Construction and expression of the prokaryotic expression vector of MTb esat6 gene

Objective:To construct prokaryotic expression vector carrying esat6 gene and express in E. coli. Methods: The MTb esat6 gene was amplified by PCR,then cloned into pQE30 plasmid, sequenced and then cloned into pET32a( + ) plasmid. Thus two kinds of prokaryotic expression vectors were constructed. Results: After being transformed into the E. coli and inducted with 1mM IPTG,no protein was expressed in the pQE30 - ESAT6 system, but a recombinant protein, about 19 kDa,was expressed in the pET32a( + ) - ESAT6 system. In the presence of 1mM IPTG for 4h,the protein was expressed to the maximum. The protein existed in cytoplasm in soluble form and represented 42% total protein of E. coli. It's antigenicity was confirmed by Westem blotting. The protein was purified through the Ni - NTA resin and the purity reached 92 % . Conclusion : The prokaryotic expression vector (pET32a( + ) - ESAT6) was constructed successfully,and the rESAT6 was obtained,providing an experimental basis for application of rESAT6.