CN1749277A - Heat shock protein 65-human SARS coronary virus epitope antigen recombinant fusion protein (HSP65SARS/3CL161-264) - Google Patents

Abstract

The present invention provides one kind of recombinant fusion protein formed through fusing heat shock protein 65 (HSP65) and human SARS coronavirus epitone antigen. The present invention also provides the amino acid sequence of the recombinant fusion protein, nucleotide sequence coding the recombinant fusion protein, expression vector containing the nucleotide sequence, host cell containing the expression vector, the preparation process of the recombinant fusion protein, the preparation process of the vaccine product containing the recombinant fusion protein and the application of the recombinant fusion protein in preparing medicine and/or reagent for treating and/or preventing SARS virus infection. The recombinant fusion protein may be used in preventing and treating human SARS coronavirus infection effectively.

Description

Heat shock protein(HSP) 65-people sars coronavirus epitope antigen recombinant fusion protein (HSP65-SARS/3CL 161-264)

Technical field

The present invention relates to a kind of recombination fusion protein, relate to the recombination fusion protein that a kind of people's of preventing and/or treating sars coronavirus infects specifically, this recombination fusion protein (HSP65-SARS/3CL _161-264) be by heat shock protein(HSP) 65 (heat shockprotein 65, HSP65) and SARS/3CL _161-264The fusion of epitope antigen polypeptide constitutes; The invention provides the aminoacid sequence of this recombination fusion protein and this recombination fusion protein of encoding nucleotide sequence, contain this nucleotide sequence expression vector, contain the host cell of this expression vector, the preparation method of this recombination fusion protein; The invention still further relates to the vaccine product that contains this recombination fusion protein and this recombination fusion protein and be used for the treatment of and/or prevent medicine that SARS virus infects and/or the application in the reagent in preparation.

Background technology

On November 16th, 2002, the SARS case that the 1st example has record has appearred in China Guangdong Province, and in short 3 months time, Guangdong Province 305 people occur and infects SARS, wherein 5 the infected's death.Since in late Febuary, 2003, severe acute respiratory syndrome spreads to the Hong Kong Special Administrative Region, Vietnam, Canada, Singapore and the U.S. and other places.The infectious atypical pneumonia of 2002～2003 years eruption and prevalences has the advantages that infectivity is strong, hazardness is big, cause 8 of 29 countries altogether, 098 example infects, 774 routine the infected's death [Stadler K, Masignani V, Eickmann M, etal.SARS-beginning to understanda new virus.Nat Rev Microbiol.2003,1 (3): 209-18.].WHO with infectious atypical pneumonia called after severe acute respiratory syndrome (severe acute respiratory syndrome, SARS).Scientists has used the time of some months just to isolate the pathogenic agent of SARS, and after Canada had at first announced the genome sequence of viral Tor2, the genome sequence of corresponding virus had all been reported in the U.S., Hong Kong, Singapore, Beijing etc. in succession.On April 16th, 2003, WHO was formally with its called after sars coronavirus (SARS Coronavirus, SARS CoV).

Sars coronavirus is a kind of newfound coronavirus, is tunicary single positive chain RNA virus.SARS virus through after the negative staining under transmission electron microscope diameter be 60～130nm, coating arranged that the distinctive projection of coronavirus can both be seen in most of virus particle surface.[Ksiazek?TG，Erdman?D，Goldsmith?CS，et?al.A?novel?coronavirusassociated?with?severe?acute?respiratory?syndrome.N?Engl?JMed，2003，348(20)：1953-1966.，Drosten?C，Gunther?S，Preiser?W，et?al.Identificationof?a?novel?coronavirus?in?patients?with?severe?acute?respiratory?syndrome.N?EnglJ?Med，2003，348(20)：1967-1976.，Peiris?JS，Lai?ST，Poon?LL，et?al.Coronavirusas?a?possible?cause?of?severe?acute?respiratory?syndrome.Lancet，2003，361(9366)：1319-1325.]。SARS virus is stronger to the resistibility of environmental factors.Under the room temperature condition, the 24h of in normal people's urine, surviving at least, the 48h of in ight soil, surviving at least.At 4 ℃ and-80 ℃, virus titer has only slight decline after 21 days.Fixing agent that 10% formaldehyde and Paraformaldehyde 96,10% Mono Chloro Acetic Acid, 75% ethanol etc. are commonly used and sterilizing agent can be with its complete inactivation [WHO First data.First data on stability and resistance of SARS coronaviruscompiled by members of WHO laboratory network.http: //www.who.int/csr/gars/survival-2003-05-04/en/print.html] in 5 minutes.

Because the hazardness of SARS CoV is very big, science personnel are stepping up to study control this viral medicine and vaccine.Virus vaccines roughly is divided into inactivated vaccine, attenuated vaccine, subunit vaccine and 4 types of nucleic acid vaccine.Development of Inactivated Vaccine is the most classical, the simplest, the most quick, is the inactivation of virus that will cultivate, is used for clinical application after preparing purifying with methods such as ultra-filtration, column chromatographies.Through China scientific and technical personnel's joint efforts, on January 19th, 2004, SARS CoV inactivated vaccine has obtained state approval and has entered I phase clinical study.The advantage of inactivated vaccine is that the lead time is short.Shortcoming is the safety condition of producing to be required high, during viral large scale culturing, in case leak huge disaster will take place.So inactivated vaccine and attenuated live vaccine will be replaced (for example genetically engineered hepatitis B vaccine replaced inactivated vaccine to be applied to clinical) by recombinant vaccine.The outstanding advantage of recombinant vaccine is to have a good security, can scale operation, and cost is low, and can design as required.We are when development SARS recombinant vaccine, and the main protein processive enzyme 3CLpro that has selected sars coronavirus is as target antigen.One of major function of 3Clpro is that coronavirus is duplicated necessary RNA polymerase and helicase is processed, this is confirmed (Marra MA by experiments such as many 3CLpro mutant and inhibitor thereof, Jones SJM, Astell CR, et al.The genome sequence of the SARS-associated coronavirus.Science, 2003,300:1399., Pinon JD, Teng H, Weiss SR.Further requirements for cleavage by the murine coronavirus3Clike proteinase:dentification of a cleavage site within ORF1b.Virology, 1999,263 (2): 471-484., Kim JC, Spence RA, Currier PF, Lu X, Deni son MR.Coronavirusprotein processing and RNA synthesis is inhibited by the cysteine proteinaseinhibitor E64d.Virology, 1995,208 (1): 1-8.), and 3Clpro all very conservative (Anand K in all coronavirus, Ziebuhr J, Wadhwani, P, the target spot of good cellular immunization is provided for the development of SARSCoV vaccine et al.2003.Coronavirus main proteinase (3CLpro) structure:Basis for design of anti-SARS drugs.Science 300:1763-1767.).

The present invention is according to the cytotoxic T lymphocyte of sars coronavirus major protein enzyme 3Clpro (cytotoxic TLymphocytes, CTL) predicting the outcome of epi-position and the CTL epi-position recombination fusion protein that makes up.CTL is the anti-infectious important immunocyte of body.Behind the virus infected cell, the viral protein of virogene coding is processed into small peptide as endogenous protein in cell, be transported to endoplasmic reticulum and combine with MHC I quasi-molecule, CTL discerns and MHCI quasi-molecule bonded virus related peptides by acceptor (TCR), and kills and wounds virus infected cell specifically.These combine with the MHCI quasi-molecule and can be called as the CTL epi-position by the polypeptide that TCR discerned.The CTL epitope peptide combines first activation signals that the complex body that forms is t cell activation and performance immunization with the MHCI quasi-molecule, clinical trial has confirmed that the CTL epiposition vaccine can induce epitope specificity ctl response (Muderspach L, Wilczynski S, Roman L, et al.A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelialneoplasia who are HPV 16 positive.Clin Cancer Res.2000; 6 (9): 3406-16.), so we can activate the specific CTL of virus antigen by CTL epi-position recombination fusion protein, reach the purpose of removing virus.

Because SARS Cov is a kind of newfound virus, the research report that does not also have the CTL epi-position to identify at present.If by traditional method screening CTL epi-position, blindness is big, cost is high, time and effort consuming.Computer epi-position prediction procedure is a kind of new technology of screening epi-position, at first selects the potential epi-position by prediction, utilizes experiment to carry out Screening and Identification again, because this technical object is clear and definite, method is advanced, has obtained the achievement that attracts people's attention now.Therefore this research has adopted this computer epi-position forecast method to predict the CTL epi-position of SARS Cov major protein enzyme 3Clpro.Because the software of epi-position prediction is a lot, the theoretical foundation of each software is also incomplete same, in order to reach the purpose of learning from other's strong points to offset one's weaknesses, we have selected three software (http://bimas.dcrt.nih.gov HLA Peptide Binding Predictions that authoritative website provides under study for action for use; Http:// www.syfpeithi.deSYFPEITH; Http:// www.bioinformatics.leeds.ac.uk) predicts respectively, choose the 161st amino acids to the 214 amino acids (SARS/3CL in the 3Clpro protein sequence that epi-position is densely distributed, scoring is higher according to predicting the outcome _161-264) as the main component of recombinating and merging.

Generally, ectogenic protein mainly enters mhc class ii submission approach, activates humoral immune reaction, can not activate CTL effectively, therefore, must manage to make recombination fusion protein to enter the effect that its activation specific CTL of approach competence exertion is offered in the processing of MHC I class antigen.

HSP65 is a member in the heat-shock protein family, in the process of immunne response, can assist the antigenicity substance of external source enter antigen presenting cell (antigen presenting cell, APC); And assist exogenous antigen to enter MHC I class processing submission approach, and then activate specific CTL; Simultaneously HSP65 can also stimulate the APC secrete cytokines and express collaborative stimulation molecule, and the second signal of potent T cell activation is provided.

