Prokaryotic expression and analysis on the mpb83-ag85b fusion gene of Mycobacterium bovis

For raising the antigenicity of Mycobacterium bovis single antigen, fusion protein of two genes was acquired. The DNA fragments of mpb83 and ag85b were fused by splicing by overlapping extension (SOE) polymerase chain reaction(PCR), and the fusion gene mpb83-ag85b were cloned into pMD18-T vector, then we got the recombinant plasmid pMD-83-85b. pMD-83-85b and pET28a(+) were digested by BamH I and EcoR I double enzymes. The purified pMD-83-85b fusion gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET-83-85b was constructed. Plasmid containing pET-83-85b 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 55 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 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 subunit vaccine and DNA vaccine against bovine tuberculosis.