CN101007168B

CN101007168B - SARS vaccine and its preparation method

Info

Publication number: CN101007168B
Application number: CN2006100017421A
Authority: CN
Inventors: ***; 冯倩; 刘莹莹
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2006-01-23
Filing date: 2006-01-23
Publication date: 2010-06-09
Anticipated expiration: 2026-01-23
Also published as: CN101007168A

Abstract

The invention discloses a SARS vaccine and preparing method, which comprises the following steps: 1) constructing external baculoviral surface display carrier of S protein of SARS coronary virus; 2) transmitting the carrier into susceptive cell with baculoviral genome plasmid Bacmid; obtaining recombinant baculoviral genome plasmid; 3) using the plasmid to infect insect cell; purifying the recombinant plasmid; obtaining the product.

Description

A kind of SARS vaccine and preparation method thereof

Technical field

The present invention relates to vaccine of virosis and preparation method thereof, particularly relate to a kind of SARS vaccine and preparation method thereof.

Background technology

(Severe Acute Respiratory Syndrome SARS) claims severe acute respiratory syndrome again to SARS (Severe Acute Respiratory Syndrome), is a kind of acute respiratory infectious disease, once is very popular in the world in 2003.At present confirmed that this disease is caused by a kind of coronavirus of variation, World Health Organization (WHO) with its called after sars coronavirus (SARScoronavirus, SARS-CoV).Still needleless is to the specific drug of SARS virus at present, and it is very necessary therefore to develop effective viral vaccine as early as possible.Traditional viral vaccine comprises inactivated vaccine and attenuated vaccine.Inactivated vaccine preparation is simple, but for the such highly pathogenic virus of SARS-Cov, all exists very high potential safety hazard in the production of inactivated vaccine and use; Attenuated vaccine is comparatively safe, but the lead time is long, is difficult to meet an urgent need.Live vector vaccine is one of focus of recombinant vaccine research in recent years, is characterized in adopting non-pathogenic type microorganism (as poxvirus, adenovirus) to contain the antigenic recombinant of virus protein as vector construction.The outstanding advantage of live vector vaccine is: (1) can bring out body fluid and the comprehensive immunne response of cell, causes good immune effect; (2) can constitute multivalence and multiple vaccines simultaneously; (3) the vaccine consumption is few, and the immunoprotection time is long, need not add adjuvant.

S albumen is an important albumen on sars coronavirus surface, it is a transmembrane glycoprotein, form by 1255 amino acid residues, the signal peptide (from aminoterminal 1-11 position) that comprises N end, a big extracellular region (from aminoterminal 12-1190 position) and one lack strides film district and intracellular region (from aminoterminal 1191-1255 position).S albumen can combine with cell receptor, plays an important role in the virus infection cell processes; In addition, S albumen protrudes in virus surface with trimerical form, also is main antigen decision site.The serial number that the nucleotides sequence of S protein gene is listed in the ncbi database is: DQ231462.

Summary of the invention

The purpose of this invention is to provide a kind of vaccine that is used to prevent SARS (Severe Acute Respiratory Syndrome) (SARS).

SARS vaccine provided by the present invention is the recombinant baculovirus that sars coronavirus S protein extracellular is arranged at surface display; Described sars coronavirus S albumen sac membrane superficial tissue territory has SEQ ID № in the sequence table: 2 amino acid residue sequence.

Wherein the baculovirus as carrier can be arbitrary baculovirus.

Second purpose of the present invention provides a kind of preparation method of above-mentioned SARS vaccine.

The preparation method of SARS vaccine provided by the present invention may further comprise the steps:

1) the baculovirus surface display carrier of structure sars coronavirus S protein extracellular; Be connected with signal peptide (gp64 SP) coded sequence of the envelope protein gp64 of baculovirus self in the described display carrier in turn from 5 ' to 3 ' end, sars coronavirus S protein extracellular (S ectodomain) coded sequence, the coded sequence of striding film district and intracellular region of the G albumen (VSV-G) of gp64 albumen or vesicular stomatitis virus;

2) the baculovirus surface display carrier of the sars coronavirus S extracellular region that step 1) is made up transforms the competent cell that contains baculovirus geneome plasmid Bacmid, obtains the recombinant baculovirus geneome plasmid by homologous recombination;

3) with step 2) the recombinant baculovirus geneome plasmid transfection insect cell that obtains, the recombinant baculovirus particle of the budding pattern sars coronavirus S protein extracellular in the culture supernatant carried out purification after, obtain the SARS vaccine.

