Mycobacterium Bovis MPB70-64Fusion Protein In The Diagnosis Of Bovine Tuberculosis ELISA

Bovine tuberculosis, caused by M. bovis, is a chronic consumptive zoonotic infection. Eversince a long time ago, bovine tuberculosis showed global upgrade tendency because ofdeficiency of prevention vaccine and specific diagnostic agents.So the development of novel,safe and high performance vaccine and sensitive and specific diagnostic antigen have becomethe research spot.MPB70and MPB64are belong to MPB family and secreted proteins inculture fluids of pathogenic M. tuberculosis can induce strong memory T cell reaction. CD4+Tcell and CD8+T cell were stimulated and activated by them and produced high level IFN-γ. SoMPB70and MPB64are the ideal antigens for developing novel diagnostic reagents andvaccines. To increase the antigenicity of the unity antigen, the DNA fragments of mpb70andmpb64were fused by SOE-PCR and expressed in E.coli. The DNA fragments of mpb70andmpb64were fused by splicing by overlapping extension (SOE) polymerase chainreaction(PCR),and the fusion gene mpb70-64were cloned into pMD18-T vector, and then wegot the recombinant plasmid pMD-mpb70-64. pMD-mpb70-64and pET28a(+) were digestedby BamH I and EcoR I double enzymes. The purified mpb70-64fusion gene was subclonedinto the expression vector pET28a(+), and the prokaryotic expression vector pET-mpb70-64wasconstructed. Plasmid containing pET-mpb70-64was transformed into competence Escherichiacoli BL21(DE3). The bacterium was induced by IPTG (isopropyl-β-D-thiogalactopyranoside)and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE),approximately65kDa exogenous protein was observed on the SDS-PAGE. Theprotein was analyzed by using Western-blotting. The results indicated that the protein was ofantigenic activity of Mycobacterium bovis.ELISA diagnostic method was initially established by useing purified fusion proteinMPB70-64as a diagnostic antigen of bovine tuberculosis，and the selection of the reagents，theconditions of the test were reasonably arranged and optimized.By processing and testing296tested serum，the results revealed that17serum were positive by ELISA，and the positive ratewas6.42%，so the coincidence ratio of the two assay was89.4%.Thus, the fusion proteinMPB70-64as a ELISA diagnostic antigen to detect bovine tuberculosis had a very goodsensitivity and specificity.