CN1472321A - Chemosynthesized SARS virus S gene segement, its expression and application - Google Patents
Chemosynthesized SARS virus S gene segement, its expression and application Download PDFInfo
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Abstract
A chemically synthetic SARS virus S gene fragment and its expression and application are disclosed. Its preparing process includes such steps as computer analysis to screen strong antigen epitope in SARS virus S protein, choosing the codon preferably to both eukaryotic and prokaryotic organisms, chemically synthesizing the brand-new gene sequence of antigen epitope, expressing the gene fragment and preparing said strong antigen epitope fragment. It can be used for vaccine, detecting the antibody or antigen of SARS virus, and preparing monoclone and polyclone of SARS virus.
Description
Technical field
The present invention is the proteic brand-new gene fragment of the SARS virus S of chemosynthesis, utilizes genetic engineering technique, preparation reorganization SARS virus S albumen.By Computer Analysis, filter out the S 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 gene order that chemosynthesis is brand-new, utilize genetic engineering technique to express, expressed proteins 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
The first routine SARS (Severe Acute RespiratorySyndrome) patient has appearred on November 16th, 2003 in China Guangzhou, SARS is a kind of potent virus sexually transmitted disease, involves more than 30 countries in the world in the some months, especially in China domestic popular attaching most importance to.Up to about 15%, the elderly's mortality ratio that infects this disease can reach 40% to 50% according to the mortality ratio of WHO (World Health Organization) statistics SARS.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.
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.By Computer Analysis, we filter out the S 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 gene order that chemosynthesis is brand-new, utilize genetic engineering technique to express, expressed proteins can be used for vaccine and SARS virus antibody or detection of antigens etc.
Summary of the invention
The present invention is the proteic brand-new gene fragment of the SARS virus S of chemosynthesis, utilizes genetic engineering technique, preparation reorganization SARS virus S protein fragments.By Computer Analysis, filter out the S protein fragments of the SARS virus that contains the strong antigen epi-position, the 460th amino acid of the 162nd amino acid 1, totally 299 amino acid, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, the gene order that chemosynthesis is brand-new utilizes genetic engineering technique to express this gene.Expressed proteins can be used for vaccine, SARS virus antibody or detection of antigens and is used for the anti-human cytomegalic inclusion disease virus monoclonal antibody of immunity preparation and how anti-etc.The SARS virus S gene fragment of chemosynthesis and expression thereof, application take following steps to implement: the screening of 1.SARS virus S protein epitope and the chemosynthesis of gene fragment thereof:
Utilize softwares such as ANTHEWIN,, find that the proteic N end of S (the 162nd amino acid-the 460th amino acid) contains stronger antigenic determinant by the proteic aminoacid sequence of Computer Analysis SARS virus S.The codon of selecting eucaryon and prokaryotic organism all to have a preference for; the gene order that chemosynthesis is brand-new; and BamHI restriction enzyme site (following setting-out part) and two protection bases (GT) have been increased at 5 ' end; increased terminator codon (TAA) and EcoRI restriction enzyme site (following setting-out part) and two protection bases (AC) at 3 ' end, made this gene fragment be easy to be cloned in plasmid pET28a (+) the interior BamHI and EcoRI restriction enzyme site.
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 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
GAATTCAC2. express SARS virus S protein fragments construction of recombinant plasmid:
Extract plasmid pET28a (+),, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis, be dissolved in the deionized water with BamHI and EcoRI double digestion.Same SARS virus S protein gene fragment with BamHI and the chemosynthesis of EcoRI double digestion, electrophoresis is dissolved in the deionized water after reclaiming.