In above-mentioned preparation method, the signal peptide of the envelope protein gp64 of baculovirus self has SEQ ID № in the sequence table in the described step 1): 3 amino acid residue sequence, sars coronavirus S protein extracellular has SEQ ID № in the sequence table: 2 amino acid residue sequence, gp64 is proteic to stride the film district and intracellular region has SEQ ID № in the sequence table: 4 amino acid residue sequence, and the G of vesicular stomatitis virus is proteic to stride the film district and intracellular region has SEQ ID, № in the sequence table: 5 amino acid residue sequence; The carrier that sets out that is used to make up the baculovirus surface display carrier of sars coronavirus S protein extracellular can be pFastBac DUAL, pFastBac I etc., is preferably pFastBacDUAL; Be the carrier that sets out with pFastBac DUAL, the baculovirus surface display carrier of the sars coronavirus S protein extracellular of structure is S-gp64-DUAL or S-vsvG-DUAL.

Step 2) competent cell that contains baculovirus geneome plasmid Bacmid in is E.coliDH10Bac, is preferably E.coli DH10Bac; With the recombinant baculovirus geneome plasmid that obtains behind the S-gp64 DUAL transfection E.coli DH10Bac is S-gp64-Bacmid; With the recombinant baculovirus geneome plasmid that obtains behind the S-vsvG-DUAL transfection E.coliDH10Bac is S-vsvG-Bacmid.

The insecticide zooblast that can be used for transfection in the step 3) is sf9 cell or sf21 etc., is preferably the sf9 cell.

Above-mentioned preparation method is applicable to all SARS vaccines that use recombinant baculovirus surface display technology to prepare that comprise AcMNPV and silkworm NPV.

The invention provides a kind of SARS vaccine and preparation method thereof.This SARS vaccine be with baculovirus (Baculovirus) as carrier, what utilize baculovirus surface display technique construction is antigenic recombinant baculovirus with SARS-CoV S protein extracellular.Specifically, the structure principle of vaccine of the present invention is, the N end of the S protein extracellular of SARS-CoV is merged the signal peptide of the envelope protein gp64 of baculovirus self, the C end merges gp64 (or the G albumen of vesicular stomatitis virus, VSV-G) stride film district and intracellular region, this recombiant protein is being sorted into behind the insect cell inner expression on the cell membrane, is packaged in the cyst membrane surface of the baculovirus of budding pattern at last, thereby reaches the purpose of surface display.Vaccine of the present invention has the following advantages: 1) safe, baculovirus this as a kind of insect viruses, in mammalian cell, can't duplicate, to mammal without any toxicity and do not possess potential danger; 2) immune protective effect is good, because the immunological characteristic of baculovirus itself, this live vector vaccine can cause the immunity system and the cell immune system of body simultaneously, the SARS-CoV S albumen that immunity system can produce being reconstituted in the baculovirus surface produces specific neutralizing antibody, the neutralizing antibody that produces can not only with the virion combination in the blood, thereby can also blocking virus and the combination blocking virus of receptor enter cell, cell immune system then can be eliminated by the cell of viral infection, in addition, utilize baculovirus surface display technology can with foreign protein complete be showed in the recombinant baculovirus surface, and can keep the native conformation of foreign protein dramatically, thereby make foreign protein have good immunogenicity, effectively cause immunoreation, experimental results show that, behind the proteic recombinant baculovirus immune mouse of SARS-CoV S of the present invention, can in mice serum, detect generation, viral infection be had suppress effect preferably at the proteic neutralizing antibody of S; 3) preparation method is simple, is easy to large-scale production.Based on above-mentioned advantage, the present invention is in medical science, and particularly the prevention and control field of SARS has higher actual application value.

The invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 is the flow chart of SARS vaccine production method

Fig. 2 is the physical map of S-gp64-DUAL and S-vsvG-DUAL

Fig. 3 infects the proteic Western blot of the S testing result that the Sf9 cell is expressed after 38 hours, 48 hours respectively for S-gp64 and S-vsvG recombinant virus

Fig. 4 is the proteic Western blot of the sharp gp64 of the S albumen testing result of S-gp64, S-vsvG recombinant virus and the wild-type baculovirus of purification

Fig. 5 is the S-gp64 of purification, the S protein immunization fluoroscopic examination result of S-vsvG recombinant virus

Fig. 6 is the S-gp64 of purification, the immuno-electron microscope testing result of S-vsvG recombinant virus

Fig. 7 be S-gp64 and S-vsvG recombinant virus to the immunity of low concentration pseudovirus infecting mouse and in and the active testing result

Fig. 8 be S-gp64 and S-vsvG recombinant virus to the immunity of high concentration pseudovirus infecting mouse and in and the active testing result

The specific embodiment

Method therefor is conventional method if no special instructions among the following embodiment, and described percent concentration is mass percentage concentration, and the primer is synthetic by Beijing AudioCodes biotechnology Co., Ltd.

