 555 560＜210〉2＜211〉1683＜212〉DNA＜213〉artificial sequence＜220〉＜221〉CDS＜222〉(1) ... (1683)＜223〉gene fragment of synthetic, the fusion rotein of encoding SARS virus S protein fragment and N protein fragments.＜220〉＜221〉mis-feature＜222〉(1) ... (777)＜223〉gene fragment of synthetic, proteic the 1st amino acid to 258 amino acid fragment of encoding SARS virus N inserts a codon glycine GGA behind the sub-ATG of first amino acid code of N albumen.＜220〉＜221〉mis-feature＜222〉(778) ... (783)＜223〉connect SARS virus S protein gene fragment and the segmental BamHI restriction enzyme site of N protein gene.＜220〉＜221〉mis-feature＜222〉(784) ... (1680)＜223〉gene fragment of synthetic, the 162nd amino acid to 460 amino acid fragment of encoding SARS virus S protein.＜220〉＜221〉mis-feature＜222〉(1681) ... (1683)＜223〉terminator codon that increases during synthetic gene.<400>2ATG?GGA?TCT?GAT?AAC?GGT?CCG?CAG?TCA?AAC?CAA?CGT?AGT?GCC?CCC?CGC?ATT?ACA?TTT?GGT 60Met?Gly?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg?Ile?Thr?Phe?Gly1 5 10 15 20GGA?CCC?ACA?GAT?TCA?ACT?GAC?AAT?AAC?CAG?AAT?GGA?GGA?CGC?AAT?GGG?GCA?AGG?CCA?AAA 120Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly?Arg?Asn?Gly?Ala?Arg?Pro?Lys

25 30 35 40CAG?CGC?CGA?CCC?CAA?GGT?TTA?CCC?AAT?AAT?ACT?GCG?TCT?TGG?TTC?ACA?GCT?CTC?ACT?CAG 180Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro?Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln

45 50 55 60CAT?GGC?AAG?GAG?GAA?CTT?AGA?TTC?CCT?CGA?GGC?CAG?GGC?GTT?CCA?ATC?AAC?ACC?AAT?AGT 240His?Gly?Lys?Glu?Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?GIy?Val?Pro?Ile?Asn?Thr?Asn?Ser

65 70 75 80GGT?CCA?GAT?GAC?CAA?ATT?GGC?TAC?TAC?CGA?AGA?GCT?ACC?CGA?CGA?GTT?CGT?GGT?GGT?GAC 300Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val?Arg?Gly?Gly?Asp

85 90 95 100GGC?AAA?ATG?AAA?GAG?CTC?AGC?CCC?AGA?TGG?TAC?TTC?TAT?TAC?CTA?GGA?ACT?GGC?CCA?GAA 360Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe?Tyr?Tyr?Leu?Gly?Thr?Gly?Pro?Glu

105 110 115 120GCT?TCA?CTT?CCC?TAC?GGC?GCT?AAC?AAA?GAA?GGC?ATC?GTA?TGG?GTT?GCA?ACT?GAG?GGA?GCC 420Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn?Lys?Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala

125 130 135 140TTG?AAT?ACA?CCC?AAA?GAC?CAC?ATT?GGC?ACC?CGC?AAT?CCT?AAT?AAC?AAT?GCT?GCC?ACC?GTG 480Leu?Asn?Thr?Pro?Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val

145 150 155 160CTA?CAA?CTT?CCT?CAA?GGA?ACA?ACA?TTG?CCA?AAA?GGC?TTC?TAC?GCA?GAG?GGA?AGC?AGA?GGC 540Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala?Glu?Gly?Ser?Arg?Gly

165 170 175 180GGC?AGT?CAA?GCC?TCT?TCT?CGC?TCC?TCA?TCA?CGT?AGT?CGC?GGT?AAT?TCA?AGA?AAT?TCA?ACT 600Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg?Ser?Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr

185 190 195 200CCT?GGC?AGC?AGT?AGG?GGA?AAT?TCT?CCT?GCT?CGA?ATG?GCT?AGC?GGA?GGT?GGT?GAA?ACT?GCC 660Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser?Pro?Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala

