Prokaryotic Expression and Identification on the ag85a and mpb70 Fusion Gene of Mycobacterium bovis

摘要

Based on splicing by overlapping extension(SOE) polymerase chain reaction(PCR) ,the ag85a and mpb70 were amplified and the fusion gene ag85a-mpb70 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-85a-70. pMD-85a-70 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified ag85a-mpb70 fusion gene was subcloned into the expression vector pET28a(+),and the prokaryotic expression vector pET-85a-70 was constructed. Plasmid containing pET-85a-70 was transformed into competence Escherichia coli BL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranosidc (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 49 kDa fusion protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that Ag85A-MPB70 was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of novel vaccine against bovine tuberculosis.