Therefore, the invention is with HSP65 and SARS/3CL _161-264Merge the formation recombination fusion protein, utilize HSP65 SARS/3CL _161-264Carry into MHCI class processing submission approach, activate the specific CTL of SARS specifically, reach and kill and wound the purpose that infects the SARS virus cell.In the present invention, help Recombinant Protein Expression in order to optimize structure, at SARS/3CL _161-264The upstream add irrelevant amino acid D (aspartic acid), E (L-glutamic acid), irrelevant amino acid E, D, D, E, D are added in the downstream, hereinafter referred is SARS/3CL _161-264

Summary of the invention

The purpose of this invention is to provide a kind of recombination fusion protein, this recombination fusion protein is with heat shock protein(HSP) 65 (HSP65) and SARS/3CL _161-264The epitope antigen polypeptide merges and is constituted, and (hereinafter referred is HSP65-SARS/3CL to this recombination fusion protein _161-264) people's SARS virus is infected the effect that prevents and/or treats.The present invention also provides a kind of expression vector that contains this nucleotide sequence.The present invention provides a kind of host cell that contains this expression vector simultaneously.

Another object of the present invention provides a kind of method for preparing this recombination fusion protein.

Another object of the present invention provides a kind of vaccine product that contains this recombination fusion protein and this recombination fusion protein and is used for the treatment of and/or prevents medicine that SARS virus infects and/or the application in the reagent in preparation

The invention provides a kind of recombination fusion protein, this recombination fusion protein is that the inventor is first pioneeringly with the 161st amino acids to the 214 amino acids (SARS/3CL in HSP65 and the 3Clpro protein sequence _161-264) merge and to have formed brand-new genetically engineered recombination fusion protein, thereby give SARS/3CL _161-264Epitope antigen can activate specificity SARS/3CL specifically _161-264The character of CTL, the specificity SARS/3CL that is activated _161-264CTL can kill and wound the cell that infects SARS virus, can infect SARS virus and effectively prevent and treat.

The present invention also provides the nucleotide sequence of this recombination fusion protein of coding and the aminoacid sequence of this recombination fusion protein.This recombination fusion protein gene has the nucleotide sequence shown in the SEQ ID NO:5, and its aminoacid sequence is shown in SEQ ID NO:6.SARS/3CL in this nucleotide sequence _161-264Epitope antigen can be a 1-5 copy, the SARS/3CL of a copy _161-264Epitope antigen has the aminoacid sequence shown in the SEQ ID NO:2, and its nucleotide coding sequence is shown in SEQ ID NO:1; The SARS/3CL that copy is optimized _161-264Epitope antigen has the aminoacid sequence shown in the SEQ ID NO:4, and its nucleotide coding sequence is shown in SEQ ID NO:3.

The nucleotide sequence or the aminoacid sequence that have 70%, 75%, 80%, 85%, 90%, 95%, 97% homology with SEQ ID NO:1-6, or under the making nucleic acid molecular hybridization condition with the nucleotide sequence of the coding nucleotide sequence of above-mentioned nucleotide sequence or above-mentioned aminoacid sequence hybridization, or the artificial point mutation of above-mentioned sequence or carry out the sequence that house of correction gets in the mode of inserting, connect other sequence and also can play the effect that is equal to above-mentioned sequence in both sides.The making nucleic acid molecular hybridization condition of indication can be as described below in the present invention, but be not confined to this: 5 * SSC, the 20mmol/L phosphoric acid buffer, 5 * Denhardt ' s solution, 10% sulfuric acid crawl glycan and 100 μ g/ml sex change salmon sperm DNA fragments, 65 ℃ of hybridization 8-16 hour, and in 2 * SSC and 0.5%SDS solution, cleaned 5 minutes under the room temperature, then in 2 * SSC and 0.1%SDS solution, cleaned 15 minutes under the room temperature, then in 0.1 * SSC and 0.1%SDS solution 65 ℃ cleaned 30-60 minute down rinsing filter membrane slightly under 0.1 * SSC room temperature at last.

It is a kind of with the vaccine product of recombination fusion protein of the present invention as effective constituent that the present invention also provides, recombination fusion protein of the present invention is optionally combined with pharmaceutically acceptable carrier or vehicle etc., put on the person of being applied by suitable route of administration and effective dose, treat and/or prevent the purpose that SARS virus infects thereby reach.

" pharmaceutically acceptable carrier or vehicle " of the present invention is meant that they can not produce disadvantageous, hypersensitive or other untoward reaction when molecule body and composition suitably are not applied the person." the pharmaceutically acceptable carrier or the vehicle " of indication of the present invention should be compatible with recombination fusion protein of the present invention, can not reduce the effect of pharmaceutical composition with its blend under normal conditions significantly.The object lesson that can be used as some materials of pharmaceutically acceptable carrier or vehicle has carbohydrate, as lactose, dextrose plus saccharose; Starch is as W-Gum and potato starch; Mierocrystalline cellulose and derivative thereof are as Xylo-Mucine, ethyl cellulose and methylcellulose gum; Fructus Hordei Germinatus; Gelatin; Talcum; Solid lubricant is as stearic acid and Magnesium Stearate; Calcium sulfate; Vegetables oil is as peanut oil, Oleum Gossypii semen, sesame oil, sweet oil, Semen Maydis oil and theobroma oil; Polyvalent alcohol is as propylene glycol, glycerine, Sorbitol Powder, mannitol and polyoxyethylene glycol; Lalgine; Emulsifying agent is as Tween; Wetting agent is as Sodium Lauryl Sulphate BP/USP; Tinting material; Seasonings; Sheet is pressed agent; Antioxidant; Sanitas; Apirogen water; Deng oozing salts solution and phosphate buffered saline buffer etc.

Vaccine product of the present invention can be made various formulations as required, and can be determined the useful dosage of the person of being applied is used according to the person's of being applied factors such as kind, age, body weight and administering mode by the doctor.Administering mode for example can adopt injection and other therapeutic modality.

Recombination fusion protein of the present invention can be produced by known method.

Heat shock protein(HSP) 65 is a kind of protein that derives from bacille Calmette-Guerin vaccine, it with SARS/3CL _161-264Epitope antigen can be assisted SARS/3CL after merging the formation fusion rotein _161-264The epitope antigen polypeptide enters the antigen presenting cell that comprises dendritic cell and enters the processing of MHC I class offers approach, and then activates at expressing SARS/3CL _161-264The CTL of epitope antigen cell.In addition, heat shock protein(HSP) 65 also can stimulate the antigen presenting cell that comprises dendritic cell to express collaborative stimulation molecule (as B7 etc.) and secrete cytokines, and these collaborative stimulation molecules (as B7 etc.) and cytokine can assist to activate CTL.

Because the strong pathogenic and highly infective of SARS virus, can only in having the laboratory of specific installation, just can directly test with SARS virus, therefore, we have made up the recombinant mammalian expressing vector pcDNA3-GFP that carries green fluorescent protein (GFP) gene, synthetic SARS/3CL _161-264Gene constructed in the downstream of GFP gene, make GFP and SARS/3CL _161-264Gene is in same reading frame, obtains eukaryotic cell recombinant expression plasmid pcDNA3-GFP-SARS/3CL _161-264With this recombinant plasmid transfection mouse melanin tumor cell strain B16, obtained expression GFP-SARS/3CL _161-264Transfectional cell.To transfection SARS/3CL _161-264The B16 cell CTL reflection be: SARS/3CL _161-264Specific CTL is to infecting killing and wounding of SARS virus cell.HSP65-SARS/3CL of the present invention _161-264Suppress transfection SARS/3CL _161-264What the B16 cell of gene was grown reflection in the mouse body is: by HSP65-SARS/3CL provided by the invention _161-264Induced specific CTL in the mouse body, CTL has killed and wounded expression SARS/3CL _161-264The B16 cell of specific antigens causes B16 quantity to reduce, and the HSP65-SARS/3CL among the present invention is described _161-264Can induce SARS/3CL mouse _161-264Specific CTL, this CTL have the stronger ability of killing and wounding the SARS virus cells infected.

In addition, it is pointed out that aspect and creationary beneficial effect that other has substantive distinguishing features of the present invention are conspicuous to those skilled in the art on the basis of the application's contextual disclosure.

Description of drawings

Fig. 1: SARS virus epi-position prediction

Utilize three kinds of software predictions to analyze the SARS/3CL epi-position, between the 161-264 amino acid sites epi-position densely distributed (SYFPEITH predict the outcome in epi-position scoring differ greatly, scope is from 0～2500000000, in order to get more information about the epi-position distribution situation, it is to map respectively with value＜30 in value＞100 that this partial results is decomposed into two portions)

Fig. 2: HSP65-SARS/3CL _161-264The recombinant protein structural analysis

As seen from the figure, proteic α spiral, β lamella, hydrophobicity and hydrophilic structure are evenly distributed, and proteic N, C-terminal are hydrophilic structure (DNA star.INC)

Fig. 3: synthetic SARS/3CL _161-264The agarose gel electrophoresis analysis of epitope antigen gene first round PCR product

Swimming lane 1: arrow is depicted as PCR product 99bp

Swimming lane 2:DL2000marker (2000/1000/750/500/250/100bp) (Takara)

Fig. 4: synthetic SARS/3CL _161-264Epitope antigen gene second is taken turns the agarose gel electrophoresis analysis of PCR product

Swimming lane 1: arrow is depicted as PCR product 183bp

Swimming lane 2:DNA2000marker (2000/1000/750/500/250/100bp) (Takara)

Fig. 5: synthetic SARS/3CL _161-264The agarose gel electrophoresis analysis of epitope antigen gene third round PCR product

Swimming lane 1: arrow is depicted as PCR product 267bp

Swimming lane 3:DNA2000marker (2000/1000/750/500/250/100bp) (Takara)

Fig. 6: synthetic SARS/3CL _161-264The agarose gel electrophoresis analysis of epitope antigen gene four-wheel PCR product

Swimming lane 1: arrow is depicted as PCR product 357bp

Swimming lane 2:DNA2000marker (2000/1000/750/500/250/100bp) (Takara)

Fig. 7: pMD-18T-SARS/3CL _161-264The enzyme of recombinant plasmid is cut evaluation

