Expression and Identification on the MPB83-MPB70 Fusion Gene of <i>Mycobacterium bovis </i>in <i>Escherichia coli</i>

Abstract

For raising the antigenicity ofMycobacterium bovissingle antigen, fusion protein of two genes was acquired. The DNA fragments ofmpb83andmpb70were fused by splicing by overlapping extension (SOE) polymerase chain reaction (PCR), and the fusion gene mpb83-mpb70 were cloned into pMD18-T vector, then we got the recombinant plasmid pMD-83-70. pMD-83-70 and pET28a (+) were digested byBamH I andEcoR I double enzymes. The purified pMD-83-70 fusion gene was subcloned into the expression vector pET28a (+), and the prokaryotic expression vector pET-83-70 was constructed. Plasmid containing pET-83-70 was transformed into competenceEscherichia coliBL21 (DE3). The bacterium was induced by isopropyl-β-D-thiogalactoside (IPTG) and the lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 41 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 ofM.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.