从结核分枝杆菌H37Rv基因组中扩增出ESAT-6基因,克隆入pET21a(+)载体。将成功构建的pET21a(+)-ESAT-6重组质粒转化入感受态BL21(DE3)中,经诱导表达后,使用SDS-PAGE和Western blot鉴定。超声破碎后发现目的蛋白以可溶性表达形式存在,经过Ni-NTA柱和DEAE-SepharoseTMFast Flow柱纯化,获得纯度约为95%的重组ESAT-6蛋白。将目的蛋白和RAW264.7细胞共孵育后,使用免疫荧光技术发现ESAT-6蛋白能够直接和RAW264.7的细胞膜结合。 The ESAT-6 gene was amplified from the Mycobacterium tuberculosis H37Rv genome and cloned into the pET21a (+) vector. The successfully constructed pET21a (+) - ESAT-6 recombinant plasmid was transformed into competent BL21 (DE3) and induced by SDS-PAGE and Western blot. After sonicated, the target protein was found to exist in soluble form. After purified by Ni-NTA column and DEAE-SepharoseTM Fast Flow column, a recombinant ESAT-6 protein of about 95% purity was obtained. After incubation of the target protein with RAW264.7 cells, it was found by immunofluorescence that ESAT-6 protein can directly bind to the membrane of RAW264.7 cells.