Expression,purification of the fusion protein Ag85B-ESAT6 of Mycobacterium tuberculosis and its antigenic activity

Ag85B and ESAT6 genes were firstly amplified by PCR from H37Rv virulent strain of Mycobacterium tuberculosis(MTB), and a flexible chain with 48bp was linked between Ag85B and ESAT6 genes, in order to ensure the correct folding of the fusion protein. The two genes were further cloned into vector pMD18-T respectively. By analysis with enzymatic digestion and DNA sequence, Ag85B and ESAT6 genes were subcloned into the prokaryotic expression vector pPROEXHTa, in turns, to produce the recombinant plasmid pPROEXHTa-Ag85B-ESAT6. The fusion protein was induced by IPTG in E.coli DH5α, and purified by Ni-NTA purification system under denaturing condition. Following analysis of SDS-PAGE and Western blotting, the protein was detected and purified efficiently. It mainly existed in inclusion body, which was fully renaturated by dialysis. The renaturated protein could react with sera of 8 cases with tuberculosis by indirect ELISA. It is evident that the prokaryotic expression vector of pPROEXHTa-Ag85B-ESAT6 was successfully constructed and the fusion protein was expressed efficiently, which enables further study on the immunogenicity and protective effect of Ag85B-ESAT6.