Above-mentioned two 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, SARS virus S protein gene fragment is inserted between carrier pET28a (+) the interior BamHI and EcoRI site, consistent with the initiator codon translation framework on the carrier, express a fusion rotein.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, extracting plasmid respectively, is template with the plasmid that extracts, pcr amplification SARS virus S protein gene fragment, contain the segmental positive recombinant plasmid of SARS virus S protein gene, the plasmid that should amplify the gene fragment that is about 916bp. will contain foreign gene carries out dna sequence analysis; SARSS,：GAA TAC ATC TCT GAC GCA TTC TCT CTG GAC GTT TCC GAA AAG TCT GGT AAC TTC AAACAC CTG CGC GAG TTC GTG TTT AAA AAC AAA GAC GGT TTC CTG TAC GTT TAC AAG GGCTAC CAG CCG ATC GAC GTA GTT CGT GAC CTG CCG TCT GGT TTT AAC ACT CTG AAA CCGATC TTC AAG CTG CCG CTG GGT ATT AAC ATC ACT AAC TTC CGC GCT ATC CTG ACT GCTTTC TCT CCG GCT CAG GAC ACT TGG GGC ACT TCT GCT GCA GCC TAC TTC GTT GGC TACCTG AAG CCA ACT ACC TTT ATG CTG AAG TAC GAC GAA AAC GGT ACT ATC ACT GAT GCTGTT GAC TGC TCT CAG AAC CCA CTG GCT GAA CTG AAA TGC TCT GTT AAG AGC TTT GAGATC GAC AAA GGT ATT TAC CAG ACC TCT AAC TTC CGT GTT GTT CCG TCT GGT GAC GTTGTG CGT 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 GCT GATTAC TCT GTG CTG TAC AAC TCT ACC TTT TTC TCT ACC TTC AAG TGC TAC GGC GTT TCTGCT ACT AAG CTG AAC GAC CTG TGC TTC TCC AAC GTT TAC GCA GAT TCT TTC GTA GTTAAG GGT GAT GAC GTA CGT CAG ATC GCT CCA GGT CAG ACT GGT GTT ATC GCT GAC TACAAC TAT AAA CTG CCG GAC GAT TTC ATG GGT TGC GTT CTG GCT TGG AAC ACT CGT AACATT GAC GCT ACT TCT ACT GGT AAC TAC AAC TAC AAA TAT CGT TAC CTG CGT CAC GGCAAA CTG CGT CCG TTC GAA CGT GAC ATC TCT AAC GTG CCG TTC TAA
The virus S protein fragment (299 amino acid) of the expression of recombinant plasmid SARS that makes up; 34 amino acid on the carrier have been merged at its N end; 333,：Met Gly Ser Ser His His His His His His Ser Ser Gly Lys Val Pro Arg Gly SerHis Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Glu Tyr Ile SerAsp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe Lys His Leu Arg GluPhe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro IleAsp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys LeuPro Leu Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro AlaGln Asp 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 Cys SerGln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp Lys GlyIle Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe ProAsn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro SerVal Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser Val LeuTyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys LeuAsn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp AspVal Arg Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys LeuPro Asp 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 Arg ProPhe Glu Arg Asp Ile Ser Asn Val Pro Phe4.：
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 the SARS virus S albumen that relative molecular weight is about 37kD, expression amount is about 30%, and contrast bacterium BL21 (DE3) does not have this protein band.5. express the proteic purifying of SARS virus S:
1) ultrasonic degradation of expression SARS virus S protein engineering bacterium
Centrifugal (8000rpm, 10min, 4 ℃) receives bacterium with the engineering bacteria of abduction delivering fusion rotein, thalline is resuspended in the lysate (50mmol/L Tris-HCl pH8.0,10mmol/L EDTA, 10mmol/LDTT) of original fluid 1/10 volume, ice-bath ultrasonic is broken bacterium 10min, centrifugal (12000rpm, 20min, 4 ℃) collects supernatant, abandons precipitation.The supernatant of collecting is used for next step ion-exchange purification.
2) DEAE-Sepharose FF anion column purifying
DEAE-Sepharose FF anion column is connected to the normal pressure chromatographic system, earlier with balance liquid (50mmol/LTris-HCl pH8.0,1mmol/L EDTA, 0.1mmol/L DTT) flushing balance, to go up the cracking supernatant of step acquisition then and directly go up sample, last sample flow velocity is 1.5ml/min, collects and passes the peak.
3) S-Sepearse FF cation seperation column purifying
To go up step DEAE-Sepharose FF negatively charged ion and pass the peak, in the dialysis tubing of packing into, dialyzate (20mmol/LPB (pH7.4), 1mmol/L EDTA, 0.1mmol/L DTT) be stirred dialysis 4h.With above-mentioned dialyzate flushing balance S-Sepharose FF cation seperation column, sample is directly gone up at the peak that passes after will dialysing then, last sample flow velocity 1.5ml/min.Fully wash post with dialyzate behind the end of the sample, more successively with contain 50,100, the dialyzate eluted protein of 1000mmol/L NaCl, collect 100mmol/L NaCl elution peak albumen, be the SARS virus S albumen of purifying.6. the SARS virus S albumen of purifying is used as Detection of antigen HCMV antibody:
Wrap by elisa plate behind SARS virus S albumen usefulness carbonate (pH9.6) doubling dilution of 50mmol/L with purifying, the indirect enzyme-linked immunosorbent method detects known anti-SARS virus positive serum and normal human serum, SARS virus S albumen can react with the anti-SARS virus positive serum,, do not illustrate that the SARS virus S albumen of virus has better antigenicity and specificity with the normal human serum reaction.
Same horseradish peroxidase (HRP) the mark SARS virus S albumen of using detects anti-SARS virus IgM or IgG antibody with pouncing on the method that obtains, and higher susceptibility and specificity are arranged.SARS virus S albumen detects anti-SARS virus antibody as antigen with golden mark method, and higher susceptibility and specificity are also arranged.7. with the SARS virus S protein fragments 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.8. synthetic SARS virus S protein gene fragment is connected with other gene fragment, expresses, prepare with the form of fusion rotein.The advantage that the present invention compared with prior art has
The S protein fragments of the SARS virus that we express has more advantage:
1. the enzyme-linked immunologic detecting kit of the SARS virus antibody of using at present, 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 can overcome above-mentioned shortcoming as antigen.
