结核分枝杆菌RpfA蛋白的表达、纯化与鉴定

目的构建结核分枝杆菌（Mycobacterium tuberculosis,MTB）RpfA的原核表达质粒,并在大肠杆菌中表达、纯化和鉴定。方法用PCR方法从结核分枝杆菌H37Rv基因组中扩增出编码RpfA蛋白的Rv0867c基因,通过克隆载体pMD18-T构建质粒载体pMD18-T-Rv0867c。经酶切和DNA序列测定证实正确后,将Rv0867c基因克隆到pET32a（＋）载体并在大肠杆菌BL21中诱导表,表达产物经SDS-PAGE和Western-blot鉴定后,用Ni-NTA亲和层析柱进行纯化。结果双酶切鉴定所切下的片段大小与理论值相符,测序结果与文献报道一致。Western-blot结果显示,在相对分子量约102 kDa处有分别与Anti-His单抗、Rpf结构域单抗和TB病人血清特异性结合带。结论成功表达、鉴定和纯化了RpfA蛋白。

Expression, identification and purification of RpfA protein from Mycobacterium tuberculosis

To construct the RpfA expression plasmid of Mycobacterium tuberculosis,and to express and purify the fusion protein in E.coli BL21,the Rv0867c gene was amplified by PCR from genomic DNA of MTB H37Rv strain,and cloned into the vector pMD18-T.Then Rv0867c was subcloned into the expression vector pET32a（＋） after identifying by restricted enzymes digestion and DNA sequencing.The constructed recombinant plasmid was transformed into E.coli BL21,and expressed the recombinant protein.The expressed protein was identified by SDS-PAGE and Western-blot,and the recombinant 6×His-fused protein was purified by Ni-NTA purification system.The size of the Rv0867c gene digested by restricted enzymes accorded with the theoretical size and the DNA sequence was identical to the reported one in literatures.As revealed by Western-blot,a specific binding band could be detected at site where relative molecular mass of 102 kDa located with Anti-His Tag mAb,Anti-Rpf domain mAb and sera of one TB patient.It is apparent that the recombinant RpfA protein was successfully expressed and purified.