CN100484570C - Gene vaccine against SARS virogene and its construction and use - Google Patents
Gene vaccine against SARS virogene and its construction and use Download PDFInfo
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- CN100484570C CN100484570C CNB2004100394599A CN200410039459A CN100484570C CN 100484570 C CN100484570 C CN 100484570C CN B2004100394599 A CNB2004100394599 A CN B2004100394599A CN 200410039459 A CN200410039459 A CN 200410039459A CN 100484570 C CN100484570 C CN 100484570C
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Abstract
The present invention relates to a gene vaccine against SARS virus, which is composed of SARS virus N albumen of 26386-227054 fragment code having login number is AY278487 in gene library, M albumen of 28120-29388 fragment code and RNA polyase gene cDNA of 2486-2935 fragment code and mammalian expression carrier pVAX1. The construction of the vaccine includes: choosing single bacterial colony, swinging bacterium, extracting clone carrier plasmid containing N albumen containing SARS virus, M albumen and RNA polyase gene cDNA fragment; designing primer, cloning the N albumen, M albumen and RNA polyase gene cDNA fragment by polyase chain manner expand method; purifying them and recombining them to mammalian special expression carrier pVAX1 to construct gene vaccine against SARS virus-pVAX1-SARS-MNRna vaccine. Compared with existing vaccine, the vaccine needs smaller amount for achieving the same immune effect, and can excite the humoral immunity and cell immunity and stronger immune effect than other immune means, and it is safety, long effect, stable and convenient in operation and can be used for preparing the medicine for defending and curing SARS.
Description
Technical field
The present invention relates to a kind of gene vaccine, more particularly, the present invention relates to the anti-SARS gene vaccine of a kind of N of containing albumen (Nucleocapsidphosphoprotein nucleocapsid phosphoprotein), M albumen (Membrane glycoprotein membrane glycoprotein) and rna polymerase gene segment cDNA.
Technical background
Human prevention of disease is specially finished with the specific immune system by non-.Along with developing rapidly of gene recombination technology and genophore transmission system, there were four laboratory (Robinson in the U.S. in 1992, Johoston, Li, David) developmental research has simultaneously gone out the blank of dna vaccination (being gene vaccine), and reports simultaneously in the vaccinology new development conference that in JIUYUE, 1992 is held.From then on the new technique of dna vaccination comes out and to cause and the great attention of countries in the world be considered as a revolution in vaccine research field by people.NIH (NIH) research work of subsidizing to carry out dna vaccination treatment HIV in 1994 with the specific project expenditure of the biological strategic development plan of country.Drugs approved by FDA in 1994 carry out the clinical trial of dna vaccination treatment AIDS at human body.May nineteen ninety-five, reported the clinical trial of the first in the world example dna vaccination, The World Health Organization's appropriation is included dna vaccination in the WHO immune development plan in the whole world.1996 Nian doctors inject healthy philtrum with new gene (coding HIV or influenza proteins matter) first, rather than inject the philtrum of having suffered from certain disease.Present five kinds of dna vaccinations of FDA approved enter clinical, and these five kinds of vaccines are: HIV (human immunodeficiency virus) vaccine, influenza virus vaccine, Hepatitis B virus vaccine, herpes simplex virus (HSV) vaccine and plasmodium virus vaccine.