2. lack the SARS virus vaccine at present.Inactivated Vaccine has obtained the exhibition of throwing into, but production cost height, dangerous big.The outermost S albumen of SARS virus, be mediation virus and host cell bonded major protein, seal 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, we have selected its strong antigen epi-position, utilize genetic engineering technique to express preparation, for the development recombinant vaccine lays the foundation.Recombinant vaccine safety, cost are low.
3. according to the S protein fragments aminoacid sequence of the SARS virus that filters out, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, the gene order that chemosynthesis is brand-new, this gene high expression level in eucaryon and prokaryotic cell prokaryocyte that suits.
4. the proteic engineering bacteria of S of the expression SARS virus that makes up of this paper, expression amount can reach 30% of tropina, is easy to purifying, but large-scale purification prepares this albumen.Do not see Table at present and reach the proteic report of this section.
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 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. 2 is the proteic construction of recombinant plasmid schema of S of expressing SARS virus.
Fig. 3 is the pcr amplification product with 5 recons of Agarose gel detection of 1.2%.M: the low-molecular-weight dna standard (TaKaRa, DL2000); C: the contrast bacterium that contains plasmid pET28a (+); 1～5: 5 transformants all amplify the target gene fragment of 916bp, i.e. the position that arrow indicates in the figure.
Fig. 4 is the SDS-PAGE analytical results of expressing the S albumen reorganization bacterium of SARS virus.M: low molecular weight protein (LMWP) standard (Pharmacia); C: contrast bacterium E.coli BL21 (DE3); 1～5: 1～No. 5 reorganization bacterium, 5 recons are all expressed relative molecular weight and are about 37000 fusion rotein, i.e. the position that arrow indicates in the figure.
Fig. 5 is the SDS-PAGE analytical results of expressing the S protein purification front and back of SARS virus.M: low molecular weight protein (LMWP) standard (Pharmacia); 1: contrast bacterium BL21 (DE3); 2: the proteic engineering bacteria of S of expressing PSARS virus; Behind the 3:S-Sepharose FF cation seperation column purifying the S albumen of SARS virus.
Embodiment
The detailed description of embodiment of the present invention:
The analysis of the S proteantigen epi-position of SARS virus, synthetic the reaching of gene are expressed
By the proteic whole aminoacid sequences of the S of Computer Analysis SARS virus, filter out the interior strong antigen epi-position of S albumen of SARS virus, select the codon of bacterium preference for use, the brand-new gene fragment of the S albumen strong antigen epi-position of chemosynthesis SARS virus.With the BamHI/EcoRI site of gene fragment clone to the plasmid pET28a (+), consistent with the translation framework of initiator codon on the carrier, can express a fusion rotein.With recombinant plasmid transformed e. coli bl21 (DE3), screening has obtained the proteic engineering bacteria of S of highly effectively expressing SARS virus, and the S albumen 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 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 German QIAGEN company product.DTT and IPTG are Promega 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 QIAGEN company plasmid purification test kit plasmid purification, with the full-automatic sequenator order-checking of DNA.Synthesizing of the S proteantigen epi-position of 1.SARS virus screening as a result and gene fragment:
Utilize the softwares such as ANTHEWIN; Whole amino acid sequence (GeneBank of the S albumen by the Computer Analysis SARS virus; Closing number:AY278488); Filter out the interior strong antigen epi-position (Fig. 1) of S albumen of SARS virus; 162460,：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
According to the epitope aminoacid sequence in the SARS virus S albumen of screening; the codon of selecting eucaryon and prokaryotic organism all to have a preference for; the gene order that chemosynthesis is brand-new; and BamHI restriction enzyme site (following setting-out part) and two protection bases (GT) have been increased at 5 ' end; increased terminator codon (TAA) and EcoRI restriction enzyme site (following setting-out part) and two protection bases (AC) at 3 ' end, made this gene fragment be easy to be cloned in plasmid pET28a (+) the interior BamHI and EcoRI restriction enzyme site.The dna sequence dna (916bp) that contains SARS virus S proteantigen epitope gene of chemosynthesis is as follows: 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 TAG 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
GAATTCAC2. express SARS virus S protein fragments construction of recombinant plasmid:
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 BamHI and EcoRI double digestion.Same SARS virus S protein gene fragment with BamHI and the chemosynthesis of EcoRI double digestion, electrophoresis is dissolved in the deionized water after reclaiming.
