牛分枝杆菌融合基因hsp65-esat6的原核表达及产物的纯化  

Prokaryotic expression of the fusion gene hsp65-esat6 of Mycobacterium bovis and purification of the expressed protein

作　　者：迟磊[1] 刘思国[1] 宫强[1] 王春来[1] 赵昆[1] 王勇[1] 刘建东[1] 刘慧芳[1] 周媛媛[1] 常月红[1] 云孟克[1] 赵福广[2] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001 [2]吉林农业大学生命科学学院,吉林长春130118

出　　处：《中国兽医科学》2007年第3期223-226,共4页Chinese Veterinary Science

基　　金：国家科技攻关计划项目(2002BA518A04)

摘　　要：以牛分枝杆菌ValleeⅢ株基因组DNA为模板扩增hsp65和esat6基因,采用重叠延伸剪接技术(SOE)获得了融合基因hsp65-esat6,将hsp65-esat6连接到原核表达载体pET32a(+)上,构建重组质粒pET65-E6,将其转化到大肠杆菌BL21(DE3)感受态细胞中,以IPTG诱导(终浓度为1 mmol/L),表达产物进行SDS-PAGE分析。以Ni2+亲和层析柱纯化表达的融合蛋白和Western-blot分析该融合蛋白的免疫活性。结果表明:Hsp65-ESAT6融合蛋白以包涵体形式表达。表达的融合蛋白裂解为两部分,即58 ku和30ku,二者相加与预测大小88 ku相符。纯化的融合蛋白能与抗牛分枝杆菌阳性血清发生反应。The DNA fragments of hsp65 and esat6 were amplified separately by PCR from genomic DNA of Mycobacterium boris Vallee m strain, and a fusion gene hsp65-esat6 was obtained by SOE（splicing-by-overlapping extension）. Then the fusion gene was inserted into pET32a（＋） to construct a recombinant plasmid pET65-E6. The recombinant plasmid was transformed into Escherichia coli BL21 （DE3） competent cells under induction of 1 mmol/L IPTG. SDS-PAGE analysis showed that the expressed fusion protein was insoluble. It was separated into two proportions,namely 58 ku and 30 ku. The total protein molecular mass of the fusion protein was in accordance with the expected 88 ku. Western blot analysis showed that the protein purified by Ni^2＋ Purification System could be recognized by sera from bovines infected with M. boris.

关 键 词：牛分枝杆菌 融合基因 原核表达 纯化 

分 类 号：S852.618[农业科学—基础兽医学] Q786[农业科学—兽医学]