Dna vaccination is an emerging a kind of new technique that carries out immunity with the recombinant DNA form nineties.The core of technology is that the DNA sequence with coding for antigens is assembled into and contains constructed dna vaccine in the carrier for expression of eukaryon that is necessary expression regulation element, in different ways recombiant plasmid is imported in the animal body then, the dna vaccination of reorganization can utilize the synthetic exogenous gene encoded protein of the intravital enzyme of host system, thereby bring out the host this foreign protein is produced immunne response, expression product both can activate humoral immunization, again can the active cell immunity, thereby can reach the effect of prevention and treatment disease, moreover, this new technique has also been avoided protein vaccine complicated and expensive technology in preparation process, has safety, long-acting, stable, characteristics such as easy and simple to handle.Therefore vaccine DNA has the potential advantages that other immunization method hardly matches.In recent years America NI H subsidizes the research of carrying out dna vaccination treatment major disease with the specific project expenditure of the biological strategic development plan of country.European Union's the 5th framework in 2002, European Union's the 6th framework life sciences part in 2003 all develop the problem of genetic immunization technology treatment tumor, AIDS with preferential subsidy.The revolution of vaccine for the third time that dna vaccination causes can't prevent for a long time for some or the prevention and the treatment of the unfavorable infectious disease of preventive effect bring hope.Many animal models and human experimentation have proved that all dna vaccination can excite effective humoral immunoresponse(HI) and cellullar immunologic response both can activate humoral immunization, again can the active cell immunity, show dna immunization technique, can not only be by producing the antibody prophylaxis of viral infections, the more important thing is that dna vaccination can induce extremely the effectively ability of specificity T killer cell, remove infected body cell, reach the purpose of treatment, and the reaction of being free from side effects, this point is the most tempting part of this technology, also fully demonstrates the great potential and the application prospect of dna vaccination.
Dna vaccination is compared with vaccine commonly used, has remarkable advantages, and at first the required immunity amount of genetic immunization is less, and the 20-100 microgram gets final product.The immunoreation that next genetic immunization and viral infection bring out is quite similar, but genetic immunization can efficiently excite humoral immunization and cellular immunization simultaneously, and is stronger than other immune means immune efficacy.The 3rd genetic immunization safety, long-acting (immunocompetence has long memory), stable, simple operation.It can produce protection antibody by the bone-marrow-derived lymphocyte immune induction, can form more efficiently T lymphocyte immunity again.
SARS virus is a kind of novel coronavirus (being RNA viruses), behind the infection human body, and the active drug of also not treating SARS at present.Now, scientist is studying SARS both at home and abroad, in the hope of finding the anti-SARS vaccin method of prevention SARS virus early.Inactivated vaccine that has just under study for action and attenuated vaccine, inactivated vaccine does not wherein solve the problem of immunopathogenesis aspect, and some inactivation of viruses enters the people and knows from experience to increase the weight of the state of an illness when poisoning intrusion usually; Attenuated vaccine then will be taken makes virus breeding many generations for a long time, and subduction virus gradually is most of active then, can not satisfy the demand, and this vaccine is also improper to the immunoprophylaxis SARS virus.WHO expert thinks: but the desirable vaccine of SARS should be efficiently, mass production that be perfectly safe, that be applicable to all kinds of crowds and be convenient to accumulating and convenient the use.Dna vaccination can satisfy these conditions just.
Summary of the invention
The objective of the invention is to overcome the blank that prior art also is not applied to gene vaccine to prevent and treat SARS virus, thereby provide a kind of gene vaccine that can prevent and treat SARS virus, this vaccine is formed by containing SARS virus N albumen, M albumen and rna polymerase gene cDNA fragment and mammalian expression vector pVAX1, described eucaryon plasmid expression vector pVAX1 is the carrier for expression of eukaryon that can be used for the people, approves through U.S. FDA.
Another object of the present invention provides the structure of this gene vaccine.
A further object of the present invention provides the purposes of this gene vaccine.
The objective of the invention is to realize by following technical scheme:
The invention provides a kind of gene vaccine of anti-SARS virus, this vaccine is that the SARS virus N albumen of 26386-227054 fragment (sequence 1 shown in Fig. 1) coding of AY278487, the M albumen of 28120-29388 fragment (sequence 2 shown in Fig. 2) coding and the rna polymerase gene cDNA and the mammalian expression vector pVAX1 of 2486-2935 fragment (sequence 3 shown in Fig. 3) coding form by accession number in the gene library, described accession number is that the gene of AY278487 is a SARS virus S protein surface antigenic determinant molecule, is provided by the Huada Gene Research Center, Beijing.