The preparation of embodiment 1, SARS vaccine

Adopt Invitrogen company the external transposon system of Bac-to-Bac (article No.: 10359-016,10608-016) and make up SARS vaccine of the present invention to specifications, the flow chart of preparation method as shown in Figure 1, concrete grammar may further comprise the steps:

One, the sub-clone of sars coronavirus S protein extracellular cDNA

According to the cDNA full length sequence of sars coronavirus S protein gene (the SEQ ID № in the sequence table: 1) design primer, pcr amplification S protein extracellular cDNA fragment, primer sequence is as follows:

Fs-12:5 '-ggg

Agtggtagtgaccttgacc-3 ' (base is a restricted enzyme BamHI recognition site in the square frame)

Rs-1190:5 '-attggg

Gttgctcatattttcccaattc-3 ' (base is a restricted enzyme Not I recognition site in the square frame)

Plasmid with the cDNA full length sequence that contains sars coronavirus S protein gene is a template, under the guiding of primers F s-12 and Rs-1190, use the cDNA fragment of high-fidelity Pyrobest enzyme (TaKaRa company) with conventional PCR method amplification sars coronavirus S protein extracellular gene, the cDNA fragment after sepharose electrophoresis purification and recovery is stand-by.

Two, make up the baculovirus surface display carrier of sars coronavirus S protein extracellular

With the donor plasmid pFastBac DUAL in the external transposon system of Bac-to-Bac is carrier, makes up the baculovirus surface display carrier of sars coronavirus S protein extracellular.Concrete grammar is: at first, (gp64 SP is that gp64 albumen is from aminoterminal 1-34 amino acids residue with the coded sequence of the signal peptide (gp64 SP) of the envelope protein gp64 of baculovirus self, has SEQ ID № in the series of tables: 3 amino acid residue sequence) with behind restricted enzyme Bgl II and the BamH I enzyme action, with be connected through the carrier pFastbac of BamH I enzyme action DUAL, be introduced into the BamHI site of pFastbac DUAL; Then, with the G albumen (VSV-G) of gp64 albumen and vesicular stomatitis virus stride film district and intracellular region (gp64 TM+CTD be gp64 albumen from aminoterminal 471-512 amino acids residue, have SEQ ID № in the sequence table: 4 amino acid residue sequence; VSV-G TM+CTD is that VSV-G albumen is from aminoterminal 441-511 amino acids residue, has SEQ ID № in the sequence table: after coded sequence 5 amino acid residue sequence amino acids) is used restricted enzyme Pst I and Hind III enzyme action, be connected with the linear carrier of the above-mentioned gp64 of the being connected with signal coding sequence of Hind III enzyme action respectively with through Pst I, be introduced into corresponding site; At last, with step 1 obtain the cDNA fragment of sars coronavirus S protein extracellular (S ectodomain) with behind restricted enzyme BamH I and the Not I enzyme action, with be connected through the above-mentioned gp64 of the being connected with signal peptide of BamH I and Not I enzyme action and the linear carrier of gp64 TM+CTD (or VSV-G TM+CTD) coded sequence, obtain the baculovirus surface display carrier of two kinds of sars coronavirus S protein extracellulars, difference called after S-gp64-DUAL and S-vsvG-DUAL, its physical map and insertion sequence position are as shown in Figure 2.

Three, the acquisition of sars coronavirus S protein extracellular recombinant baculovirus

S-gp64-DUAL that step 2 is made up and S-vsvG-DUAL donor plasmid change in the E.coliDH10Bac competent cell (Invitrogen) with the heat shock method respectively and carry out homologous recombination, obtain two kinds of baculovirus geneome plasmids after the reorganization, difference called after S-gp64-Bacmid and S-vsvG-Bacmid, again above-mentioned two kinds of baculovirus geneome plasmids are passed through liposome mediated-method transfection Sf9 cell respectively after purified, collect the transfectional cell supernatant after 72 hours, the recombinant baculovirus particle that promptly contains the sars coronavirus S protein extracellular of budding pattern in the supernatant, called after S-gp64 and S-vsvG respectively.With the infection cell supernatant 5000g of above-mentioned collection, low-speed centrifugal was removed cell debris (precipitation), 4 ℃, 100 again, 000g ultracentrifugation 2 hours, the purified virus of slightly being carried (precipitation) in 10 minutes.With the PBS suspension precipitation of 1/100 centrifugal front volume, and carry out sucrose density gradient centrifugation, take out viral band, after dialysis, promptly can be used as the SARS vaccine that is used for human immunity.

