CN1449826A - Gene vaccine for anti SARS coronal virus and use thereof - Google Patents
Gene vaccine for anti SARS coronal virus and use thereof Download PDFInfo
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Abstract
本发明公开了一种抗SARS冠状病毒的基因疫苗及应用，含有SARS冠状病毒S蛋白的全长基因序列的全部或部分基因片段的真核表达质粒；编码SARS冠状病毒S蛋白的核苷酸序列可操作地连接于一个启动子序列后，以便体内表达所编码的SARS冠状病毒S蛋白的完整或部分多肽。将所述基因疫苗用于包括人类和啮齿类动物如小鼠在内的宿主进行免疫，使其产生特异性保护性的体液免疫和细胞免疫，以抵抗引起非典型性肺炎传染病的SARS冠状病毒的感染。本发明稳定性好，生产方便，价格便宜，能诱导机体同时产生针对引起非典传染病的SARS冠状病毒S蛋白的特异性体液免疫和细胞免疫，为治疗和预防非典型性肺炎提供了有效且价廉的疫苗。The invention discloses an anti-SARS coronavirus gene vaccine and its application, a eukaryotic expression plasmid containing all or part of the gene fragment of the full-length gene sequence of the SARS coronavirus S protein; a nucleotide sequence encoding the SARS coronavirus S protein It is operably linked to a promoter sequence, so that the whole or partial polypeptide of the encoded SARS coronavirus S protein can be expressed in vivo. The genetic vaccine is used to immunize hosts including humans and rodents such as mice to produce specific protective humoral immunity and cellular immunity to resist the SARS coronavirus that causes atypical pneumonia infectious diseases infection. The invention has good stability, convenient production and low price, can induce the body to simultaneously produce specific humoral immunity and cellular immunity against the SARS coronavirus S protein that causes SARS infectious diseases, and provides an effective and cost-effective method for treating and preventing atypical pneumonia. cheap vaccines.
Description
Technical field
The present invention relates to prevent and treat the field of the severe acute respiratory syndrome vaccine that causes by sars coronavirus, relate in particular to the vaccine of the proteic nucleotide sequence of encoding SARS coronavirus S.Also relate to the purposes of this vaccine in SARS coronary virus resistant.
Background technology
Be called severe acute respiratory syndrome (Severe Acute Respiratory Sydrome, SARS) more than 20 countries and regions generation of atypical pneumonia in the whole world.According to World Health Organization's statistics, by 22 Geneva time of May 17:00, the accumulative total reported cases 8046 of the whole world, source, death 682; Whole nation accumulative total interiorly reports the atypical pneumonia case 5285 examples, dead 303 examples.This precipitate disaster give the people of the world particularly Chinese people brought huge personnel to go up and loss economically.
Till May 22, the chemicals that improves symptom is only arranged, as " sivelestat sodium ", be used to improve the acute lung injury that systemic inflammatory reaction syndrome and idiopathic pulmonary fibrosis etc. cause, with the human body immunity improving function, as glucocorticoid, and broad-spectrum antiviral medicament " genetic engineering human omega interferon ", do not see the medicine listing of the severe acute respiratory syndrome that specific treatment is arranged and prevent to cause by sars coronavirus.A kind of prevention of this feasible development and treatment seem very urgent and important by the vaccine of the severe acute respiratory syndrome that sars coronavirus causes.
In the development of vaccine, exist three kinds of selections.First kind of mode is the severe acute respiratory syndrome vaccine that adopts the sars coronavirus of attenuation or deactivation to make, and it is the method for sars coronavirus by physics or chemistry lowered or to eliminate its infectivity, directly uses as vaccine then.It still has the regressive potential probability of virulence, has very large danger.
The second way is to adopt engineered protein or polypeptide as vaccine, be incomplete antigen gene with sars coronavirus at cell inner expressions such as yeast, escherichia coli, the protein that obtains through purification as vaccine.But often can not obtain correct translation post-treatment owing to express the albumen obtain, use the antigenicity of the vaccine that this albumen makes often relatively poor, can not make body produce the excellent protection immunoreation; The main component of this kind vaccine is a protein in addition, and is very unstable, need remain " cold chain transportation " in selling and transporting, and this has increased its production cost greatly, and this kind vaccine effective percentage is reduced greatly.
