| Mycobacterium bovis can cause tuberculosis in cattle and a variety of domestic,wild mammals,and humans.Bovine tuberculosis infections are widespread throughout the world and pose major challenges to human and animal health,the economic sustainability of agriculture,and the protection of wildlife.Although many developed countries are implementing tuberculosis control eradication programs,the use of wild animals as hosts may cause difficulties in the implementation of eradication programs.Prevention is currently an important direction for the prevention and treatment of tuberculosis.BCG is the only tuberculosis preventive vaccine recognized in the world for clinical.However,its protection efficiency is only for young populations with differences for different ages(ranging from 0 to 85%),and BCG vaccination has a certain impact on the routine quarantine of livestock,so the development of new efficient tuberculosis vaccine is the first priority for prevention and control.Viral vector vaccine is a popular direction for vaccine development now.Adenovirus has become an advantageous vector for human gene therapy because it is easy to produce high titers,efficiently introduce DNA into host cells,and is not easy to be inactivated by complement in the body.Therefore,in this study,Ad was used as a vector to construct a recombinant adenovirus expressing the fusion genes CFP10,ESAT6 and CFP7,which are important extracellular secreted antigens of M.bovis,and to analyze the immunogenicity of the recombinant adenovirus in a mouse model.This will lay the foundation for the development of new bovine tuberculosis vaccine.In this study,the p MD-cfp10-esat6-cfp7 plasmid was used as a template to amplify the fusion gene cfp10-esat6-cfp7 with the upstream primer of gene cfp10 and the downstream primer of gene cfp7.After purification,it was digested with Hind III and Eco R I and recovered.Then it was ligated with the adenovirus shuttle vector pac Ad5 CMVK-Np A,and the recombinant shuttle plasmid pac Ad5 CMV-cfp10-esat6-cfp7 was successfully constructed,and the constructed recombinant shuttle plasmid was linearized with the adenovirus backbone plasmid pac Ad59.2-100.Transfected into human kidney-derived cells HEK293,the recombinant adenovirus r Ad5CMV-cfp10-esat6-cfp7 was successfully packaged.The genomic DNA was further extracted and the fusion gene cfp10-esat6-cfp7 recombinant adenovirus was detected by PCR.It was proved by RT-PCR,indirect immunofluorescence and other technologies that the target gene cfp10-esat6-cfp7 was successfully expressed at the m RNA and protein level.The obtained recombinant adenovirus r Ad5 CMV-cfp10-esat6-cfp7 was immunized into mice,and based on flow cytometry analysis,the levels of the recombinant adenovirus r Ad5 CMV-cfp10-esat6-cfp7 immunized mice with CD4~+T and CD8~+T lymphocyte is similar with the BCG group and not significantly different(P>0.05).The number of IL-2 cells secreted by recombinant adenovirus r Ad CMV-cfp10-esat6-cfp7 group was slightly lower than that of BCG group,the difference was not significant(P>0.05),higher than 0.9%Na Cl group and r Ad5 CMVK-Np A group,the difference was significant(P<0.05).The amount of TNF-?secreted was higher than that of BCG group,the difference was not significant(P>0.05),and significantly higher than those in the 0.9%Na Cl group(P<0.05).The amount of IL-12 secreted was higher than that of BCG and 0.9%Na Cl group,the difference was significant(P<0.05).The levels of Ig G antibodies in the serum of mice immunized with recombinant adenovirus r Ad5 CMV-cfp10-esat6-cfp7 were significantly higher than those in 0.9%Na Cl group and r Ad5 CMVK-Np A group(P<0.05)by the detection of Enzyme Linked Immuno Sorbent Assay.It can be seen that the recombinant adenovirus r Ad5 CMV-cfp10-esat6-cfp7 can induce a certain level of cellular and humoral immunity in mice,providing a basis for the development of new bovine tuberculosis vaccines. |
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