Expression&Immune Stimulatingactivity Of The MPT63Recombinant Protein For Mycobacterium Bovis

Bovine tuberculosis (bovine Tuberculosis) is a very important zoonotic disease, mainly caused by Mycobacterium bovis (Mycobacterium bovis, M. bovis) infection by inhalation the bacterial aerosol or edible unsterilized milk transmitted to humans. Prevention and treatment and the total eradication of bovine tuberculosis need to be sensitive and effective diagnostic method, a widely used intradermal allergy test and IFN-gamma release assay depends on the PPD as a source of stimulation, there is the detection of specific defects. In recent years, looking for the PPD an alternative source of stimulation study, in order to improve the detection specificity becomes a hotspot. Compare pathogenic mycobacteria and non-pathogenic mycobacterial genome, it was found that the RD region genes differ.Therefore, the RD region protein is expected to replace the PPD is used to distinguish pathogenic mycobacteria and non-pathogenic mycobacteria infection. Prokaryotic expression system to construct an expression of Mycobacterium bovis antigen MPT63, and purified by nickel affinity chromatography purified protein replacement to toxins PBS buffer system with the removal of the Triton X-114, with a purity above95%, the endotoxin content is less than2EU/mg of recombinant protein.By IFN-gamma release assay, guinea pig intradermal allergy test and leather allergy test evaluation of the recombinant protein T cell activity. Results show the MPT63not cause inactivation of Mycobacterium bovis DTH reactions in guinea pigs sensitized, but can be specific stimulation of the TB-positive cattle T lymphocytes to release a certain level of IFN-gamma, having certain T cell activity, additional antigen as CE potential.Due to the absence of bovine tuberculosis positive standard serum on the market, and different cattle infected with Mycobacterium individuals produce antibodies against different antigens from clinical screening367TB-positive bovine serum and47negative health bovine serum by indirect ELISA the method of analysis of the B cell activity of the recombinant protein MPT63. The results show that the recombinant protein has a certain B cell activity, but the detection rate of the recombinant protein by indirect ELISA detection method3.15-47.5%.