[Immunogenicity evaluation of Mycobacterium tuberculosis MPT83 protein and establishment of serological diagnostic method for bovine tuberculosis detection].

ABSTRACT

OBJECTIVE: The aim of this study was to express Mycobacterium tuberculosis MPT83 protein and to evaluate its immunogenicity in murine model as well as the serological diagnosis potential value for bovine tuberculosis. METHODS: The fragment of mpt83 gene was amplified and constructed into pET30a(+)-mpt83 recombinant plasmid. MPT83 fusion protein was purified with His affinity chromatography column from strain of BL21(DE3)-pET30a(+)-mpt83 after induced by IPTG, and then used to evaluate its immunogenicity in mice and the potential application in ELISA assay for the detection of bovine tuberculosis. RESULTS: SDS-PAGE and Western blot results show that MPT83 fusion protein was expressed successfully and possessed a good immunological reactivity. Flow cytometry (FCM) analysis displayed decreased expression of CD80 on dendritic cells and up-regulation of CD69 expression on both splenic CD4+ and CD8+ T cells. Meanwhile, more IL-4 specific secreting cell spots rather than those of IFN-γ were detected by ELISPOT assay in C57BL/6 mice injected with the fusion protein. Total 200 serum samples were detected by indirect ELISA based on MPT83 as antigen and the results showed 48.6% positive coincidence rate and 90% negative's compared to results of peripheral blood specific IFN-γ release assay in bovine tuberculosis detection. CONCLUSIONS: MPT83 fusion protein was expressed successfully with capability of eliciting Th2 immune response in mice and could be used for ELISA assay to detect bovine tuberculosis as a serological diagnosis antigen.