Swimming lane 1: arrow is depicted as the 345bp fragment that plasmid discharges after EcoRI and the two enzymic digestions of HindIII

Swimming lane 2: complete pMD18T-SARS/3CL _161-264Plasmid

Swimming lane 3:DNA2000marker (2000/1000/750/500/250/100bp) (Takara)

Fig. 8: the agarose gel electrophoresis analysis of HSP65 encoding gene

Swimming lane 1:marker (2000/1000/750/500/250/100bp) (Takara)

Swimming lane 2: by heat shock protein(HSP) 65 (HSP65) encoding gene (1638bp) of PCR acquisition

Fig. 9: eukaryotic cell transfection plasmid pcDNA3-GFP-SARS/3CL _161-264Enzyme cut evaluation

Swimming lane 1: complete pcDNA3-GFP-SARS/3CL _161-264Plasmid

Swimming lane 2: plasmid is identified with the HindIII single endonuclease digestion and is discharged about 1100bp fragment

Swimming lane 3:DNA2000marker (2000/1000/750/500/250/100bp) (Takara)

Figure 10: stable transfection GFP-SARS/3CL _161-264The evaluation of gene B16 cell

Visible transfectional cell sends green fluorescence under the 10a Laser Scanning Confocal Microscope:

10b FACS identifies: compare with the cell of untransfected, have the transfectional cell of fluorescence to move to right

Figure 11: expression plasmid pET28a-HSP65-SARS/3CL _161-264Enzyme cut evaluation

Swimming lane 1: arrow is depicted as the 345bp fragment of plasmid through EcoRI and the release of HindIII double digestion

Swimming lane 2: complete pET28a-HSP65-SARS/3CL _161-264Plasmid

Swimming lane 3:DNA2000marker (2000/1000/750/500/250/100bp) (Takara)

Figure 12: the recombination fusion protein HSP65-SARS/3CL behind expression, the purifying _161-264SDS-PAGE identifies and Western blot identification and analysis

12a HSP65-SARS/3CL _161-264Western blot identify

Swimming lane 1: arrow is depicted as HSP65-SARS/3CL _161-264Express band, 80KD

Swimming lane 2:Protein marker, 14,20,33,45,66,90KD (Takara)

Swimming lane 3: arrow is depicted as HSP65-SARS/3CL _161-264Albumen Western blot, one anti-is the his-tag monoclonal antibody

HSP65-SARS/3CL behind 12b expression, the purifying _161-264SDS-PAGE

Swimming lane 1: arrow is depicted as Ni ²⁺Affinity chromatography obtains the HSP65-SARS/3CL of purifying _161-264, 80KD

Swimming lane 2:Protein marker, 33,45,66,90KD (Takara)

Swimming lane 3: arrow is depicted as the HSP65-SARS/3CL before the purifying _161-264

Figure 13: HSP65-SARS/3CL _161-264The specific inhibitory effect that in the mouse body, excites

Figure 14: HSP65-SARS/3CL _161-264The comparison of experimental group and PBS control group mice tumor weight

PBS injection group

HSP65-SARS/3CL _161-264Immune group

Figure 15: HSP65-SARS/3CL _161-264Immunity back mouse SARS/3CL _161-264Specific CTL induces

The effector cell is the splenocyte of mouse, and target cell is transfection SARS/3CL _161-264The B16 cell of epitope antigen, untransfected SARS/3CL _161-264The B16 cell of epitope antigen, imitating the target ratio is that 200: 1,100: 1,50: 1,25: the 1 o'clock specific killing rates to target cell all are higher than the PBS control group.

HSP65-SARS/3CL _161-264Immune mouse spleen cell kills and wounds transfection SARS/3CL _161-264The B16 cell of gene

HSP65-SARS/3CL _161-264Immune mouse spleen cell kills and wounds the B16 cell

PBS injection mice spleen cell kills and wounds transfection SARS/3CL _161-264The B16 cell of gene

PBS injection mice spleen cell kills and wounds the B16 cell

Figure 16: HSP65-SARS/3CL _161-264To SARS/3CL _161-264The lymphocytic hormesis of mouse memory

HSP65-SARS/3CL _161-264Immune group

: PBS injection group

Figure 17: HSP65-SARS/3CL _161-264Inducing of vitro human cytotoxic T lymphocyte

The effector cell is for to use CD4, CD14, and CD56, HLA-DR, TCR, Glycoglin A magnetic bead is purifying CD8 in succession ⁺The T cell.Target cell is for loading the T2 cell of 12h with the HPV antigen peptide.CD8 ⁺The T cell is through 100ug/ml

HSP65-SARS/3CL _161-264Or the external immunity of people's mature dendritic cell of cytokine stimulation.

Load HSP65-SARS/3CL _161-264People's mature dendritic cell.

Negative control is people's mature dendritic cell that cytokine stimulates.

Embodiment

In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art, Molecular Cloning one book (Joseph.Sambrook, David W.Russell.Molecular cloning:ALaboratory Manual, 3 for example ^RdEd.Cold Spring Harbor Laboratory Press, 2001) described method.

The prediction of embodiment 1SARS virus CTL epi-position

When the target antigen of option table position prediction, we are the target spot of the conservative protein enzyme 3Clpro that duplicates of control sars coronavirus as SARS CoV vaccine.Selection accounts for the epi-position of the HLA-A2 restriction of population ratio maximum, the software that provides with three websites carries out the epi-position prediction of HLA specific combination:

(1)http：//bimas.dcrt.nih.gov：HLA?Peptide?Binding?Predictions

(2)http：//www.syfpeithi.de：SYFPEITH

(3)http：//www.bioinformatics.leeds.ac.uk

Predict the outcome according to area of computer aided HLA restricted epitope and to be figure (Fig. 1) with EXCEL, the zone of choosing the epi-position dense distribution is as the candidate antigens composition, trial has designed multiple scheme, and various schemes Sequence Analysis Software software analysis, the 161-264 amino acids that found that 3Clpro is the zone that epi-position is concentrated, albumen secondary structure (α spiral, the β lamella) distributes rationally, biological parameters such as close and distant pool ideal (Fig. 2), we select this section as target protein, and it is blended in the downstream of HSP65, carried out the expression and the function test of recombination fusion protein.In order to improve its expression amount in intestinal bacteria, in the design of recombination fusion protein encoding gene, all used the prokaryotic gene preference codon.Simultaneously in order further to optimize this proteic structure, at SARS/3CL _161-264Upstream and downstream added 2 amino acid DE and 5 amino acid EDDED respectively.

SARS/3CL after the optimization _161-264Aminoacid sequence as follows:

DEYMHHMELPTGVHAGTDLEGKFYGPFVDRQTAQAAGTDTTITLNVLAWLYAAVINGDRWFLNRFT

TTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMEDDED

In order to detect the biological function of recombination fusion protein, we have synthesized 3 SARS/3CL according to the epi-position prediction result _161-264Epitope peptide:

Peptide A:NH2-vlawlyaav

Peptide B:NH2-flnrftttl

Peptide C:NH2-ttlndfnlv

Embodiment 2 single copy SARS/3CL _161-264Synthesizing of epitope antigen gene

1, first round PCR (two primer template increase) each other

The sequence of primer 1 is (SEQ ID NO.7):

5′-ACTATCACTCTTAATgTTCTggCTTggCTgTACgCTgCTgTTATTAACggTgACCgC

The sequence of primer 1 ' is (SEQ ID NO.8):

5′-gTTgAAATCgTTCAgggTAgTAgTAAAgCggTTCAggAACCAgCggTCACCgTTAAT

The sequence of first round PCR synthetic product is:

ACTATCACTCTTAATGTTCTGGCTTGGCTGTACGCTGCTGTTATTAACGGTGACCGCTGGT

TCCTGAACCGCTTTACTACTACCCTGAACGATTTCAAC

The agarose gel electrophoresis of first round PCR product is seen Fig. 3.

2, be that template is carried out second and taken turns PCR with first round PCR product

The sequence of primer 2 is (SEQ ID NO.9):

5′-TTCgTTgACCgTCAgACTgCACAggCTgCTggTACTgACACTACTATCACTCTTAATg

Primer 2 ' sequence be (SEQ ID NO.10):

5′-gTCTTgAgTCAgCggTTCgTAgTTgTACTTCATAgCAACCAggTTgAAATCgTTCAg

The sequence of product is:

5′

TTCGTTGACCGTCAGACTGCACAGGCTGCTGGTACTGACACTACTATCACTCTTAATGTT

CTGGCTTGGCTGTACGCTGCTGTTATTAACGGTGACCGCTGGTTCCTGAACCGCTTTACT

ACTACCCTGAACGATTTCAACCTGGTTGCTATGAAGTACAACTACGAACCGCTGACTCA

AGAC3′

Second agarose gel electrophoresis of taking turns the PCR product is seen Fig. 4.

3, taking turns the PCR product with second is that template is carried out third round PCR

Primer 3 sequences are (SEQ ID NO.11):

5′-gTTCACgCAggTACTgACCTggAAggTAAATTTTATggTCCgTTCgTTgACCgTCAg

Primer 3 ' sequence be (SEQ ID NO.12):

5′-gATACCAgTCTgAgCAgACAgTggACCCAgAATgTCTACgTggTCTTgAgTCAgCgg

The sequence of product is:

5′

GTTCACGCAGGTACTGACCTGGAAGGTAAATTTTATGGTCCGTTCGTTGACCGTCAGACT

GCACAGGCTGCTGGTACTGACACTACTATCACTCTTAATGTTCTGGCTTGGCTGTACGCT

GCTGTTATTAACGGTGACCGCTGGTTCCTGAACCGCTTTACTACTACCCTGAACGATTTC

AACCTGGTTGCTATGAAGTACAACTACGAACCGCTGACTCAAGACCACGTAGACATTCT

GGGTCCACTGTCT GCTCAGACTGGTATC?3′

The agarose gel electrophoresis of third round PCR product is seen Fig. 5.

