人结核分支杆菌HSP70的原核表达质粒的构建及诱导表达

Construction and expression of prokaryotic plasmid of mycobacterium tuberculosis HSP70

目的 构建编码人结核分支杆菌 (TB)热休克蛋白 70 (HSP70 )的Dnak基因的重组原核表达质粒 ,并研究其表达动力学。方法 质粒pMT70用限制性内切酶消化、核酸体外扩增 (PCR)及末端修饰后可获得Dnak基因全长 ,与大肠杆菌质粒 pRSET连接重组 ,阳性克隆经酶切分析鉴定后即为质粒 pMT70 3,转入受体菌BL2 1DE3中 ,经诱导剂异丙基硫代 - β -D-半乳糖 (IPTG)诱导 ,由SDS -PAGE、Western -blot鉴定 ,并以薄层扫描分析。结果 重组表达质粒 pMT70 3构建成功 ,可在大肠杆菌中高效表达 ,诱导后 1h即开始表达 ,16h达高峰期。表达量占菌体总蛋白的 6 5 % ,可溶性产物占总表达量的 90 %。结论 pMT70 3质粒不仅构建成功且更有利于对分支杆菌HSP70蛋白的纯化 ,方便对其抗结核。

Objective To construct recombinant prokaryotic plasmid DNA encoding mycobacterium tuberculosis heat shock protein 70(HSP70)-Dnak gene which was obtained from pMT70.Methods Amplified by polymerase chain reaction (PCR) and with terminal modification, the Dnak gene was cloned into the vector with T7 promoter and was confirmed by restriction endonuclease digestion. So a new plasmid pMT703 was constructed and transformed in E.Coli. strain- BL21DE3. The engineering bacteria were induced by IPTG. SDS-PAGE, Western-blot and scanning were used to analyze the results.Result The Dnak gene could be highly expressed in E.Coli as soluble protein. The expressing efficiency was 65% of the total cell protein and the soluble protein was 90% of the expressed protein.Conclusion The successful construction of plasmid pMT703 may assist in the functional characterization of HSP70 and its role in the therapy for tumor or tuberculosis is explored.