结核分枝杆菌分泌蛋白ESAT-6的表达、纯化和鉴定

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pMD18一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Westem—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。

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

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.