EV71中和表位嵌合病毒样颗粒的表达纯化

Expression and purification of the chimeric virus-like particles displaying enterovirus 71 epitopes

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（HBeAg）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人大肠埃希菌诱导表达，经DEAE离子交换层析、CsC1垫层离心以及密度梯度离心后得到纯化的重组VLPs，并通过WesternBlot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HBe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HBe-SP70VLPs，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 vaceinet. Methods The fusion protein hepatitis B core （HBc）-SP70 was constructed by inserting SPT0 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 EVT1 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.