结核分枝杆菌热休克蛋白60编码基因的克隆与原核表达

Cloning and prokaryotic expression of heat shock protein 60 gene of Mycobacterium tuberculosis

目的克隆结核分枝杆菌热休克蛋白60(Hsp60)编码基因,并在大肠杆菌中表达。方法利用PCR技术从结核杆菌H37Rv株中扩增hsp60基因,并将其与pZero-T载体连接,进行DNA测序。将得到的hsp60基因亚克隆到表达载体pProEXHTb中,构建重组原核表达质粒pProEXHTb-TBhsp60,并在大肠杆菌BL21中诱导表达。结果成功地克隆了结核分枝杆菌hsp60基因。DNA测序证实,与GenBank公布的序列一致。含pProEXHTb-TBhsp60基因表达质粒的大肠杆菌经IPTG诱导后,能够高效表达相对分子质量(KD)约为60KD的融合蛋白。结论获得了结核分枝杆菌hsp60基因,成功地构建了原核表达质粒pProEXHTb-TBhsp60,并在大肠杆菌得到表达,为研究其免疫学特性奠定了基础。

Objective To clone the heat shock protein 60 (Hsp60) gene of Mycobacterium tuberculosis and express it in E.coli.Methods hsp60 gene was amplified by PCR from Mycobacterium tuberculosis H37Rv genome DNA,and then connected with pZero-T vector and sequenced.The confirmed hsp60 DNA fragment was furthe subcloned into the prokaryotic expression vector pProEXHTb.The recombinant expression plasmid pProEXHTb-TBhsp60 was constructed and expre ssed in E.coli BL21 strain.Results hsp60 gene was obtained form Mycobacterium tuberculosis and its sequence was identical with that reported in GenBank.The E.coli BL21 strain containing pProEXHTb-TBhsp60 over expressed a about 60000 kDa protein after induction of IPTG.Conclusion A confirmed hsp60 gene was cloned and expressed in E.coli successfully,which lays the foundation for research the immunological characterisitics of Hsp60 protein.