The invention provides a kind of structure of gene vaccine of described anti-SARS virus, comprise the steps:
1) plasmid that will contain SARS virus N albumen, M albumen and SARS RNA polymerase cDNA fragment and pGEM-tEasyvector carrier is introduced the DH5a bacterial strain, choose single bacterium colony of containing SARS virus N albumen, M albumen and rna polymerase gene cDNA fragment cloning carrier according to a conventional method respectively, shake bacterium, carry and contain SARS virus N albumen, M albumen and the segmental cloning vehicle of rna polymerase gene cDNA; Described SARS virus N albumen, M albumen and SARS RNA polymerase cDNA fragment are respectively 26386-227054,28120-29388 and the 2486-2935 coded by said gene of AY278487 by accession number in the gene library;
2) increase SARS virus N albumen, M albumen and the segmental primer sequence of rna polymerase gene cDNA of being used to of design is respectively:
M albumen
Forward primer: 5 '-CGCGGATCCACC ATGGCAGACAACGGTACTATTACC-3 '
Downstream primer: 5 '-CGCGGATCC TTACTGTACTAGCAAAGCAA-3 '
N albumen
Forward primer: 5 '-CGCGGATCCACC ATGTCTGATAATGGACCCCAATCAA-3 '
Downstream primer: 5 '-CGCGGATCC TTATGCCTGAGTTGAATCAG-3 '
RNA polymerase
Forward primer: 5 '-CCGTATTCATGGGTGATTCACATGACA-3 '
Downstream primer: 5 '-ATAACAAACTGGTTGTAAAGTCTTC-3 '
3) use the polymerase chain amplification method, the plasmid clone that obtains from step 1) obtains SARS virus N albumen, M albumen and rna polymerase gene cDNA fragment respectively;
4) with N albumen, M albumen and the rna polymerase gene cDNA fragment of step 3) amplification, utilize
PCR Preps.DNA purification system test kit reclaims purification;
5) SARS virus N albumen, M albumen and the rna polymerase gene cDNA fragment of step 4) purification are recombinated among the mammalian special expression pVAX1, be built into the gene vaccine-pVAX1-SARS-MNRna vaccine of anti-SARS virus.
The polymerase chain amplification method of described step 3) was: 95 ℃ of reactions 5 minutes; Continue 1 minute at 94 ℃, continue 1 minute, continue 1 minute, cooling totally 40 circulations of heating repeatedly at 72 ℃ at 56 ℃; Extended 10 minutes at 71 ℃ at last.
The gene vaccine that the present invention further provides described anti-SARS virus is used for preventing the application of the medicine of SARS in preparation.
The gene vaccine that the present invention also provides described anti-SARS virus is used for the treatment of application in the medicine of SARS in preparation.
The gene vaccine advantage of anti-SARS provided by the invention is: the present invention obtains target gene-N albumen, M albumen and rna polymerase gene cDNA fragment through clone, screening, and prepared the gene vaccine that can be used for anti-SARS first with this, this vaccine have virus easy to identify, high degree of specificity is arranged, the other biological body is unexistent and can causes that again body produces the characteristic of efficient immunne response;
This vaccine can not only produce protection antibody by bone-marrow-derived lymphocyte immune induction body, and can induce the extremely effectively ability of specificity T killer cell, this anti-SARS novel vaccine is killed SARS virus by transferring the intravital immune system of patient, reach the purpose of treatment SARS, thereby reach the purpose of prevention and treatment SARS virus; Compare with current vaccine commonly used, it is little that this gene vaccine reaches the required amount of identical immune effect, and the 20-100 microgram gets final product; This gene vaccine can efficiently excite humoral immunization and cellular immunization simultaneously, and is stronger than immune means immune effect commonly used; Use this dna gene vaccine safety, long-acting, stable, simple operation.This gene vaccine can transport at normal temperatures.
Description of drawings
Fig. 1 is a sequence 1---accession number is the 26398-27063 fragment of AY278487 in the gene library;
Fig. 2 is a sequence 2---accession number is the 28120-29388 fragment of AY278487 in the gene library;
Fig. 3 is a sequence 3---accession number is the 2486-2935 fragment of AY278487 in the gene library.
