| Bovine brucellosis is a zoonotic infectious disease which is widespread in the world.Brucella bovis is particularly resilient,adapts quickly to new hosts,and can be transmitted directly or indirectly to both primary and susceptible hosts.Mycobacteriosis bovis is a chronic disease of cattle that often results in reduced productivity and poses a significant public health risk.The existing vaccines have the risk of poor immune effect and strong virulence.Therefore,it is necessary to develop a safer and more effective vaccine against bovine brucellosis and mycobacteriosis bovis.In this study,epitopes of Brucella bovis outer membrane protein Omp25 and Mycobacterium bovis secreted protein Ag85A were screened,and the constructed peptides were named NewⅠand NewⅡ,respectively.The antigenicity,hydrophilicity and stability of NewⅠand NewⅡwere analyzed,and NewⅠ,Omp25,NewⅡand Ag85A were connected with self-shear peptides T2A,P2A and E2A in order to construct a new peptide segment named COE.Through homologous recombination,the COE peptide gene was inserted into the eukaryotic expression vector GV658.HEK293 cells were transfected with GV658-COE and GV658respectively.The effects of the recombinant plasmid on cell activity and the safety of the recombinant plasmid were evaluated by CCK-8 method.Western Blot was used to verify the expression of exogenous proteins after transfection.The results showed that NewⅠand NewⅡhad good immunogenicity.The recombinant plasmid was identified by PCR and double enzyme digestion,and the size of the target gene was consistent with that of the recombinant plasmid.After transfecting HEK293 cells with recombinant plasmid,green fluorescence could be observed under FITC channel of fluorescence microscope,indicating that the constructed recombinant plasmid could successfully transfect cells.The results of CCK-8 assay showed that the recombinant plasmid GV658-COE had no significant inhibitory effect on the activity of HEK293 cells and had high safety.Western Blot results showed that exogenous proteins NewⅠ,Omp25,NewⅡand Ag85A could be successfully expressed in HEK293 cells,and the specific protein bands were consistent with the actual protein sizes.The recombinant plasmid GV658-COE carrying multiple epitope genes was immunized three times in the SD rat animal model,once every 14 days and three times in total.Blood was collected through the caudal vein before each immunization.The changes of T cell subsets were detected by flow cytometry.Specific antibodies and cytokines in peripheral blood serum were detected by Elisa.The effects of recombinant plasmid GV658-COE on rat organs were evaluated by specific organ gravity and pathological tissue sections.Flow cytometry showed that CD4~+and CD8~+T cells in GV658-COE group were significantly different from those in PBS control group(P<0.01).The number of CD4~+and CD8~+T cells in the recombinant plasmid group was significantly increased compared with that in the no-loaded plasmid group and the control group.Serum antibody results showed that:There were significant differences in Brucellosis Ig G antibody levels in the recombinant plasmid GV658-COE group compared with that in the no-loaded plasmid GV658 group(P<0.05),and very significant differences in brucellosis antibody levels with PBS control group(P<0.01).The antibody levels of Mycobacterium Ig G in recombinant plasmid GV658-COE group and no-loaded plasmid GV658 group were significantly different(P<0.05),and IL-12,IL-4,TNF-αand IFN-γshowed an increasing trend.The specific gravity of organs and pathological section results showed no difference between groups.In conclusion,the recombinant biepitope gene vaccine GV658-COE constructed in this study can induce animal body to produce specific antibodies,which can produce protective effects against bovine brucellosis and mycobacteriosis.It is expected to provide theoretical basis and research basis for the research of Brucella and Mycobacterium bovis vaccines. |
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