Fusion Expression Of Cfp 10-Esat6 And Detection Of Mycobacterium Bovis

Bovine tuberculosis is a chronic consumptive disease that is mainly caused by mycobacterium bovis. It is wildly prevalent in many countries, particularly in developing countries. The disease not only caused vasty economic loss to husbandry industry, but also exerted serious threat to human health. As the rapid growth of our milk industry, bovine tuberculosis of dairy cow has spread to the whole country in recent years. This situation impeded the sustainable development of our nation's milk industry and threatened the food safety and public health severely. Thus, the control of bovine tuberculosis is important to the prevention and cure of human tuberculosis. However, there has not specific therapeutic method to cure tuberculosis radically yet. So it's more urgent and realistic to detect, diagnose and prevent tuberculosis in initial stage. Purified protein derivative (PPD) of tuberculin, which is used in skin test and immunological diagnosis of tuberculosis, is a mixture of multiple antigens. The antigens exist in pathogenic mycobacteria, environmental mycobacteria and Bacilli Chalmette-Guerin (BCG). Due to the cross reaction, the diagnostic value of PPD skin test is limited.Gene CFP-10-Esat6 was expressed by polymerase chain reaction (PCR), using the genome of bovine mycobacteria as template, the fusion gene Cfp10-Esat6 was amplified and plasmid pMAL-p2X was expressed in E.coli. The results of SDS-PAGE showed that recombination strain expressed interest protein MBP-EC separately, which weighs about 65kD. The interest protein is soluble and account 30.3% of the total supernatant soluble proteins.The expressed protein was purified by Amylose resin chromatography. Serum antibodies of tubercle infected and healthy guinea pig were detected by enzyme linked immunosorbent assay (ELISA) and the purified fusion protein MBP-EC was used as antigen. Mycobacterium sensitized Guinea pig model was established. PPD and recombination protein were used in DTH skin test. After 24h, we measured the diameter of DTH, to valuate the diagnosis application of the recombination protein. The recombination protein has comparatively high antigenic specificity and immunologic characteristic, so it may become the specific antigen in tuberculosis immunological diagnosis.