结核分枝杆菌分泌蛋白ESAT-6的表达、纯化和鉴定  

Expression,Purification and Identification of the Mycobacterium Tuberculosis Fosters Filtrate Protein ESAT-6

作　　者：季林[1] 赵勇[1] 毛峰峰[1] 赵善民[1] 张彩勤[1] 白冰[1] 师长宏[1] 

机构地区：[1]第四军医大学实验动物中心,陕西西安710032

出　　处：《现代生物医学进展》2011年第23期4409-4411,共3页Progress in Modern Biomedicine

基　　金：国家"863"专项课题(2007AA02Z473);国家自然科学基金(30972767)

摘　　要：目的:克隆结核分枝杆菌分泌蛋白ESAT-6基因,并在大肠杆菌中进行表达和纯化。方法:用PCR方法从结核分枝杆菌H37Rv基因组扩增出ESAT-6基因片段,克隆至pMD18-T载体中,序列测定正确后,将其亚克隆到表达载体pGEX-4T-1并在大肠杆菌DH5α中表达,表达蛋白经SDS-PAGE及Western-blot分析后,亲和层析法纯化蛋白。结果:成功克隆了ESAT-6基因,并对其在E.coli中进行了表达,SDS-PAGE及Western-blot分析表明表达产物正确。通过GST纯化系统获得34kD纯化蛋白,与文献报道相符。结论:成功获得了纯化的ESAT-6蛋白,为进一步研究ESAT-6蛋白的致病机理提供了实验依据。Objective： To clone the gene of Mycobacterium Tuberculosis fosters filtrate protein ESAT-6,then express the fusion protein of ESAT-6 in Escherichia. Coli DH5α （E.coli） and purify the expressed protein. Methods： The ESAT-6 gene was amplified from the genome of M.tuberculosis H37RV by PCR and cloned into plasmid pMD 18-T. The fragment sequenced correctly was subcloned into the expression vector pGEX-4T-1 and expressed in E.coli DH5α . The expressed protein was identified by SDS-PAGE analysis and Western blot method, and subsequently purified it by GST-tag purification system. Results： The gene of ESAT-6 was successfully cloned and expressed in E.coli, and the expressed protein was identified correctly by SDS-PAGE analysis and Western blot analysis. Conclusion： The ESAT-6 was successfully expressed and purified, which provided material foundation for its pathogenic mechanism study.

关 键 词：结核分枝杆菌 ESAT-6 表达 纯化 

分 类 号：R378.911[医药卫生—病原生物学]