The specific embodiment
The structure of the gene vaccine of embodiment 1. anti-SARS virus of the present invention-pVAX1-SARS-MNRna vaccine
The construction step of pVAX1-SARS-MNRna gene vaccine provided by the invention is as follows:
1) plasmid that will contain SARS virus N albumen, M albumen and SARS RNA polymerase cDNA fragment and pGEM-tEasyvector carrier is introduced the DH5a bacterial strain, choose single bacterium colony of containing SARS virus N albumen, M albumen and rna polymerase gene cDNA fragment cloning carrier according to a conventional method respectively, shake bacterium, carry and contain SARS virus N albumen, M albumen and the segmental cloning vehicle of rna polymerase gene cDNA; Described SARS virus N albumen, M albumen and SARS RNA polymerase cDNA fragment are 26386-227054 (sequence 1 shown in Figure 1), the 28120-29388 (sequence 2 shown in Figure 2) of AY278487 and the gene code of 2486-2935 (sequence 3 shown in Figure 3) by accession number in the gene library respectively, provide by the Huada Gene Research Center, Beijing;
2) increase SARS virus N albumen, M albumen and the segmental primer sequence of rna polymerase gene cDNA of being used to of design is respectively:
M albumen
Forward primer: 5 '-CGCGGATCCACC ATGGCAGACAACGGTACTATTACC-3 '
Downstream primer: 5 '-CGCGGATCC TTACTGTACTAGCAAAGCAA-3 '
N albumen
Forward primer: 5 '-CGCGGATCCACC ATGTCTGATAATGGACCCCAATCAA-3 '
Downstream primer: 5 '-CGCGGATCC TTATGCCTGAGTTGAATCAG-3 '
RNA polymerase
Forward primer: 5 '-CCGTATTCATGGGTGATTCACATGACA-3 '
Downstream primer: 5 '-ATAACAAACTGGTTGTAAAGTCTTC-3 '
3) use the polymerase chain amplification method, the plasmid clone that obtains from step 1) obtains SARS virus N albumen, M albumen and rna polymerase gene cDNA fragment respectively;
The PCR program:
95℃?5min
↓
72 ℃ of 1min of 56 ℃ of 1min of 94 ℃ of 1min totally 40 circulations
↓
71℃?10min
4) with N albumen, M albumen and the rna polymerase gene cDNA fragment of step 3) amplification, utilize
PCR Preps.DNA purification system test kit reclaims purification;
5) SARS virus N albumen, M albumen and the rna polymerase gene cDNA fragment of step 4) purification are recombinated among the mammalian special expression pVAX1, be built into the gene vaccine-pVAX1-SARS-MNRna vaccine of anti-SARS virus;
The cDNA fragment of rmCG β full length amino acid coded sequence is recombinated in the pVAX1 eukaryon expression plasmid:, get bacterium liquid coating LB (the containing ampicillin) flat board that 80ul transforms, in 37 ℃ of overnight incubation with coupled reaction liquid transformed competence colibacillus cell Top10F '; Picking colony shakes bacterium and spends the night, and extracts plasmid DNA with the alkali cracking method, i.e. the pVAX1-SARS-MNRna of present embodiment carries out enzyme action and identifies and positive recombiant plasmid is checked order; To have forward to insert the pulsating recombinant clone plasmid of SARS-MNRna amplified production foreign DNA utilizes cloned plasmids sequencing primer M13 reverse primer (reverse primer) and T7 promoter primer (promoter primer) to the two-way order-checking of pVAX1-SARS-MNRna amplified production after purified;
6) the pVAX1-SARS-MNRna gene vaccine of the anti-SARS of Gou Jianing, the inside and outside mRNA of detection bodies expresses behind immune mouse and the transfection HeLa cell,
The RT-PCR program is:
45℃?55min
↓
94℃?5min
↓
68 ℃ of 1min of 55 ℃ of 1min of 94 ℃ of 1min totally 40 circulations
↓
64℃?10min
The reverse transcription of described step 6)-polymerase chain amplification method comprises: reverse transcription reaction was 45 ℃ of reactions 55 minutes, then 94 ℃ of deactivations 5 minutes, and start reverse transcription-polymerase chain amplification method amplified reaction: add polymerase when first denaturing step, denaturation temperature is 94 ℃, continues 1 minute, annealing temperature is since 55 ℃, continue 1 minute, reduce to 55 ℃ with the speed of 1 ℃ of per two circulation decline then, continue 1 minute, elongating temperature is 68 ℃, continues 40 seconds; Continuing 15 circulations of amplification in 60 ℃ minutes with annealing temperature at last; Extended 10 minutes at 64 ℃ at last.
