Prokaryotic Expression Of Mycobacterium Bovis Membrane-associated Proteins And Antigenicity Identification

Bovine tuberculosis is highly contagious zoonosis caused by the positive intracellular bacteria of Mycobacterium bovis. As an important zoonosis, it is a great threat to human health and animal husbandry. WHO indicated that if a country was contaminated by Bovine tuberculosis, the human beings will always be threatened by this bacterium. If they do not take actions to eliminate it, the control of human tuberculosis will be unsuccessful. Nowadays, some developed countries and regions, such as USA, Australia and north Europe etc. have eliminated Bovine tuberculosis in some extents, but the prevalence of human beings and wildlife tuberculosis made these countries detect it all the time. But in some developing countries, Bovine tuberculosis still prevails severely. In recent years, with the development and application of molecular biology and immunology, we have more deeply known about the gene sequence, the function of important genes, pathogenic and immunological mechanism of Mycobacterium bovis than before, this provide a foundation for the prevention and treatment of Bovine tuberculosis. In recent years, the diagnostic methods were improved gradually, more specific antigens and genes of Mycobacterium bovis were found. The diagnostic methods and products of Bovine tuberculosis were developed by the cooperation of traditional bacterium inspection procedure, immunology and molecular biological methods.It was well known that Mycobacterium bovis outer membrane protein had the immunogenic and immuno-protective effects. In this study the genomic sequence of Mycobacterium bovis was analyzed, the outer membrane proteins were selected, and 20 cell wall protein-coding genes were obtained. By the signal peptide and transmembrane structure of the target gene analyzed, the result showed that no transmembrane structures existed and all the proteins located in the extramembrane. The primers were designed with Primer 5.0 software. The genome of Mycobacterium bovis was extracted from the strain Vallee III, and then 20 genes of omps were amplified by touch down PCR. The products of PCR were ligased directly to the expression vector-PET-32a(+) at 16℃over night, then the products were transformed into DH5αand 20 recombinant expression strains were obtained. The recombinant expression vectors were identified by PCR, restriction enzyme analysis and sequencing. After induction by IPTG, 13 combinant fusion proteins were expressed in E.coli BL21 (DE3) induced. 7 recombination proteins PET-32a(+)/OMPs were specially recognized by serum from Mycobacterium bovis infected naturally dairy cows, this means the outer membrane proteins or cell wall proteins have strong immunogenicity and reaction. In the meanwhile, the expressions of these proteins provide the foundation for further research of the associated protein function.