Expression of MPT83 gene from Mycobacterium bovis and purification of its recombinant protein

The total DNA was extracted from the culture of Mycobacterium bovis. MPT83 gene fragment was amplified by PCR, the PCR product was cloned into pMD 18-T Simple Vector and then se-quenced. The confirmed MPT83 gene was cloned into plasmid pET-32a( + ) and then transformed into BL21(DE3) where it was induced to express by IPTG. The expressed protein was purified through affinity chromatography. Sequencing results revealed that target sequence was identical to that of the gene available in GenBank. The expression vector pET-32a( + ) was constructed and verified by PCR amplification and digestion with endonucleases BamHⅠ and HindⅢ. After induction with IPTG, there was a specific protein band of approximately 42 ku on SDS-PAGE gel. Cells induced for 4 hours by IPTG were harvested. Results of Western-blotting analysis indicated that the recombinant protein could react with polyclonal antibody against M. bovis. After sonication and affinity chromatography, SDS-PAGE showed only one specific band. These results demonstrated that the MPT83 gene was successfully cloned, its prokaryotic expression vector successfully constructed and purified recombinant protein was obtained through affinity chromatography.