Embodiment 2, S-gp64 and the proteic evaluation of S-vsvG recombinant virus surface S

One, Western blot and identified by immunofluorescence result

After S-gp64 that embodiment 1 is obtained and S-vsv6 recombinant virus infect the Sf9 cell respectively, method by Westernblot and immunofluorescence is carried out Preliminary detection to the S albumen of expressing in the Sf9 cell and S-gp64, the S-vsvG recombinant virus of purification, is contrast with the wild-type baculovirus.Infect the proteic Western blot of the S testing result expressed in the Sf9 cell after back 38 hours, 48 hours (wt Bac is a wild-type baculovirus) as shown in Figure 3, in the Sf9 cell of infection after back 38 hours, 48 hours, all can detect the S protein band of about 175KDa, and infect that the proteic expression of S significantly improves after back 48 hours.(A is S Protein Detection result, and B is gp64 Protein Detection result as shown in Figure 4 for the S albumen of the S-gp64 of purification, S-vsvG recombinant virus and wild-type baculovirus and the proteic Western blot of gp64 testing result; Wt Bac is a wild-type baculovirus), the result only detects S albumen in the S-gp64 of purification, S-vsvG recombinant virus, and all can detect the gp64 albumen of about 64KDa at S-gp64, S-vsvG recombinant virus and the wild-type baculovirus of purification.The immunofluorescence testing result that infects the Sf9 cell that S-gp64, S-vsvG recombinant virus are arranged respectively is (the Sf9 cell is contrast) as shown in Figure 5, and S albumen is distributed on the plasma membrane of Sf9 transfectional cell.

Two, immuno-electron microscope qualification result

S albumen to S-gp64, S-vsvG recombinant virus and the wild-type baculovirus of purification under immuno-electron microscope is identified, qualification result as shown in Figure 6, colloid gold particle is distributed in the recombinant virus surface, in S-gp64, be distributed in an end (the black arrow among a) of virus, in S-vsvG, then be distributed in the structure of whole virus surface (the black arrow among the b) and similar film (the white arrow among the b), at wild-type baculovirus (c) with only use that (d) then do not have specific colloid gold particle to distribute in the S-gp64 virus of gold colloidal two anti-labellings.

Above-mentioned qualification result shows that the correct displaying of S-gp64 and S-vsvG recombinant virus surface has S albumen.

The mouse immune of embodiment 3, S-gp64 and S-vsvG recombinant virus and in and active testing

Get the S-gp64 and the female Mus of S-vsvG recombinant virus subcutaneous injection 6 week Balb/c in age of 0.1mL purification respectively, the 11st day behind the initial immunity with the same dose booster immunization once, detected the activity of neutralizing antibody in the mice serum on the 25th day, the neutralization activity is by measuring mouse resisting anteserum the infection inhibition effect of a kind of S albumen-HIV pseudovirus to be measured, with the negative contrast of Mus serum (wt Bac) of wild-type baculovirus immunity, it infects pseudovirus does not have inhibitory action; (Positive, dilution factor is 1 in the experiment: 200-3200), it infects pseudovirus and has the obvious suppression effect with the positive contrast of SARS rehabilitation patient's serum.(A is the S-gp64 experimental group to the active testing result of the neutralization of low concentration pseudovirus infection (viral infection concentration is the 0.2ng/ hole) as shown in Figure 7, B is the S-vsvG experimental group), (A is the S-gp64 experimental group to the active testing result of the neutralization of high concentration pseudovirus infection (viral infection concentration is the 10ng/ hole) as shown in Figure 8, B is the S-vsvG experimental group), under 1: 20 dilution factor level, all recombinant virus immunity antiserums all can suppress about 80% pseudovirus infection, show that it contains the proteic neutralizing antibody at S, has very high neutralization activity, and under 1: 80 to 1: 160 dilution factor level, the sero-fast inhibition ability drop to 50% of recombinant virus immunity.Showing that with the active testing result baculovirus of recombinant s protein of the present invention can cause the generation of neutralizing antibody effectively in above-mentioned, is a kind of effective candidate vaccine at SARS-CoV.

Sequence table

<160>5

<210>1

<211>3768

<212>DNA

＜213〉sars coronavirus (SARS coronavirus, Coronaviridae)

