Construction of prokaryotic recombinant plasmid and high expression in E.coli of Mycobacterium tuberculosis ESAT-6

Objective To obtain the high expressed early secretory antigentic target-6 (ESAT-6)protein of mycobacterium tuberculosis.Methods ESAT-6 gene was amplified by PCR from mycobacterium tuberculosis and the digestion fragment was cloned into pET-21b(+).After being verified with restriction endonuclease digestion and DNA sequencing,the positive recombinant vectors were transformed into E.coli BI21(DE3)which was then induced with IPTG and examined the expression of recombinant by SDS-PAGE and western-blotting.The fusion protein was purified by affinity chromatograph.Results ESAT-6 gene was amplified and the pET-ESAT-6 prukaryotlc recombinant plasmid was obtained.The recombinant protein was highly expressed by the induction of IPTG and can be recognized by serum of patient with Tuberculosis.Conclusion pET prokaryotic expression system can be used to construct the ESAT-6 recombinant and highly expressed the ESAT-6 protein.Its antigen specificity was confirmed as it could be recognized by antigen-specific antibody(anti-ESAT-6).