结核分枝杆菌ESAT-6的原核表达与纯化及免疫原性检测  

Prokaryotic expression,purification and antigenicity analysis of Mycobacterium tuberculosis ESAT-6 protein

作　　者：石洁[1] 马晓光[1] 李辉[1] 邢进[1] 闫国蕊[1] 吴彩霞[1] 靳晓伟 

机构地区：[1]河南省疾病预防控制中心结核病防治所,河南郑州450016 [2]新密市疾病预防控制中心,河南新密452370

出　　处：《中华医院感染学杂志》2015年第3期481-484,共4页Chinese Journal of Nosocomiology

基　　金：国际合作项目"中澳卫生与艾滋病项目"基金资助项目(EID23);河南省重点科技攻关基金资助项目(122102310053)

摘　　要：目的构建及鉴定含有GST标签的结核分枝杆菌esat-6基因的原核表达载体,并在大肠埃希菌中高效表达融合蛋白。方法 PCR扩增结核分枝杆菌esat-6基因,并构建到pGEX4T-1上,重组质粒经测序鉴定后进行诱导表达,采用聚丙烯酰胺凝胶电泳和免疫印迹分析重组蛋白。结果扩增出了结核分枝杆菌esat-6基因,构建了具有正确基因序列的质粒载体pGEX4T-1-ESAT-6,转化大肠埃希菌BL21后经诱导产生了高水平的表达产物,蛋白纯化后可被结核病患者血清特异性识别。结论构建了质粒载体pGEX4T-1-ESAT-6,并经诱导后高效表达了ESAT-6融合蛋白,为进一步研究结核菌的免疫机制奠定了基础。OBJECTIVE To construct and identify a glutathione S-transferase （GST）-tag prokaryotic expression plasmid for the esat-6 gene of Mycobacterium tuberculosis, and to express the fusion proteins efficiently in Esche- richia coli BL21. METHODS The esat-6 gene was amplified by PCR and was cloned into pGEX4T-1 expression vector. The recombinant vector conformed by sequencing was induced to express recombinant proteins. The prokaryotic expression proteins were analyzed by SDS-PAGE and immunoblot. RESULTS The esat-6 gene was amplified by PCR and the recombinant expressive vector pGEX4T-1-ESAT-6 with correct sequence was constructed. The E. coli BL21 strains with recombinant vector showed high level expressions of ESAT-6 fusion protein after IPTG induction. The purified protein could be specifically identified by sera of patients with tuberculosis. CONCLUSION The plasmid vector pGEX4T-1-ESAT-6 was constructed and the expression of recombinant ESAT-6 protein laid a basis for further studies on immunological mechanism of M. tuberculosis.

关 键 词：结核分枝杆菌 esat-6基因 原核表达 抗结核免疫原性 

分 类 号：R378.91[医药卫生—病原生物学]