结核分枝杆菌复活促进因子E的原核表达及纯化

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。

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

Objective To construct the prokaryotic expression vector of resuscitation-promoting factor(Rpf) E of Mycobacterium tuberculosis and purify the expressed product.Methods Amplify Rpf 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 plasmid,identify by restriction analysis and DNA sequencing,then transform to E.coli 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 pET32a( )-RpfEv was successfully constructed,and recombinant Rpf E protein with a hight purity was obtained.It laid a foundation of further study on Rpf E.