4, be that template is carried out four-wheel PCR with third round PCR product

Primer 4 sequences are (SEQ ID NO.13):

5′-gAATTCgATgAgTACATgCACCACATggAACTgCCgACCggTgTTCACgCAggTACTg

Primer 4 ' sequence be (SEQ ID NO.14):

5′-CTCgAgTCACTAAAgCTTgTCTTCgTCgTCCTCCATATCCAgAACAgCgATACCAgTCTgAgC

The sequence of product is:

5′

GAATTCGATGAGTACATGCACCACATGGAACTGCCGACCGGTGTTCACGCAGGTACTGA

CCTGGAAGGTAAATTTTATGGTCCGTTCGTTGACCGTCAGACTGCACAGGCTGCTGGTA

CTGACACTACTATCACTCTTAATGTTCTGGCTTGGCTGTACGCTGCTGTTATTAACGGTGA

CCGCTGGTTCCTGAACCGCTTTACTACTACCCTGAACGATTTCAACCTGGTTGCTATGAA

GTACAACTACGAACCGCTGACTCAAGACCACGTAGACATTCTGGGTCCACTGTCTGCTC

AGACTGGTATCGCTGTTCTGGATATGGAGGACGACGAAGACAAGCTTTAGTGACTCGAG

3′

The agarose gel electrophoresis of four-wheel PCR product is seen Fig. 6.

5 of four-wheel PCR product ' end has EcoRI enzyme point of contact, and downstream primer 3 ' end has Hind III, XhoI enzyme point of contact.This sequence is the sars coronavirus epitope antigen sequence of the optimization of a copy.

5, four-wheel PCR product cloning is gone into carrier pMD-18T (Takara)

The clone of PCR product:

Adopt TA cloning process clone PCR products, method see (test kit, Takara).

The sequential analysis of PCR product:

(Joseph.Sambrook, David W.Russell.Molecular cloning:A LaboratoryManual, 3 according to a conventional method ^RdThe ed.SDS alkaline lysis prepares plasmid DNA, 27-30.Cold Spring Harbor Laboratory Press, 2001) extract plasmid, adopt the terminal cessation method of two deoxidations, carry out sequencing to inserting fragment, its aminoacid sequence is shown in SEQ IDNO:4, and corresponding nucleotide coding sequence is shown in SEQ ID NO:3.

Fig. 7 is that enzyme is cut evaluation pMD-18T-SARS/3CL _161-264The agarose gel electrophoresis figure of recombinant plasmid.

The acquisition of embodiment 3 heat shock protein(HSP) 65KD (HSP65) encoding genes

1, bacille Calmette-Guerin vaccine: derive from Changchun Biological Products Institute.Adopt the logical potato culture of Soviet Union to cultivate bacille Calmette-Guerin vaccine, the temperature of cultivation is a 37-39 ℃ of bacille Calmette-Guerin vaccine that grows, and presents the lurid mycoderm of shriveling.Collect mycoderm, therefrom extract bcg genomic dna.

2, extract bcg genomic dna: method is with reference to Molecular Cloning one book (Joseph.Sambrook, David W.Russell.Molecular cloning:A Laboratory Manual, 3 ^RdEd. from mammalian cell, separate high molecular weight DNA, 463-470 with Proteinase K with phenol.Cold?Spring?Harbor?LaboratoryPress，2001))。

3, separate HSP65 structure gene: adopt PCR method to separate HSP65 structure gene from bacille Calmette-Guerin vaccine.5 ' the end primer sequence that adopts is 5 ' CCATG GCC AAG ACA ATT GCG3 ', 3 ' (SEQ ID NO:15); 3 ' end primer sequence is 5 ' ACCGAA TTC GCT AGC CAT ATG GAA ATC CAT GCC ACC CAT, 3 ' (SEQ ID NO:16).

The PCR schedule of operation

In one 500 μ l Eppendorf tubes, add following reagent:

Template cDNA (mmol/L) 5 μ l

10 * PCR damping fluid (containing magnesium chloride), 5 μ l

dNTPs(10mmol/L) 1μl

5 ' end and 3 ' each 0.5 μ l of end primer (0.01mmol/L)

Taq archaeal dna polymerase (5u/ μ l) 0.25 μ l

Deionized water adds to final volume 50 μ l

Mix the back and add 3 in mineral oil

Reaction conditions: 94 ℃, 30 "; 55 ℃, 1 '; 72 ℃, behind 2 ', 30 loop cycles, 72 ℃ are extended 10min, and 5 of PCR product ' end has Nco I restriction enzyme site, and downstream primer 3 ' end has the EcoRI restriction enzyme site.

4, clone PCR products: adopt the TA cloning process, obtained to carry the plasmid pMD-18T-HSP65 of HSP65 gene.Method see (test kit, Takara).(Joseph.Sambrook, David W.Russell.SDS alkaline lysis prepares plasmid DNA, Molecularcloning:A Laboratory Manual, 3 according to a conventional method ^RdEd.27-30, Cold Spring HarborLaboratory Press, 2001) extract plasmid, adopt the terminal cessation method of two deoxidations, carry out sequencing (ABIPrism310 to inserting fragment ^TM, USA).

The sequence of HSP65 encoding gene is as follows:

ATGGCCAAGA?CAATTGCGTA?CGACGAAGAG?GCCCGTCGCG?GCCTCGAGCG 50

GGGCTTGAAC?GCCCTCGCCG?ATGCGGTAAA?GGTGACATTG?GGCCCCAAGG 100

GCCGCAACGT?CGTCCTGGAA?AAGAAGTGGG?GTGCCCCCAC?GATCACCAAC 150

GATGGTGTGT?CCATCGCCAA?GGAGATCGAG?CTGGAGGATC?CGTACGAGAA 200

GATCGGCGCC?GAGCTGGTCA?AAGAGGTAGC?CAAGAAGACC?GATGACGTCG 250

CCGGTGACGG?CACCACGACG?GCCACCGTGC?TGGCCCAGGC?GTTGGTTCGC 300

GAGGGCCTGC?GCAACGTCGC?GGCCGGCGCC?AACCCGCTCG?GTCTCAAACG 350

CGGCATCGAA?AAGGCCGTGG?AGAAGGTCAC?CGAGACCCTG?CTCAAGGGCG 400

CCAAGGAGGT?CGAGACCAAG?GAGCAGATTG?CGGCCACCGC?AGCGATTTCG 450

GCGGGTGACC?AGTCCATCGG?TGACCTGATC?GCCGAGGCGA?TGGACAAGGT 500

GGGCAACGAG?GGCGTCATCA?CCGTCGAGGA?GTCCAACACC?TTTGGGCTGC 550

AGCTCGAGCT?CACCGAGGGT?ATGCGGTTCG?ACAAGGGCTA?CATCTCGGGG 600

TACTTCGTGA?CCGACCCGGA?GCGTCAGGAG?GCGGTCCTGG?AGGACCCCTA 650

CATCCTGCTG?GTCAGCTCCA?AGGTGTCCAC?TGTCAAGGAT?CTGCTGCCGC 700

TGCTCGAGAA?GGTCATCGGA?GCCGGTAAGC?CGCTGCTGAT?CATCGCCGAG 750

GACGTCGAGG?GCGAGGCGCT?GTCCACCCTG?GTCGTCAACA?AGATCCGCGG 800

CACCTTCAAG?TCGGTGGCGG?TCAAGGCTCC?CGGCTTCGGC?GACCGCCGCA 850

AGGCGATGCT?GCAGGATATG?GCCATTCTCA?CCGGTGGTCA?GGTGATCAGC 900

GAAGAGGTCG?GCCTGACGCT?GGAGAACGCC?GACCTGTCGC?TGCTAGGCAA 950

GGCCCGCAAG?GTCGTGGTCA?CCAAGGACGA?GACCACCATC?GTCGAGGGCG 1000

CCGGTGACAC?CGACGCCATC?GCCGGACGAG?TGGCCCAGAT?CCGCCAGGAG 1050

ATCGAGAACA?GCGACTCCGA?CTACGACCGT?GAGAAGCTGC?AGGAGCGGCT 1100

GGCCAAGCTG?GCCGGTGGTG?TCGCGGTCAT?CAAGGCCGGT?GCCGCCACCG 1150

ACGTCGAACT?CAAGGAGCGC?AAGCACCGCA?TCGAGGATGC?GGTTCGCAAT 1200

GCCAAGGCCG?CCGTCGAGGA?GGGCATCGTC?GCCGGTGGGG?GTGTGACGCT 1250

GTTGCAAGCG?GCCCCGACCC?TGGACGAGCT?GAAGCTCGAA?GGCGACGAGG 1300

CGACCGGCGC?CAACATCGTG?AAGGTGGCGC?TGGAGGCCCC?GCTGAAGCAG 1350

ATCGCCTTCA?ACTCCGGGCT?GGAGCCGGGC?GTGGTGGCCG?AGAAGGTGCG 1400

CAACCTGCCG?GCTGGCCACG?GACTGAACGC?TCAGACCGGT?GTCTACGAGG 1450

ATCTGCTCGC?TGCCGGCGTT?GCTGACCCGG?TCAAGGTGAC?CCGTTCGGCG 1500

CTGCAGAATG?CGGCGTCCAT?CGCGGGGCTG?TTCCTGACCA?CCGAGGCCGT 1550

CGTTGCCGAC?AAGCCGGAAA?AGGAGAAGGC?TTCCGTTCCC?GGTGGCGGCG 1600

ACATGGGTGG?CATGGATTTC?CATATGGCTA?GCGAATTC

The agarose gel electrophoresis of the HSP65 gene that obtains with PCR is seen Fig. 8

5, construction recombination plasmid pET28a-HSP65

Digest pET28a (Invitrogen) plasmid and the pMD-18T-HSP65 plasmid that carries the HSP65 gene simultaneously with NCoI and EcoRI, reclaim HSP65 gene and linearizing pET28a plasmid respectively, use T ₄Dna ligase with after linearizing pET28a plasmid is connected, forms the pET28a-HSP65 recombinant plasmid with the HSP65 gene.With pET28a-HSP65 recombinant plasmid transformed host bacterium JM109, get positive transformed bacteria and cultivate, extract plasmid.After EcoRI and NCoI enzymic digestion, in 2% agarose gel electrophoresis, positive colony is identified that plasmid discharges about 1638bp fragment after with enzymic digestion.

