SARS-CoV主要结构蛋白的基因表达及DNA疫苗构建

发布时间：2018-06-26 21:05

本文选题：SARS冠状病毒 + 蛋白表达　；参考：《华南热带农业大学》2005年硕士论文

【摘要】：严重急性呼吸道综合症(severe acute respiratory sydrome, SARS)的病原体为SARS冠状病毒,SARS-CoV中参与病毒组装和细胞侵染过程的物质主要为4种结构蛋白(S、M、N、E),这些蛋白是决定SARS-CoV独特的生物学行为的物质基础,正确表达这四种结构蛋白对于SARS的诊断,试剂开发和疫苗的构建有着深远的意义。本课题研究的目的是用大肠杆菌表达的SARS冠状病毒的主要结构蛋白S、M、N、E作为抗原检测SARS-CoV抗体的可行性,及其这4个结构蛋白基因构建的DNA疫苗诱发小鼠产生抗SARS-CoV的IgG水平。研究方法:首先克隆了SARS-CoV主要结构蛋白(S、M、N、E)的编码基因,以pET-15b、pET-22b、pQE-2为载体,并分别在原核系统中表达,表达的蛋白均以可溶的形式存在,根据融合蛋白的性质采用亲和层析的方法获得纯化的重组蛋白M15b、M22b、rN、rS1、rS2、E15b。同时将这四个主要蛋白的编码基因克隆入真核表达载体pSecTagB,构建DNA疫苗,免疫小鼠后获得抗血清,并进行了一系列的ELISA检测。结果发现以重组蛋白作为抗原进行ELISA检测能与免疫了DNA疫苗的小鼠血清以及SARS病人恢复期血清均可发生特异性的抗原抗体反应;另外,用SARS全病毒作为抗原(华大公司SARS检测试剂盒)也可以和免疫鼠血清发生抗原抗体特异性反应。重组蛋白可以在大肠杆菌中得到高效表达,并可作为SARS检测的抗原。构建的DNA疫苗可以在小鼠体内产生抗SARS的特异性抗体,为SARS-CoV DNA疫苗的开发提供借鉴作用。
[Abstract]:The pathogen of severe Acute Respiratory Syndrome (SARS) is SARS-CoV SARS-CoV, which is involved in the assembly and infection of SARS-CoV mainly by four structural proteins, which are the material basis for determining the unique biological behavior of SARS-CoV. The correct expression of these four structural proteins is of great significance for SARS diagnosis, reagent development and vaccine construction. The purpose of this study was to detect the SARS-CoV antibody by using the main structural protein of SARS coronavirus expressed in Escherichia coli as antigen, and the level of IgG against SARS-CoV in mice induced by the DNA vaccine constructed by these four structural protein genes. Methods: firstly, the encoding gene of SARS-CoV main structural protein (SARS-CoV) was cloned. The vector pET-15bPET-22bPQE-2 was expressed in prokaryotic system, and the expressed proteins were all in soluble form. According to the properties of the fusion protein, the purified recombinant protein M15bS1, rS1 and E15b was obtained by affinity chromatography. At the same time, the four major protein coding genes were cloned into eukaryotic expression vector pSecTagB. the DNA vaccine was constructed, and the antiserum was obtained after immunizing mice, and a series of Elisa tests were carried out. The results showed that Elisa with recombinant protein as antigen could react with the sera of mice immunized with DNA vaccine and the convalescent sera of SARS patients. SARS-CoV as antigen (SARS-CoV kit) can also react specifically with the sera of immunized mice. The recombinant protein can be highly expressed in E. coli and can be used as an antigen for SARS detection. The constructed DNA vaccine can produce specific antibodies against SARS in mice and provide reference for the development of SARS-CoV DNA vaccine.
【学位授予单位】：华南热带农业大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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