EV71中和表位嵌合病毒样颗粒的表达纯化

摘要

目的 通过原核系统表达纯化嵌合肠道病毒71型(EV71)中和表位SP70的病毒样颗粒(VLPs),作为备选的EV71基因重组亚单位疫苗.方法 将SP70插入乙肝病毒核心抗原(HBcAg)截短序列的主要免疫显性区域,合成融合蛋白基因后连接表达质粒转化入大肠埃希菌诱导表达,经DEAE离子交换层析、CsCl垫层离心以及密度梯度离心后得到纯化的重组VLPs,并通过Western Blot和ELISA实验鉴定其抗原性.结果 成功构建重组质粒pHBc-SP70,在大肠埃希菌中经IPTG低温诱导后高效表达,可溶性目的蛋白经离子交换层析、CsCl垫层离心和密度梯度离心三步纯化后纯度可达90％以上;纯化的融合蛋白可自发折叠组装为病毒样空心颗粒,与抗SP70单克隆抗体和抗HBc单克隆抗体均可特异性结合.结论 在原核系统中成功高效表达和纯化了表面展示EV71中和表位的嵌合HBc-SP70 VLPs,并证实其具有良好的抗原性,为该EV71基因重组疫苗的免疫效果评价奠定了基础.%Objective To study the expression and purification of the chimeric virus-like particles displaying epitopes of EV71 as a candidate of enterovirus 71 gene recombined vaccinet.Methods The fusion protein hepatitis B core (HBc)-SP70 was constructed by inserting SP70 into the main immunogenic region of truncated hepatitis B core antigen (HBcAg) sequence,expressed in E.Coli,and purified through sonication,ion exchange chromatography,CsC1 cushion centrifugation and density gradient centrifugation.Then its antigenicity was detected by ELISA and Western blot assay.Results Recombinant plasmid pHBc-SP70 was successfully constructed.And the soluble fusion protein was efficiently expressed induced by IPTG.The purity of the chimeric virus-like particles (VLPs) was up to 90％ after the purification process described in method.The purified fusion protein HBc-SP70 could be spontaneously folded and assembled into empty virus-like particles and react with the monoclonal antibodies against HBc and SP70.Conclusions The chimeric VLPs displaying epitopes of EV71 were efficiently expressed and purified in E.Coli.with excellent antigenicity,which laid a foundation for evaluation of the immune effect evoked by this EV71 gene recombined vaccine.