Embodiment 4 reorganization SARS/3CL _161-264The structure of epitope antigen gene recombinant mammalian expressing vector

Digest the pMD-18T-SARS/3CL that embodiment 1 makes up simultaneously with EcoRI and XhoI _161-264Plasmid and the pcDNA3-GFP plasmid that carries the GFP gene reclaim SARS/3CL respectively _161-264Gene fragment and linearizing pcDNA3-GFP plasmid are used T ₄Dna ligase is with SARS/3CL _161-264Gene fragment forms recombinant plasmid pcDNA3-GFP-SARS/3CL with after linearizing pcDNA3-GFP plasmid is connected _161-264, use recombinant plasmid pcDNA3-GFP-SARS/3CL _161-264Transform host bacterium JM109, get positive transformed bacteria and cultivate, extract plasmid.After enzymic digestion, in 2% agarose gel electrophoresis, positive colony is identified pcDNA3-GFP-SARS/3CL _161-264Plasmid discharges about 1100bp fragment after with the single enzymic digestion of HindIII.Enzyme is cut the evaluation electrophoresis and is seen Fig. 9.The result of dna sequencing shows the construction of recombinant plasmid success.

Embodiment 5SARS/3CL _161-264The preparation of gene transfection murine melanoma B16 cell

Though SARS Cov can vitro culture, hazardness that should virus is very big, can only be in having the laboratory of special equipment amplification cultivation.Owing to there is not to be fit to the virus infected cell model of the daily research usefulness in other laboratories, therefore, we utilize the recombinant mammalian expressing vector pcDNA3-GFP that carries green fluorescent protein GFP gene, synthetic SARS/3CL _161-264Gene constructed in the downstream of GFP gene, make GFP and SARS/3CL _161-264Gene is in together in the reading frame, has obtained reorganization pcDNA3-GFP-SARS/3CL _161-264Recombinant mammalian expressing vector.With this plasmid transfection mouse melanin tumor cell strain B16, obtained expression GFP-SARS/3CL _161-264Transfectional cell, this cell is detected HSP65-SARS/3CL by us as the cell model that SARS Cov infects _161-264Activity.

1, the transfection of cell

(1) prepare following reagent:

Solution A: the 100 μ l serum-free IMDM that contain 1-2 μ g plasmid DNA.

Solution B: the 100 μ l serum-free IMDM that contain 10-20 μ l Liposome reagent.

(2) solution A slowly is added in the solution B, mixes gently, room temperature (being generally about 20-25 ℃) is hatched 45min, and floss may occur in the mixed solution this moment, is normal phenomenon.

(3), cell is washed once between incubation period at mixed solution with 2ml serum-free IMDM nutrient solution.

(4) 0.8ml serum-free IMDM nutrient solution is added in the A/B mixed solution, mixes gently.

(5) the A/B mixed solution is joined in the culture hole that contains cell gently.

(6) 37 ℃, 5%CO ₂Cultivated 5 hours.

(7) directly in culture hole, add the IMDM that 1ml contains 20%FBS, continue to cultivate 18-24 hour.

(8) abandon nutrient solution, add the fresh IMDM that 2ml contains 10%FBS, continue to cultivate 48-72 hour, collecting cell, the expression that is used for recombinant protein is identified.

2, pcDNA3-GFP-SARS/3CL _161-264The evaluation of transfection murine melanoma B16 cell

Collect pcDNA3-GFP-SARS/3CL _161-264The B16 cell of transfection is identified stable transfection GFP-SARS/3CL to positive colony with Laser Scanning Confocal Microscope and FACS technology _161-264The murine melanoma B16 cell of gene cell under Laser Scanning Confocal Microscope presents green, and the FACS detected result shows in the transfectional cell stronger green fluorescence, and qualification result is seen Figure 10.

Embodiment 6HSP65-SARS/3CL _161-264The structure of fusion gene, expression and purifying

1, fusion protein expression plasmid pET28a-HSP65-SARS/3CL _161-264Structure:

The pMD-18T-SARS/3CL that embodiment 1 makes up _161-264Plasmid reclaims the dna fragmentation of 345bp after EcoRI and the two enzymic digestions of HindIII.Subclone is connected to the pET28a-HSP65 linear carrier with same digestion with restriction enzyme and recovery.The experimental technique literary composition that sees before.The subclone recombinant plasmid after the two enzymic digestions of EcoRI and HindIII, electrophoresis in 1% sepharose, as seen about 345bp fragment (Figure 11), dna sequencing confirms to express plasmid construction successfully.Fusion rotein HSP65-SARS/3CL _161-264Nucleotide sequence shown in SEQ ID NO:5, its amino acid sequence corresponding is shown in SEQ ID NO:6.

2, HSP65-SARS/3CL _161-264Expression and purifying

Adopt 10 liters fermentor cultivation to contain HSP65-SARS/3CL _161-264The intestinal bacteria BL DE 3 (Novagen) of encoding gene expression plasmid is an inductor with the lactose.After splitting bacterium, behind nickel affinity chromatography, desalination, ion exchange chromatography, desalination, obtain HSP65-SARS/3CL _161-264Get that sample carries out 12%SDS-PAGE on the 10 μ l samples, behind the coomassie brilliant blue staining, HSP65-SARS/3CL _161-264Appear as a single district band, do not have assorted band.Get 10 μ l samples and be HPLC analysis, HSP65-SARS/3CL _161-264Purity be 96.1%.HSP65-SARS/3CL _161-264SDS-PAGE and Western Blot identify and to see Figure 12.

The specificity growth-inhibiting that embodiment 7 infects the SARS virus cell

1, method: the HSP65-SARS/3CL that sets up PBS control group and 5,15,30 μ g various dose _161-264Experimental group, every group of 8 6-8 age in week, female C57BL/6 mouse.Gave experimental group and control group mice inoculation transfection in the 0th day

SARS/3CL _161-264The B16 cell, 1.5 * 10 ⁵Individual cell/mouse, inoculation position are that the right side, back of mouse is subcutaneous.In the 2nd day and the 16th day, inject HSP65-SARS/3CL respectively for 3 experimental mice _161-264, injected dose is respectively 5,15,30 μ g, and the injection site is that the mouse four limbs are entad held subcutaneous.Control group mice injection PBS.In the 25th day, put to death the mouse of bearing tumor, the separation tumour is also weighed.

2, experimental result: compare the HSP65-SARS/3CL of 3 various dose with PBS group mouse _161-264SARS/3CL in the experimental mice body _161-264The positive tumor growth is slow, and volume is little, and weight in average is 0.52g, and the weight in average of PBS group mouse tumor is 4.01g.HSP65-SARS/3CL _161-264Experimental mice and PBS control group mice tumor weight relatively see Figure 14.Experimental result shows: HSP65-SARS/3CL _161-264At the mouse vivo activation SARS/3CL _161-264The specific CTL of epitope antigen has suppressed the SARS/3CL that expresses _161-264The growth of epitope antigen cell (Figure 13).

3, conclusion: HSP65-SARS/3CL _161-264Can suppress mouse expression in vivo SARS/3CL _161-264The growth of cell.HSP65-SARS/3CL _161-264Can suppress to infect the growth of SARS virus cell.

Embodiment 8 mouse SARS/3CL _161-264Inducing of specific CTL

1, method: set up HSP65-SARS/3CL _161-264Experimental group and PBS control group, every group of 8 6-8 age in week, female C57BL/6 mouse in the 0th, 14,21 day, are given experimental mice immunity HSP65-SARS/3CL _161-264, it is subcutaneous that immune position is that the mouse four limbs are entad held, and immunizing dose is every mouse 10 μ gHSP65-SARS/3CL _161-264To control group mice injection PBS, the same experimental mice in injection site.Immunity finishes back the 5th day, separates the splenocyte action effect cell of mouse, transfection SARS/3CL _161-264B16 cell and untransfected SARS/3CL _161-264The B16 cell as target cell, carry out the CTL fragmentation test according to a conventional method.

2, result: compare HSP65-SARS/3CL with PBS group mouse _161-264Immune group mice spleen T lymphocyte can kill and wound transfection SARS/3CL specifically _161-264Target cell, and can not kill and wound untransfected SARS/3CL _161-264Target cell.This shows HSP65-SARS/3CL _161-264Immunity has produced at expressing SARS/3CL mouse _161-264The CTL of epitope antigen cell.

3, conclusion: HSP65-SARS/3CL _161-264Can induce mouse SARS/3CL _161-264Specific CTL.

HSP65-SARS/3CL _161-264The CTL that induces can kill and wound the SARS virus cells infected specifically.

Figure 15 expresses SARS/3CL _161-264The B16 cells in vitro specific killing experimental result of epitope antigen.

Embodiment 9HSP65-SARS/3CL _161-264To the lymphocytic hormesis of mouse memory

The virus antigen of invasion body can inductor in the lymphocyte of memory, when same antigen stimulates immune anamnestic reaction can take place when body runs into once more.

In order to observe HSP65-SARS/3CL _161-264Whether have such function, get two respectively through HSP65-SARS/3CL _161-264The lymph-node cell of immune mouse and two PBS injection mouse is made the lymphocyte suspension, sets up HSP65-SARS/3CL _161-264Albumen, SARS/3CL _161-2643 groups of synthetic epitope peptide, nutrient solutions add HSP65-SARS/3CL respectively _161-264Albumen, SARS/3CL _161-264Synthetic epitope peptide, nutrient solution.Cultivate after 72 hours, every hole adds 1 μ Ci ³H-TdR continues to cultivate 16 hours, and collecting cell is measured ³The H-TdR incorporation.The result shows that the lymph-node cell of protein immunization group is at HSP65-SARS/3CL _161-264Albumen, SARS/3CL _161-264Under the stimulation of synthetic epitope peptide A, B and C ³The H-TdR incorporation is higher than the nutrient solution control group, and the result shows lymphocytic specificity hyperplasia has taken place. ³H-TdR mixes result of experiment and is analyzed as follows: 1. peptide A and PBS group, medium group relatively have notable difference (P＜0.05) 2. peptide B and PBS group, medium group relatively have a notable difference (P＜0.01) 3. peptide C organizes with PBS, medium organizes notable difference (P＜0.05) is relatively arranged.

