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 rectangle and EcoR rectangle 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-beta-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 productin the development of subunit vaccine, DNA vaccine and diagnostic reagents against bovine tuberculosis.