肠道病毒71型VP1的原核表达及生物活性初步研究  

Prokaryotic expression of EV71 VP1 and initial evaluation of the biological activity

作　　者：李志会[1] 宋楠楠[1] 岳盈盈[1] 李鹏[1] 纪璇[1] 曹银光[2] 孟红[1] 

机构地区：[1]山东省医学科学院基础医学研究所,山东省罕少见病重点实验室,济南250062 [2]聊城市人民医院

出　　处：《中华微生物学和免疫学杂志》2012年第11期972-976,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（81000720）;山东省自然科学基金资助项目（2009ZRC03096）

摘　　要：目的原核表达系统表达肠道病毒71型（Enterovirus71，Ev71）VP1蛋白，并探讨该基因工程蛋白阻断病毒感染及其制备抗体的中和活性。方法构建pET30a（＋）-VP1表达载体、IPTG诱导表达，Westernblot鉴定。His亲和纯化后，阻断试验分析该重组蛋白阻断病毒感染活性，免疫小鼠制备多抗分析多抗中和活性。结果构建的重组菌可有效表达VP1，用其免疫小鼠制备多抗ELISA效价达到1：3200，而中和活性只有1：16，并且该工程蛋白没有表现出阻断活性。结论本研究原核表达的VPl蛋白能刺激小鼠产生抗体，且抗体具有一定的中和活性，但是该蛋白没有阻断活性，表明该蛋白在原核表达系统中未能形成天然构象从而导致与受体结合的能力较差。Objective To express EV71 VP1 in prokaryotic expression system, initially evaluate the ability of blocking EVT1 infection and the neutralizing activity of its polyclonal antibody. Methods Con- struct the prokaryotic expression plasmid pET30a （ ＋ ） -VP1. Induced expression in Transetta （ DE3 ） with IPTG and identified by Western blot. After purified with HisBind Protein Purification Kit, its ability of bloc- king EV71 infection and the neutralizing activity of its po]yclonal antibody were analyzed. Results Plasmid PET-30a（ ＋ ）-VP1 was constructed successfully and the objective protein was expressed effectively. The ELISA titer of the polyclonal antibody was 1：3200 while neutralizing titer was 1：16 and the recombination protein lost the ability of blocking EV71 infection. Conclusion The recombination protein can stimulate mice to produce antibodies and the polyclonal antibody shew neutralizing activity but the recombination pro- tein lost the binding activity to receptors probably due to the wrong advanced structure.

关 键 词：肠道病毒71型 VP1 原核表达 生物活性 

分 类 号：R3[医药卫生—基础医学]