And gene vaccine is the third generation vaccine that grew up in recent years.It is exogenous gene cloning to carrier for expression of eukaryon, the vector gene with reorganization is injected directly in the animal body then, and exogenous gene is in vivo expressed, the antibody of generation activates the immune system of body, causes immunne response.Lot of experiment results shows that gene vaccine not only can be used for human diseases, but also can be widely used in zoonosis and animals and plants disease.Gene vaccine not only has prophylactic effect, also has the effect of treatment disease simultaneously.
Can only induce body to produce humoral immunization with the engineered protein vaccine compares, gene vaccine can induce body to produce powerful humoral immunization and cellular immunization simultaneously, this is inducing body to create antagonism pathogenic infection particularly in the viral infection, have in the very big antibody capable that advantage and application prospect-humoral immunization produced and blood and tissue fluid in virion; And cellular immunization can make all kinds of killer cell identifications and kill infected host cell, and host cell just that it is parasitic before the virion maturation kills, and makes virus to increase, and discharges, and invasion is the host cell of infection not, thereby reaches the purpose of treatment.
Summary of the invention
The object of the present invention is to provide a kind of gene vaccine of SARS coronary virus resistant, this vaccine is stable, preserves at normal temperatures, can transport easily, stores, and can cause simultaneously that human body is at sars coronavirus humoral immunization and cellular immunization.
Another object of the present invention is to provide a kind of application of gene vaccine in prevention and treatment severe acute respiratory syndrome of SARS coronary virus resistant.
The present invention relates to polynucleotide, comprise DNA, carry out immunity, make the body opposing, relate to the proteic full-length gene of sars coronavirus S or its fragment that contain in the gene vaccine that is useful on described purpose by the caused severe acute respiratory syndrome of sars coronavirus.Among the present invention, some recombiant plasmid have been made up, so that comprise the proteic nucleotide sequence of encoding SARS coronavirus S.Technical scheme implementation step provided by the invention is: a. adopts the synthetic method of gene or obtain sars coronavirus S protein part or full-length gene fragment by the method that RT-PCR increases from the patients serum.B. the resulting genetic fragment of a step is inserted into the promoter that has the early stage immediately cytomegalovirus (CMV) of the mankind, or in the commercial carrier for expression of eukaryon of other similar eukaryotic expression promoter.C. with the resulting recombinant eukaryon expression vector that has the resulting genetic fragment of a step, use digestion with restriction enzyme method or PCR method through identifying.D. the recombinant eukaryon expression vector that the amplification c step obtains in escherichia coli is removed impurity wherein, as the host bacteria chromosomal DNA, and RNA and protein.E. the recombinant eukaryon expression vector that d is gone on foot resulting purification joins in the appropriate formulation, as normal saline, the concentration of regulating recombinant eukaryon expression vector is to suitable immunity concentration, scope is 0-100mg/ml, promptly obtains can be used for the gene vaccine of the object of the invention SARS coronary virus resistant.
The proteic nucleotide sequence of encoding SARS coronavirus S is seen sequence table, and the proteic nucleotide sequence of sars coronavirus S involved in the present invention comprises the complete sequence shown in the table 1 and its fragment.
After the proteic nucleotide sequence of encoding SARS coronavirus S is operably connected to a promoter sequence, so that the coded proteic complete or part of polypeptide of sars coronavirus S of expression in vivo.Described promoter sequence can be mankind's promoteres of early stage cytomegalovirus (CMV) immediately, also can use any other common promoter, comprise constitutive promoter, as sarcoma virus LTRs, with bring out the type promoter, as metallothionein promoter and tissue-specific promoter.
Use technical scheme provided by the invention to obtain containing the gene vaccine of sars coronavirus S protein part or full-length gene order.The gene vaccine that wherein contains sars coronavirus S albumen full-length gene order, promptly recombiant plasmid pCDNA-S is stored among escherichia coli Escherichia coli DH5 α-pCDNA-S, CCTCC No:M203044.
Above-mentioned eukaryotic expression recombinant plasmid contains some basic framework units: protokaryon replication origin fi origin and pPUCorigin; Eucaryon replication origin SV40; Neomycin resistance gene; The penbritin resistant gene; And several have the non-coding immunostimulatory sequence (ISS) of immunologic adjuvant function.
