流行性乙型脑炎病毒E蛋白主要抗原表位的原核表达  

Prokaryotic expression of major antigenic epitope of Japanese encephalitis virus

作　　者：李云云 郭万柱[2] 

机构地区：[1]赤峰农牧学校畜牧兽医系,内蒙古赤峰024031 [2]四川农业大学动物生物技术中心,四川雅安625014

出　　处：《黑龙江畜牧兽医》2014年第1期14-16,20,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(30500019);"长江学者和创新团队发展计划"创新团队项目(IRT0848);四川省教育厅重点实验室项目(07ZZ027)

摘　　要：为了克隆乙型脑炎病毒E蛋白主要抗原片段基因的原核表达系统，试验采用RT—PCR方法扩增乙型脑炎病毒E蛋白上2个重要抗原域73～88aa和292～402aa，并将其定向连接至原核表达载体pET-32a（＋），构建重组质粒pET—EAB，再将重组质粒转入大肠杆菌Rosetta（DE3）感受态细胞，用IPTG进行诱导表达，应用SDS—PAGE和Western—blot技术对表达产物进行检测与鉴定。结果表明：经SDS—PAGE分析，表达出的目的蛋白主要以包涵体形式存在；再使用Western—blot技术对纯化复性后的包涵体进行检测，证实其具有免疫活性。To clone a prokaryotic expression system of major antigenic segment of Japanese encephalitis virus （ JEV ） E protein gene. A iragment about the E protein on the two major antigenic domain of 73 ~ 88 aa and 292 N 402 aa was amplified by RT - PCR, and then the fragment was cloned into the prokaryotic expression vector pET - 32a （ ＋ ） to construct prokaryotic expression plasmid pET - EAB. After confirmed positive by enzyme digestion, the recombinant plasmid was transformed into E. coli Rosetta （ DE3 ）, and then was induced using IPTG for the expression of EAB protein. The expression products were detected and identified using SDS - PAGE and Western - blot. The results showed that the ex- pressed target protein existed mainly in the form of inclusion body using SDS - PAGE analysis ; and then the Western - blot analysis was used to determine the inclusion body after purified and refolded. It is confirmed that the EAB protein has immune activity.

关 键 词：乙型脑炎病毒 E蛋白主要抗原域 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]