Study On Cloning, Expression And Immunity Of Major Protective Antigen Genes Of Mycobacterium Bovis

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 raised 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 raised 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 DNA vaccine, in this study, the mainly study contents as follows: 1. the clone of the mainly protective antigen genes of M. bovisIn this study, we filtered seven protective antigen genes of secreted proteins:Ag85A, Ag85B, MPB51, MPB63, MPB70, MPB83 and ESAT-6. These mature protein genes were amplified from M. bovis Vallee111 chromosomal DNA by using PCR technique, the target gene is 888bp, 858bp, 801bp, 390bp, 492bp, 603bp and 288bp respectively. Through T-A clone, the PCR products were cloned into pGEM-T Vector system. The recombinant clonewas identified by a -complementation, enzyme digestion, PCR identification and sequential analysis, then these recombinant plasmids pGEM-T-85A, pGEM-T-85B, pGEM-T-51, pGEM-T-63, pGEM-T-70, pGEM-T-83 and pGEM-T-ESAT-6 were constructed successfully. Sequential and homogeneous analysis indicated that between M. bovis Valleelll and other strains of M bovis, the related mature protein genes are homogeneous in large extents.2. the prokaryotic expression of the mainly protective antigen genesTo acquire the expression products of seven protective antigen genes of M. bovis, these seven protective antigen genes that were cloned into pGEM-T system were subcloned into prokaryotic expression vector pET28a and pGEX-4T-3, so prokaryotic expression plasmids pET28a-85A, pET28a-85B, pET28a-51, pET28a-63, pET28a-70, pET28a-83 and pGEX-4T-3-ESAT-6 were constructed successfully. These recombinant expression plasmids were transformed into E. coli BL21 (DE3), then through the induction of IPTG and SDS-PAGE analysis, these fused target proteins such as 32kDa, 30kDa, 30kDa, 18kDa, 25kDa, 26kDa and 34kDa were acquired respectively. Western-blot indicated that these target proteins have the antigenicity of M. bovis. Furthermore, except MPB63, which was expressed as the soluble proteins, others were expressed as inclusion body. These bacteria were broken by the cooling sonifier, then were washed by Triton X-100, so the inclusion body were abstracted. These target proteins were purified by SDS-PAGE, then were analyzed by SDS-PAGE, these purified proteins only presented a band, so they can be acted as the identified antigens for the eukaryotic expression plasmids.3. 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 pVAXl, then these seven eukaryotic expression plasmids pVAXl-85A, pVAXl-85B, pVAXl-51, pVAXl-63, pVAXl-70, pVAXl-83 and pVAXl-ESAT-6 were constructed successfully. These recombinant plasmids were transfected into BHK-21 cells with liposome, these expressions were detected with immunofluorescence and RT-PCR, which indicated that these target genes were expressed in BHK-21.4. Immunogenicity of eukaryotic expression plasmidsTo explore protective efficacy of eukaryotic expression plasmids, BALB/c mice was vaccinated with pVAXl-85A, PVAX1-85B, pVAXl-51, pVAXl-63, pVAXl-70, pVAXl-83 and pVAXl-ESAT-6 DNA vaccine respectively and combined vaccinated by pVAXl-85B, pVAXl-70, pVAXl-83 and pVAXl-ESAT-6 DNA vaccines, moreover, pVAXl, physiological saline and BCG immunization as control. The protective efficacy was detected by indirectELISA. The detection results indicated that the immunized mice harvested the special antibody against Ag85A, Ag85B, MPB51, MPB63, MPB70, MPB83 and ESAT-6, the antibody titer increased with the increase of immunity. Furthermore, the serum antibody titer of combined immunization was higher than the mono-immunization obviously (P<0.05), but the immunization efficacy was not as good as BCG, which is not the same with international reports that antibody titer of combined immunization was higher than BCG obviously. Lymphocyte transformation test indicated that except the pVAXl-85A, other recombinant plasmids induced cell meditated immunity that was as high as or higher than BCG. CD4+T and CD8+T cells determination results demonstrated that the recombinant plasmids immunization induced the mice to produce obvious cell immunity (combined immunity group), and the CDg+T cell was the chief aspect (PO.05). These results indicated that the expressed protein antigens by recombinant plasmids were mainly presented by MHC I molecule, CDg+T cells were activated. The protein antigen expressed by pVAXl-51 can be also presented by MHC II molecule and CD/T cells were activated, so the high level cell immunity can be induced. Furthermore, the cavy was vaccinated by pVAXl-70, pVAXl-83 respectively and combined immunized by pVAXl-70 and pVAXl-83, then pVAXl and BCG as control. Lymphocyte transformation test indicated that recombinant plasmids induced cell meditated immunity that was as high as or higher than BCG, the result was as same as that of little mice. The allergy quarantine of PPD indicated that it was different in large extents between the recombinant plasmids and BCG (PO.01, p=0.0001), this result is as same as the international reports.Those above results indicated that in this study, we acquired the prokaryotic expression products of seven protective antigen genes: Ag85A, Ag85B, MPB51, MPB63, MPB70, MPB83 and ESAT-6. So this laid a solid foundation for the study of biologic specialty and the new diagnosis reagents. The eukaryotic expression plasmids induced the experimental animals to produce special humoral immunity and cell immunity. So these results laid a solid foundation for the study of the protective efficacy of these genes, then we can filter the protective genes against M. bovis and develop the new and effective DNA vaccine.