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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 ESAT-6 gene of Mycobacterium tuberculosis secreted protein and express and purify it in Escherichia coli. Methods: ESAT-6 gene fragment was amplified from the Mycobacterium tuberculosis H37Rv genome by PCR and cloned into pMD18-T vector. After the sequence was determined correctly, it was subcloned into expression vector pGEX-4T-1 and transformed into E.coli DH5α, the expressed proteins were analyzed by SDS-PAGE and Western-blot, and the proteins were purified by affinity chromatography. Results: The ESAT-6 gene was successfully cloned and expressed in E. coli. SDS-PAGE and Western-blot analysis showed that the expression product was correct. The 34kD purified protein was obtained by GST purification system, which was reported in the literature. CONCLUSION: The purified ESAT-6 protein was successfully obtained and provided an experimental basis for further study on the pathogenesis of ESAT-6 protein.