结核分枝杆菌复活促进因子E的原核表达及纯化

Prokaryotic Expression and Purification of Resuscitation-promoting Factor E of Mycobacterium tuberculosis

目的构建结核分枝杆菌复活促进因子E(RpfE)的原核表达质粒,在大肠杆菌中表达并纯化。方法采用PCR方法从结核分枝杆菌H37Rv基因组DNA中扩增出RpfE基因,双内切酶消化后,与同样酶消化的pET32a(+)载体连接,转化大肠杆菌Top10。阳性克隆经酶切和DNA测序鉴定正确后,将重组质粒转化至大肠杆菌BL21中,经IPTG诱导,由T7启动子调控表达RpfE蛋白,并对表达蛋白进行鉴定和纯化。结果双酶切鉴定所切下的片段大小与预期相符,测序结果与文献报道一致。经SDS-PAGE分析和Western blot鉴定,在相对分子质量41000处均可见特异性蛋白条带。经Ni2+-NTA金属螯合层析后,重组蛋白纯度可达90%以上。结论已成功地克隆并构建了RpfE基因的重组表达质粒pET32a(+)-RpfEv,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 prokaryotic expression vector of resuscitation-promoting factor（Rpf） E of Mycobacterium tuberculosis and purify the expressed product. Methods Amplify Rift E gene from the genomic DNA of M. tuberculosis H37Rv by PCR,insert into expression vector pET32a（ ＋ ） and transform to E. coli Top10. Screen positive clones,extract recombinant plnsmid, identify by restriction analysis and DNA sequencing,then transform to E. coil BL21 for expression under induction of IPTG. Identify the expressed product by SDS-PAGE and Western blot and purify by Ni2 ＋ -NTA agarose column chromatography. Results The length of amplified Rpf E gene fragment was consistent with that expected,and the sequence was identical to that reported. SDS-PAGE and Western blot showed specific protein band with a relative molecular weight of 41 000. The expressed protein reached a purity of more than 90% after purification. Conclusion The prokaryotic expression vector pE332a（ ＋ ）-BpfEv was successfully constructed,and recombinant Bpf E protein with a hight purity was obtained. It laid a foundation of further study on Bpf E.