The severe acute respiratory syndrome vaccine that the present invention and the severe acute respiratory syndrome vaccine that adopts attenuation and inactivated vaccine to make and engineered protein or polypeptide are made is compared, and contains the segmental gene vaccine of sars coronavirus S protein gene and has the following advantages and make it have very big advantage in the severe acute respiratory syndrome infectious disease that prevention and treatment are caused by sars coronavirus:
(1) can cause humoral immunization and the cellular immunization of human body simultaneously at sars coronavirus.
(2) preparation is easy, time saving and energy saving.The exogenous antigen gene is easy to the clone and advances expression vector, can increase in a couple of days and purification.
(3) compare safety.After containing the segmental gene vaccine inoculation of sars coronavirus S protein gene, in mammalian cell, energy high level expression virus antigen gene does not relate to infectious factor, does not rely on the assembling of virion, has avoided adopting the danger of live-virus vaccine.
(4) can provide the effective vaccine of the wide spectrum of multiple Strain.Because carrier itself is induce immune response not, therefore can set up the gene vaccine simultaneous inoculation immunity that contains multiple antigen dna.
(5) contain the inoculation of the segmental gene vaccine of sars coronavirus S protein gene after because the proteic time of antigen expressed is the 1-2 month, can strengthen the memory of B cell and T cell, cause persistent body fluid and cellullar immunologic response.
(6) it is more stable to contain the segmental gene vaccine of sars coronavirus S protein gene, can preserve at normal temperatures, can transport easily, store.
(7) low price.
Show that by the homology contrast sars coronavirus S albumen is the key protein of the film fusion process in mediation sars coronavirus recognition of host cell and the phagocytic process, closely related with the pathogenic course of sars coronavirus invasion human body.Sars coronavirus S albumen is positioned at the virus envelope surface, the interaction of mediation virus and host cell membrane surface receptor when poisoning intrusion, and stay cell surface, become the surface marker of the cell that is infected by the virus, thereby become the target of antibody and lymphocytic attack, thereby can be as the important gene of severe acute respiratory syndrome prevention with treatment.Utilization bioinformatics means are analyzed the segmental antigenicity of sars coronavirus S protein gene, the result shows that it has A, B, three very high zones of antigenicity evaluation of estimate of C, B wherein, what the C district was arranged in sars coronavirus S Protein S 2 subunits is positioned at the outside carboxylic acid end polypeptide peptide section of virus, length is respectively 130aa, its antigenicity is very strong, has 4 above MHCII binding site molecule points respectively, is the vaccine gene fragment with strong antigen.The zone that one of them or two antigenicities is good is together as the antigen gene of vaccine, and constructed vaccine can cause that body produces powerful protective immunity effect.
The gene vaccine that contains proteic part of sars coronavirus S or full length amino acid sequence that the present invention makes has Heat stability is good, convenient for production, low price, can induce human body to produce, thereby play prevention and treat the effect that is caused the SARS infectious disease by sars coronavirus at the proteic specific humoral immunity of sars coronavirus S and the cellular immunization that cause the SARS infectious disease.
Description of drawings
Fig. 1 is the design of graphics of recombiant plasmid pCDNA-SARS-S2.
The specific embodiment
The present invention is described in further detail in conjunction with the accompanying drawings:
1. make up the carrier for expression of eukaryon of the nucleotide sequence that contains the segmental 2752bp-3162bp of sars coronavirus S albumen full-length gene, the gene order of the segmental 2752bp-3162bp of sars coronavirus S albumen full-length gene sees Table 2.
The genetic fragment of the synthetic segmental 2752bp-3162bp of sars coronavirus S albumen full-length gene is right with primer by the PCR method then:
5’ggggaattcgacatggaatcacttacaacaacatcaactgc?3’
5’cccggatcctactaagcttgctcctgggatggcacatacg 3’
Be primer, the genetic fragment of the segmental 2752bp-3162bp of synthetic sars coronavirus S albumen full-length gene is a template, and amplification obtains the genetic fragment that two ends add the segmental 2752bp-3162bp of sars coronavirus S albumen full-length gene of EcoRI and BamHI restriction enzyme site respectively.PCR reaction cumulative volume is 50 μ l, and reaction condition is: 94 ℃ of pre-degeneration 5 minutes; The PCR loop parameter: 94 ℃ 60 seconds, 58 ℃ 30 seconds, 72 ℃ 20 seconds, circulate in 72 ℃ for the last time and extended 7 minutes.
