关键词: 结核, 热休克蛋白质70, 基因表达, 原核细胞, 蛋白质类∕分离与提纯

Abstract: Objective To clone the heat shock protein 70 (HSP70) gene of mycobacterium tuberculosis bovis,and construct prokaryotic plasmid of mycobacterium tuberculosis HSP70 (Mtb HSP70) and express it in E. coli,further more,to obtain high pure Mtb HSP70. Methods The whole Mtb HSP70 DNA sequence was amplified from mycobacterium tuberculosis bovis genome by polymerase chain reaction (PCR).Then the sequence was cloned into plasmid pGEM-T easy and sequenced. The recombinant expression plasmid PQE30-Mtb HSP70 was constructed and expressed in E.coli M15,and Mtb HSP70 was purified by Ni-NTA affinity chromatography column. Results The sequence of Mtb HSP70 was amplified and identical with that published in GenBank. The prokaryotic expresstion plasmid of Mtb HSP70 was constructed success fully.With induction of IPTG, a new fusion protein with relative molecular mass of 70 400 was expressed and mainly located in inclusion bodies,the expressed 6×his-Mtb HSP70 fusion proteins were identified by Western blotting with anti-His monoclonal antibody. Conclusion A  confirmed Mtb Hsp70 gene is cloned and expressed in E. coli successfully,and high pure Mtb HSP70 is obtained.

Key words: uberculosis , heat-shock proteins 70 , gene expression , prokaryotic cells, proteins, isolation and purfication