Expression and analysis of Mycobacterium bovis mpb63–64 fusion gene in Escherichia coli

The DNA fragments of mpb63 and mpb64 were fused by splicing by overlapping extension(SOE) polymerase chain reaction(PCR), and the fusion gene mpb63-64 was cloned into pMD18-T vector, then we got the recombinant plasmid pMD-63-64. pMD-63-64 and pET28a(+) were digested by BamH□and EcoR□ double enzymes.The purified mpb63-64 fusion gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET-63-64 was constructed. Plasmid containing pET-63-64 was transformed into competence Escherichia coli BL21(DE3). The bacterium was induced by isopropyl-β-D-thiogalactopyranoside(IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE), approximately 40 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic reactivity 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 subunit vaccine, DNA vaccine and diagnostic reagents against bovine tuberculosis.