Above-mentioned two 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, SARS virus S protein gene fragment is inserted between carrier pET28a (+) the interior BamHI and EcoRI site, consistent with the initiator codon translation framework on the carrier, express a fusion rotein (make up flow process and see Fig. 2).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.5 transformant bacterium colonies of random choose next day (being labeled as respectively 1-5 number) and 1 contrast bacterium (plasmid pET28a transformed bacteria), be inoculated into respectively and contain 4ml liquid LB substratum (containing kantlex 60 μ g/ml) 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) 2 μ l as pcr template, pcr amplification inserts and carries intravital SARS virus S protein gene fragment, the PCR reaction density is: plasmid template 2 μ l, each 1 μ l of segmental normal chain P1 of SARS virus S protein gene (GAATACATCTCTGACGCATTC) and minus strand primer P2 (TTAGAACGGCACGTTAGAGAT), 10xBuffer 5.0 μ l, 2.5mmol/L dNTP 4.0 μ l, Taq archaeal dna polymerase 0.5 μ l (2.5U), deionized water 36.5 μ l, cumulative volume 50 μ l.Amplification condition is: 94 ℃ 30 seconds, 65 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations; Last 72 ℃ were extended 7 minutes.Get pcr amplification product 5 μ l, the Agarose gel detection with 1.2%, the result, 5 transformants all amplify the target gene fragment (see figure 3) of 900bp, and the contrast bacterium that contains plasmid pET28a (+) does not amplify this gene fragment.Tentative confirmation, these 5 transformants all contain SARS virus S protein gene fragment.
Extract the plasmid of No. 1 recon; Measure the SARS virus S protein gene sequence in the plasmid; Dna sequence analysis confirms; SARSS,：GAA TAC ATC TCT GAC GCA TTC TCT CTG GAC GTT TCC GAA AAG TCT GGT AAC TTC AAACAC CTG CGC GAG TTC GTG TTT AAA AAC AAA GAC GGT TTC CTG TAC GTT TAC AAG GGCTAC CAG CCG ATC GAC GTA GTT CGT GAC CTG CCG TCT GGT TTT AAC ACT CTG AAA CCGATC TTC AAG CTG CCG CTG GGT ATT AAC ATC ACT AAC TTC CGC GCT ATC CTG ACT GCTTTC TCT CCG GCT CAG GAC ACT TGG GGC ACT TCT GCT GCA GCC TAC TTC GTT GGC TACCTG AAG CCA ACT ACC TTT ATG CTG AAG TAC GAC GAA AAC GGT ACT ATC ACT GAT GCTGTT GAC TGC TCT CAG AAC CCA CTG GCT GAA CTG AAA TGC TCT GTT AAG AGC TTT GAGATC GAC AAA GGT ATT TAC CAG ACC TCT AAC TTC CGT GTT GTT CCG TCT GGT GAC GTTGTG CGT 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 GCT GATTAC TCT GTG CTG TAC AAC TCT ACC TTT TTC TCT ACC TTC AAG TGC TAC GGC GTT TCTGCT ACT AAG CTG AAC GAC CTG TGC TTC TCC AAC GTT TAC GCA GAT TCT TTC GTA GTTAAG GGT GAT GAC GTA CGT CAG ATC GCT CCA GGT CAG ACT GGT GTT ATC GCT GAC TACAAC TAT AAA CTG CCG GAC GAT TTC ATG GGT TGC GTT CTG GCT TGG AAC ACT CGT AACATT GAC GCT ACT TCT ACT GGT AAC TAC AAC TAC AAA TAT CGT TAC CTG CGT CAC GGCAAA CTG CGT CCG TTC GAA CGT GAC ATC TCT AAC GTG CCG TTC TAA
The virus S protein fragment (299 amino acid) of the expression of recombinant plasmid SARS that makes up; 34 amino acid on the carrier have been merged at its N end; 333,：Met Gly Ser Ser His His His His His His Ser Ser Gly Lys Val ProArg Gly SerHis Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Glu Tyr Ile SerAsp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe Lys His Leu Arg GluPhe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro IleAsp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys LeuPro Leu Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro AlaGln Asp 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 Cys SerGln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp Lys GlyIle Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe ProAsn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro SerVal Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser Val LeuTyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys LeuAsn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp AspVal Arg Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys LeuPro Asp 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 Arg ProPhe Glu Arg Asp Ile Ser Asn Val Pro Phe4.SARSS：
5 positive transformants and 1 contrast bacterium (plasmid pET28a transformed bacteria) 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 the SARS virus S albumen that relative molecular weight is about 37 kD, expression amount is about 30%, and contrast bacterium BL21 (DE3) does not have this protein band (Fig. 4).