<400>1

atgtttattt?tcttattatt?tcttactctc?actagtggta?gtgaccttga?ccggtgcacc 60

acttttgatg?atgttcaagc?tcctaattac?actcaacata?cttcatctat?gaggggggtt 120

tactatcctg?atgaaatttt?tagatcagac?actctttatt?taactcagga?tttatttctt 180

ccattttatt?ctaatgttac?agggtttcat?actattaatc?atacgtttgg?caaccctgtc 240

atacctttta?aggatggtat?ttattttgct?gccacagaga?aatcaaatgt?tgtccgtggt 300

tgggtttttg?gttctaccat?gaacaacaag?tcacagtcgg?tgattattat?taacaattct 360

actaatgttg?ttatacgagc?atgtaacttt?gaattgtgtg?acaacccttt?ctttgctgtt 420

tctaaaccca?tgggtacaca?gacacatact?atgatattcg?ataatgcatt?taattgcact 480

ttcgagtaca?tatctgatgc?cttttcgctt?gatgtttcag?aaaagtcagg?taattttaaa 540

cacttacgag?agtttgtgtt?taaaaataaa?gatgggtttc?tctatgttta?taagggctat 600

caacctatag?atgtagttcg?tgatctacct?tctggtttta?acactttgaa?acctattttt 660

aagttgcctc?ttggtattaa?cattacaaat?tttagagcca?ttcttacagc?cttttcacct 720

gctcaagaca?tttggggcac?gtcagctgca?gcctattttg?ttggctattt?aaagccaact 780

acatttatgc?tcaagtatga?tgaaaatggt?acaatcacag?atgctgttga?ttgttctcaa 840

aatccacttg?ctgaactcaa?atgctctgtt?aagagctttg?agattgacaa?aggaatttac 900

cagacctcta?atttcagggt?tgttccctca?ggagatgttg?tgagattccc?taatattaca 960

aacttgtgtc?cttttggaga?ggtttttaat?gctactaaat?tcccttctgt?ctatgcatgg 1020

gagagaaaaa?aaatttctaa?ttgtgttgct?gattactctg?tgctctacaa?ctcaacattt 1080

ttttcaacct?ttaagtgtta?tggcgtttct?gccactaagt?tgaatgatct?ttgcttctcc 1140

aatgtctatg?cagattcttt?tgtagtcaag?ggagatgatg?taagacaaat?agcgccagga 1200

caaactggtg?ttattgctga?ttataattat?aaattgccag?atgatttcat?gggttgtgtc 1260

cttgcttgga?atactaggaa?cattgatgct?acttcaactg?gtaattataa?ttataaatat 1320

aggtatctta?gacatggcaa?gcttaggccc?tttgagagag?acatatctaa?tgtgcctttc 1380

tcccctgatg?gcaaaccttg?caccccacct?gctcttaatt?gttattggcc?attaaatgat 1440

tatggttttt?acaccactac?tggcattggc?taccaacctt?acagagttgt?agtactttct 1500

tttgaacttt?taaatgcacc?ggccacggtt?tgtggaccaa?aattatccac?tgaccttatt 1560

aagaaccagt?gtgtcaattt?taattttaat?ggactcactg?gtactggtgt?gttaactcct 1620

tcttcaaaga?gatttcaacc?atttcaacaa?tttggccgtg?atgtttctga?tttcactgat 1680

tccgttcgag?atcctaaaac?atctgaaata?ttagacattt?caccttgctc?ttttgggggt 1740

gtaagtgtaa?ttacacctgg?aacaaatgct?tcatctgaag?ttgctgttct?atatcaagat 1800

gttaactgca?ctgatgtttc?tacagcaatt?catgcagatc?aactcacacc?agcttggcgc 1860

atatattcta?ctggaaacaa?tgtattccag?actcaagcag?gctgtcttat?aggagctgag 1920

catgtcgaca?cttcttatga?gtgcgacatt?cctattggag?ctggcatttg?tgctagttac 1980

catacagttt?ctttattacg?tagtactagc?caaaaatcta?ttgtggctta?tactatgtct 2040

ttaggtgctg?atagttcaat?tgcttactct?aataacacca?ttgctatacc?tactaacttt 2100

tcaattagca?ttactacaga?agtaatgcct?gtttctatgg?ctaaaacctc?cgtagattgt 2160

aatatgtaca?tctgcggaga?ttctactgaa?tgtgctaatt?tgcttctcca?atatggtagc 2220

ttttgcacac?aactaaatcg?tgcactctca?ggtattgctg?ctgaacagga?tcgcaacaca 2280