The vivoexpression of the external source mRNA of embodiment 2.pVAX1-SARS-MNRna vaccine and the evaluation of expression contents
Set up the Hela cell, the external transient expression system of Chinese hamster ovary celI, with the pVAX1-SARS-MNRna expression plasmid that makes up among the embodiment 1 through liposome transfection, respectively at 24 hours, 48 hours, collected culture fluid in 72 hours, adopt RT-PCR, methods such as laser co-focusing indirect immunofluorescence detection are identified recombinant eukaryon expression vector external transient expression of pVAX1-SARS-MNRna vaccine and content, the pVAX1-SARS-MNRna eukaryon expression plasmid is at the Hela cell as can be known, can efficiently express on the mRNA level in the external transient expression system of Chinese hamster ovary celI, the expression of mRNA level exceeds 151% than matched group (empty plasmid).
The evaluation that the external protein level of embodiment 3.pVAX1-SARS-MNRna vaccine is expressed
Set up the Hela cell, the external transient expression system of Chinese hamster ovary celI, with the pVAX1-SARS-MNRna eukaryon expression plasmid that makes up among the embodiment 1 through liposome transfection, respectively at 24 hours, 48 hours, added in 72 hours, BALB/C mice serum after the pVAX1-SARS-MNRna immunity, adopt laser co-focusing indirect immunofluorescence (immunofluorescence) to detect, RT-PCR, ELISA methods such as (elisa) is identified recombinant eukaryon expression vector external transient expression of pVAX1-SARS-MNRna vaccine and content, the pVAX1-SARS-MNRna eukaryon expression plasmid is at the Hela cell as can be known, can efficiently express on protein level in the external transient expression system of Chinese hamster ovary celI, the expression of protein level is higher by 168% than matched group (empty plasmid).
Embodiment 4.pVAX1-SARS-MNRna is the detection of the mRNA expression of RNA in vivo
The total RNA of muscular tissue that collects when extracting the 6th week behind the pVAX1-SARS-MNRna immune mouse muscle cell, the primer and the RT-PCR method that adopt when utilizing amplification SARS virus RNA polymerase cDNA fragment, amplification cDNA band, by testing result as can be known, behind the pVAX1-SARS-MNRna dna gene vaccine, RNA expresses on the mRNA level in the muscle cell, and the expression of mRNA level exceeds 243% than matched group (empty plasmid).
Embodiment 5. elisa (ELISA), western blotting (Western-Blotting) and laser co-focusing indirect immunofluorescence detect the humoral immunoresponse(HI) behind the mouse immune
The normal serum that doubly dilutes with 1:50 in contrast, the dilution proportion that the serum of collection pVAX1-SARS-MNRna immune mouse is pressed 1:100 and 1:1000, adopt method evaluation recombinant eukaryon expression vector pVAX1-SARS-MNRna vaccine internal antibody expression and content such as the detection of laser co-focusing indirect immunofluorescence, by testing result as can be known, pVAX1-SARS-MNRna dna vaccination immunity inoculation BALB/C mice, the dosage of 20 μ g can produce intensive humoral immunoresponse(HI) by inducing mouse.From the 2nd week of detecting, immunoreactive titre continues to raise, and rises near the peak when the 6th week, and antibody titer reaches as high as 1:4200, is significantly higher than matched group.
Obtain N albumen at prokaryotic expression system DL21 (PET21-N) and eukaryotic expression system CHO respectively, and obtained the N albumen of purification.Behind Western-Blotting and elisa technique assay certificate fusion pVAX1-SARS dna vaccination immune mouse, induce and activate the proteic specific antibody of body immune system generation anti-SARS virus N.The result shows that the pVAX1-SARS-MNRna dna vaccination can effectively excite mice to produce the humoral immunoresponse(HI) of antigenic specificity.
