SARS新型核酸疫苗的实验研究

发布时间：2018-06-26 09:47

本文选题：SARS-CoV + 核酸疫苗　；参考：《中国人民解放军军事医学科学院》2005年博士论文

【摘要】：严重急性呼吸系统综合征(Severe Acute Respiratory Syndrome,SARS)又称传染性非典型肺炎,是由SARS冠状病毒(SARS-associated coronavirus,SARS-CoV)感染引起的一种严重急性呼吸系统传染病。2002～2003年间SARS的暴发流行,涉及数十个国家和地区,全球病例高达9096例。目前尚无安全有效的SARS疫苗。核酸疫苗是SARS疫苗研究的重点。核酸疫苗不但兼有减毒活疫苗诱导全面免疫应答和重组亚单位疫苗安全的特点,而且具有研制周期短,生产工艺简单,成本低等优点,代表了SARS疫苗研发的发展方向。目前SARS核酸疫苗的研究主要以其结构蛋白S、N和M作为靶标构建重组质粒DNA,免疫小鼠则均可诱导产生体液免疫应答和细胞免疫应答。但核酸疫苗主要的缺点是免疫原性较低。为此,本研究对SARS-CoV的S和N基因进行密码子优化,观察其在小鼠中的免疫原性,并进一步采用原核表达的重组N蛋白与含有N基因的重组质粒DNA进行联合免疫,分析其诱导免疫应答水平的差异,观察对靶抗原基因密码子优化以及与重组蛋白联合免疫这两种策略提高SARS核酸疫苗免疫原性的效果,为研制具有高免疫原性的SARS核酸疫苗提供依据。首先,我们采用RT-PCR扩增获得长为2307bp、1632bp和1269bp的cDNA分子,分别编码SARS-CoV的S1(1～768aa)、S2(712～1255aa)和全长的N蛋白。将扩增产物分别克隆至pVAX1真核表达载体,获得重组质粒pVS1、pVS2和pVN。同时对SARS-CoV的S基因和N基因进行密码子偏好分析,发现其与人冠状病毒OC43株和229E株较为相近,但与哺乳动物则差异较大。以哺乳动物偏好密码子对S2和N蛋白进行反编译后获得优化S/N基因(opt-S2和opt-N),其G+C%含量有明显的提高。Opt-N和opt-S2基因序列的G+C%分别由48%提高至70%和由41%提高至64%,密码子第三位的G+C%则均提高至100%。将优化基因人工合成后同样克隆至pVAX1真核表达载体获得重组质粒pVeS2和pVeN。然后,分别将上述5种重组质粒DNA转染BHK_(21)真核细胞,48hr后通过间接
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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