Express the proteic purifying of SARS virus S
According to expressing the proteic aminoacid sequence of SARS virus S, analyze its physicochemical property, determine suitable purification process.Machine analysis as calculated, expressing the proteic iso-electric point of SARS virus S is pH8.5, so our decision with DEAE-SepharoseFF negatively charged ion purifying, is collected and worn the slide peak in pH is the damping fluid of 8.0 Tris-HCl.And then 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:
DEAE-SepharoseFF negatively charged ion and S-SepharoseFF positively charged ion gel are Pharmacia company product, and IPTG, DTT are Promega company product.Other reagent is homemade or the import analytical reagent.2. express the proteic ultrasonic degradation of S of SARS virus:
Centrifugal (the 8000rpm of the proteic engineering bacteria of S with the expression SARS virus of cultivating, l0mins, 4 ℃), abandon supernatant, thalline is resuspended in the lysate (50mmol/L Tris-HCl pH8.0,10mmol/L EDTA, 10mmol/L DTT) of original fluid 1/10 volume, and ice-bath ultrasonic is broken bacterium 10mins, centrifugal (12000rpm, 20mins, 4 ℃) collect supernatant, abandon precipitation.The supernatant of collecting is used for next step ion-exchange purification.3.DEAE-Sepharoe FF anion column purifying:
With DEAE-Sepharose FF anion column (the high 20cm of glue, diameter 1.2cm) is connected to Bio-Rad normal pressure chromatographic system, earlier with balance liquid (50mmol/LTris-HCl pH8.0,1mmol/L EDTA, 0.1mMmol/L DTT) flushing balance, to go up the cracking supernatant of step acquisition then and directly go up sample, last sample flow velocity is 1.5ml/min.The peak is passed in collection.4.S-Sepharose FF cation seperation column purifying:
To go up step DEAE-Sepharose FF negatively charged ion and pass the peak, in the dialysis tubing of packing into, phosphoric acid salt dialyzate (20mmol/L PB, 1mmol/L EDTA, 0.1mmol/L DTT) the stirring dialysis 4h of pH7.4.With above-mentioned dialyzate flushing balance S-Sepharose FF cation seperation column, sample is directly gone up at the peak that passes after will dialysing then, last sample flow velocity 1.5ml/min.Fully wash post with dialyzate behind the end of the sample, make stylus be back to baseline, more successively with contain 50,100, the dialyzate eluted protein of 1000mmol/L NaCl, collect each elution peak albumen and carry out SDS-PAGE and detect.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 5), the S albumen of SARS virus exists only in the 100mmol/L NaCl elution peak, the S protein band (37kD place) of a dense SARS virus of colour developing, the S protein band of no obvious SARS virus in other elution peak.
Proteic evaluation of the S of purifying SARS virus and application
The recombinant human cytomegalovirus fusion protein of purifying is used as antigen,, detects anti-SARS virus antibody (IgM or IgG) positive and negative serum, to identify proteic antigenicity of SARS virus S and the specificity of expressing by the indirect ELISA test method.Experimental result shows that this recombinant protein has good antigenicity and specificity, can be used as the Detection of antigen anti-SARS virus antibody.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 Sigama 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: usefulness 50mmol/L carbonate solution (pH9.6) dilutes the S albumen bag of SARS virus by elisa plate, every hole 100 μ l, and 4 ℃ are spent the night.Inferior daily confining liquid (10mmol/L phosphate buffered saline buffer, pH7.4,10% lowlenthal serum, 20% calf serum, 0.5%casein, 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, 0.5%casein, 0.05% sulphur sulphur mercury, 0.5mmol/L NaCl) dilution back (IgG dilution in 1: 20, IgM 1: 100 dilution), add in the enzyme linked plate holes after the sealing every hole 100 μ l respectively, each sample adds 2 holes, 37 ℃ of reaction 30min are with PBST liquid (10mmol/L phosphate buffered saline buffer, pH7.4,0.5% tween 20) wash 5 times after, the anti-human IgG or the anti-people μ chain monoclonal antibody that add the horseradish peroxidase-labeled of dilution in 1: 5000, every hole 100 μ l, 37 ℃ of reaction 30min, PBST washes 5 times, add substrate 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.1. indirect ELISAs detect anti-SARS virus antibody (IgG or IgM) in the serum as a result
With bovine serum albumin (the 5th part) co-electrophoresis of the S albumen of the SARS virus of purifying and known different concns, colour developing back relatively, determine that the S protein concentration of the SARS virus of purifying is about 1.5mg/ml.By elisa plate, detect known 2 parts of anti-SARS virus antibody (IgG) positive serums and 1 part of anti-SARS virus antibody (IgM) positive serum with 1: 1000 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 0.857 and 0.731.Color reaction also appears in 1 part of anti-SARS virus antibody (IgM) positive serum, and the A450 value is 0.557.Detected 180 portions of normal human serums simultaneously, their A450 value illustrates that all less than 0.05 the S albumen of SARS virus has antigenicity and specificity preferably.
