Abstract：This study was to express the fusion protein of the culture filter protein 10 (CFP10) and earlier secreted antigen target 6 (ESAT6) of Mycobacterium tuberculosis in the yeast Pichia pastoris system and to evaluate its potential in bovine tuberculosis (bTB) detection. The fusion fragment cfp10-esat6 was amplified by PCR and constructed into the recombinant plasmid pPICZαA-(cfp10-esat6), which was transformed into the yeast P. pastoris GS115 by high voltage electroporation. The positive recombinant yeast was induced for 3 d in the culture medium containing 1% methanol. The fusion protein of CFP10-ESAT6 (eCE) was analyzed by SDS-PAGE and then purified by nickel ions affinity chromatography column which targets the histidine (His) tag. Immunological reactivity of eCE was also appraised by Western blot. Furthermore, the application potential of eCE in bTB detection was evaluated via using it as stimulator in bovine peripheral blood interferon gamma release test in vitro (IFN-γ release test). The results showed that eCE successfully expressed in the recombinant yeast and secreted into the culture supernatant after induced by methanol. Results of Western blot demonstrated that only one specific band appeared when the fusion protein reacted with monoclonal antibodies of CFP10, ESAT6, His tag and c-Myc proteins, respectively, indicating that eCE proved an efficacious immunological reactivity. In the IFN-γ release test for 165 cow blood samples, eCE had a positive coincidence rate of 82.3% as well as a negative coincidence rate of 78.8% compared with purified protein derivative (PPD) used as stimulator in bTB detection. In summary, eCE expressed successfully in the yeast P. pastoris system and exhibited an excellent biological activity. It can be used as stimulator like PPD in IFN-γ release test for the diagnosis of bTB, as well as improving the sensitivity and specificity of the test.
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