cgtgaagtgt?tcgctcaagt?caaacaaatg?tacaaaaccc?caactttgaa?atattttggt 2340

ggttttaatt?tttcacaaat?attacctgac?cctctaaagc?caactaagag?gtcttttatt 2400

gaggacttgc?tctttaataa?ggtgacactc?gctgatgctg?gcttcatgaa?gcaatatggc 2460

gaatgcctag?gtgatattaa?tgctagagat?ctcatttgtg?cgcagaagtt?caatggactt 2520

acagtgttgc?cacctctgct?cactgatgat?atgattgctg?cctacactgc?tgctctagtt 2580

agtggtactg?ccactgctgg?atggacattt?ggtgctggcg?ctgctcttca?aatacctttt 2640

gctatgcaaa?tggcatatag?gttcaatggc?attggagtta?cccaaaatgt?tctctatgag 2700

aaccaaaaac?aaatcgccaa?ccaatttaac?aaggcgatta?gtcaaattca?agaatcactt 2760

acaacaacat?caactgcatt?gggcaagctg?caagacgttg?ttaaccagaa?tgctcaagca 2820

ttaaacacac?ttgttaaaca?acttagctct?aattttggtg?caatttcaag?tgtgctaaat 2880

gatatccttt?cgcgacttga?taaagtcgag?gcggaggtac?aaattgacag?gttaattaca 2940

ggcagacttc?aaagccttca?aacctatgta?acacaacaac?taatcagggc?tgctgaaatc 3000

agggcttctg?ctaatcttgc?tgctactaaa?atgtctgagt?gtgttcttgg?acaatcaaaa 3060

agagttgact?tttgtggaaa?gggctaccac?cttatgtcct?tcccacaagc?agccccgcat 3120

ggtgttgtct?tcctacatgt?cacgtatgtg?ccatcccagg?agaggaactt?caccacagcg 3180

ccagcaattt?gtcatgaagg?caaagcatac?ttccctcgtg?aaggtgtttt?tgtgtttaat 3240

ggcacttctt?ggtttattac?acagaggaac?ttcttttctc?cacaaataat?tactacagac 3300

aatacatttg?tctcaggaaa?ttgtgatgtc?gttattggca?tcattaacaa?cacagtttat 3360

gatcctctgc?aacctgagct?tgactcattc?aaagaagagc?tggacaagta?cttcaaaaat 3420

catacatcac?cagatgttga?tcttggcgac?atttcaggca?ttaacgcttc?tgtcgtcaac 3480

attcaaaaag?aaattgaccg?cctcaatgag?gtcgctaaaa?atttaaatga?atcactcatt 3540

gaccttcaag?aattgggaaa?atatgagcaa?tatattaaat?ggccttggta?tgtttggctc 3600

ggcttcattg?ctggactaat?tgccatcgtc?atggttacaa?tcttgctttg?ttgcatgact 3660

agttgttgca?gttgcctcaa?gggtgcatgc?tcttgtggtt?cttgctgcaa?gtttgatgag 3720

gatgactctg?agccagttct?caagggtgtc?aaattacatt?acacataa 3768

<210>2

<211>1179

<212>PRT

＜213〉sars coronavirus (SARS coronavirus, Coronaviridae)

<400>2

Ser?Gly?Ser?Asp?Leu?Asp?Arg?Cys?Thr?Thr?Phe?Asp?Asp?Val?Gln?Ala

1 5 10 15

Pro?Asn?Tyr?Thr?Gln?His?Thr?Ser?Ser?Met?Arg?Gly?Val?Tyr?Tyr?Pro

20 25 30

Asp?Glu?Ile?Phe?Arg?Ser?Asp?Thr?Leu?Tyr?Leu?Thr?Gln?Asp?Leu?Phe

35 40 45

Leu?Pro?Phe?Tyr?Ser?Asn?Val?Thr?Gly?Phe?His?Thr?Ile?Asn?His?Thr

50 55 60

Phe?Gly?Asn?Pro?Val?Ile?Pro?Phe?Lys?Asp?Gly?Ile?Tyr?Phe?Ala?Ala

65 70 75 80

Thr?Glu?Lys?Ser?Asn?Val?Val?Arg?Gly?Trp?Val?Phe?Gly?Ser?Thr?Met

85 90 95

Asn?Asn?Lys?Ser?Gln?Ser?Val?Ile?Ile?Ile?Asn?Asn?Ser?Thr?Asn?Val

100 105 110

Val?Ile?Arg?Ala?Cys?Asn?Phe?Glu?Leu?Cys?Asp?Asn?Pro?Phe?Phe?Ala

115 120 125

Val?Ser?Lys?Pro?Met?Gly?Thr?Gln?Thr?His?Thr?Met?Ile?Phe?Asp?Asn

130 135 140

Ala?Phe?Asn?Cys?Thr?Phe?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Trp?Pro?Leu?Asn?Asp?Tyr?Gly?Phe?Tyr?Thr?Thr?Thr?Gly?Ile?Gly?Tyr