Use restricted enzyme EcoRI and BamHI that this PCR product and carrier for expression of eukaryon pCDNA3.1 (-) are carried out double digestion then, make both become cohesive end in two ends.In 16 ℃ under the effect of T4 dna ligase two fragments coupled together, connect product and transform competent bacillus coli DH 5 alpha thereafter.From transforming picking list bacterium colony on the plate, be inoculated in the LB fluid medium overnight incubation that 5mL contains 100 μ g/mL Amp.Press a small amount of alkaline process that people such as Sambrook describes next day and extract plasmid [Sambrook J, et al, 1989].Making enzyme action with restricted enzyme EcoRI identifies, select and contain the bigger recombiant plasmid of fragment, make enzyme action with restricted enzyme EcoRI and BamHI then and identify, choose the recombiant plasmid of the genetic fragment that wherein can cut out the segmental 2752bp-3162bp of sars coronavirus S albumen full-length gene.Add that according to the aforementioned fragment two ends of giving the PCR method in EcoRI and BamHI site carries out PCR, identify further that to containing the segmental recombiant plasmid of SARS S2 the recombiant plasmid that can amplify the genetic fragment of the segmental 2752bp-3162bp of sars coronavirus S albumen full-length gene is named and is pCDNA-SARS-S2.
2. the gene vaccine of the genetic fragment of the segmental 2752bp-3162bp of encoding SARS coronavirus S albumen full-length gene is that the preparation of recombiant plasmid pCDNA-SARS-S2 immune formulation and positive control sample and negative control sample is a sars coronavirus gene vaccine candidate sample with recombiant plasmid pCDNA-SARS-S2; To have the positive contrast of eukaryon expression plasmid pCMV-S of hepatitis B surface antigen; The negative contrast of empty carrier pCDNA3.1 (-) with the genetic fragment that do not contain the segmental 2752bp-3162bp of sars coronavirus S albumen full-length gene: with 0.9% normal saline is another negative control;
The recombination bacillus coli that will contain each plasmid enlarges the plasmid [Sambrook J, et al, 1989] that extracts in the 200mL culture fluid by a small amount of alkaline process that people such as Sambrook describe by equal proportion.After with RNaseA digestion RNA, use phenol, phenol successively: protein is removed in chloroform, chloroform extracting.The 3mol/L sodium acetate that adds 1/10 volume in the supernatant, the two volumes dehydrated alcohol ,-20 ℃ of precipitations are spent the night.Next day, the centrifugal 15min results of 10000g plasmid.After washing drying, use physiological saline solution.Use spectrophotometer, measure A260/A280 ratio and determine plasmid purity, the A260 value is determined the concentration of sample, regulates concentration to 1.0 μ g/ μ L with normal saline, as the dna immunization injected sample.
3. carry out immune BALB/c mouse with four kinds of immunization wayses
Four kinds of immunization wayses are: a. intramuscular injection; B. particle gun subcutaneous injection; C. spray nasal membrane immunity; D. particle gun subcutaneous injection initial immunity, the spray nasal membrane is strengthened
Every kind of immunization ways choose 40 six age in week female BALB/c white mice, wherein 10 as negative control group with the normal saline immunity, 10 as positive controls with recombiant plasmid pCMV-S immunity, 10 as test group with recombiant plasmid pCDNA-SARS-S2 immunity; 10 as negative control group with empty carrier pCDNA3.1 (-) immunity.Every injection 100 μ gDNA inoculation samples.Behind the initial immunity fortnight, booster immunization once once more.The mice peripheral blood is adopted every seven days eye sockets in twice back of booster immunization, adds anticoagulant heparin.
4. the detection of immune effect
BALB/c mouse is being accepted immunity back the 7th, 14,21,28,35 days, there is situation by plasmid in the quantitative PCR detection mice body, by immunoblotting testing goal antigen presentation situation, detect the body fluid immune state by ELISA, detect mice CD4+T lymphocyte and CD8+T lymphocyte situation to estimate the specific cellular immunity situation by flow cytometer.
The result shows that BALB/c mouse is after the immunity of accepting multiple mode; keeping gene vaccine in a long time in body in varying degrees is recombiant plasmid; and the genetic fragment of the segmental 2752bp-3162bp of sars coronavirus S albumen full-length gene in the recombinant expression; the mice body is produced at proteic cellular immunization of sars coronavirus S and humoral immunization, make mice obtain the protective immunity that antagonism causes the sars coronavirus infection of severe acute respiratory syndrome.
