Construction And Immunogenicity Of Eukaryotic Expression Vector Of Mycobacterium Bovis MPB64 Gene

Mycobacterium bovis is the causive agent of bovine tuberculosis in a range of animal species and human beings. The prevalence and circulation of this disease not only influence the development of stock raising severely, but also menace the health of human beings, so it was recognized as B infection by OIE. More than 10% of human tuberculosis is caused by M. bovis. In recent years, the incidence of human tuberculosis has risen in large extents; the death toll has reached 3 millions every year, which is the accumulation of death toll of other infections, so it is the ringleader among all the infections. Because of the prevalence of bovine tuberculosis, human tuberculosis can not be eradicated. Recently, the incidence of bovine tuberculosis has also risen largely, especially in the developing countries. In our country, the positive incidence is about 10%. As regards as the prevention of bovine tuberculosis, BCG is the only vaccine that can be used, but after the incubation of BCG, the immunized effects is not obvious, but it will disturb the allergy test of PPD, so we can not differentiate the natural infection and artificial immunization. Moreover, it made the routine quarantine more difficult. So the new vaccine for the diagnosis and prevention of the bovine tuberculosis has been the hot spot.M. bovis is a kind of parasitic bacterium in the cell. In the immunity of anti-mycobacterium, cell mediated immunity exerts leading effect. Some studies indicated that the secreted proteins of living bacterium exerted the mainly protective immunity, so the secreted proteins were protective antigens. DNA vaccine can induce permanent humoral and cell immunity, but also the expressed protein antigen in the cell is presented by MHC I molecule, the activation of CTL and the kill of target cell are the main mechanisms of DNA vaccine, this is necessary for the prevention of tuberculosis. Other studies indicated that the incubation of DNA vaccine do not disturb the allergy quarantine of tuberculosis. For the development of sensitive and special diagnosis and new and effective prevention, especially the DNAvaccine, in this study, the mainly study contents as follows:1. eukaryotic expression of the mainly protective antigen genesFor the construction of DNA vaccine, these seven protective antigen genes that were cloned into pGEM-T system were subcloned into eukaryotic expression vector pVAX1, then these seven eukaryotic expression plasmids pVAX1-64 were constructed successfully. These recombinant plasmids were transfected into BHK-21 cells with liposome; these expressions were detected with immunofluorescenceand RT-PCR, which indicated that these target genes were expressed in BHK-21.2. Immunogenicity of eukaryotic expression plasmids...