人肠道病毒71型衣壳蛋白VP1、VP2、VP3、VP4的构建、表达及抗原性研究

发布时间：2018-05-08 06:36

本文选题：衣壳蛋白 + VP1　；参考：《上海交通大学》2011年硕士论文

【摘要】：人肠道病毒71型(EV71)属于小RNA病毒科、肠道病毒属的成员,是手足口病的主要病原体。手足口病和中枢神经感染是EV71病毒感染引起的两大常见临床症状。目前针对EV71病毒感染的治疗还没有特异和高效的药物,也没有预防性疫苗上市。灭活疫苗的研究还处于Ⅰ期临床试验阶段;基因工程重组蛋白疫苗尚处于动物实验阶段,需要寻找好的有效抗原蛋白。因此,本论文在原核表达系统E.coli中高效表达了EV71四种衣壳蛋白:VP1、VP2、VP3、VP4,表达产物均以包涵体形式存在;包涵体经过纯化、复性后的SDS-PAGE电泳纯度平均达到90%以上。动物实验结果显示VP1、VP4蛋白具有良好的免疫原性,产生高抗体滴度;VP2、VP3无明显的免疫原性,没有检测到抗体滴度;仅VP1免疫血清具有中和活性。通过本论文实验,加深了对EV71四种衣壳蛋白的认识;证实了VP4因为没有产生中和抗体,不具有成为基因工程重组蛋白疫苗候选抗原的潜力。同时证实了原核表达的VP2、VP3抗原蛋白经过去变性剂初步复性后,与EV71病毒颗粒表面的VP2、VP3蛋白在空间构象上有差异,需要进一步深入研究;证实了VP1具有成为基因工程重组蛋白疫苗候选抗原的特性。这为今后EV71疫苗候选抗原的筛选提供了重要依据。
[Abstract]:Human enterovirus 71 (EV71), a member of the genus RNA, is a major pathogen of hand, foot and mouth disease. Hand, foot and mouth disease and central nervous system infection are two common clinical symptoms caused by EV71 virus infection. Currently, there are no specific and effective drugs for the treatment of EV71 infection, and no prophylactic vaccine is available. The research of inactivated vaccine is still in the stage of phase I clinical trial, and the recombinant protein vaccine is still in animal experiment stage, so it is necessary to search for a good effective antigen protein. Therefore, in the prokaryotic expression system E.coli, the four capsid proteins of EV71, 1 / VP1 / VP2 / VP3 / VP4, were highly expressed in the prokaryotic expression system, and all the expressed products were in the form of inclusion bodies, and the purity of SDS-PAGE after renaturation was over 90% on average. The results of animal experiments showed that VP1mVP4 protein had good immunogenicity, and the antibody titer of VP2VP3 was not detected, but that of VP1 immunized serum had neutralizing activity. Through the experiments in this paper, four kinds of capsid proteins of EV71 were further recognized, and it was confirmed that VP4 did not have the potential to be a candidate antigen for recombinant protein vaccine because it did not produce neutralizing antibody. At the same time, it was confirmed that the VP2VP3 protein expressed in prokaryotic cells was different from the VP2VP3 protein on the surface of EV71 virus after denaturalization, and needed further study. It is confirmed that VP1 is a candidate antigen for recombinant protein vaccine. This provides an important basis for the screening of EV71 vaccine candidate antigens in the future.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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