Sequence table
Gene vaccine and application＜130 of the complete genome sequence table of table, 1 sars coronavirus S albumen＜110〉Wuhan University＜120〉SARS coronary virus resistant〉gene vaccine and application＜160 of SARS coronary virus resistant〉1＜170〉PatentIn version 3.1＜210〉1＜211〉3768＜212〉DNA＜213〉SARS coronavirus＜300〉＜301〉Marra; M.A.; Jones; S.J.; Astell; C.R.; Holt; R.A.; Brooks-Wilson; A.,＜302〉The Genome Sequence of the SARS-Associated Coronavirus＜303〉Science＜304〉2003＜305〉0＜306〉13＜307〉2003-05-01＜308〉GI：29836496＜309〉2003-05-22＜313〉 ( 1 ) .. ( 3768 )＜400〉1atgtttattt tcttattatt tcttactctc actagtggta gtgaccttga ccggtgcacc 60acttttgatg atgttcaagc tcctaattac actcaacata cttcatctat gaggggggtt 120tactatcctg atgaaatttt tagatcagac actctttatt taactcagga tttatttctt 180ccattttatt ctaatgttac agggtttcat actattaatc atacgtttgg caaccctgtc 240atacctttta aggatggtat ttattttgct gccacagaga aatcaaatgt tgtccgtggt 300tgggtttttg gttctaccat gaacaacaag tcacagtcgg tgattattat taacaattct 360actaatgttg ttatacgagc atgtaacttt gaattgtgtg acaacccttt ctttgctgtt 420tctaaaccca tgggtacaca gacacatact atgatattcg ataatgcatt taattgcact 480ttcgagtaca tatctgatgc cttttcgctt gatgtttcag aaaagtcagg taattttaaa 540cacttacgag agtttgtgtt taaaaataaa gatgggtttc tctatgttta taagggctat 600caacctatag atgtagttcg tgatctacct tctggtttta acactttgaa acctattttt 660aagttgcctc ttggtattaa cattacaaat tttagagcca ttcttacagc cttttcacct 720gctcaagaca tttggggcac gtcagctgca gcctattttg ttggctattt aaagccaact 780acatttatgc tcaagtatga tgaaaatggt acaatcacag atgctgttga ttgttctcaa 840aatccacttg ctgaactcaa atgctctgtt aagagctttg agattgacaa aggaatttac 900cagacctcta atttcagggt tgttccctca ggagatgttg tgagattccc taatattaca 960aacttgtgtc cttttggaga ggtttttaat gctactaaat tcccttctgt ctatgcatgg 1020gagagaaaaa aaatttctaa ttgtgttgct gattactctg tgctctacaa ctcaacattt 1080ttttcaacct ttaagtgcta tggcgtttct gccactaagt tgaatgatct ttgcttctcc 1140aatgtctatg cagattcttt tgtagtcaag ggagatgatg taagacaaat agcgccagga 1200caaactggtg ttattgctga ttataattat aaattgccag atgatttcat gggttgtgtc 1260cttgcttgga atactaggaa cattgatgct acttcaactg gtaattataa ttataaatat 1320aggtatctta gacatggcaa gcttaggccc tttgagagag acatatctaa tgtgcctttc 1380tcccctgatg gcaaaccttg caccccacct gctcttaatt gttattggcc attaaatgat 1440tatggttttt acaccactac tggcattggc taccaacctt acagagttgt agtactttct 1500tttgaacttt taaatgcacc ggccacggtt tgtggaccaa aattatccac tgaccttatt 1560aagaaccagt gtgtcaattt taattttaat ggactcactg gtactggtgt gttaactcct 1620tcttcaaaga gatttcaacc atttcaacaa tttggccgtg atgtttctga tttcactgat 1680tccgttcgag atcctaaaac atctgaaata ttagacattt caccttgcgc ttttgggggt 1740gtaagtgtaa ttacacctgg aacaaatgct tcatctgaag ttgctgttct atatcaagat 1800gttaactgca ctgatgtttc tacagcaatt catgcagatc aactcacacc agcttggcgc 1860atatattcta ctggaaacaa tgtattccag actcaagcag gctgtcttat aggagctgag 1920catgtcgaca cttcttatga gtgcgacatt cctattggag ctggcatttg tgctagttac 1980catacagttt ctttattacg tagtactagc caaaaatcta ttgtggctta tactatgtct 2040ttaggtgctg atagttcaat tgcttactct aataacacca ttgctatacc tactaacttt 