Embodiment 6. cellular immunization detect
Detect the antigenic specificity killing activity of CTL with 8 all T lymphocytes after the pVAX1-SARS-MNRna dna vaccination immunity inoculation BALB/C mice.The active testing result of the lymphocytic CTL of T after handling through specific antigen protein M albumen, the proteic sensitization of N: the antigenic specificity killing activity of T cellulotoxic lymphocyte is in different E:T ratios: 50:1 reaches 35%, reach 24% during 25:1, compare significant difference (p＜0.01) with matched group, presentation of results pVAX1-SARS-MNRna dna vaccination is induced the cellullar immunologic response that has produced antigenic specificity in the BALB/c mouse body.Cell proliferation experiment also shows the breeder reaction that specific antigen can induction of lymphocyte.
Embodiment 7. cytokines measurement
After handling with the SARS virus sensitization of specific antigen protein M albumen, the proteic sensitization processing of N or complete deactivation respectively, IL-2 in the pVAX1-SARS-SR mice, IFN-γ, IILL-4, TNF-α, TGF-β all has expression.
The external SARS virus experiment of embodiment 8. vaccines (finishing) by the CDC cooperation
We give the serum of 30 parts of pVAX1-SARS-MNRnaDNA vaccine immune mouses of CDC virosis institute (CDC), and wherein control serum samples is 6 parts.In the external SARS live virus and experiment confirm: merge the serum of pVAX1-SARS-MNRna dna vaccination immune mouse can be fully in and SARS virus.Can produce the specific antibody of anti-SARS virus behind Western-Blotting and the elisa technique detection confirmation fusion pVAX1-SARS-MNRna dna vaccination immune mouse.
SARS virus experiment (being cooperated) in the embodiment 9. vaccine bodies by Chinese Academy of Medical Sciences's zooscopy
1. Rhesus Macacus SARS animal model experiment
7 Rhesus Macacus are divided into two groups: one group of injecting normal saline is organized (N=3) in contrast, and one group (N=4) injection fusion pVAX1-SARS-MNRna dna vaccination, 50 μ g inject once totally three times week about.Injection was merged behind the pVAX1-SARS-MNRna dna vaccination 40 days, 7 Rhesus Macacus are injected the SARS live virus with dosage respectively, get throat swab from 7 monkeys in second day behind the counteracting toxic substances and separate SARS virus, vaccinate group and matched group all are positive, the vaccinate group all is negative (promptly separate then do not see SARS virus) after 15 days, 20 days, and matched group all be positive (after promptly separating SARS virus being arranged); Got the separating plasma SARS virus from 7 monkeys respectively in second day and the 5th day behind the counteracting toxic substances, vaccinate group and matched group all are positive, the vaccinate group all is negative (promptly separate then do not see SARS virus) after 15 days, 20 days, and matched group all be positive (after promptly separating SARS virus being arranged); Got lung, kidney,spleen,liver, lymph node respectively from 7 monkeys of peaceful and comfortable execution in the 21 day, the vaccinate group all is negative.Routine blood test detects, and it is normal that the vaccinate group all is.
2. Microtus brandti SARS animal model experiment
16 Microtus brandtis are two groups: (matched group, N=5), another group (N=11) injection is merged pVAX1-SARS-MNRna dna vaccination 20 μ g totally three times to one group of injecting normal saline.Injection was merged behind the pVAX1-SARS dna vaccination 30 days, 16 Microtus brandtis are delivered to zooscopy institute of the Chinese Academy of Medical Sciences, inject SARS live virus respectively with dosage, matched group is dead 4 after 5 days, be dyspnea before the animal dead, the tangible SARS symptom of the hemorrhage grade of mouth and nose, and the vaccinate group is not seen death, and euthanasia is not also seen other abnormal symptom after surviving 15 days always.
Embodiment 10. safeties detect
By long-term observation, the mice of vaccinate does not have variations such as obvious diet, behavior, hair; Pathological section shows that organs such as the heart, liver, spleen, lung, kidney, brain do not have obvious pathological change.
The heterologous nucleic acid vaccine is incorporated into probability on the chromosome in theory than little many of nucleic acid vaccine of the same race, and this has just effectively solved the integration safety problem of long-term puzzlement nucleic acid vaccine to a certain extent.
F1 does not detect plasmid DNA for mice.