205 210 215 220CTC?GCG?CTA?TTG?CTG?CTA?GAC?AGA?TTG?AAC?CAG?CTT?GAG?AGC?AAA?GTT?TCT?GGT?AAA?GGC 720Leu?Ala?Leu?Leu?Leu?Leu?Asp?Arg?Leu?Ash?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly

225 230 235 240CAA?CAA?CAA?CAA?GGC?CAA?ACT?GTC?ACT?AAG?AAA?TCT?GCT?GCT?GAG?GCA?TCT?AAA?AAG?GGA 780Gln?Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala?Ser?Lys?Lys?Gly

245 250 255 260TCC?GAA?TAC?ATC?TCT?GAC?GCA?TTC?TCT?CTG?GAC?GTT?TCC?GAA?AAG?TCT?GGT?AAC?TTC?AAA 840Ser?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly?Ash?Phe?Lys

265 270 275 280CAC?CTG?CGC?GAG?TTC?GTG?TTT?AAA?AAC?AAA?GAC?GGT?TTC?CTG?TAC?GTT?TAC?AAG?GGC?TAC 900His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr

285 290 295 300CAG?CCG?ATC?GAC?GTA?GTT?CGT?GAC?CTG?CCG?TCT?GGT?TTT?AAC?ACT?CTG?AAA?CCG?ATC?TTC 960Gln?Pro?Ile?Asp?Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe

305 310 315 320AAG?CTG?CCG?CTG?GGT?ATT?AAC?ATC?ACT?AAC?TTC?CGC?GCT?ATC?CTG?ACT?GCT?TTC?TCT?CCG 1020Lys?Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro

325 330 335 340GCT?CAG?GAC?ACT?TGG?GGC?ACT?TCT?GCT?GCA?GCC?TAC?TTC?GTT?GGC?TAC?CTG?AAG?CCA?ACT 1080Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr

345 350 355 360ACC?TTT?ATG?CTG?AAG?TAC?GAC?GAA?AAC?GGT?ACT?ATC?ACT?GAT?GCT?GTT?GAC?TGC?TCT?CAG 1140Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln

365 370 375 380AAC?CCA?CTG?GCT?GAA?CTG?AAA?TGC?TCT?GTT?AAG?AGC?TTT?GAG?ATC?GAC?AAA?GGT?ATT?TAC 1200Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr

385 390 395 400CAG?ACC?TCT?AAC?TTC?CGT?GTT?GTT?CCG?TCT?GGT?GAC?GTT?GTG?CGT?TTC?CCT?AAC?ATC?ACT 1260Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr

405 410 415 420AAC?CTG?TGC?CCG?TTT?GGT?GAA?GTT?TTC?AAC?GCT?ACT?AAA?TTC?CCT?TCT?GTC?TAC?GCA?TGG 1320Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp

425 430 435 440GAG?CGT?AAA?AAA?ATT?TCT?AAC?TGC?GTT?GCT?GAT?TAC?TCT?GTG?CTG?TAC?AAC?TCT?ACC?TTT 1380Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe

445 450 455 460TTC?TCT?ACC?TTC?AAG?TGC?TAC?GGC?GTT?TCT?GCT?ACT?AAG?CTG?AAC?GAC?CTG?TGC?TTC?TCC 1440Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser

465 470 475 480AAC?GTT?TAC?GCA?GAT?TCT?TTC?GTA?GTT?AAG?GGT?GAT?GAC?GTA?CGT?CAG?ATC?GCT?CCA?GGT 1500Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly

485 490 495 500CAG?ACT?GGT?GTT?ATC?GCT?GAC?TAC?AAC?TAT?AAA?CTG?CCG?GAC?GAT?TTC?ATG?GGT?TGC?GTT 1560Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe?Met?Gly?Cys?Val

505 510 515 520CTG?GCT?TGG?AAC?ACT?CGT?AAC?ATT?GAC?GCT?ACT?TCT?ACT?GGT?AAC?TAC?AAC?TAC?AAA?TAT 1620Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr

525 530 535 540CGT?TAC?CTG?CGT?CAC?GGC?AAA?CTG?CGT?CCG?TTC?GAA?CGT?GAC?ATC?TCT?AAC?GTG?CCG?TTC 1680Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe

545 550 555 560TAA?1683

Claims

1. the fusion rotein of SARS virus S protein fragments and N protein fragments, it is the fusion rotein of proteic the 1st amino acid to 258 amino acid fragment of N of proteic 460 amino acid fragments of the 162nd amino acid to the of S of SARS virus and SARS virus, the N protein fragments is at the N of fusion rotein end, the S protein fragments is at the C of fusion rotein end, be connected with Serine with glycine between two protein fragments, behind first amino acid methionine of N protein fragments, insert a glycine, 560 amino acid of fusion rotein total length, aminoacid sequence is as follows:

Met?Gly?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg

1 5 10 15

Ile?Thr?Phe?Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly

20 25 30

Gly?Arg?Asn?Gly?Ala?Arg?Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro

35 40 45

Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln?His?Gly?Lys?Glu

50 55 60

Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn?Thr?Asn?Ser

65 70 75 80

Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val

85 90 95

Arg?Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe

100 105 110

Tyr?Tyr?Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn

115 120 125

Lys?Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro

130 135 140

Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val

145 150 155 160

Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala?Glu

165 170 175

Gly?Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg?Ser

180 185 190

Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser

195 200 205

Pro?Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu

210 215 220

Leu?Leu?Asp?Arg?Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly

225 230 235 240

Gln?Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala

245 250 255

Ser?Lys?Lys?Gly?Ser?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val

260 265 270

Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys

275 280 285

Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp

290 295 300

Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe

305 310 315 320

Lys?Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr

325 330 335

Ala?Phe?Ser?Pro?Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr

340 345 350

Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu

355 360 365

Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala

370 375 380

Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr

385 390 395 400

Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe

405 410 415

Pro?Asn?Ile?Thr?Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr

420 425 430

Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys

435 440 445

Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe

450 455 460

Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser

465 470 475 480

Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln

485 490 495

Ile?Ala?Pro?Gly?Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu

500 505 510

Pro?Asp?Asp?Phe?Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile

515 520 525

Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg

530 535 540

His?Gly?Lys?Leu?Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe

2. the fusion rotein of described SARS virus S protein fragments of claim 1 and N protein fragments utilizes genetic engineering technique to express, prepare this albumen, and concrete grammar is as follows:

The screening of SARS virus S albumen and N proteantigen epi-position:

By Computer Analysis SARS virus S albumen and the proteic aminoacid sequence of N, find that the proteic N of S holds 460 amino acid of the 162nd amino acid to the to contain stronger antigenic determinant, proteic the 1st amino acid to 258 amino acid of N contains stronger antigenic determinant, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, chemosynthesis comprises S albumen and the proteic gene order of N of above-mentioned antigenic determinant respectively.NcoI restriction enzyme site and 8 protection bases have been increased at the segmental 5 ' end of synthetic N protein gene, and after initiator codon ATG, inserted a codon glycine GGA, BamHI restriction enzyme site and 3 protection bases have been increased at 3 ' end, BamHI restriction enzyme site and two protection bases have been increased at the segmental 5 ' end of synthetic S protein gene, increased terminator codon TAA and EcoRI restriction enzyme site and two protection bases at 3 ' end, made two gene fragments of synthetic be easy to series connection and be cloned in plasmid pET28a (+) the interior NcoI and EcoRI restriction enzyme site;

SARSN ( 1aa-258aa ) ：Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile Thr PheGly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly Arg Asn Gly AlaArg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp PheThr Ala Leu Thr Gln His Gly Lys Glu Glu Leu Arg Phe Pro Arg Gly Gln GlyVal Pro Ile Asn Thr Asn Ser Gly Pro Asp Asp Gln Ile Gly Tyr Tyr Arg ArgAla Thr Arg Arg Val Arg Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro ArgTrp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly AlaAsn Lys Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro LysAsp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu Gln LeuPro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg Gly GlySer Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Gly Asn Ser Arg Asn SerThr Pro Gly Ser Ser Arg Gly Asn Ser Pro Ala Arg Met Ala Ser Gly Gly GlyGlu Thr Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser LysVal Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser AlaAla Glu Ala Ser Lys Lys