The SARS virus S gene fragment order table of chemosynthesis＜110〉Li Yue Xi Taokaihua＜120〉the SARS virus S gene fragment of chemosynthesis and expression thereof, application＜160〉2＜210〉1＜211〉299＜212〉PRT＜213〉SARS virus S protein fragments＜220〉＜223 contain the S protein fragments of the SARS virus of strong antigen epi-position, 460 amino acid of the 162nd amino acid-Di, totally 299 amino acid.<400>1Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly1 5 10 15Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe
20 25 30Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp?Leu
35 40 45Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys?Leu?Pro?Leu?Gly
50 55 60Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro?Ala65 70 75 80Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr?Leu
85 90 95Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr
100 105 110Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser
115 120 125Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn?Phe
130 135 140Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr?Asn145 150 155 160Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser?Val
165 170 175Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr?Ser
180 185 190Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val
195 200 205Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala?Asp
210 215 220Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly?Gln225 230 235 240Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe?Met
245 250 255Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser?Thr
260 265 270Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg
275 280 285Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe
290 295＜210〉2＜211〉900＜212〉DNA＜213〉artificial sequence＜220〉＜221〉CDS＜222〉(1) ... (897)＜223〉the brand-new gene fragment of synthetic, encoding SARS virus S protein fragment.＜220〉＜221〉mis-feature＜222〉(898) ... (900)＜223〉terminator codon that increases during synthetic gene.<400>2GAA?TAC?ATC?TCT?GAC?GCA?TTC?TCT?CTG?GAC?GTT?TCC?GAA?AAG?TCT?GGT?AAC?TTC?AAA?CAC 60Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys?His1 5 10 15 20CTG?CGC?GAG?TTC?GTG?TTT?AAA?AAC?AAA?GAC?GGT?TTC?CTG?TAC?GTT?TAC?AAG?GGC?TAC?CAG 120Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln
25 30 35 40CCG?ATC?GAC?GTA?GTT?CGT?GAC?CTG?CCG?TCT?GGT?TTT?AAC?ACT?CTG?AAA?CCG?ATC?TTC?AAG 180Pro?Ile?Asp?Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys
45 50 55 60CTG?CCG?CTG?GGT?ATT?AAC?ATC?ACT?AAC?TTC?CGC?GCT?ATC?CTG?ACT?GCT?TTC?TCT?CCG?GCT 240Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro?Ala
65 70 75 80CAG?GAC?ACT?TGG?GGC?ACT?TCT?GCT?GCA?GCC?TAC?TTC?GTT?GGC?TAC?CTG?AAG?CCA?ACT?ACC 300Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr?Thr
85 90 95 100TTT?ATG?CTG?AAG?TAC?GAC?GAA?AAC?GGT?ACT?ATC?ACT?GAT?GCT?GTT?GAC?TGC?TCT?CAG?AAC 360Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn
105 110 115 120CCA?CTG?GCT?GAA?CTG?AAA?TGC?TCT?GTT?AAG?AGC?TTT?GAG?ATC?GAC?AAA?GGT?ATT?TAC?CAG 420Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr?Gln
125 130 135 140ACC?TCT?AAC?TTC?CGT?GTT?GTT?CCG?TCT?GGT?GAC?GTT?GTG?CGT?TTC?CCT?AAC?ATC?ACT?AAC 480Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr?Asn
145 150 155 160CTG?TGC?CCG?TTT?GGT?GAA?GTT?TTC?AAC?GCT?ACT?AAA?TTC?CCT?TCT?GTC?TAC?GCA?TGG?GAG 540Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu
165 170 175 180CGT?AAA?AAA?ATT?TCT?AAC?TGC?GTT?GCT?GAT?TAC?TCT?GTG?CTG?TAC?AAC?TCT?ACC?TTT?TTC 600Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe
185 190 195 200TCT?ACC?TTC?AAG?TGC?TAC?GGC?GTT?TCT?GCT?ACT?AAG?CTG?AAC?GAC?CTG?TGC?TTC?TCC?AAC 660Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn
205 210 215 220GTT?TAC?GCA?GAT?TCT?TTC?GTA?GTT?AAG?GGT?GAT?GAC?GTA?CGT?CAG?ATC?GCT?CCA?GGT?CAG 720Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly?Gln
225 230 235 240ACT?GGT?GTT?ATC?GCT?GAC?TAC?AAC?TAT?AAA?CTG?CCG?GAC?GAT?TTC?ATG?GGT?TGC?GTT?CTG 780Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe?Met?Gly?Cys?Val?Leu
245 250 255 260GCT?TGG?AAC?ACT?CGT?AAC?ATT?GAC?GCT?ACT?TCT?ACT?GGT?AAC?TAC?AAC?TAC?AAA?TAT?CGT 840Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg
265 270 275 280TAC?CTG?CGT?CAC?GGC?AAA?CTG?CGT?CCG?TTC?GAA?CGT?GAC?ATC?TCT?AAC?GTG?CCG?TTC?TAA 900Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe
285 290 295
Claims (5)