The result shows HSP65-SARS/3CL _161-264Lymphocyte that can the inducing mouse memory, these lymphocytes can be discerned us by prediction synthetic A, B, a C3 epitope peptide external.(Figure 16)

Embodiment 10 the external of people CTL induce

(Ali method):

One, separation of human peripheral blood lymphocytes

1. in the 50ml centrifuge tube, add the 12.5ml urografic acid methylglucamine salt.

2. drawing 50ml PBS/EDTA damping fluid moves in the flask.

3. the blood bag is after 70% ethanol wiping, and wherein blood is moved in the flask that contains the PBS/EDTA damping fluid mixing.

4. drawing the immigration of 25ml mixed solution from flask contains in the 50ml test tube of 12.5ml urografic acid methylglucamine salt.Note not destroying the interface of urografic acid methylglucamine salt and mixed solution.

The above-mentioned test tube that contains mixed solution is centrifugal 5., room temperature, 3000rpm/min (not apply the brakes) 20-25min.

6. after centrifugal, draw the interfacial layer cell, move in the new centrifuge tube.

7. contain to this that to add the PBS/EDTA damping fluid in centrifuge tube of branch confluent monolayer cells centrifugal to the 45ml, 1800rpm/min, 4 ℃, centrifugal 10min.This is flushing for the first time; When washing for the second time is 1200rpm/min, 4 ℃, and centrifugal 7min.

8. collect the cell mass of test tube bottom, add PBS/EDTA/ human serum damping fluid re-suspended cell.

9. the test tube that will contain cell is centrifugal, 1200rpm/min, 4 ℃, centrifugal 7min (need not brake).

10. with the cold PBS/EDTA/ human serum damping fluid re-suspended cell of 6ml, get 2 test tubes, respectively add the cold Percol of 6ml 52%.

11. drawing the 3ml cell suspension slowly moves into (not stirring Percol) and contains in the cold 52%Percol test tube of 6ml 2000rpm/min under no brake condition, 4 ℃, centrifugal 20min.

12. draw the interfacial layer cell, move in the new test tube; Other cells are moved into carry out the HLA somatotype in another pipe.

13. divide confluent monolayer cells with cold PBS/EDTA/ human serum damping fluid washing, 4 ℃, 1300rpm/inm does not have the centrifugal 10min of brake.

14. filter cause death and agglomerating branch confluent monolayer cells with nylon wire.

15. tally sheet karyocyte number.

Two, induce immature dendritic cell

1. with IMDM dilution monocyte, adjust cell count and reach 2 * 10 ⁶/ ml.

2. each hole adds 1ml cell suspension and 1mlIMDM in 12 orifice plates.In 37 ℃, 5%CO ₂Cultivate 2h in the incubator.

3.2h after, wash cell in 12 orifice plates with IMDM, with moving 12 orifice plates of have gentle hands jog, remove every Kong Zhongwei adherent cell.

4. add the IMDM that 2ml contains 100ng (the 200U)/GM-CSF of ml and the IL-4 of 200U/ml in every hole.

5. in 37 ℃, 5%CO ₂Cultivate 5d in the incubator, every 2-3d changes the IMDM nutrient solution one time.

Three, with reorganization HSP65-SARS/3CL _161-264Fusion rotein loads dendritic cell

In the 5th day with HSP65-SARS/3CL _161-264Directly be added on and make its final concentration reach 100 μ g/ml in the immature dendritic cell (DC), continue to cultivate 2d.

2. gather in the crops immature DC in 7d.

Four, from people's peripheral blood separation of C D8 ⁺The T lymphocyte

1. separate peripheral blood mononuclear cell (PBMC) from the human plasma leukocytic cream.

2. wash isolated mononuclearcell and counting with PBS/EDTA/ human serum damping fluid.

3. prepare antibody-solutions according to the cell quantity that obtains: antibody-solutions is with PBS/EDTA/ human serum damping fluid Antibody Preparation to be become 1: 1000 diluent, contained antibody is mouse anti human anti-CD4, anti-CD14, anti-CD56, anti-CD19, anti-CD45RO, HLA-DR, γ δ-TCR, per 20 * 10 ⁶Individual cell needs the 1ml antibody-solutions.

4. use the antibody-solutions re-suspended cell.

5. with cell and the antibody suspension is put in the cold house or 30min is cultivated in concussion on ice.

6. centrifugal cell harvesting will be resuspended in the cell of antibodies in the 25ml PBS/EDTA/ human serum damping fluid.

7. prepare anti-mouse IgG magnetic bead, magnetic bead is given a baby a bath on the third day after its birth inferior with PBS (pH7.4).

8. according to the ratio of cell/magnetic bead=1/4 cell is resuspended in and contains magnetic bead in vitro, put on ice and on two-way shaking table, cultivate 20min.

9. centrifugal results supernatant promptly contains needed CD8 in the supernatant ⁺The T lymphocyte, cell counting.

10. in 96 hole U templates, add 2 * 10 ⁵CD8 ⁺T lymphocyte/hole.

Five, with loading recombination fusion protein HSP65-SARS/3CL _161-264Dendritic cell stimulate CD8 ⁺The T lymphocyte

1. with CD8 ⁺T lymphocyte and loaded recombination fusion protein HSP65-SARS/3CL _161-264Dendritic cell (DCs) mix, cultivate 6d-7d.

2. repetitive stimulation CD8 according to the method described above ⁺T lymphocyte twice is cultivated 6d at every turn.

3.3 after week, results SARS/3CL _161-264Specific CD8 ⁺The T lymphocyte.

Six, T2 cell marking and SARS/3CL _161-264The loading of specific antigens peptide

1. contain in the culturing bottle of T2 cell, add ⁵¹Cr makes its final concentration reach 1 μ Ci/ml, will contain the T2 cell and ⁵¹The culturing bottle of Cr places 37 ℃, 5%CO ₂Cultivated 1-2 hour in the incubator.Shake mixing occasionally several times during this time, then with nutrient solution washed cell 3 times.

2. to containing mark ⁵¹In the T2 Tissue Culture Flask of Cr, add 10-100 μ g SARS/3CL respectively _161-264Epitope peptide A, B, C, in 37 ℃, 5%CO ₂Cultivated 1-2 hour in the incubator.

Seven, CTL analyzes

1. in 96 orifice plates, add effector cell and target cell with different effect target ratios.

2. set up control group:

Naturally discharge contrast target cell 50 μ l+ substratum 100 μ l

The maximum contrast target cell 50 μ l+10%Triton X-100 100 μ l that discharge

3. 96 orifice plates that added effector cell and target cell are centrifugal, 350r/min, 5min.

4. 96 orifice plates are placed 37 ℃, 5%CO ₂Cultivate 4-6h in the incubator.

5. cultivate the back and use Skkatron to filter system's harvested cell suspension, each sample is counted 1min on γ-counter.

6. calculate release rate (%) as follows:

Specific killing %=experimental port (cpm)-spontaneous release aperture (cpm)/maximum release aperture (cpm)-spontaneous release aperture (cpm) %

Eight, result

The result shows: load recombination fusion protein HSP65-SARS/3CL _161-264Dendritic cell can stimulate CD8 ⁺The T lymphocyte is expressed SARS/3CL at external specific killing _161-264Tumour cell (seeing Figure 17).

Sequence table

＜110〉Beijing DiWeiHuaYu Biological Technology Co., Ltd

＜120〉heat shock protein(HSP) 65-people sars coronavirus epitope antigen recombinant fusion protein (HSP65-SARS/3CL _161-264)

<160>16

<210>1

<211>312

<212>DNA

＜213〉people (Homo sapiens)

<400>1

tacatgcacc?acatggaact?gccgaccggt?gttcacgcag?gtactgacct?ggaaggtaaa 60

ttttatggtc?cgttcgttga?ccgtcagact?gcacaggctg?ctggtactga?cactactatc 120

actcttaatg?ttctggcttg?gctgtacgct?gctgttatta?acggtgaccg?ctggttcctg 180

aaccgcttta?ctactaccct?gaacgatttc?aacctggttg?ctatgaagta?caactacgaa 240

ccgctgactc?aagaccacgt?agacattctg?ggtccactgt?ctgctcagac?tggtatcgct 300

gttctggata?tg 312

<210>2

<211>104

<212>PRT

＜213〉people (Homo sapiens)