SARSS ( 162aa-460aa ) ：Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn PheLys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val TyrLys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn ThrLeu Lys Pro Ile Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg AlaIle Leu Thr Ala Phe Ser Pro Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala AlaTyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu AsnGly Thr Ile Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu LysCys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn PheArg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn Leu CysPro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr Ala Trp GluArg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser ThrPhe Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp LeuCys Phe Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val ArgGln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu ProAsp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala ThrSer Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu ArgPro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe

The dna sequence dna (796bp) that contains SARS virus N proteantigen epitope gene of chemosynthesis: GCG AGT CA C CAT GGG ATC TGA TAA CGG TCC GCA GTC AAA CCA ACG TAG TGC CCCCCG CAT TAC ATT TGG TGG ACC CAC AGA TTC AAC TGA CAA TAA CCA GAA TGG AGGACG CAA TGG GGC AAG GCC AAA ACA GCG CCG ACC CCA AGG TTT ACC CAA TAA TACTGC GTC TTG GTT CAC AGC TCT CAC TCA GCA TGG CAA GGA GGA ACT TAG ATT CCCTCG AGG CCA GGG CGT TCC AAT CAA CAC CAA TAG TGG TCC AGA TGA CCA AAT TGGCTA CTA CCG AAG AGC TAC CCG ACG AGT TCG TGG TGG TGA CGG CAA AAT GAA AGAGCT CAG CCC CAG ATG GTA CTT CTA TTA CCT AGG AAC TGG CCC AGA AGC TTC ACTTCC CTA CGG CGC TAA CAA AGA AGG CAT CGT ATG GGT TGC AAC TGA GGG AGC CTTGAA TAC ACC CAA AGA CCA CAT TGG CAC CCG CAA TCC TAA TAA CAA TGC TGC CACCGT GCT ACA ACT TCC TCA AGG AAC AAC ATT GCC AAA AGG CTT CTA CGC AGA GGGAAG CAG AGG CGG CAG TCA AGC CTC TTC TCG CTC CTC ATC ACG TAG TCG CGG TAATTC AAG AAA TTC AAC TCC TGG CAG CAG TAG GGG AAA TTC TCC TGC TCG AAT GGCTAG CGG AGG TGG TGA AAC TGC CCT CGC GCT ATT GCT GCT AGA CAG ATT GAA CCAGCT TGA GAG CAA AGT TTC TGG TAA AGG CCA ACA ACA ACA AGG CCA AAC TGT CACTAA GAA ATC TGC TGC TGA GGC ATC TAA AAA G GG ATC CGC G

The dna sequence dna (916bp) that contains SARS virus S proteantigen epitope gene of chemosynthesis: GT GGATCCGAA TAC ATC TCT GAC GCA TTC TCT CTG GAC GTT TCC GAA AAG TCT GGTAAC TTC AAA CAC CTG CGC GAG TTC GTG TTT AAA AAC AAA GAC GGT TTC CTG TACGTT TAC AAG GGC TAC CAG CCG ATC GAC GTA GTT CGT GAC CTG CCG TCT GGT TTTAAC ACT CTG AAA CCG ATC TTC AAG CTG CCG CTG GGT ATT AAC ATC ACT AAC TTCCGC GCT ATC CTG ACT GCT TTC TCT CCG GCT CAG GAC ACT TGG GGC ACT TCT GCTGCA GCC TAC TTC GTT GGC TAC CTG AAG CCA ACT ACC TTT ATG CTG AAG TAC GACGAA AAC GGT ACT ATC ACT GAT GCT GTT GAC TGC TCT CAG AAC CCA CTG GCT GAACTG AAA TGC TCT GTT AAG AGC TTT GAG ATC GAC AAA GGT ATT TAC CAG ACC TCTAAC TTC CGT GTT GTT CCG TCT GGT GAC GTT GTG CGT TTC CCT AAC ATC ACT AACCTG TGC CCG TTT GGT GAA GTT TTC AAC GCT ACT AAA TTC CCT TCT GTC TAC GCATGG GAG CGT AAA AAA ATT TCT AAC TGC GTT GCT GAT TAC TCT GTG CTG TAC AACTCT ACC TTT TTC TCT ACC TTC AAG TGC TAC GGC GTT TCT GCT ACT AAG CTG AACGAC CTG TGC TTC TCC AAC GTT TAC GCA GAT TCT TTC GTA GTT AAG GGT GAT GACGTA CGT CAG ATC GCT CCA GGT CAG ACT GGT GTT ATC GCT GAC TAC AAC TAT AAACTG CCG GAC GAT TTC ATG GGT TGC GTT CTG GCT TGG AAC ACT CGT AAC ATT GACGCT ACT TCT ACT GGT AAC TAC AAC TAC AAA TAT CGT TAC CTG CGT CAC GGC AAACTG CGT CCG TTC GAA CGT GAC ATC TCT AAC GTG CCG TTC TAA GAATTCAC expresses the fusion rotein construction of recombinant plasmid of SARS virus S protein fragments and N protein fragments:

