| Mycobacterium bovis is the causive agent of bovine tuberculosis in a range of animal species and human beings. The cross circulation of human and bovine tuberculosis became one of the important reasons for the extensive prevalence of tuberculosis. Because of the nearly relation between bovis and human beings, more than 10% of human tuberculosis is caused by Mycobacterium bovis, so bovine tuberculosis will menace the health of human beings severely. The prevalence of this disease will influence the development of stock raising directly. Moreover,it will infect human beings through many pathways, so we must take actions to prevent tuberculosis which is imminent. Since 1990s,the incidence of human tuberculosis has raised broadly, the death toll has reached 3 millions every year and become the maximum among many infections. Moreover, it became the maximal record for recent years. Up to 2005,the death toll of tuberculosis will surpass 5 millions. Now, there are 6 millions tuberculous people in our country which is one of the 22 countries for bearing tuberculosis.Together with the mobile population, multi-drug resistance(MDR) of Mycobacterium bovis, combined infection of AIDS and many social and economic factors, they have made the diagnosis and cure more difficult and influenced the prevention of tuberculosis severely, so now people are more attentive to tuberculosis than before. In memory of Kock who found Mycobacterium bovis in march 24 1882,WHO decided that march 24 of every year became the prevention day of tuberculosis since 1996,and that WHO indicated that if a country is contaminated by bovine tuberculosis, the human beings will always be menaced by it. 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, Austrilia and north Europe etc have eliminated bovine tuberculosis to some extents, but the prevalence of tuberculosis of human beings and wildlife 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, pathogenic and immunological mechanism of Mycobacterium bovis than before, so the prevention of bovine tuberculosis can base on it.Mycobacterium bovis is a kind of parasitic bacterium in the cell. In the immunity of anti-Mycobacterium, cell mediated immunity exerts leading effect. Some studiesindicated that the secreted proteins of living bacterium exerted the mainly protective immunity and induced T cell to produce strong cell mediated immunity. Culture fluids after growth of Mycobacterium bovis contained actively secreted proteins and these proteins can be identified by CD4+ and CD8+ T cells. Moreover, they can protect the laboratorial animals. The protective antigens of early culture fluids are Ag85B* MPB64* ESAT-6 and so on, which can regulate the action between Mycobacterium bovis and immunitive system, especially in the early stage of infection. Through the study of special immunity, it indicated that IFN which was produced by TLC was related with some secreted proteins directly. MPB64 mature protein is one of the proteins found in culture fluids and a mainly protective antigen of Mycobacterium bovis. Furthermore, MPB64 is a kind of exported lipid proteins presenting on the surface of Mycobacterium bovis, maybe it is the main target protein that can be identified by antibody as soon as the infection begin. The plasmid DNA encoding MPB64 can induce strong T-cell immunoreaction, mixing antibody of IgGl and IgG2 and protection of anti-Mycobacterium bovis if little mice* cavy and bovis were incubated with the plasmid.In this study, based on the analysis of national and international human tuberculosis, the sequential determination of MPB64 gene and its expression in E.coli BL21(DE3) were the main contents, so we can study the molecularly biological characteristic of Mycobacterium bovis and lay a solid foundation for the study of new vaccine. The MPB64 mature protein gene was amplified from Mycobacterium bovis Valleelll chromosomal DNA by using PCR technique and cloned into pGEM-T vector system. Recombinant clone was identified by a-complementarity, enzyme digestion and PCR identification, it supported that the recombinant plasmid pGEM-T-64 was successfully constructed. Moreover,through sequential determination and DNASTAR analysis,between the cloned MPB64 gene and that of Mycobacterium bovis BCG Tokyo strain, the sequential homogeneity reached 100%,and the amino acid did not mutate. The preceding analysis indicated that MPB64 gene was very conservative in Mycobacterium bovis. MPB64 gene that was cloned into pGEM-T vector system was subcloned into pET28a(+) procaryotic expression system,then the procaryotic expression plasmid pET28a-64 was constructed successfully. The plasmid was transformed into E.coli BL21(DE3) and induced by IPTQ when it was induced to 4 hours, its expression quantity reached the peak. Shallow scan indicated that the expression quantity has reached 28%. The expression protein was analyzed by usingWestern-blot to be proved that it had the antigenic activity of Mycobacterium bovis. So these results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and DNA vaccine against bovine tuberculosis.
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