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 DH5a (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 pMD18-T. The fragment sequenced correctly was subcloned into the expression vector pGEX-4T-l and expressed in E.coli DH5a . 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.
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