Extract plasmid pET28a (+), with Nco I and EcoR I double digestion, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis, be dissolved in the deionized water, SARS virus N protein gene fragment with Nco I and the chemosynthesis of BamH I double digestion, with the SARS virus S protein gene fragment of BamHI and the chemosynthesis of EcoR I double digestion, after electrophoresis reclaims respectively, be dissolved in the deionized water;

Above-mentioned three kinds of enzymes of volumetric molar concentration such as get and cut the back dna fragmentation, in same centrifuge tube, connect with the T4 dna ligase, make SARS virus N protein gene fragment and S protein gene fragment after BamH I enzyme is connected, be inserted between the Nco I and EcoR I site in the carrier pET28a (+), express the fusion rotein of a SARS virus S protein fragments and N protein fragments;

The screening of recombinant plasmid and evaluation:

With recombinant plasmid transformed e. coli bl21 (DE3); Coating contains the LB flat board of 60 μ g/ml kanamycins; 37 ℃ of of of of of spends the of Putting night; Next day, picking transformed bacterium colony and the contrast bacterium that contains pET28a (+) plasmid at random; Extract respectively plasmid; Take the plasmid that extracts as template; With the primer of the primer of N GFP fragment 5 ' end (5 '-GCGAGTCACCATGGGATCTGATAACGGTCCGCAGTCAAACCAACG-3 ') and S GFP fragment 3 ' end (5 '-GTGAATTCTTAGAAAGGCACATTAGATATGT-3 ') composition primer pair; The SARS Nucleocapsid genetic fragment that pcr amplification links together and S GFP fragment; The positive restructuring plasmid that contains two genetic fragments of connecting; Should amplify the tandem gene fragment that is about 1701bp; The plasmid that will contain tandem gene carries out dna sequence analysis; SARSNS,：ATG GGA TCT GAT AAC GGT CCG CAG TCA AAC CAA CGT AGT GCC CCC CGC ATT ACATTT GGT GGA CCC ACA GAT TCA ACT GAC AAT AAC CAG AAT GGA GGA CGC AAT GGGGCA AGG CCA AAA CAG CGC CGA CCC CAA GGT TTA CCC AAT AAT ACT GCG TCT TGGTTC ACA GCT CTC ACT CAG CAT GGC AAG GAG GAA CTT AGA TTC CCT CGA GGC CAGGGC GTT CCA ATC AAC ACC AAT AGT GGT CCA GAT GAC CAA ATT GGC TAC TAC CGAAGA GCT ACC CGA CGA GTT CGT GGT GGT GAC GGC AAA ATG AAA GAG CTC AGC CCCAGA TGG TAC TTC TAT TAC CTA GGA ACT GGC CCA GAA GCT TCA CTT CCC TAC GGCGCT AAC AAA GAA GGC ATC GTA TGG GTT GCA ACT GAG GGA GCC TTG AAT ACA CCCAAA GAC CAC ATT GGC ACC CGC AAT CCT AAT AAC AAT GCT GCC ACC GTG CTA CAACTT CCT CAA GGA ACA ACA TTG CCA AAA GGC TTC TAC GCA GAG GGA AGC AGA GGCGGC AGT CAA GCC TCT TCT CGC TCC TCA TCA CGT AGT CGC GGT AAT TCA AGA AATTCA ACT CCT GGC AGC AGT AGG GGA AAT TCT CCT GCT CGA ATG GCT AGC GGA GGTGGT GAA ACT GCC CTC GCG CTA TTG CTG CTA GAC AGA TTG AAC CAG CTT GAG AGCAAA GTT TCT GGT AAA GGC CAA CAA CAA CAA GGC CAA ACT GTC ACT AAG AAA TCTGCT GCT GAG GCA TCT AAA AAG。