甲型流感病毒NP和M2e融合蛋白高效表达和纯化  

Efficient expression and purification of nucleoprotein and M2e fusion protein of influenza A virus in Escherichia coli

作　　者：黄保英[1] 王秀平[1] 谭文杰[1] 王文玲[1] 阮力[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所冠状病毒和痘病毒相关疾病室,北京102206

出　　处：《中华实验和临床病毒学杂志》2015年第3期270-272,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家863计划（2006AA02A203）;国家自然科学基金项目（31200127）

摘　　要：目的 探索甲型流感病毒NP-M2e融合蛋白在大肠埃希菌中可溶性高效表达和纯化的条件.方法 将NP-M2e融合基因（NM2e）经密码子优化后插入pET30a后获得pET30a-NM2e原核表达质粒,通过转化BL21（DE3）后的克隆筛选、诱导温度与诱导时间等条件的优化实现NM2e蛋白在大肠埃希菌中的可溶性高效表达;表达产物经离子交换层析与分子筛层析纯化并通过Western-Blot鉴定其抗原性.结果 NM2e基因正确插入pET30a中并能在大肠埃希菌中表达;25℃温度诱导下NM2e蛋白出现可溶性表达,诱导时间由4h延长至10 h可明显提高蛋白表达量;可溶性NM2e蛋白经阴离子交换和凝胶过滤层析两步纯化后的纯度可达90％,纯化产物能特异性结合NP鼠多抗及M2e鼠单抗.结论 甲型流感病毒NM2e融合蛋白能在大肠埃希菌中高效表达和纯化并保持良好的免疫反应活性。Objective To efficiently express NP-M2e fusion protein of influenza A virus A/Jingke/ 30/95 （H3N2） in E.coli.Methods NP-M2e fusion structure （NM2e） was codon-optimized and pET30a-NM2e recombinant plasmids were constructed.Comparative studies were carried out on the gene expression efficiency,induction temperature and time,purification process and immune reactivity.Results NM2e gene was correctly inserted into pET-30a.SDS-PAGE showed that NM2e could be efficiently expressed in E.coli.The lower temperature （T =25℃） and longer time induction （t =10 h） were necessary for high-level expression of soluble NM2e.The purity of NM2e was up to 90% through two-step purification process with anion-exchange and gel filtration chromatography,and purified NM2e reacted well with serum from NP immunized mice and monoclonal antibody against M2e.Conclusions NM2e with specific immune reactivity can be efficiently expressed and purified from supernatant of E.coli lysate.

关 键 词：流感病毒A型 病毒融合蛋白质类 原核细胞 蛋白质工程 

分 类 号：R373[医药卫生—病原生物学]