1. the genetic fragment of the SARS virus S albumen of a chemical synthesis; This genetic fragment coding contains the S protein fragments of the SARS virus of strong antigen epi-position; 460 amino acid of the 162nd amino acid-Di; 299,DNA：GAA TAC ATC TCT GAC GCA TTC TCT CTG GAC GTT TCC GAA AAG TCT GGT AAC TTCAAA 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 ACTCTG AAA CCG ATC TTC AAG CTG CCG CTG GGT ATT AAC ATC ACT AAC TTC CGC GCTATC CTG ACT GCT TTC TCT CCG GCT CAG GAC ACT TGG GGC ACT TCT GCT GCA GCCTAC TTC GTT GGC TAC CTG AAG CCA ACT ACC TTT ATG CTG AAG TAC GAC GAA AACGGT ACT ATC ACT GAT GCT GTT GAC TGC TCT CAG AAC CCA CTG GCT GAA CTG AAATGC TCT GTT AAG AGC TTT GAG ATC GAC AAA GGT ATT TAC CAG ACC TCT AAC TTCCGT GTT GTT CCG TCT GGT GAC GTT GTG CGT TTC CCT AAC ATC ACT AAC CTG TGCCCG TTT GGT GAA GTT TTC AAC GCT ACT AAA TTC CCT TCT GTC TAC GCA TGG GAGCGT AAA AAA ATT TCT AAC TGC GTT GCT GAT TAC TCT GTG CTG TAC AAC TCT ACCTTT TTC TCT ACC TTC AAG TGC TAC GGC GTT TCT GCT ACT AAG CTG AAC GAC CTGTGC TTC TCC AAC GTT TAC GCA GAT TCT TTC GTA GTT AAG GGT GAT GAC GTA CGTCAG 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 ACTTCT ACT GGT AAC TAC AAC TAC AAA TAT CGT TAC CTG CGT CAC GGC AAA CTG CGTCCG TTC GAA CGT GAC ATC TCT AAC GTG CCG TTC TAA
2. the proteic gene fragment order of the SARS virus S of the described chemosynthesis of claim 1 utilizes genetic engineering technique to express this sequence, and concrete grammar is as follows: the screening of SARS virus S proteantigen epi-position:
By the proteic aminoacid sequence of Computer Analysis SARS virus S; Find that the proteic N of S holds 460 amino acid of the 162nd amino acid to the to contain stronger antigenic determinant; The codon of selecting eucaryon and prokaryotic organism all to have a preference for; The gene order that chemosynthesis is brand-new; And BamHI restriction enzyme site and two protection bases G T have been increased at 5 ' end; Increased terminator codon TAA and EcoRI restriction enzyme site and two protection base AC at 3 ' end, the epitope amino acid sequence in the SARS virus S albumen of made this gene fragment be easy to be cloned in plasmid pET28a (+) the interior BamHI and EcoRI restriction enzyme site. screening; 162460,299：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 (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 SARS virus S protein fragments construction of recombinant plasmid:
Extract plasmid pET28a (+),, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis with BamHI and EcoRI double digestion, be dissolved in the deionized water, same SARS virus S protein gene fragment with BamHI and the chemosynthesis of EcoRI double digestion, electrophoresis is dissolved in the deionized water after reclaiming;
Above-mentioned two 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, SARS virus S protein gene fragment is inserted between carrier pET28a (+) the interior BamHI and EcoRI site, consistent with the initiator codon translation framework on the carrier, express a fusion rotein; 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 plasmid pET28a (+) at random; Extract respectively plasmid; Take the plasmid that extracts as template; Pcr amplification SARS virus S GFP fragment; The positive recombinant plasmid that contains SARS virus S GFP fragment; Should amplify the genetic fragment that is about 916bp; The plasmid that will contain foreign gene carries out dna sequence analysis; SARSS,：GAA TAC ATC TCT GAC GCA TTC TCT CTG GAC GTT TCC GAA AAG TCT GGT AAC TTCAAA 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 ACTCTG AAA CCG ATC TTC AAG CTG CCG CTG GGT ATT AAC ATC ACT AAC TTC CGC GCTATC CTG ACT GCT TTC TCT CCG GCT CAG GAC ACT TGG GGC ACT TCT GCT GCA GCCTAC TTC GTT GGC TAC CTG AAG CCA ACT ACC TTT ATG CTG AAG TAC GAC GAA AACGGT ACT ATC ACT GAT GCT GTT GAC TGC TCT CAG AAC CCA CTG GCT GAA CTG AAATGC TCT GTT AAG AGC TTT GAG ATC GAC AAA GGT ATT TAC CAG ACC TCT AAC TTCCGT GTT GTT CCG TCT GGT GAC GTT GTG CGT TTC CCT AAC ATC ACT AAC CTG TGCCCG TTT GGT GAA GTT TTC AAC GCT ACT AAA TTC CCT TCT GTC TAC GCA TGG GAGCGT AAA AAA ATT TCT AAC TGC GTT GCT GAT TAC TCT GTG CTG TAC AAC TCT ACCTTT TTC TCT ACC TTC AAG TGC TAC GGC GTT TCT GCT ACT AAG CTG AAC GAC CTGTGC TTC TCC AAC GTT TAC GCA GAT TCT TTC GTA GTT AAG GGT GAT GAC GTA CGTCAG 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 ACTTCT ACT GGT AAC TAC AAC TAC AAA TAT CGT TAC CTG CGT CAC GGC AAA CTG CGTCCG TTC GAA CGT GAC ATC TCT AAC GTG CCG TTC TAA。