GGA TCCGAA TAC ATC TCT GAC GCA TTC TCT CTGGAC GTT TCC GAA AAG TCT GGT AAC TTC AAA CAC CTG CGC GAG TTC GTG TTT AAAAAC AAA GAC GGT TTC CTG TAC GTT TAC AAG GGC TAC CAG CCG ATC GAC GTA GTTCGT GAC CTG CCG TCT GGT TTT AAC ACT CTG AAA CCG ATC TTC AAG CTG CCG CTGGGT ATT AAC ATC ACT AAC TTC CGC GCT ATC CTG ACT GCT TTC TCT CCG GCT CAGGAC ACT TGG GGC ACT TCT GCT GCA GCC TAC TTC GTT GGC TAC CTG AAG CCA ACTACC TTT ATG CTG AAG TAC GAC GAA AAC GGT ACT ATC ACT GAT GCT GTT GAC TGCTCT CAG AAC CCA CTG GCT GAA CTG AAA TGC TCT GTT AAG AGC TTT GAG ATC GACAAA GGT ATT TAC CAG ACC TCT AAC TTC CGT GTT GTT CCG TCT GGT GAC GTT GTGCGT TTC CCT AAC ATC ACT AAC CTG TGC CCG TTT GGT GAA GTT TTC AAC GCT ACTAAA TTC CCT TCT GTC TAC GCA TGG GAG CGT AAA AAA ATT TCT AAC TGC GTT GCTGAT TAC TCT GTG CTG TAC AAC TCT ACC TTT TTC TCT ACC TTC AAG TGC TAC GGCGTT TCT GCT ACT AAG CTG AAC GAC CTG TGC TTC TCC AAC GTT TAC GCA GAT TCTTTC GTA GTT AAG GGT GAT GAC GTA CGT CAG ATC GCT CCA GGT CAG ACT GGT GTTATC GCT GAC TAC AAC TAT AAA CTG CCG GAC GAT TTC ATG GGT TGC GTT CTG GCTTGG AAC ACT CGT AAC ATT GAC GCT ACT TCT ACT GGT AAC TAC AAC TAC AAA TATCGT TAC CTG CGT CAC GGC AAA CTG CGT CCG TTC GAA CGT GAC ATC TCT AAC GTGCCG TTC TAA

The virus S protein fragment of the expression of recombinant plasmid SARS that makes up and the fusion of N protein fragments; 560 amino acid of total length; Be the N protein fragments at its N end; Long 259 amino acid; The C end is the S protein fragments; Long 299 amino acid; ,：Met Gly Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile ThrPhe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly Arg Asn GlyAla Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser TrpPhe Thr Ala Leu Thr Gln His Gly Lys Glu Glu Leu Arg Phe Pro Arg Gly GlnGly Val Pro Ile Asn Thr Asn Ser Gly Pro Asp Asp Gin Ile Gly Tyr Tyr ArgArg Ala Thr Arg Arg Val Arg Gly Gly Asp Gly Lys Met Lys Glu Leu Ser ProArg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr GlyAla Asn Lys Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr ProLys Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu GlnLeu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg GlyGly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Gly Asn Ser Arg AsnSer Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro Ala Arg Met Ala Ser Gly GlyGlv Glu Thr Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu SerLys Val Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys SerAla Ala Glu Ala Ser Lys Lys Gly Ser Glu Tyr Ile Ser Asp Ala Phe Ser LeuAsp Val Ser Glu Lys Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe LysAsn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val ValArg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro LeuGly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro Ala GlnAsp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr Leu Lys Pro ThrThr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp Ala Val Asp CysSer Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile AspLys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val ValArg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala ThrLys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val AlaAsp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr GlyVal Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp SerPhe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln Thr Gly ValIle Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met Gly Cys Val Leu AlaTrp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys TyrArg Tyr Leu Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn ValPro Phe

