Preparation Of Nucleic Acid Vaccines PVAX1-FliA In Yanbian Strain Of Coustridium Chauvoei And Estimate Of Their Efficiencies Of Protective Immunity

Clostridium chauvoei is black leg's pathogen that is spore gram positive bacterium.Clostridium chauvoei cause fatal disease of cattle and sheep,and ruminant animals grazing on the pasture that spore contaminated is more prone to infection.Clinical touble spots often is muscles,and clinical symptoms are emphysematous gangrene inflammation when crepitus was detected.It is the high mortality desease.Animals do not be infect by clostridium chauvoei except ruminant.Newborn ruminant that are grazing is the most sensitive animal.The clostridium chauvoei is usually in soil and deject in endemic areas.The infections generally occur every year once pastures are severely contaminated.In recent years,there has many outbreaks of black leg disease in Yanbian,it is grim that the situation which brings losses to the Yanbian Yellow Cattle farmers and their property,and it is harm for people's safe production.It is urgent that need for effective prevention and control of the disease.Designed a pair of specific primers based on the reference sequence of the Genbank flagellation gene(accession number:AB058932),and we used the specific primers to amplify the FliA gene including a complete open reading frame.The FliA gene of which length is 1267 bp,amplifying by PCR,was cloned into pMD19-T simple vector.The pMD-19t-FliA vetor that is correct by identification was sequenced.The FliA gene of pMD-19t-FliA vetor that is sequenced successfully was ligated to eukaryotic expression vector.The pVAX1-FliA eukaryotic expression recombinant plasmid that is correct by identification was transferred into Vero cells,and used the FITC technology to detect the product of the pVAX1-FliA.The pVAX1-FliA recombinant plasmid was greatly purified by Plasmid Extending Kit for preparation of the Yanbian clostridium chauvoei Flagellum gene nucleic acid vaccine.Using the prepared nucleic acid vaccine,BALB/c mice were injected into the nucleic acid vaccine according to the pVAXl-FliA group,the pVAX-1 empty vector control group,and the PBS control group.They were detected that IgG,IgG1 and IgG2a antibodies by indirect ELISA;The content of IL-4 and IFN-y were detected by indirect ELISA kits.The results showed that the FliA gene fragment was 1267 bp.Making the sequencing of FliA gene to compare with 6 types of clostridium chauvoei on NCBI,the homology of nucleotide sequence was 93%?100%.The homology by comparing with the German strain 12S0467 reached 100%.The pVAX1-FliA eukaryotic expression recombinant plasmid can be stably expressed in Vero cells.The BALB/c mices that were received pVAXl-FliA recombinant plasmid intramuscularly induced protective cell-mediated and humoral immune responses.The flagellar antigen genes of local isolating chauvoei clostridium in Yanbian was sequenced and analyzed in this study,and it provided a reference for the prevention and control to local clostridium chauvoei.and it laid solid foundation for the development of nucleic acid vaccine from Yanbian's clostridium chauvoei.In order to prepare a safe and effective new type of clostridium chauvoei vaccine and effectively control the outbreak of clostridium chauvoei in Yanbian,the nontoxic and safe eukaryotic expression vector pVAX1-FliA was established as DNA vaccine.The immune effect of the nucleic acid vaccine assessed with mice.