Molecular Cloning Of Heat Shock Protein 60 Gene Of Mycobacterium Tuberculosis And Expression In E.coli

1. ObjectiveHeat shock proteins (Hsps) belong to the most highly conserved protein families in nature. under thermal stress condition,They have large expression and consequently referred to as Hsp.increased synthesis of Hsp occurs under a variety of conditions of stress such as hypoxia,nutrient deprivation, viral infection, phagocytosis and transformation. Mycobacterium tuberculosis, the causative agent of tuberculosis. Mycobacterium tuberculosis genome carries a duplicate set of GroEL genes,GroEL1 and GroEL2,they encode Hsp60(cpn60.1)and Hsp65(cpn60.2),respective. when the human infect with mycobacterium tuberculosis,Hsp60 of M.tuberculosis have been shown to be highly antigenic,eliciting strong B cell and T cell immune responses. Thus,we clone the heat shock protein 60 (Hsp60) gene of Mycobacterium tuberculosis and express it in E.coli.and which lays the foundation for purify and research the immunological characterisitics of Hsp60 protein.2. MethodsHsp60 gene was amplified by PCR from Mycobacterium tuberculosis H37Rv genome DNA, and then connected with pZero-T vector and sequenced. The gene of hsp60 was cut form the plasmid TBhsp60-pZero-T by sestriction endonuclease digestion with EcoRI+XbaI,and was correctly inserted into sites cut with the same restriction endonucleases of prokaryotic expression vector pProEXHTb. The recombinant expression plasmid pProEXHTb-TBhsp60 was constructed.and transformed in different E.coli strain(BL21, DH5α, JM109), it has expressed the recombinant proteins by different molarity of IPTG.3. ResultsHsp60 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.4.ConclusionA confirmed hsp60 gene was cloned and expressed in E. coli successfully, which lays the foundation for purify and research the immunological characterisitics of Hsp60 protein. The plasmid was transformed into E.coli BL21 and induced by IPTG when it was induced to 4 hours; its expression quantity reached the peak and the augmentation of expression while the time addition,but that had no effect by IPTG..