FPI04035?sequence?list.txt
SEQUENCE?LISTING
＜110〉Institute of Zoology, Academia Sinica Beijing Hua Daji state research center
＜120〉a kind of gene vaccine of anti-SARS virus and structure thereof and application
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FPI04035?sequence?list.txt
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Claims (8)
1, a kind of gene vaccine of anti-SARS virus, this vaccine is made up of the SARS virus N albumen of sequence 1 coding, the M albumen of sequence 2 codings and the rna polymerase gene cDNA and the expression vector pVAX1 of sequence 3 codings.
2, the gene vaccine of anti-SARS virus as claimed in claim 1 is characterized in that, described sequence 1 is the 26398-27063 fragment of AY278487 for accession number in the gene library.
3, the gene vaccine of the described anti-SARS virus of claim 1 is characterized in that, described sequence 2 is the 28120-29388 fragment of AY278487 for accession number in the gene library.
4, the gene vaccine of the described anti-SARS virus of claim 1 is characterized in that, described sequence 3 is the 2486-2935 fragment of AY278487 for accession number in the gene library.
5, the construction method of the gene vaccine of the described anti-SARS virus of a kind of claim 1 comprises the steps:
1) plasmid that will contain SARS virus N albumen, M albumen and SARS RNA polymerase cDNA fragment and pGEM-tEasyvector carrier is introduced the DH5a bacterial strain, choose single bacterium colony of containing SARS virus N albumen, M albumen and rna polymerase gene cDNA fragment cloning carrier according to a conventional method respectively, shake bacterium, carry and contain SARS virus N albumen, M albumen and the segmental cloning vehicle of rna polymerase gene cDNA; Described SARS virus N albumen, M albumen and SARS RNA polymerase cDNA fragment are respectively 26386-227054,28120-29388 and the 2486-2935 coded by said gene of AY278487 by accession number in the gene library;
2) increase SARS virus N albumen, M albumen and the segmental primer sequence of rna polymerase gene cDNA of being used to of design is respectively:
M albumen
Forward primer: 5 '-CGCGGATCCACC ATGGCAGACAACGGTACTATTACC-3 '
Downstream primer: 5 '-CGCGGATCC TTACTGTACTAGCAAAGCAA-3 '
N albumen
Forward primer: 5 '-CGCGGATCCACC ATGTCTGATAATGGACCCCAATCAA-3 '
Downstream primer: 5 '-CGCGGATCC TTATGCCTGAGTTGAATCAG-3 '
RNA polymerase
Forward primer: 5 '-CCGTATTCATGGGTGATTCACATGACA-3 '
Downstream primer: 5 '-ATAACAAACTGGTTGTAAAGTCTTC-3 '
3) use the polymerase chain amplification method, the plasmid clone that obtains from step 1) obtain respectively SARS virus N albumen,
M albumen and rna polymerase gene cDNA fragment;
4) with N albumen, M albumen and the rna polymerase gene cDNA fragment of step 3) amplification, utilize Wizard
PCR Preps.DNA purification system test kit reclaims purification;
5) SARS virus N albumen, M albumen and the rna polymerase gene cDNA fragment of step 4) purification are recombinated among the mammalian special expression pVAX1, be built into the gene vaccine-pVAX1-SARS-MNRna vaccine of anti-SARS virus.
6, the construction method of the gene vaccine of anti-SARS virus as claimed in claim 5 is characterized in that: the polymerase chain amplification method of described step 3) was: 95 ℃ of reactions 5 minutes; Continue 1 minute at 94 ℃, continue 1 minute, continue 1 minute, cooling totally 40 circulations of heating repeatedly at 72 ℃ at 56 ℃; Extended 10 minutes at 71 ℃ at last.
7, the gene vaccine of the described anti-SARS virus of a kind of claim 1 is used for preventing the application of the medicine of SARS in preparation.
8, the gene vaccine of the described anti-SARS virus of a kind of claim 1 is used for the treatment of application in the medicine of SARS in preparation.
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| SARS相关冠状病毒基因工程重组蛋白质疫苗的研制. 徐安龙等.广东医学，第S1期. 2003 * |
| 预防SARS病毒核酸疫苗的构建. 易艳萍，李平，李楚芳，刘萱，黄维，靳彦文，李晓荣，马清钧，曹诚.生物技术通讯，第3期. 2003 * |
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