| Bovine tubereulosis (bTB), caused by Mycobacterium bovis, is a zoonotic disease. It caused an important health problem and significant losses in economies worldwide. In many countries, the control measure for bTB is based on test and slaughter policies. In spite of widespread control programmes, bTB remains biologically present, even in countries officially considered free of the disease, and it appears very difficult to eradicate.Immune responses against mycobacterial infections are predominantly cellular, at least initially. For testing, cell mediated immune(CMI)-based assays are the primary surveillance and disease control tests for bTB, including tuberculin skin test (TST) and the interferon gamma (IFN-γ) assay. The most common diagnostic method, based on intradermal tests, has limitations regarding both sensitivity and specificity. The Gamma-interferon assay has been evaluated as a primary diagnostic method in many countries. IFN-γ assay measures the IFN-γ released in vitro in response to specific antigens in whole-blood culture. IFN-γ assay used as a supplement method for TST in disease control tests. Purified protein derivative (PPD) tuberculins is prepared by precipitation of heat-killed cultures of Mycobacteria and consists of an ill-defined mixture of proteins. So the cross-reactions always cause PPD resulting in lower specificities. Mycobacterium tubereulosis specific antigen CFP10and ESAT6, whose encoding genes existent in the genome of virulent Mycobacterium tubereulosis, but absent in M.avium and M.paratuberculosis, have considerable potential as diagnosis antigens for bTB. In this study, we expressed and purified CFP10-ESAT6fusion protein in the yeast Pichia pastoris. Meanwhile, its potential capacity in the detection of bovine tuberculosis was evaluated. 1. Expression and indentification of Mycobacterium tubereulosis CFP10-ESAT6fusion protein in the yeast Pichia pastorisThe fusion gene cfp10-esat6was cloned and identified by PCR amplification and sequencing analysis, then cloned into pPICZaA vector and named as pPICZaA-(cfp10-esat6). The recombinant plasmid was transformed into the yeast Pichia pastoris GS115. After induced by methanol, fusion protein was purified by the method of affinity chromatography from the yeast culture supernatant. Further more, the immunoreactivity of the fusion protein was analyzed in Western bloting assay. SDS-PAGE analysis demonstrated that the fusion protein was successfully expressed and secreted. Results of Western blotting assay which only one specific band appeared for monoclonal antibody (McAb) CFP10and McAb ESAT6indicated that the purified protein proved its efficacious immunoreactivity. The production of the fusion protein was18.3mg/L under optimal inducing condition.2. The application of CFP10-ESAT6fusion protein in the detection of bovine tubereulosisThe skin tests were assayed with the purified recombinant CFP10-ESAT6proteins expressed from yeast Pichia pastoris and E.coli. The results indicated that the specificity of fusion proteins was as well as the comparative cervical test (CCT). The index coincidence of the results between eukaryotic protein skin tests and CCT was59.3%, higher than prokaryotic protein skin tests. For the negative samples of CCT, the coincidence rate of the results between the eukaryotic protein assay and CCT was83.3%, higher than the prokaryotic protein (54.5%).The results of IFN-y assay with CFP10-ESAT6were similar as the results of IFN-y assay with PPD during these low disease prevalence samples. Than the eukaryotic protein and prokaryotic protein were applied to IFN-y ELISA test during these hight disease prevalence samples, respectively, the sensitivity of recombinant proteins were efficacious. The index coincidence between eukaryotic protein and bovine PPD was83.7%, higher than the prokaryotic protein (78.9%). These results shows that IFN-y assay with the recombinant proteins could distinguish these samples which influenced by cross reaction caused BOVIGAMTM kit was inability. |
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