Construction Of Eukaryotic Expression Vector Of Clostridium Chauvoei NanA Gene And Preliminary Study On Mice Immunity

Clostridium chauvoei is an acute,febrile,septic infection that affects ruminants.The common typical symptoms are the occurrence of inflammation and gas swelling in the fleshy parts of the diseased animals,as well as the contact pressure and twisting pronunciation.Gangrene occurs when the skin is dry and hard.The animals lost their appetite and had difficulty breathing.The prevalence of the disease has certain local characteristics and seasonality,and it mostly occurs in wet valleys and pastures,and in hot and rainy seasons.In recent years,there are many reports of the outbreak of Clostridium chauvoei,which bring huge impact and economic loss to the development of animal husbandry and breeding industry,and also gradually threaten the safety and production of human beings.For the preparation of safety and efficient new nucleic acid vaccine of Clostridium chauvoei.to effectively control the occurrence of the disease.according to the published Clostridium chauvoei NanA gene sequence in GenBank,this study designed synthesis a pair of specific primers,and the total DNA of Clostridium chauvoei was used as the template.NanA gene was amplified and cloned into pMD 18-t simple vector and analyzed the polymorphisms of the sequenced NanA genes.The cloned plasmid pMD 18-T-NanA was double digested and linked with pVAX-1 eukaryotic expression vector.The eukaryotic expression recombinant plasmid pVAX-NanA was constructed and transfected into Vero cells,and its expression was identified by indirect immunofluorescence.BALB/c mice were immunized with the constructed pVAX-NanA plasmid after massive extraction.Set group as pVAX-NanA nucleic acid vaccine group.pVAX1 group and PBS control group.Blood samples were collected from mice and serum was isolated.The serum levels of IgG,IgG1 and Ig G2a antibodies.IFN-y and IL-4 cytokines were determined by ELISA.Two weeks after the third immunization.CD4+ and CD8+ in mouse spleen lymphocytes were determined by flow cytometry.The humoral and cellular immune responses of mice were evaluated by antibody level.T cell subpopulation content and cytokine level.The results showed that the NanA gene of clostridium pneumoniae was successfully amplified in this Study.The fragment size is 1950hp.Comparcd with the reference sequence,there were 3 nucleotide mutations,and the homology of nucleotide and amino acid sequences was 99.8%.With a secondary structure dominated by-folding and random curling,and multiple epitope regions.Inoculated the successfully constructed pVAX-NanA nucleic acid vaccine in BALB/c mice,the antibody levels of IgG,IgG1 and IgG2a were significantly higher in the pVAX1 empty vector group and the PBS control group(P<0.05).The levels of IFN-? and IL-4 cytokines in serum of mice were significantly higher than those of pVAX1 empty vector group and PBS control group(P<0.01).CD4+and CD8+analysis of lymphocyte subsets showed that the CD4+and CD8+level were higher than that of pVAX1 group and PBS control group(P<0.05).The CD4+and CD8+ratio of pVAX-NanA nucleic acid vaccine group was higher than that of pVAX1 empty vector group and PBS control group,and there was no significant difference between pVAX1 empty vector group and PBS control group.This experiment provides a reference for the in-depth study of the function of NanA protein and lays a certain foundation for the research and development of the NanA nucleic acid vaccine of Clostridium chauvoei.