2100tcaattagca ttactacaga agtaatgcct gtttctatgg ctaaaacctc cgtagattgt 2160aatatgtaca tctgcggaga ttctactgaa tgtgctaatt tgcttctcca atatggtagc 2220ttttgcacac aactaaatcg tgcactctca ggtattgctg ctgaacagga tcgcaacaca 2280cgtgaagtgt tcgctcaagt caaacaaatg tacaaaaccc caactttgaa atattttggt 2340ggttttaatt tttcacaaat attacctgac cctctaaagc caactaagag gtcttttatt 2400gaggacttgc tctttaataa ggtgacactc gctgatgctg gcttcatgaa gcaatatggc 2460gaatgcctag gtgatattaa tgctagagat ctcatttgtg cgcagaagtt caatggactt 2520acagtgttgc cacctctgct cactgatgat atgattgctg cctacactgc tgctctagtt 2580agtggtactg ccactgctgg atggacattt ggtgctggcg ctgctcttca aatacctttt 2640gctatgcaaa tggcatatag gttcaatggc attggagtta cccaaaatgt tctctatgag 2700aaccaaaaac aaatcgccaa ccaatttaac aaggcgatta gtcaaattca agaatcactt 2760acaacaacat caactgcatt gggcaagctg caagacgttg ttaaccagaa tgctcaagca 2820ttaaacacac ttgttaaaca acttagctct aattttggtg caatttcaag tgtgctaaat 2880gatatccttt cgcgacttga taaagtcgag gcggaggtac aaattgacag gttaattaca 2940ggcagacttc aaagccttca aacctatgta acacaacaac taatcagggc tgctgaaatc 3000agggcttctg ctaatcttgc tgctactaaa atgtctgagt gtgttcttgg acaatcaaaa 3060agagttgact tttgtggaaa gggctaccac cttatgtcct tcccacaagc agccccgcat 3120ggtgttgtct tcctacatgt cacgtatgtg ccatcccagg agaggaactt caccacagcg 3180ccagcaattt gtcatgaagg caaagcatac ttccctcgtg aaggtgtttt tgtgtttaat 3240ggcacttctt ggtttattac acagaggaac ttcttttctc cacaaataat tactacagac 3300aatacatttg tctcaggaaa ttgtgatgtc gttattggca tcattaacaa cacagtttat 3360gatcctctgc aacctgagct tgactcattc aaagaagagc tggacaagta cttcaaaaat 3420catacatcac cagatgttga tcttggcgac atttcaggca ttaacgcttc tgtcgtcaac 3480attcaaaaag aaattgaccg cctcaatgag gtcgctaaaa atttaaatga atcactcatt 3540gaccttcaag aattgggaaa atatgagcaa tatattaaat ggccttggta tgtttggctc 3600ggcttcattg ctggactaat tgccatcgtc atggttacaa tcttgctttg ttgcatgact 3660agttgttgca gttgcctcaa gggtgcatgc tcttgtggtt cttgctgcaa gtttgatgag 3720gatgactctg agccagttct caagggtgtc aaattacatt acacataa 37682
Gene order table＜110 of the segmental 2752bp-3162bp of sars coronavirus S albumen full-length gene〉Wuhan University＜120〉gene vaccine and application＜130 of SARS coronary virus resistant〉gene vaccine and application＜160 of SARS coronary virus resistant〉1＜170〉PatentIn version 3.1＜210〉1＜211〉411＜212〉DNA＜213〉SARS coronavirus＜300〉＜301〉Marra, M.A., Jones, S.J., Astell, C.R., Holt, R.A., Brooks-Wilson, A.,＜302〉The Genome Sequence of the SARS-Associated Coronavirus＜303〉Science＜304〉2003＜305〉0＜306〉13＜307〉2003-05-01＜308〉GI:29836496＜309〉2003-05-22＜313〉(1) .. (411)＜400〉1gaatcactta caacaacatc aactgcattg ggcaagctgc aagacgttgt taaccagaat 60gctcaagcat taaacacact tgttaaacaa cttagctcta attttggtgc aatttcaagt 120gtgctaaatg atatcctttc gcgacttgat aaagtcgagg cggaggtaca aattgacagg 180ttaattacag gcagacttca aagccttcaa acctatgtaa cacaacaact aatcagggct 240gctgaaatca gggcttctgc taatcttgct gctactaaaa tgtctgagtg tgttcttgga 300caatcaaaaa gagttgactt ttgtggaaag ggctaccacc ttatgtcctt cccacaagca 360gccccgcatg gtgttgtctt cctacatgtc acgtatgtgc catcccagga g 411
