Construction and expression efficiency of recombinant plasmid pGEX-ESAT-6 of Mycobacterium tuberculosis

Objective To investigate expression efficiency of the recombinant plasmid pGEX ESAT-6 of Mycobacterium tuberculosis in Escherichia coli BL21(DE3).Methods ESAT-6 antigen gene were amplified by PCR.Then the gene was cloned into prokaryotic expression vector pGEX-1λT containing gluathione-S-transferaze(GST) gene.The recombinant plasmid(pGEX-ESAT-6) was analyzed with restriction-endonuclease digestion.The expression of pGEX-ESAT-6 was induced with isopropyl-β-D-thiogalactopyranosid(IPTG) and protein ESAT-6 /GST was examined by SDS-PAGE and Western blot techniques.Results 288 bp gene of ESAT-6 was amplified by PCR and cloned into pGEX-1λT by restriction analysis,the recombinant plasmid pGEX-ESAT-6 was constructed.It was demonstrated with SDS-PAGE and Western blot that the protein ESAT-6 /GST were expressed in E.coli BL21(DE3).Conclusion The gene ESAT-6 of M.tuberculosis was highly expressed in E.coli in fused form with GST and this kind of protein shows specific antigenicity.