肠道病毒71型外壳蛋白VP1的可溶性表达、纯化及活性鉴定  

Expression,purification and characterization of the capsid protein VP1 of enterovirus 71 in E.coli

作　　者：周亚萍 史伟峰[2] 朱荫昌 曹利民 朱思梅 史梅[2] 李坤[2] 姜庆波[2] 

机构地区：[1]常州二十一世纪生物技术研究所有限公司,江苏常州213164 [2]常州市第一人民医院检验科,江苏常州213003

出　　处：《热带医学杂志》2011年第9期1018-1020,1035,共4页Journal of Tropical Medicine

基　　金：常州市卫生局重大科技项目(ZD200912)

摘　　要：目的克隆、表达和鉴定肠道病毒71型(EV71)VP1基因,得到可溶性的蛋白VP1,为制备EV71的抗体和诊断试剂的开发打下基础。方法优化EV71VP1蛋白基因,克隆并构建重组表达质粒pET15b/VP1,转化大肠杆菌BL21。使用Ni2+亲和层析柱对重组蛋白进行纯化,并用Westernblotting检测目的蛋白。以重组蛋白VP1为抗原,ELISA检测抗原活性。结果重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量为36000,与预计大小一致。ELISA实验证实,重组蛋白具有良好的抗原性。结论本研究成功克隆和表达了EV71VP1蛋白,并得到可溶性的蛋白,对肠道病毒71型诊断试剂的开发有进一步潜在的应用价值。Objective To clone,express and characterize the recombinant protein of the capsid protein VP1 of enterovirus 71（EV71）,and lay the foundation for preparation of antibodies and diagnose reagents of EV71.Methods The VP1 gene was cloned into pET15b vector to construct the recombinant plasmid pET15b/VP1.Recombinant VP1 protein was expressed in E.coli BL21 and purified by metal（Ni2＋） chelating affinity chromatography.The recombinant protein was determined by Western blot.ELISA was used to determine the antigenicity of the recombinant protein.Results The recombinant VP1 protein can be over expressed in E.coli.The molecular mass was estimated as 36 kDa by SDS-PAGE,and was consistent with the expected size.The antigenicity of the recombinant protein was demonstrated by ELISA.Conclusion The capsid protein VP1 of EV71 was successful cloned and expressed,which could be useful for developing diagnose reagents of EV71.

关 键 词：肠道病毒71型 重组蛋白VP1 可溶性抗原 

分 类 号：R373.2[医药卫生—病原生物学]