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| WO2004085650A1 (en) * | 2003-03-24 | 2004-10-07 | The University Of Hong Kong | A high-troughput diagnostic assay for the human virus causing severe acute respiratory syndrome (sars) |
| WO2005001096A1 (en) * | 2003-06-17 | 2005-01-06 | Bio-Pharm Science & Technology Co., Ltd. | Vaccines against sars |
| WO2005054473A1 (en) * | 2003-12-05 | 2005-06-16 | The University Of Hong Kong | GENETICALLY MODIFIED PLANTS COMPRISING SARS-CoV VIRAL NUCLEOTIDE SEQUENCES AND METHODS OF USE THEREOF FOR IMMUNIZATION AGAINST SARS |
| WO2005027963A3 (en) * | 2003-09-15 | 2005-08-25 | Us Health | METHODS AND COMPOSITIONS FOR THE GENERATION OF A PROTECTIVE IMMUNE RESPONSE AGAINTS SARS-CoV |
| CN100360557C (en) * | 2003-07-10 | 2008-01-09 | 国家人类基因组南方研究中心 | SARS virus S protein, vaccine and method for screening drugs |
| CN100484570C (en) * | 2003-06-16 | 2009-05-06 | 中国科学院动物研究所 | Gene vaccine against SARS virogene and its construction and use |
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| CN113264991A (en) * | 2020-01-31 | 2021-08-17 | 上海桀蒙生物技术有限公司 | Preparation method and pharmaceutical composition of virus vaccine |
| CN113521272A (en) * | 2021-07-28 | 2021-10-22 | 四川大学 | Novel coronavirus pneumonia DNA nano-vaccine and preparation method thereof |
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- 2003-05-27 CN CN 03128042 patent/CN1449826A/en active Pending
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| WO2004085650A1 (en) * | 2003-03-24 | 2004-10-07 | The University Of Hong Kong | A high-troughput diagnostic assay for the human virus causing severe acute respiratory syndrome (sars) |
| CN100484570C (en) * | 2003-06-16 | 2009-05-06 | 中国科学院动物研究所 | Gene vaccine against SARS virogene and its construction and use |
| WO2005001096A1 (en) * | 2003-06-17 | 2005-01-06 | Bio-Pharm Science & Technology Co., Ltd. | Vaccines against sars |
| CN100360557C (en) * | 2003-07-10 | 2008-01-09 | 国家人类基因组南方研究中心 | SARS virus S protein, vaccine and method for screening drugs |
| WO2005027963A3 (en) * | 2003-09-15 | 2005-08-25 | Us Health | METHODS AND COMPOSITIONS FOR THE GENERATION OF A PROTECTIVE IMMUNE RESPONSE AGAINTS SARS-CoV |
| WO2005054473A1 (en) * | 2003-12-05 | 2005-06-16 | The University Of Hong Kong | GENETICALLY MODIFIED PLANTS COMPRISING SARS-CoV VIRAL NUCLEOTIDE SEQUENCES AND METHODS OF USE THEREOF FOR IMMUNIZATION AGAINST SARS |
| CN113264991A (en) * | 2020-01-31 | 2021-08-17 | 上海桀蒙生物技术有限公司 | Preparation method and pharmaceutical composition of virus vaccine |
| CN113264991B (en) * | 2020-01-31 | 2023-05-16 | 上海桀蒙生物技术有限公司 | Preparation method of virus vaccine and pharmaceutical composition |
| CN111378629A (en) * | 2020-04-09 | 2020-07-07 | 西北大学 | A kind of bionic 2019 novel coronavirus, its preparation method and application |
| CN113521272A (en) * | 2021-07-28 | 2021-10-22 | 四川大学 | Novel coronavirus pneumonia DNA nano-vaccine and preparation method thereof |
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