The Screening and Identification of expressed fusion protein engineering bacteria:

The positive transformant that will contain recombinant plasmid, be seeded in the LB liquid nutrient medium that contains kanamycin 60 μ g/ml, 37 ℃ of shaking culture 3h, add IPTG to final concentration 0.5mmol/L, continue shaking culture and induce 6h, centrifugal collection thalline carries out SDS-PAGE and detects, and recon is expressed SARS virus S albumen and the proteic fusion rotein of N that relative molecular weight is about 60kD, and contrast bacterium BL21 (DE3) does not have this protein band; Express the purifying of SARS virus S albumen and the proteic fusion rotein of N:

Centrifugal (8000rpm, 10min, 4 ℃) receives bacterium with the engineering bacteria of abduction delivering fusion rotein, thalline is resuspended in the bacterial lysate (20mmol/L PB pH7.4,10mmol/L EDTA, 1mmol/L DTT, 5% glycerine) of original fluid 1/10 volume, ice-bath ultrasonic is broken bacterium, centrifugal collection supernatant;

Accurately measure the supernatant volume, in supernatant, add an amount of saturated sulfuric acid amine aqueous solution, the limit edged stirs, making the final concentration of sulfuric acid amine is 30%, puts in the ice bath and spends the night, centrifugal collection supernatant, measurement volumes, add an amount of solid sulfur acid amide powder, the limit edged stirs, and the final concentration that makes sulfuric acid amine is 50%, put in the ice bath and spend the night, next day, centrifugal collecting precipitation was used balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2mmol/L DTT) suspension precipitation is in the dialysis tubing of packing into, to the balance liquid dialysed overnight, change liquid 1 time, centrifugal collection supernatant is directly gone up S-Sepearse FF cation seperation column purifying purifying;

3) S-Sepearse FF cation seperation column purifying

With balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2 flushing balance S-Sepharose FF cation seperation column mmol/L DTT), to go up the good direct upper prop of supernatant of step dialysis then, fully wash post with balance liquid liquid behind the end of the sample, again successively with the balance liquid eluted protein that contains gradient concentration NaCl, collect each elution peak albumen, with each peak albumen of SDS-PAGE electrophoresis detection, determine which elution peak contains the SARS virus S albumen and the proteic fusion rotein of N of expression, 100mmol/L NaCl eluted protein peak is SARS virus S albumen and the proteic fusion rotein of N; The SARS virus S albumen of purifying and the proteic fusion rotein of N are used as Detection of antigen SARS virus antibody:

SARS virus S albumen and the proteic fusion rotein of N with renaturation, with wrapping behind the carbonate buffer solution doubling dilution of 50mmol/L by elisa plate, the indirect enzyme-linked immunosorbent method detects known anti-SARS virus positive serum and normal human serum, the proteic fusion rotein of SARS virus S albumen and N can react with the anti-SARS virus positive serum, not with the normal human serum reaction, illustrate that the SARS virus S albumen and the proteic fusion rotein of N of expressing have better antigenicity and specificity.

3. described SARS virus S albumen of claim 1 and the proteic fusion rotein of N are used for vaccine, SARS virus antibody or detection of antigens and are used for immunity preparing anti-SARS virus monoclonal antibody and how anti-.

4. the fusion rotein of described SARS virus S protein fragments of claim 1 and N protein fragments by gene recombination technology, utilizes bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.

5. the fusion rotein of described SARS virus S protein fragments of claim 1 and N protein fragments, SARS virus S protein fragments is connected with any of N protein fragments in the fusion rotein, the N protein fragments is expressed, is prepared with the form of fusion rotein at the N of fusion rotein end or C end.