465 470 475 480

Gln?Pro?Tyr?Arg?Val?Val?Val?Leu?Ser?Phe?Glu?Leu?Leu?Asn?Ala?Pro

485 490 495

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

500 505 510

Cys?Val?Asn?Phe?Asn?Phe?Asn?Gly?Leu?Thr?Gly?Thr?Gly?Val?Leu?Thr

515 520 525

Pro?Ser?Ser?Lys?Arg?Phe?Gln?Pro?Phe?Gln?Gln?Phe?Gly?Arg?Asp?Val

530 535 540

Ser?Asp?Phe?Thr?Asp?Ser?Val?Arg?Asp?Pro?Lys?Thr?Ser?Glu?Ile?Leu

545 550 555 560

Asp?Ile?Ser?Pro?Cys?Ser?Phe?Gly?Gly?Val?Ser?Val?Ile?Thr?Pro?Gly

565 570 575

Thr?Asn?Ala?Ser?Ser?Glu?Val?Ala?Val?Leu?Tyr?Gln?Asp?Val?Asn?Cys

580 585 590

Thr?Asp?Val?Ser?Thr?Ala?Ile?His?Ala?Asp?Gln?Leu?Thr?Pro?Ala?Trp

595 600 605

Arg?Ile?Tyr?Ser?Thr?Gly?Asn?Asn?Val?Phe?Gln?Thr?Gln?Ala?Gly?Cys

610 615 620

Leu?Ile?Gly?Ala?Glu?His?Val?Asp?Thr?Ser?Tyr?Glu?Cys?Asp?Ile?Pro

625 630 635 640

Ile?Gly?Ala?Gly?Ile?Cys?Ala?Ser?Tyr?His?Thr?Val?Ser?Leu?Leu?Arg

645 650 655

Ser?Thr?Ser?Gln?Lys?Ser?Ile?Val?Ala?Tyr?Thr?Met?Ser?Leu?Gly?Ala

660 665 670

Asp?Ser?Ser?Ile?Ala?Tyr?Ser?Asn?Asn?Thr?Ile?Ala?Ile?Pro?Thr?Asn

675 680 685

Phe?Ser?Ile?Ser?Ile?Thr?Thr?Glu?Val?Met?Pro?Val?Ser?Met?Ala?Lys

690 695 700

Thr?Ser?Val?Asp?Cys?Asn?Met?Tyr?Ile?Cys?Gly?Asp?Ser?Thr?Glu?Cys

705 710 715 720

Ala?Asn?Leu?Leu?Leu?Gln?Tyr?Gly?Ser?Phe?Cys?Thr?Gln?Leu?Asn?Arg

725 730 735

Ala?Leu?Ser?Gly?Ile?Ala?Ala?Glu?Gln?Asp?Arg?Asn?Thr?Arg?Glu?Val

740 745 750

Phe?Ala?Gln?Val?Lys?Gln?Met?Tyr?Lys?Thr?Pro?Thr?Leu?Lys?Tyr?Phe

755 760 765

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

770 775 780

Lys?Arg?Ser?Phe?Ile?Glu?Asp?Leu?Leu?Phe?Asn?Lys?Val?Thr?Leu?Ala

785 790 795 800

Asp?Ala?Gly?Phe?Met?Lys?Gln?Tyr?Gly?Glu?Cys?Leu?Gly?Asp?Ile?Asn

805 810 815

Ala?Arg?Asp?Leu?Ile?Cys?Ala?Gln?Lys?Phe?Asn?Gly?Leu?Thr?Val?Leu

820 825 830

Pro?Pro?Leu?Leu?Thr?Asp?Asp?Met?Ile?Ala?Ala?Tyr?Thr?Ala?Ala?Leu

835 840 845

Val?Ser?Gly?Thr?Ala?Thr?Ala?Gly?Trp?Thr?Phe?Gly?Ala?Gly?Ala?Ala

850 855 860

Leu?Gln?Ile?Pro?Phe?Ala?Met?Gln?Met?Ala?Tyr?Arg?Phe?Asn?Gly?Ile

865 870 875 880

Gly?Val?Thr?Gln?Asn?Val?Leu?Tyr?Glu?Asn?Gln?Lys?Gln?Ile?Ala?Asn

885 890 895

Gln?Phe?Asn?Lys?Ala?Ile?Ser?Gln?Ile?Gln?Glu?Ser?Leu?Thr?Thr?Thr

900 905 910

Ser?Thr?Ala?Leu?Gly?Lys?Leu?Gln?Asp?Val?Val?Asn?Gln?Asn?Ala?Gln

915 920 925

Ala?Leu?Asn?Thr?Leu?Val?Lys?Gln?Leu?Ser?Ser?Asn?Phe?Gly?Ala?Ile

930 935 940

Ser?Ser?Val?Leu?Asn?Asp?Ile?Leu?Ser?Arg?Leu?Asp?Lys?Val?Glu?Ala

945 950 955 960

Glu?Val?Gln?Ile?Asp?Arg?Leu?Ile?Thr?Gly?Arg?Leu?Gln?Ser?Leu?Gln

965 970 975

Thr?Tyr?Val?Thr?Gln?Gln?Leu?Ile?Arg?Ala?Ala?Glu?Ile?Arg?Ala?Ser

980 985 990

Ala?Asn?Leu?Ala?Ala?Thr?Lys?Met?Ser?Glu?Cys?Val?Leu?Gly?Gln?Ser

995 1000 1005

Lys?Arg?Val?Asp?Phe?Cys?Gly?Lys?Gly?Tyr?His?Leu?Met?Ser?Phe

1010 1015 1020

Pro?Gln?Ala?Ala?Pro?His?Gly?Val?Val?Phe?Leu?His?Val?Thr?Tyr

1025 1030 1035

Val?Pro?Ser?Gln?Glu?Arg?Asn?Phe?Thr?Thr?Ala?Pro?Ala?Ile?Cys

1040 1045 1050

His?Glu?Gly?Lys?Ala?Tyr?Phe?Pro?Arg?Glu?Gly?Val?Phe?Val?Phe

1055 1060 1065

Asn?Gly?Thr?Ser?Trp?Phe?Ile?Thr?Gln?Arg?Asn?Phe?Phe?Ser?Pro

1070 1075 1080

Gln?Ile?Ile?Thr?Thr?Asp?Asn?Thr?Phe?Val?Ser?Gly?Asn?Cys?Asp

1085 1090 1095

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

1100 1105 1110

Pro?Glu?Leu?Asp?Ser?Phe?Lys?Glu?Glu?Leu?Asp?Lys?Tyr?Phe?Lys

1115 1120 1125

Asn?His?Thr?Ser?Pro?Asp?Val?Asp?Leu?Gly?Asp?Ile?Ser?Gly?Ile

1130 1135 1140

Asn?Ala?Ser?Val?Val?Asn?Ile?Gln?Lys?Glu?Ile?Asp?Arg?Leu?Asn

1145 1150 1155

Glu?Val?Ala?Lys?Asn?Leu?Asn?Glu?Ser?Leu?Ile?Asp?Leu?Gln?Glu

1160 1165 1170