<400>2

Tyr?Met?His?His?Met?Glu?Leu?Pro?Thr?Gly?Val?His?Ala?Gly?Thr

5 10 15

Asp?Leu?Glu?Gly?Lys?Phe?Tyr?Gly?Pro?Phe?Val?Asp?Arg?Gln?Thr

20 25 30

Ala?Gln?Ala?Ala?Gly?Thr?Asp?Thr?Thr?Ile?Thr?Leu?Asn?Val?Leu

35 40 45

Ala?Trp?Leu?Tyr?Ala?Ala?Val?Ile?Asn?Gly?Asp?Arg?Trp?Phe?Leu

50 55 60

Asn?Arg?Phe?Thr?Thr?Thr?Leu?Asn?Asp?Phe?Asn?Leu?Val?Ala?Met

65 70 75

Lys?Tyr?Asn?Tyr?Glu?Pro?Leu?Thr?Gln?Asp?His?Val?Asp?Ile?Leu

80 85 90

Gly?Pro?Leu?Ser?Ala?Gln?Thr?Gly?Ile?Ala?Val?Leu?Asp?Met

95 100 104

<210>3

<211>333

<212>DNA

＜213〉artificial sequence

<400>3

gatgagtaca?tgcaccacat?ggaactgccg?accggtgttc?acgcaggtac?tgacctggaa 60

ggtaaatttt?atggtccgtt?cgttgaccgt?cagactgcac?aggctgctgg?tactgacact 120

actatcactc?ttaatgttct?ggcttggctg?tacgctgctg?ttattaacgg?tgaccgctgg 180

ttcctgaacc?gctttactac?taccctgaac?gatttcaacc?tggttgctat?gaagtacaac 240

tacgaaccgc?tgactcaaga?ccacgtagac?attctgggtc?cactgtctgc?tcagactggt 300

atcgctgttc?tggatatgga?ggacgacgaa?gac 333

<210>4

<211>111

<212>PRT

＜213〉artificial sequence

<400>4

Asp?Glu?Tyr?Met?His?His?Met?Glu?Leu?Pro?Thr?Gly?Val?His?Ala

5 10 15

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

20 25 30

Gln?Thr?Ala?Gln?Ala?Ala?Gly?Thr?Asp?Thr?Thr?Ile?Thr?Leu?Asn

35 40 45

Val?Leu?Ala?Trp?Leu?Tyr?Ala?Ala?Val?Ile?Asn?Gly?Asp?Arg?Trp

50 55 60

Phe?Leu?Asn?Arg?Phe?Thr?Thr?Thr?Leu?Asn?Asp?Phe?Asn?Leu?Val

65 70 75

Ala?Met?Lys?Tyr?Asn?Tyr?Glu?Pro?Leu?Thr?Gln?Asp?His?Val?Asp

80 85 90

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

95 100 105

Met?Glu?Asp?Asp?Glu?Asp

110?111

<210>5

<211>1977

<212>DNA

＜213〉artificial sequence

<400>5

atggccaaga?caattgcgta?cgacgaagag?gcccgtcgcg?gcctcgagcg?gggcttgaac 60

gccctcgccg?atgcggtaaa?ggtgacattg?ggccccaagg?gccgcaacgt?cgtcctggaa 120

aagaagtggg?gtgcccccac?gatcaccaac?gatggtgtgt?ccatcgccaa?ggagatcgag 180

ctggaggatc?cgtacgagaa?gatcggcgcc?gagctggtca?aagaggtagc?caagaagacc 240

gatgacgtcg?ccggtgacgg?caccacgacg?gccaccgtgc?tggcccaggc?gttggttcgc 300

gagggcctgc?gcaacgtcgc?ggccggcgcc?aacccgctcg?gtctcaaacg?cggcatcgaa 360

aaggccgtgg?agaaggtcac?cgagaccctg?ctcaagggcg?ccaaggaggt?cgagaccaag 420

gagcagattg?cggccaccgc?agcgatttcg?gcgggtgacc?agtccatcgg?tgacctgatc 480

gccgaggcga?tggacaaggt?gggcaacgag?ggcgtcatca?ccgtcgagga?gtccaacacc 540

tttgggctgc?agctcgagct?caccgagggt?atgcggttcg?acaagggcta?catctcgggg 600

tacttcgtga?ccgacccgga?gcgtcaggag?gcggtcctgg?aggaccccta?catcctgctg 660

gtcagctcca?aggtgtccac?tgtcaaggat?ctgctgccgc?tgctcgagaa?ggtcatcgga 720

gccggtaagc?cgctgctgat?catcgccgag?gacgtcgagg?gcgaggcgct?gtccaccctg 780

gtcgtcaaca?agatccgcgg?caccttcaag?tcggtggcgg?tcaaggctcc?cggcttcggc 840

gaccgccgca?aggcgatgct?gcaggatatg?gccattctca?ccggtggtca?ggtgatcagc 900

gaagaggtcg?gcctgacgct?ggagaacgcc?gacctgtcgc?tgctaggcaa?ggcccgcaag 960

gtcgtggtca?ccaaggacga?gaccaccatc?gtcgagggcg?ccggtgacac?cgacgccatc 1020

gccggacgag?tggcccagat?ccgccaggag?atcgagaaca?gcgactccga?ctacgaccgt 1080

gagaagctgc?aggagcggct?ggccaagctg?gccggtggtg?tcgcggtcat?caaggccggt 1140

gccgccaccg?acgtcgaact?caaggagcgc?aagcaccgca?tcgaggatgc?ggttcgcaat 1200

gccaaggccg?ccgtcgagga?gggcatcgtc?gccggtgggg?gtgtgacgct?gttgcaagcg 1260

gccccgaccc?tggacgagct?gaagctcgaa?ggcgacgagg?cgaccggcgc?caacatcgtg 1320

aaggtggcgc?tggaggcccc?gctgaagcag?atcgccttca?actccgggct?ggagccgggc 1380

gtggtggccg?agaaggtgcg?caacctgccg?gctggccacg?gactgaacgc?tcagaccggt 1440

gtctacgagg?atctgctcgc?tgccggcgtt?gctgacccgg?tcaaggtgac?ccgttcggcg 1500

ctgcagaatg?cggcgtccat?cgcggggctg?ttcctgacca?ccgaggccgt?cgttgccgac 1560

aagccggaaa?aggagaaggc?ttccgttccc?ggtggcggcg?acatgggtgg?catggatttc 1620

catatggcta?gcgaattcga?tgagtacatg?caccacatgg?aactgccgac?cggtgttcac 1680

gcaggtactg?acctggaagg?taaattttat?ggtccgttcg?ttgaccgtca?gactgcacag 1740

gctgctggta?ctgacactac?tatcactctt?aatgttctgg?cttggctgta?cgctgctgtt 1800

attaacggtg?accgctggtt?cctgaaccgc?tttactacta?ccctgaacga?tttcaacctg 1860

gttgctatga?agtacaacta?cgaaccgctg?actcaagacc?acgtagacat?tctgggtcca 1920

ctgtctgctc?agactggtat?cgctgttctg?gatatggagg?acgacgaaga?caagctt 1977

poly-histag

<210>6

<211>657

<212>PRT

＜213〉artificial sequence

<400>6

Met?Ala?Lys?Thr?Ile?Ala?Tyr?Asp?Glu?Glu?Ala?Arg?Arg?Gly?Leu

5 10 15

Glu?Arg?Gly?Leu?Asn?Ala?Leu?Ala?Asp?Ala?Val?Lys?Val?Thr?Leu

20 25 30

Gly?Pro?Lys?Gly?Arg?Asn?Val?Val?Leu?Glu?Lys?Lys?Trp?Gly?Ala

35 40 45

Pro?Thr?Ile?Thr?Asn?Asp?Gly?Val?Ser?Ile?Ala?Lys?Glu?Ile?Glu

50 55 60

Leu?Glu?Asp?Pro?Tyr?Glu?Lys?Ile?Gly?Ala?Glu?Leu?Val?Lys?Glu

65 70 75

Val?Ala?Lys?Lys?Thr?Asp?Asp?Val?Ala?Gly?Asp?Gly?Thr?Thr?Thr

80 85 90

Ala?Thr?Val?Leu?Ala?Gln?Ala?Leu?Val?Arg?Glu?Gly?Leu?Arg?Asn

95 100 105

Val?Ala?Ala?Gly?Ala?Asn?Pro?Leu?Gly?Leu?Lys?Arg?Gly?Ile?Glu

110 115 120

Lys?Ala?Val?Glu?Lys?Val?Thr?Glu?Thr?Leu?Leu?Lys?Gly?Ala?Lys

125 130 135

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

140 145 150

Ala?Gly?Asp?Gln?Ser?Ile?Gly?Asp?Leu?Ile?Ala?Glu?Ala?Met?Asp

155 160 165

Lys?Val?Gly?Asn?Glu?Gly?Val?Ile?Thr?Val?Glu?Glu?Ser?Asn?Thr

170 175 180

Phe?Gly?Leu?Gln?Leu?Glu?Leu?Thr?Glu?Gly?Met?Arg?Phe?Asp?Lys

185 190 195

Gly?Tyr?Ile?Ser?Gly?Tyr?Phe?Val?Thr?Asp?Pro?Glu?Arg?Gln?Glu

200 205 210

Ala?Val?Leu?Glu?Asp?Pro?Tyr?Ile?Leu?Leu?Val?Ser?Ser?Lys?Val

215 220 225

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

230 235 240

Ala?Gly?Lys?Pro?Leu?Leu?Ile?Ile?Ala?Glu?Asp?Val?Glu?Gly?Glu

245 250 255

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

260 265 270

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

175 280 285

Met?Leu?Gln?Asp?Met?Ala?Ile?Leu?Thr?Gly?Gly?Gln?Val?Ile?Ser

290 295 300

Glu?Glu?Val?Gly?Leu?Thr?Leu?Glu?Asn?Ala?Asp?Leu?Ser?Leu?Leu

305 310 315

Gly?Lys?Ala?Arg?Lys?Val?Val?Val?Thr?Lys?Asp?Glu?Thr?Thr?Ile

320 325 330

Val?Glu?Gly?Ala?Gly?Asp?Thr?Asp?Ala?Ile?Ala?Gly?Arg?Val?Ala

335 340 345

Gln?Ile?Arg?Gln?Glu?Ile?Glu?Asn?Ser?Asp?Ser?Asp?Tyr?Asp?Arg

350 355 360

Glu?Lys?Leu?Gln?Glu?Arg?Leu?Ala?Lys?Leu?Ala?Gly?Gly?Val?Ala

365 370 375

Val?Ile?Lys?Ala?Gly?Ala?Ala?Thr?Asp?Val?Glu?Leu?Lys?Glu?Arg

380 385 390

Lys?His?Arg?Ile?Glu?Asp?Ala?Val?Arg?Asn?Ala?Lys?Ala?Ala?Val

395 400 405

Glu?Glu?Gly?Ile?Val?Ala?Gly?Gly?Gly?Val?Thr?Leu?Leu?Gln?Ala

410 415 420

Ala?Pro?Thr?Leu?Asp?Glu?Leu?Lys?Leu?Glu?Gly?Asp?Glu?Ala?Thr

425 430 435

Gly?Ala?Asn?Ile?Val?Lys?Val?Ala?Leu?Glu?Ala?Pro?Leu?Lys?Gln

440 445 450

Ile?Ala?Phe?Asn?Ser?Gly?Leu?Glu?Pro?Gly?Val?Val?Ala?Glu?Lys

455 460 465

Val?Arg?Asn?Leu?Pro?Ala?Gly?His?Gly?Leu?Asn?Ala?Gln?Thr?Gly

470 475 480

Val?Tyr?Glu?Asp?Leu?Leu?Ala?Ala?Gly?Val?Ala?Asp?Pro?Val?Lys

485 490 495

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

500 505 510

Phe?Leu?Thr?Thr?Glu?Ala?Val?Val?Ala?Asp?Lys?Pro?Glu?Lys?Glu

515 520 525

Lys?Ala?Ser?Val?Pro?Gly?Gly?Gly?Asp?Met?Gly?Gly?Met?Asp?Phe

530 535 540

His?Met?Ala?Ser?Glu?Phe?Asp?Glu?Tyr?Met?His?His?Met?Glu?Leu

545 550 555

Pro?Thr?Gly?Val?His?Ala?Gly?Thr?Asp?Leu?Glu?Gly?Lys?Phe?Tyr

560 565 570

Gly?Pro?Phe?Val?Asp?Arg?Gln?Thr?Ala?Gln?Ala?Ala?Gly?Thr?Asp

575 580 585

Thr?Thr?Ile?Thr?Leu?Asn?Val?Leu?Ala?Trp?Leu?Tyr?Ala?Ala?Val

590 595 600

Ile?Asn?Gly?Asp?Arg?Trp?Phe?Leu?Asn?Arg?Phe?Thr?Thr?Thr?Leu

605 610 615

Asn?Asp?Phe?Asn?Leu?Val?Ala?Met?Lys?Tyr?Asn?Tyr?Glu?Pro?Leu

620 625 630

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

635 640 645

Gly?Ile?Ala?Val?Leu?Asp?Met?Glu?Asp?Asp?Glu?Asp?poly-histag

650 655 657

<210>7

<211>57

<212>DNA

＜213〉artificial sequence

<400>7

actatcactc?ttaatgttct?ggcttggctg?tacgctgctg?ttattaacgg?tgaccgc 57

<210>8

<211>57

<212>DNA

＜213〉artificial sequence

<400>8

gttgaaatcg?ttcagggtag?tagtaaagcg?gttcaggaac?cagcggtcac?cgttaat 57

<210>9

<211>58

<212>DNA

＜213〉artificial sequence

<400>9

ttcgttgacc?gtcagactgc?acaggctgct?ggtactgaca?ctactatcac?tcttaatg 58

<210>10

<211>57

<212>DNA

＜213〉artificial sequence

