结核分枝杆菌RpfA蛋白的原核表达、鉴定和纯化

Expression,identification and purification of the resuscitation-promoting factor A of mycobacterium tuberculosis

目的:构建结核分枝杆菌(Mycobacterium tuberculosis,MTB)复活促进因子A(Resuscitation-promoting factor,ARpfA)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)方法从结核分枝杆菌H37Rv基因组DNA中扩增出RpfA基因(1 224bp),然后用双内切酶消化后,与同样酶消化的pET32aα(+)载体连接,转入大肠杆菌Top10;阳性克隆用酶切和DNA测序鉴定。测序正确后,将重组质粒转化到的大肠杆菌BL21中,经IPTG诱导,由T7启动子调控表达了RpfA蛋白;利用His-tag in-gel Stain对表达蛋白进行鉴定,最后对目的蛋白进行纯化。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。经SDS-PAGE分析和His-tag in-gel Stain鉴定均发现66ku处有特异性蛋白条带。结论:成功克隆并构建了RpfA基因的重组表达质粒pET32α(+)-RpfA,并获得了高纯度的重组蛋白,为以后的深入研究奠定了基础。

Objective：To construct the Resuscitation-promoting factor A（RpfA） expression plasmid of Mycobacterium tuberculosis, and express and purify it in E.coli. Methods ：RpfAgene of Mycobacterium tuberculosis was amplified with PCR method from the H37Rv,cloned into pET32a（＋） vector,and then transformed into E. coli Topl0. After identifying by restriction digestion and DNA sequencing,the constructed recombinant plasmid was transformed into E.coli BI21,and expressed the recombinant protein under the control initiated by T7 after induction with IPTG.The target protein was identified by His-tag in-gel Stain and purified by using affinity chromatograghy. Results：The size of the RpfA gene digested by restricted enzymes accorded with the theoretical size and the DNA sequence with the reported one in literatures. 66ku protein was found by SDS-PAGE and His-tag in-gel Stain. Conclusion：The recombinant expression plasmid pE332a（＋）-RpfA was successfully cloned and constructed, and the recombinant protein with high purity was obteined; which laid the basis for further study.