牛分枝杆菌mpb64-ag85b-esat6融合基因的分子克隆及表达  

Cloning and expression of mpb64-ag85b-esat6 fusion gene of Mycobacterium bovis in E.coli

作　　者：迟磊[1] 刘思国[1] 王春来[1] 宫强[1] 赵昆[1] 王勇[1] 刘建东[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/动物细菌病研究室,黑龙江哈尔滨150001 [2]吉林农业大学生物技术学院,吉林长春130118

出　　处：《中国预防兽医学报》2007年第8期588-590,共3页Chinese Journal of Preventive Veterinary Medicine

摘　　要：以牛分枝杆菌valleeⅢ株基因组DNA为模板,PCR扩增mpb64、ag85b和esat6 3个目的基因片段,采用重叠延伸剪接技术(SOE)和基因重组技术获得融合基因mpb64-ag85b,将mpb64-ag85b和esat6串联到同一原核表达载体pET32a(+)中获得重组质粒pET64-85b-e6。将其转化到大肠杆菌感受态细胞BL21(DE3)中,以IPTG(终浓度1 mmol/L)进行诱导,SDS-PAGE电泳分析表达情况,以Ni^(21)亲和层析柱纯化表达的融合蛋白和Western blot分析融合蛋白的免疫活性。结果表明:表达的融合蛋白大约为76 Ku,与预测大小相符,以Ni^(21)亲和层析柱纯化的融合蛋白能与牛结核病阳性血清发生反应。The DNA fragments of mpb64, ag85b and esat6 genes were amplified by PCR from Mycobacterium boris Vallee III genome DNA, The PCR products of mpb64 and ag85b were fused by SOE-PCR （splicing by overlapping extension）, The fusion gene mpb64-ag85b was inserted into the pET32a （＋） to produce pET64-85b-e6, and transformed into E.coli BL21 （DE3） cells. Protein expression was induced by IPTG induction and analyzed by SDS-PAGE, The expressed protein was mainly in an insoluble form and could be purified by Ni2＋ purification system, Western blot analysis showed that the protein could be recognized by antibodies from bovines infected with M.bovis.

关 键 词：牛分枝杆菌 融合基因 重组表达 

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