Leu?Gly?Lys?Tyr?Glu?Gln

1175

<210>3

<211>34

<212>PRT

＜213〉baculovirus (Autographa californica nucleopolyhedrovirus, AcMNPV,

Baculoviridae)

<400>3

Met?Val?Ser?Ala?Ile?Val?Leu?Tyr?Val?Leu?Leu?Ala?Ala?Ala?Ala?His

1 5 10 15

Ser?Ala?Phe?Ala?Ala?Glu?His?Cys?Asn?Ala?Gln?Met?Lys?Thr?Gly?Pro

20 25 30

Tyr?Lys

<210>4

<211>42

<212>PRT

＜213〉baculovirus (Autographa californica nucleopolyhedrovirus, AcMNPV,

Baculoviridae)

<400>4

Met?Ala?Glu?Gly?Glu?Leu?Ala?Ala?Lys?Leu?Thr?Ser?Phe?Met?Phe?Gly

1 5 10 15

His?Val?Val?Asn?Phe?Val?Ile?Ile?Leu?Ile?Val?Ile?Leu?Phe?Leu?Tyr

20 25 30

Cys?Met?Ile?Arg?Asn?Arg?Asn?Arg?Gln?Tyr

35 40

<210>5

<211>71

<212>PRT

＜213〉vesicular stomatitis virus (Vesicular stomatitis virus, Rhabdoviridae)

<400>5

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

1 5 10 15

Trp?Phe?Ser?Ser?Trp?Lys?Ser?Ser?Ile?Ala?Ser?Phe?Phe?Phe?Ile?Ile

20 25 30

Gly?Leu?Ile?Ile?Gly?Leu?Phe?Leu?Val?Leu?Arg?Val?Gly?Ile?His?Leu

35 40 45

Cys?Ile?Lys?Leu?Lys?His?Thr?Lys?Lys?Arg?Gln?IIe?Tyr?Thr?Asp?Ile

50 55 60

Glu?Met?Asn?Arg?Leu?Gly?Lys

65 70

Claims

1. a SARS vaccine is the recombinant baculovirus that sars coronavirus S protein extracellular is arranged at surface display; The amino acid residue sequence of described sars coronavirus S protein extracellular is SEQ ID № in the sequence table: 2.

2. the preparation method of the described SARS vaccine of claim 1 may further comprise the steps:

1) the baculovirus surface display carrier of structure sars coronavirus S protein extracellular; Be connected with the signal coding sequence of the envelope protein gp64 of baculovirus self in the described display carrier in turn from 5 ' to 3 ' end, sars coronavirus S protein extracellular coded sequence, the proteic coded sequence of striding film district and intracellular region of the G of gp64 albumen or vesicular stomatitis virus; The aminoacid sequence of described sars coronavirus S protein extracellular is SEQ ID № in the sequence table: 2;

2) the baculovirus surface display carrier of the sars coronavirus S protein extracellular that step 1) is made up transforms the competent cell that contains baculovirus geneome plasmid Bacmid, obtains the recombinant baculovirus geneome plasmid;

3. the preparation method of SARS vaccine according to claim 2, it is characterized in that: the amino acid residue sequence of the signal peptide of the envelope protein gp64 of baculovirus self is SEQ ID № in the sequence table in the described step 1): 3, the proteic amino acid residue sequence of striding film district and intracellular region of gp64 is SEQ ID № 4 in the sequence table, and the proteic amino acid residue sequence of striding film district and intracellular region of the G of vesicular stomatitis virus is SEQ ID № in the sequence table: 5.

4. according to the preparation method of claim 2 or 3 described SARS vaccines, it is characterized in that: the baculovirus surface display carrier of described sars coronavirus S protein extracellular is S-gp64-DUAL or S-vsvG-DUAL.

5. the preparation method of SARS vaccine according to claim 4 is characterized in that: described recombinant baculovirus geneome plasmid is S-gp64-Bacmid or S-vsvG-Bacmid.

6. according to the preparation method of claim 2 or 3 described SARS vaccines, it is characterized in that: the insect cell that is used for transfection in the described step 3) is sf9 cell or sf21 cell.