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For raising the antigenicity of Mycobacterium bovis single antigen, fusion protein of two genes was acquired. The DNA fragments of ag85b and mpb64 were fused by splicing by overlapping extension (SOE) polymerase chain reaction(PCR), and the fusion gene ag85b-mpb64 were cloned into pMD-18-T vector, then we got the recombinant plasmid pMD-85b-64. pMD-85b-64 and pET28a(+) were digested by BamH I and EcoR I double enzymes. The purified pMD-85b-64 fusion gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET-85b-64 was constructed. Plasmid containing pET-85b-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 58 ku 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.