Clone of Mycobacterium tuberculosis Ag85A gene and its prokaryotic expression

Objective:To clone the Ag85A antigen gene of Mycobacterium tuberculosis and express it in E.coli k802.Methods:The Ag85A gene was amplified from the genome of Mycobacterium tuberculosis by PCR and was inserted into prokaryotic expressing vector pGEX5T to construct pGEX5T-Ag85A recombinant plasmid.The expression of Ag85A protein in E.coli k802 was induced by IPTG.The expressed protein was purified by affinity chromatography and the antigenicity of the expressed protein was confirmed by Western blotting assay.Results:A band about 0.9 kb in length was obtained by PCR and the recombinant plasmid pGEX5T-Ag85A was successfully constructed.A new band about 58 000 in length was observed after IPTG induction in E.coli.The relative molecular weight of the expressed protein was consistent with that expected.A single protein band of 58 000 in length was obtained after purification.The expressed protein could be specifically recognized by the sera of patients with tuberculosis patients.Conclusion:The Ag85A gene of Mycobacterium tuberculosis has been successfully cloned and expressed in E.coli k802,which paves a way for further studies on diagnosis and therapy of tuberculosis.