| Bovine tuberculosis is a disease caused by Mycobacterium bovis is widespread between humans and livestock.The disease has a wide Host range and can affect most warm-blooded vertebrates.Tuberculosis can be infected by exposure to sick animals and contaminated milk products.Hence,the spread of bovine TB seriously influences the health of human and animals and causes huge economic losses every year.At present,tuberculosis is mainly based on prevention.BCG is the only preventable vaccine available,but its immune effect is uncertain for adults and affects routine quarantine for cattle.Therefore,it is imperative to develop effective vaccines.The antigens secreted by bacteria are valued for their ability to activate the immune response.CFP-10,ESAT-6 and CFP-7 are early secreted proteins of M.bovis,and immunogenic proteins encoding ESAT-6 and CFP-10 only exist in RD-1 region of mycobacterium tuberculosis genome,but not in BCG strain.Therefore,the fusion of CFP-10,ESAT-6 and CFP-7 protein genes is expected to produce more effective immune effects,which is of great significance for the development of new bovine tuberculosis vaccine.As a natural carrier for presenting foreign antigen and a food-grade safe microorganism antibiotics addition during screening,Lactobacillus NZ3900 has been widely used in the fields of biological pharmacy and vaccine research and development.Therefore,this study used pNZ8149 as the carrier to express the fusion gene cfp10-esat6-cfp7 of mycobacterium bovine in lactobacillus NZ3900,and analyzed the immunogenicity of the constructed recombinant lactobacillus in a mouse model to lay a foundation for the development of new bovine tuberculosis vaccine.By using pUC-cfp10-esat6-cfp7 plasmidas a template the fusion gene was carried out PCR amplification,and then ligated with pMD18-T Simple Vector to construct the recombinant cloning plasmid pMD18-T(S)-cfp10-esat6-cfp7.pMD18-T(S)-cfp10-esat6-cfp7 and pNZ8149 were double-digested with Kpn I and Nco I restriction enzymes,and purified by gel extraction.The purified fusion gene cfp10-esat-6-cfp7 and the expression vector pNZ8149 were ligated to construct a pNZ8149-cfp10-esat6-cfp7 recombinant expression plasmid.pNZ8149-cfp10-esat6-cfp7 was electroporated into the host strain NZ3900 to construct pNZ8149-cfp10-esat6-cfp7/NZ3900 to obtain a recombinant lactic acid bacterium carrying the gene of interest cfp10-esat6-cfp7.After Nisin induction,SDS-PAGE and indirect immunofluorescence identification experiments,the expression of the fusion gene cfp10-esat-6-cfp7 was successfully expressed.Subsequently,mice were immunized with pNZ8149-cfp10-esat6-cfp7/NZ3900 recombinant lactic acid bacteria,and T cell subsets in spleen cells of immunized mice were detected by flow cytometry and cytokines such as IL-2,IFN-?and TNF-?were produced.The proportion of lymphocytes,as well as the use of indirect enzyme-linked immunosorbent assay(ELISA)to detect IgG levels in the serum of immunized mice.In this study,the M.bovis fusion gene cfp10-esat6-cfp7 was successfully amplified and ligated with pMD18-T Simple Vector,and the cloning plasmid pMD18-T(S)-cfp10-esat6-cfp7 was successfully constructed.The purified fusion gene cfp10-esat-6-cfp7 was ligated to the expression vector pNZ8149,and the recombinant lactic acid bacteria pNZ8149-cfp10-esat6-cfp7/NZ3900 was successfully constructed.The recombinant lactic acid bacteria pNZ8149-cfp10-esat6-cfp7/NZ 3900 was induced by Nisin,and the fusion gene cfp10-esat6-cfp7 was expressed in NZ3900 to express a foreign protein of 33 kDa by SDS-PAGE and indirect immunofluorescence assay.After immunization of mice,the results of flow cytometry showed that proportion of spleen cells of CD4~+T in immune group of recombinant lactic acid bacteria pNZ8149-cfp10-esat6-cfp7/NZ3900 was slightly highly than that of pNZ8149/NZ3900 group,PBS group and BCG group,but there was not statistical difference(P>0.05);proportion of CD8~+T cells was slightly higher than that of the pNZ8149/NZ3900 group,which was significantly higher than that of the PBS group and the BCG group,but it failed to reach the significant level(P>0.05);The proportion of cells producing IL-2 was slightly higher than that of pNZ8149/NZ3900group,which was significantly higher than that of PBS group and BCG group,but the difference was still not significant(P>0.05).The proportion of cells producing IFN-?was slightly higher than that of the PBS group and the pNZ8149/NZ3900 group,but it was far less than In the BCG group,the difference was significant(P<0.05);proportion of cells producing TNF-?was basically the same as that in the BCG group and the pNZ8149/NZ3900 group,and there was no difference(P>0.05).The level of IgG antibody in the serum of recombinant lactic acid bacteria pNZ8149-cfp10-esat6-cfp7/NZ3900 was slightly higher than that of PBS group and pNZ8149 group,which was lower than that of BCG group,and the difference was not significant(p>0.05).It can be seen that the recombinant lactic acid bacteria pNZ8149-cfp10-esat6-cfp7/NZ3900 can induce a certain level of cellular immunity in mice to lay a foundation for the development of a new vaccine for bovine tuberculosis. |
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