本文選題：SARS-CoV + 核酸疫苗��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年博士論文

【摘要】：嚴(yán)重急性呼吸系統(tǒng)綜合征(Severe Acute Respiratory Syndrome,SARS)又稱傳染性非典型肺炎,是由SARS冠狀病毒(SARS-associated coronavirus,SARS-CoV)感染引起的一種嚴(yán)重急性呼吸系統(tǒng)傳染病。2002～2003年間SARS的暴發(fā)流行,涉及數(shù)十個(gè)國家和地區(qū),全球病例高達(dá)9096例。目前尚無安全有效的SARS疫苗。 核酸疫苗是SARS疫苗研究的重點(diǎn)。核酸疫苗不但兼有減毒活疫苗誘導(dǎo)全面免疫應(yīng)答和重組亞單位疫苗安全的特點(diǎn),而且具有研制周期短,生產(chǎn)工藝簡單,成本低等優(yōu)點(diǎn),代表了SARS疫苗研發(fā)的發(fā)展方向。目前SARS核酸疫苗的研究主要以其結(jié)構(gòu)蛋白S、N和M作為靶標(biāo)構(gòu)建重組質(zhì)粒DNA,免疫小鼠則均可誘導(dǎo)產(chǎn)生體液免疫應(yīng)答和細(xì)胞免疫應(yīng)答。但核酸疫苗主要的缺點(diǎn)是免疫原性較低。 為此,本研究對SARS-CoV的S和N基因進(jìn)行密碼子優(yōu)化,觀察其在小鼠中的免疫原性,并進(jìn)一步采用原核表達(dá)的重組N蛋白與含有N基因的重組質(zhì)粒DNA進(jìn)行聯(lián)合免疫,分析其誘導(dǎo)免疫應(yīng)答水平的差異,觀察對靶抗原基因密碼子優(yōu)化以及與重組蛋白聯(lián)合免疫這兩種策略提高SARS核酸疫苗免疫原性的效果,為研制具有高免疫原性的SARS核酸疫苗提供依據(jù)。 首先,我們采用RT-PCR擴(kuò)增獲得長為2307bp、1632bp和1269bp的cDNA分子,分別編碼SARS-CoV的S1(1～768aa)、S2(712～1255aa)和全長的N蛋白。將擴(kuò)增產(chǎn)物分別克隆至pVAX1真核表達(dá)載體,獲得重組質(zhì)粒pVS1、pVS2和pVN。同時(shí)對SARS-CoV的S基因和N基因進(jìn)行密碼子偏好分析,發(fā)現(xiàn)其與人冠狀病毒OC43株和229E株較為相近,但與哺乳動物則差異較大。以哺乳動物偏好密碼子對S2和N蛋白進(jìn)行反編譯后獲得優(yōu)化S/N基因(opt-S2和opt-N),其G+C%含量有明顯的提高。Opt-N和opt-S2基因序列的G+C%分別由48%提高至70%和由41%提高至64%,密碼子第三位的G+C%則均提高至100%。將優(yōu)化基因人工合成后同樣克隆至pVAX1真核表達(dá)載體獲得重組質(zhì)粒pVeS2和pVeN。然后,分別將上述5種重組質(zhì)粒DNA轉(zhuǎn)染BHK_(21)真核細(xì)胞,48hr后通過間接
[Abstract]:Severe Acute respiratory syndrome (SARS), also known as SARS, is an outbreak of severe acute respiratory disease (SARS) caused by SARS-associated coronavirus (SARS-associated coronavirus) infection, involving dozens of countries and regions. There are as many as 9096 cases worldwide. There is no safe and effective SARS vaccine. Nucleic acid vaccine is the focus of SARS vaccine research. Nucleic acid vaccine not only has the characteristics of inducing total immune response induced by live attenuated vaccine and the safety of recombinant subunit vaccine, but also has the advantages of short development period, simple production process and low cost, which represents the development direction of SARS vaccine research and development. At present, the recombinant plasmid DNA was constructed by using the structural proteins Son N and M as targets of SARS nucleic acid vaccine, and the humoral and cellular immune responses were induced in mice immunized with SARS-CoV nucleic acid vaccine. But the main disadvantage of nucleic acid vaccine is its low immunogenicity. In this study, the codon of S and N genes of SARS-CoV was optimized to observe the immunogenicity of SARS-CoV in mice, and the recombinant N protein expressed by prokaryotic expression was further immunized with recombinant plasmid DNA containing N gene. The difference of immune response level induced by SARS vaccine was analyzed, and the effect of optimizing the codon of target antigen gene and combining with recombinant protein to improve the immunogenicity of SARS nucleic acid vaccine was observed. To provide the basis for the development of SARS nucleic acid vaccine with high immunogenicity. Firstly, we obtained the cDNA of 2307bpn1632bp and 1269bp by RT-PCR, encoding S _ 1 (1~768aa) S _ 2 (712~1255aa) and N protein of SARS-CoV, respectively. The amplified products were cloned into eukaryotic expression vector pVAX1, and the recombinant plasmids pVS1 pVS2 and pVN were obtained. Codon preference analysis of the S and N genes of SARS-CoV showed that SARS-CoV was similar to OC43 and 229E strains of human coronavirus, but was different from that of mammals. After decompilation of S2 and N proteins with mammalian preference codon, the optimized SP-N gene (opt-S2 and opt-N) was obtained. The G% of G C% in the sequence of Opt-N and opt-S2 increased from 48% to 70% and from 41% to 64%, respectively. In the third place, the percentage of G C% increased to 100%. The optimized gene was also cloned into the eukaryotic expression vector pVAX1 to obtain the recombinant plasmids pVeS2 and pVeN. Then, the five recombinant plasmids were transfected into BHK21 eukaryotic cells for 48hr, respectively.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R392

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