<400>10

gtcttgagtc?agcggttcgt?agttgtactt?catagcaacc?aggttgaaat?cgttcag 57

<210>11

<211>57

<212>DNA

＜213〉artificial sequence

<400>11

gttcacgcag?gtactgacct?ggaaggtaaa?ttttatggtc?cgttcgttga?ccgtcag 57

<210>12

<211>57

<212>DNA

＜213〉artificial sequence

<400>12

gataccagtc?tgagcagaca?gtggacccag?aatgtctacg?tggtcttgag?tcagcgg 57

<210>13

<211>58

<212>DNA

＜213〉artificial sequence

<400>13

gaattcgatg?agtacatgca?ccacatggaa?ctgccgaccg?gtgttcacgc?aggtactg 58

<210>14

<211>63

<212>DNA

＜213〉artificial sequence

<400>14

ctcgagtcac?taaagcttgt?cttcgtcgtc?ctccatatcc?agaacagcga?taccagtctg 60

agc 63

<210>15

<211>20

<212>DNA

＜213〉artificial sequence

<400>15

ccatggccaa?gacaattgca 20

<210>16

<211>39

<212>DNA

＜213〉artificial sequence

<400>16

accgaattcg?ctagccatat?ggaaatccat?gccacccat 39

Claims (20)

1. recombination fusion protein, this recombination fusion protein are to be merged and the fusion rotein that forms by heat shock protein(HSP) 65 (HSP65) and people's sars coronavirus epitope antigen polypeptide of optimizing.

2. the described recombination fusion protein of claim 1, wherein said single copy people sars coronavirus epitope antigen contain and are selected from following arbitrary aminoacid sequence:

(1) aminoacid sequence shown in the SEQ ID NO:2;

(2) homology of the aminoacid sequence shown in its aminoacid sequence and the SEQ ID NO:2 is greater than 80%;

(3) its nucleotide coding sequence can be hybridized with the nucleotide coding sequence of the aminoacid sequence shown in the coding SEQ ID NO:2 under the nucleic acid hybridization condition;

(4) comprise that those carry out artificial point mutation to the sequence shown in the SEQ ID NO:2 or insert other sequence or transform the resulting sequence in back in the mode that both sides connect other sequence.

3. the described recombination fusion protein of claim 1, wherein said single copy people sars coronavirus epitope antigen contain and are selected from following arbitrary nucleotide sequence:

(1) nucleotide sequence shown in the SEQ ID NO:1;

(2) nucleotide sequence that has degeneracy with the nucleotide sequence of SEQ ID NO:1;

(3) nucleotide sequence that has 70% above homology with the nucleotide sequence of SEQ ID NO:1;

(4) nucleotide sequence that can under the nucleic acid hybridization condition, hybridize with the nucleotide sequence of SEQ ID NO:1;

(5) comprise that those carry out artificial point mutation to the sequence shown in the SEQ ID NO:1 or insert other sequence or transform the resulting sequence in back in the mode that both sides connect other sequence.

4. the described recombination fusion protein of claim 1, single copy people sars coronavirus epitope antigen of described optimization contain and are selected from following arbitrary aminoacid sequence:

(1) aminoacid sequence shown in the SEQ ID NO:4;

(2) homology of the aminoacid sequence shown in its aminoacid sequence and the SEQ ID NO:4 is greater than 80%;

(3) its nucleotide coding sequence can be hybridized with the nucleotide coding sequence of the aminoacid sequence shown in the coding SEQ ID NO:4 under the nucleic acid hybridization condition;

(4) comprise that those carry out artificial point mutation to the sequence shown in the SEQ ID NO:4 or insert other sequence or transform the resulting sequence in back in the mode that both sides connect other sequence.

5. the described recombination fusion protein of claim 1, single copy people sars coronavirus epitope antigen of wherein said optimization contain and are selected from following arbitrary nucleotide sequence:

(1) nucleotide sequence shown in the SEQ ID NO:3;

(2) nucleotide sequence that has degeneracy with the nucleotide sequence of SEQ ID NO:3;

(3) nucleotide sequence that has 70% above homology with the nucleotide sequence of SEQ ID NO:3;

(4) nucleotide sequence that can under the nucleic acid hybridization condition, hybridize with the nucleotide sequence of SEQ ID NO:3;

(5) comprise that those carry out artificial point mutation to the sequence shown in the SEQ ID NO:3 or insert other sequence or transform the resulting sequence in back in the mode that both sides connect other sequence.

6. the described recombination fusion protein of claim 1, it contains and is selected from following arbitrary aminoacid sequence:

(1) aminoacid sequence shown in the SEQ ID NO:6;

(2) homology of the aminoacid sequence shown in its aminoacid sequence and the SEQ ID NO:6 is greater than 80%;

(3) its nucleotide coding sequence can be hybridized with the nucleotide coding sequence of the aminoacid sequence shown in the coding SEQ ID NO:6 under the nucleic acid hybridization condition;

(4) comprise that those carry out artificial point mutation to the sequence shown in the SEQ ID NO:6 or insert other sequence or transform the resulting sequence in back in the mode that both sides connect other sequence.

7. according to the described recombination fusion protein of claim 1, people's sars coronavirus epitope antigen of wherein said optimization can be 1-5 copy.

8. according to the described recombination fusion protein of claim 1, wherein HSP65 is positioned at the aminoterminal of this recombination fusion protein, and people's sars coronavirus epitope antigen is positioned at the carboxyl terminal of this recombination fusion protein.

9. the nucleotide sequence of any described recombination fusion protein in the claim 1 to 8 of encoding.

10. according to the described nucleotide sequence of claim 9, it contains and is selected from following arbitrary sequence:

(1) nucleotide sequence shown in the SEQ ID NO:5;

(2) nucleotide sequence that has degeneracy with the nucleotide sequence of SEQ ID NO:5;

(3) nucleotide sequence that has 70% above homology with the nucleotide sequence of SEQ ID NO:5;

(4) nucleotide sequence that can under the nucleic acid hybridization condition, hybridize with the nucleotide sequence of SEQ ID NO:5;

(5) comprise that those carry out artificial point mutation to the sequence shown in the SEQ ID NO:5 or insert other sequence or transform the resulting sequence in back in the mode that both sides connect other sequence.

11. contain the carrier of each nucleotide sequence among the claim 9-10.

12. according to the carrier of claim 11, it is a plasmid vector.

13. contain the host cell of any described carrier among each described nucleotide sequence among the claim 9-10 or the claim 11-12.

14. according to the host cell of claim 13, it is a prokaryotic cell prokaryocyte.

15. according to the host cell of claim 14, wherein said prokaryotic cell prokaryocyte is intestinal bacteria.

16. according to the host cell of claim 13, it is an eukaryotic cell.

17. according to the host cell of claim 16, wherein said eukaryotic cell is the B16 cell.

18. the method for any recombination fusion protein among the production claim 1-8 is included in and cultivates any one host cell among the claim 13-17 under the conditions suitable, and reclaims required recombination fusion protein.

19. recombinant protein vaccine goods, it comprises each described recombination fusion protein and pharmaceutically acceptable carrier or vehicle among the claim 1-8 of significant quantity.

20. among the claim 1-8 among any one recombination fusion protein, the claim 9-10 among any one nucleotide sequence, the claim 11-12 among any one carrier and/or the claim 13-17 any one host cell be used for the treatment of and/or prevent medicine that SARS virus infects and/or the application in the reagent in preparation.

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