| In order to develop an indirect ELISA for diagnosing bovine tuberculosis, the DNA fragments of mpb70, mpb83 and esat-6 were amplified from the genome DNA of Mycobacterium bovis Vallee III. Then the DNA fragments of mpb70 and mpb83 were spliced by overlapping extension (SOE), yielding a fusion gene mpb70-83. This fragment and esat-6 were each inserted into pMD18-T vectors, and then subcloned into pET32a (+) to construct a recombinant plasmid pET70-83-E6 coding all three genes. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The recombinant protein was expressed with two hexohistidine tags induced by IPTG It was purified by nickel-NTA chromatography. The immunogenicity of the protein was tested by immunoblotting. The purified protein was found to be immunogenic and useful for serodiagnosis.A series of tests were carried out to determine the optimal confining solution and coat situation. L9 (3~4) orthogonal experiments were designed for determination of the optimal dilution of antigen, sera and goat-anti-rabbit IgG, and their reaction time. 150 sera from healthy cattle were tested in order to determine the critical values, the S/P values above 0.5 were considered as positive, below 0.4 as negative and between 0.5 and 0.4 as ambiguous.Sera from 85 cattle with bovine tuberculosis and 100 cattle without bovine tuberculosis were tested. The results show that the sensitivity is 69.41% (59/85) and the specificity is 96% (96/100). 117 sera (67 of them from PPD skin test positive cattle and 50 of them from PPD skin test negative cattle) were also tested and the results were compared with PPD skin test results. The total agreement between ELISA and PPD skin test was 82.05%.An ELISA kit was developed and applied in field test for industrialization. This study indicates that the ELISA kit is a valuable tool for diagnosis of bovine tuberculosis and has industrialization potential.
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