Construction And Immunogenicity Research Of Recombinant Adenovirus Of Mycobacterium Bovis Ag85a?mpb70 And Bovine IFNG Fusion Gene

Bovine tuberculosis is a zoonotic disease,which is prevalent all over the world and causes great economic losses to animal husbandry.By far the most common methods for prevention of bovine tuberculosis is the BCG vaccine,is also the most widely used vaccine to prevent tuberculosis,but due to the differences between individuals,store environment and the mode of transportation,the effectiveness of BCG vaccines will be decreased over time.Thus,the development of a new and effective tuberculosis vaccine has become the focus of research.With the further study of viral vectors,adenovirus vector Ad5 has gradually become the current research hotspot.Adenovirus vector has the characteristics of high titer,strong safety and high carrier capacity,which makes it become the most widely used viral vector.Therefore,in this study,Ad5 was used as the vector to construct the recombinant adenovirus with the fusion gene of Mycobacterium bovis ag85a,mpb70 and bovine IFNG,and the immunogenicity of the recombinant adenovirus was analyzed in the mouse model.In this study,p VAX1-ag85a-mpb70-IFN-?plasmid was used as a template,and part of Mycobacterium bovis ag85a and bovine IFNG sequence were used as the upstream primer and the downstream primer to amplify the target gene ag85a-mpb70-IFN-?by PCR.By double digestion of pac Ad5 CMV K-Np A and target gene ag85a-mpb70-IFN-?with Eco R?and Hind?,the recombinant adenovirus shuttle plasmid pac Ad5 CMV-85a-70-?was constructed under the action of the T4 DNA ligase.pac Ad5 CMV-85a-70-?and pac Ad5 9.2-100 plasmids were linearized by Pac?enzyme,and were co-transfected into HEK293 cells with Lipo Fiter^TMtransfection reagent according to the volume ratio(3:1)of pac Ad5 CMV-85a-70-?and pac Ad59.2-100.The recombinant adenovirus was successfully packaged and was named r Ad5CMV-85a-70-?after entering into cells under the action of transfection reagent and completing assembly.The purified recombinant adenovirus was used for immunoassay in mice.Cytokines(IL-2,IL-12,IFN-?,TNF-?),CD4⁺and CD8⁺T lymphocytes in spleen cells of mice were detected by flow cytometry,and serum Ig G level was detected by ELISA.Results showed that the recombinant adenovirus r Ad5 CMV-85a-70-?produced significantly more IFN-?than BCG(P<0.05),indicated that the produce ability of r Ad5 CMV-85a-70-?to IFN-?was better than BCG.The recombinant adenovirus r Ad5 CMV-85a-70-?could only produce a similar level of cellular immunity to BCG in T lymphocytes and other cytokines such as IL-2,IL-12 and TNF-?.The level of Ig G produced by recombinant adenovirus r Ad5 CMV-85a-70-?was significantly higher than that of BCG(P<0.05),indicating that recombinant adenovirus r Ad5 CMV-85a-70-?could stimulate humoral immunity higher than BCG vaccine.In conclusion,the recombinant adenovirus r Ad5 CMV-85a-70-?immunized mice can stimulate cellular and humoral immunity similar to BCG.This study laid a foundation for screening the dominant antigen of bovine tuberculosis and development research of new bovine tuberculosis vaccine by constructing a fusion antigen adenovirus vector vaccine.