乙脑病毒E蛋白中和表位与结核杆菌hsp70在大肠杆菌中的融合表达  

Expression of Chimeric JEV Neutralizing Epitope-hsp70 Plasmid in E.coli

作　　者：葛菲菲[1] 刘佩红[1] 王建[1] 沈莉萍[1] 徐锋[1] 孙泉云[1] 

机构地区：[1]上海市畜牧兽医站,上海201103

出　　处：《吉林农业大学学报》2008年第2期208-212,共5页Journal of Jilin Agricultural University

基　　金：上海市农业"四新"技术推广项目[沪农科推字(2005)第3-4号]

摘　　要：根据大肠杆菌偏嗜性密码子原则,人工合成了乙脑病毒E蛋白C末端的中和表位,位于E蛋白上373-399 aa,通过EcoRⅠ和HindⅢ连接构建原核表达载体pET-32a-epitope。将hsp70通过HindⅢ和XhoⅠ连接到载体pET-32a-epitope上中和表位的3′端,构建融合表达载体pet-32a-epitope-hsp70。诱导表达后,将上清和沉淀分别进行SDS-PAGE,结果表明该融合蛋白以包含体形式存在。将包含体在8 mol/L的尿素中过夜溶解,然后将上清过柱,获得良好的纯化结果。融合蛋白的分子量为94 kD。Western blot显示该蛋白兼具对JEV和结核杆菌热休克蛋白70（M.Thsp70）的特异抗原性。The neutralizing epitope exists on the 373-399 aa of JE virus E protein which were 27 amino acids. According to the frequency of E. coli codons, an E. coli favoriate DNA fragment, encoding identical amino acids sequence to that of the wild type, the neutralizing epitope was designed and synthesized. The plasmid pet-32a-epitope was created by subcloning the neutralizing epitope into the expression vector pET-32a（ ＋ ） at the EcoR Ⅰ site and HindⅢ site. It was reported that Mycobacterium tuberculo- sis hsp70 potentiates specific immune response to some antigenic peptides fused to it. In our study, DNA encoding M. T hsp70 was then subcloned into pet-32a-epitope at the Hind Ⅲ site and Xho Ⅰ site to generate pet-32a-epitope-hsp70. The recombinant protein expression was induced in E. coli BL21. The pellet and supematant were analyzed through SDS - PAGE and the result suggested that the fusion protein was in inclusion body form, soluble in 8 mol/L ureal for a night. The supemata-nt was purified on an Niaffinity chromatography column. Mr of fused protein was 94 kD as expected. Western blot indicated that the fusion protein had specific antigenicity of both JEV and M. T hsp70. The use of hsp70 fused to the neutralizing epitope may play a role in the design of JEV vaccine for the protection of swine from JE infection.

关 键 词：乙脑病毒E蛋白中和表位 大肠杆菌 结核杆菌hsp70 融合 包含体 

分 类 号：Q786[生物学—分子生物学] S852.6[农业科学—基础兽医学]