Construction And Immunogenicity Research Of Recombinant Adenovirus Of Mycobacterium Bovis Mpb51-mpb63 Fusion Gene

Bovine tuberculosis is a chronic wasting infectious disease that has a serious impact on human health and animal husbandry development.It is classified as a Class B disease by the World Organisation for Animal Health.BCG?BCG?is a live attenuated vaccine made of Mycobacterium bovis and is currently the only widely approved vaccine for tuberculosis prevention.Studies have shown that the protective effect of BCG is age-dependent,and the protective power is weakened with age.At the same time,BCG cannot be applied to individuals with immunodeficiency diseases,and also to the tuberculin skin test?Tuberculin?.Skin test,TST)was positive and interfered with the detection of bovine tuberculosis.Therefore,there is an urgent need for a new vaccine against bovine tuberculosis.Human adenovirus type 5?Ad5?is a replication-deficient virus expression vector deleted from the E1 region and can only be propagated and amplified in HEK293 cells.The adenoviral shuttle vector inserted into the foreign gene can pass the same in HEK293 cells.The source recombination is integrated into the adenoviral vector genome and the recombinant adenovirus is packaged,and finally the recombinant adenovirus can express a large amount of foreign protein in HEK293 cells.MPB51 and MPB63 are two major secreted proteins of M.bovis,which have good immunoprotective properties and are ideal candidate antigens for the development of bovine tuberculosis vaccines.Therefore,Ad5 was used as a vector to construct a recombinant adenovirus of M.bovis mpb51-mpb63fusion gene,and the immunogenicity of the fusion protein MPB51-MPB63 expressed by recombinant adenovirus was studied in a mouse model,which laid a foundation for the development of a novel vaccine for bovine tuberculosis..In this study,the fusion gene mpb51-mpb63 was amplified by PCR using pMD-51-63 plasmid as a template.After purification,it was digested with Hind III and EcoR I and recovered,and then ligated with the adenovirus shuttle vector pacAd5CMV K-NpA.The pacAd5 CMV-51-63 recombinant shuttle plasmid was constructed.The recombinant shuttle plasmid pacAd5 CMV-51-63 and the adenoviral backbone plasmid pacAd5 9.2-100 were linearized with Pac I restriction endonuclease respectively,and co-transfected into HEK293 cells for recombination under the action of the transfection reagent LipoFiter^TM.The adenovirus was packaged to obtain recombinant adenovirus rAd5 CMV-51-63.Reverse transcription-PCR?RT-PCR?was used to verify the expression of the fusion gene mpb51-mpb63,and the expression of the fusion protein MPB51-MPB63 was further detected by indirect immunofluorescence assay?IFA?and Western Blot.The recombinant adenovirus rAd5 CMV-51-63 was amplified in large amounts and purified,and the titer of the purified rAd5 CMV-51-63 virus was determined using the TCID50 method.rAd5CMV-51-63 recombinant adenovirus,rAd5 CMV K-NpA,PBS buffer,BCG were immunized by intramuscular injection of 7-week-old male mice,boosted two weeks later,and mice were sacrificed two weeks later.Cell cytometry was used to detect the secretion of T lymphocyte subsets and IL-2,IFN-?and other cytokines in mouse spleen.The IgG level in serum was detected by enzyme-linked immunosorbent assay?ELISA?,and the recombinant adenovirus rAd5 CMV-51-63 was analyzed.Immunogenicity of the fusion protein MPB51-MPB63 expressed.The fusion gene mpb51-mpb63 was successfully obtained in this study.After recombinant digestion,the recombinant adenoviral shuttle plasmid pacAd5CMV-51-63 was successfully constructed.The pacAd5 CMV-51-63 and pacAd59.2-100 were linearized and co-transfected,and the recombinant adenovirus rAd5CMV-51-63 was successfully constructed in HEK293 by genomic PCR.Furthermore,it was confirmed by RT-PCR,IFA and Western Blot that the MPB51-MPB63 fusion protein was expressed in the recombinant adenovirus rAd5 CMV-51-63.The percentage of CD4+T cells in the splenocytes of the recombinant adenovirus rAd5CMV-51-63 immunized mice was slightly higher than that of the rAd5 CMV-K-NpA,PBS and BCG groups,but there was no significant difference?P>0.05?.The percentage of CD8+T cells in BCG group was slightly lower than that in unloaded adenovirus group,but the difference was not significant?P>0.05?,and significantly higher than that in PBS group?P<0.05?.The percentage of IL-2 producing cells in BCG group was lower than that in BCG group,and was significantly higher than that in empty adenovirus group and PBS group?P<0.05?.The percentage of IFN-gamma producing cells in BCG group was slightly lower than that in PBS group?P>0.05?,but significantly higher than that in PBS group and empty adenovirus group?P<0.05?.The level of IgG antibody in serum of mice immunized with recombinant adenovirus rAd5 CMV-51-63 was significantly higher than that of mice immunized with PBS or empty adenovirus?P<0.05?.Therefore,the recombinant adenovirus rAd5 CMV-51-63 can induce a certain level of cellular and humoral immunity in mice,which lays the foundation for the development of new vaccine against bovine tuberculosis.