299 amino acid fragments of the expression of recombinant plasmid SARS virus S albumen that makes up; 34 amino acid on the carrier have been merged at its N end; 333,：Met Gly Ser Ser His His His His His His Ser Ser Gly Lys Val ProArg Gly SerHis Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Glu Tyr IleSer Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe Lys His LeuArg Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly TyrGln 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 ThrAla Phe Ser Pro Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe ValGly Tyr Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Glv Thr IleThr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser ValLys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val ValPro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe GlyGlu Val Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys LysIle Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe SerThr Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe SerAsn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile AlaPro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp PheMet Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr GlyAsn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg Pro Phe GluArg Asp Ile Ser Asn Val Pro Phe：
The positive transformant that will contain recombinant plasmid, be seeded in the LB substratum that contains kanamycin 60 μ g/mL, 37 ℃ of shaking culture 3h, add IPTG to final concentration 0.5-1.0mmol/L, continue shaking culture and induce 4-6h, centrifugal collection thalline carries out SDS-PAGE and detects, and recon is expressed the SARS virus S albumen that relative molecular weight is about 37kD, expression amount is about 30%, and contrast bacterium BL21 (DE3) does not have this protein band; Express the proteic purifying of SARS virus S:
1) ultrasonic degradation of expression SARS virus S protein engineering bacterium
With the centrifugal receipts of the engineering bacteria of abduction delivering fusion rotein bacterium, thalline is resuspended in the lysate (50mmol/LTris-HCl pH8.0,10mmol/L EDTA, 10mmol/L DTT), ice-bath ultrasonic is broken bacterium 10min, centrifugal collection supernatant, abandon precipitation, the supernatant of collection is used for next step ion-exchange purification;
2) DEAE-Sepharose FF anion column purifying
DEAE-Sepharose FF anion column is connected to the normal pressure chromatographic system,, will goes up the cracking supernatant of step acquisition then and directly go up sample, collect and pass the peak earlier with the flushing of Tris-HCl damping fluid;
3) S-Sepearse FF cation seperation column purifying
To go up step DEAE-Sepharose FF negatively charged ion and pass the peak, pack in the dialysis tubing, phosphoric acid salt dialyzate to pH7.4 stirs dialysis 4h, with above-mentioned dialyzate flushing balance S-Sepharose FF cation seperation column, sample is directly gone up at the peak that passes after will dialysing then, and fully washes post with dialyzate behind the end of the sample, with the NaCl eluant solution albumen that contains gradient concentration, collect 100mmol/L NaCl elution peak albumen, be the SARS virus S albumen of purifying; The SARS virus S albumen of purifying is used as Detection of antigen HCMV antibody:
Wrap by elisa plate behind the SARS virus S albumen usefulness carbonate solution doubling dilution with purifying, the indirect enzyme-linked immunosorbent method detects known anti-SARS virus positive serum and normal human serum, SARS virus S albumen can react with the anti-SARS virus positive serum,, do not illustrate that the SARS virus S albumen of virus has better antigenicity and specificity with the normal human serum reaction;
Same with horseradish peroxidase-labeled SARS virus S albumen, detect anti-SARS virus IgM or IgG antibody with pouncing on the method that obtains, higher susceptibility and specificity are arranged, SARS virus S albumen is as antigen, detect anti-SARS virus antibody with gold mark method, higher susceptibility and specificity are also arranged.
3. the proteic gene fragment order of the SARS virus S of the described chemosynthesis of claim 1 utilizes bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.
4. the protein fragments of the proteic gene fragment expression of SARS virus S of the described chemosynthesis of claim 1 is used for vaccine, SARS virus antibody or detection of antigens and is used for immunity preparing anti-SARS virus monoclonal antibody and how anti-.
5. the proteic gene fragment order of the SARS virus S of the described chemosynthesis of claim 1 is connected with other gene fragment, expresses, prepares with the form of fusion rotein.
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| WO2005012337A3 (en) * | 2003-07-15 | 2006-03-16 | Crucell Holland Bv | Antigenic peptides of sars coronavirus and uses thereof |
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| CN112592390A (en) * | 2020-04-21 | 2021-04-02 | 苏州系统医学研究所 | Novel coronavirus specific antigen peptide and use thereof |
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