| Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis.The disease is not easy to purify due to the wide distribution,multiple transmission modes and slow growth rate of Mycobacterium bovis.At present,the most recognized prevention method for bovine tuberculosis in the world is BCG vaccination.BCG is an attenuated live vaccine,which has the risk of returning to the virus.Because it only has a protective effect on young individuals,the protective effect on adult and elderly individuals is not significant.Therefore,it is urgent to develop a new bovine tuberculosis vaccine.Mycobacterium bovis has a variety of specific antigens.Among them,Ag85 complex can induce Th1 immune response and plays an important role in regulating the intracellular infection of Mycobacterium bovis.MPT63 is a virulence-related small molecule protein secreted by Mycobacterium bovis.HSP65 is the main target of immune response in Mycobacterium bovis and the key component of Mycobacterium tuberculosis adhesion to macrophages.Multi-epitope vaccine,also known as cocktail vaccine,can carry multiple auxiliary epitopes of target antigens at the same time.Compared with traditional vaccines,multi-epitope vaccines can be recognized by a variety of MHC molecules,and antigen presentation is more efficient.At the same time,it has unique cellular immune advantages,and its immune function is not affected by pathogenic microbial variation.In addition,the study found that after the Salmonella was attenuated at the gene level,the attenuated Salmonella was attenuated but its invasiveness to cells was still intact.Therefore,it is very suitable as a vaccine vector.In this study,three specific antigens Ag85B,MPT63 and HSP65 of bovine tuberculosis were selected.Firstly,the B-cell epitopes and T-cell epitopes were analyzed by bio-online software IEBD and Bepi Pred.Then,the epitope peptides were screened and connected to construct a new epitope AMH.Next,Vaxi Jen,Expay,Sopma and MODEL were used to analyze the immunogenicity,physical and chemical properties,secondary structure and tertiary structure of the epitope sequence AMH.The constructed epitope gene AMH and the three specific antigen genes of bovine tuberculosis were ligated by T2A or P2A and constructed into the eukaryotic expression vector p EGFP,and then the recombinant plasmid was double digested and identified by PCR.After identification,the successfully constructed recombinant plasmid p EGFP-AMH-Ag85B-MPT63-HSP65 was transfected into HEK293cells to detect the transfection of the recombinant plasmid in the cells,and reverse transcription and WB tests were performed to verify the expression of the recombinant plasmid at the cellular level.The results showed that the constructed epitope protein AMH had good immunogenicity,hydrophilicity and stability.The successfully constructed recombinant plasmid had good transfection efficiency in cells,and the target gene could be successfully reverse transcribed into RNA in cells.The target protein was also expressed.The recombinant expression vector p EGFP-AMH-Ag85B-MPT63-HSP65 was transferred into attenuated Salmonella by heat shock.After PCR and sequencing,the recombinant attenuated Salmonella was tested in vitro and in vivo.In vitro,the growth curve,in vitro stability and infection cell test of recombinant attenuated Salmonella were carried out.In vivo experiments,mice were orally immunized with recombinant attenuated Salmonella to detect the immune effect of recombinant attenuated Salmonella in mice.By collecting the serum of mice at different time periods,the changes of related antibodies and cytokines in serum were detected.The mice were sacrificed 14 days after the second immunization,and the relevant organs were dissected and weighed to calculate the organ-body ratio.The spleen lymphocytes were extracted for typing experiments,and the relevant organs were taken for HE staining.The results of in vitro experiments showed that the introduction of exogenous genes had no significant effect on the growth of attenuated Salmonella,and the recombinant attenuated Salmonella could stably carry exogenous genes in the environment containing Kan,and the recombinant attenuated Salmonella could transfer exogenous genes at the cellular level.The results of in vivo experiments showed that the recombinant attenuated Salmonella could stimulate the production of Ig G and s Ig G in mice,stimulate the production of cytokines IFN-γ,IL-4 and IL-10,and increase the proportion of CD3~+,CD4~+and CD8~+in mice,and oral recombinant attenuated Salmonella had no significant effect on the growth of mice.In summary,the cellular and humoral immune function was initiated by the gene of the stimulating site,and the specific immune response was initiated by the three specific antigen genes Ag85B,MPT63 and HSP65 of bovine tuberculosis.The results showed that the recombinant attenuated Salmonella constructed by epitope protein had high safety and good immune effect,which provided experimental basis and theoretical support for the study of gene vaccine of bovine binding disease. |
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