3种结核分枝杆菌复活促进因子基因的克隆、原核表达与纯化鉴定

Cloning and prokaryotic expression of genes encoding resuscitation-promoting factors from Mycobacterium tuberculosis and its purification

目的构建结核分枝杆菌复活促进因子B(rpfB)、D(rpfD)、E(rpfE)的原核表达质粒,并在大肠杆菌中表达和纯化。方法采用聚合酶链反应(PCR)以结核分枝杆菌H37Rv基因组DNA为模板扩增出rpfB(1089bp),rpfD(465bp)和rpfE片段(519bp),并连接在pET32a(+)载体上,重组质粒用酶切和DNA测序鉴定后,转化到大肠杆菌BL21中,经IPTG诱导表达了rpfB,rpfD和rpfE3种蛋白;利用Western-blot鉴定重组蛋白后,纯化目的蛋白。结果双酶切鉴定所切下的片段大小与预计相符,测序结果与文献报道一致。经SDS-PAGE分析和Western-blot鉴定均发现分别在61kDa、39kDa、41kDa处有特异性蛋白条带。结论成功构建了3种重组表达质粒pET32a(+)-rpfBv、pET32a(+)-rpfDv、pET32a(+)-rpfEv,并获得了3种高纯度的重组蛋白,为以后的深入研究奠定了基础。

To express and purify the genes encoding resuscitation-promoting factor B （rpfB）,D（rpfD）,E（rpfE） of Mycobacterium tuberculosis ,these genes were amplified by PCR from the genome of M. tuberculosis H37Rv strain, and cloned into pET32a（＋） vector, then transformed into E. coli Top10. After identifying by restriction digestion and DNA sequencing, the constructed recombinant plasmid was transformed into E. coli BL21 and the recombinant proteins were expressed after induction with IPTG. The target proteins were identified by Western-blot and purified by using affinity chromatograghy. The experimental results showed that 3 recombinant expression vectors for genes encoding RpfB, RpfD and RpfE were successfully constructed and the fusion proteins with high purity was obtained. These results would constitute the basis for the future studies for these resuscitation-promoting factors.