Preparation And Immune Efficacy Enaluation Of DNA Vaccine Against CctA Gene In Bovine Clostridium Chauvoei

Emphysematous carbuncle is an acute septic infectious disease in animals including cattle and sheep caused by an obligate anaerobic bacterium Clostridium chauvoei.The most common pathological features include inflammatory symptoms,gas swelling,and crepitant rales on palpation in affected muscle groups,with regional and seasonal prevalence.The disease occurs frequently in summer,especially in wet valley pastures and marshes.Yanbian Korean Autonomous Prefecture,located in the east of Jilin province,is characterized by dense jungle and abundant rainfall in the mountain area,where the incidence of bovine emphysematous carbuncle is relatively high in recent years.This greatly impacts the development of local animal husbandry and aquaculture industry and threatens the normal life of local residents.In order to control the incidence of emphysematous carbuncle in Yanbian area,it is necessary to prepare a safe and effective nucleic acid vaccine against Clostridium chauvoei.As the basis for the present study,one pair of specific primers was designed according to the DNA sequence of Clostridium chauvoei toxin A(CctA)gene from the NCBI database.CctA gene was amplified by PCR using the other pair of primers and the fragment obtained was cloned into a eukaryotic expression vector,the pMD19-T Simple Vector.After identified by DNA sequencing,the CctA gene was sub-cloned into the eukaryotic expression vector pcDNA3.1-1 and then transfected into Vero cells.Indirect immunofluorescence method and Western blot were applied to determine the expression of CctA gene.After the concentration was measured,the expression plasmid was used to immunize mice,and the levels of IgG1 and IgG2a were measured by indirect ELISA and that of IL-4 and IFN-y were determined by an ELISA kit.The results showed that the CctA gene fragment amplified was 519 bp in size.Homology comparison of nucleotide and amino acid sequences was made by aligning the sequence of the obtained CctA gene to the Clostridium chauvoei ATCC 10092 strain,and 3 point mutations were found.Amino acid comparison of this CctA sequence to the other 6 strains showed mutations at 2 amino acid positions.The homology of these amino acid sequences was 98.8%and that of the nucleotide sequence was about 99.4%.Green fluorescence was observed in the cells transfected with PcDNA3.1-CctA,and the recombinant protein expressed by the pcDNA3.1-CctA was about 19 kD,demonstrating that CctA protein was successfully expressed by the pcDNA3.1-CctA plasmid in vero cells.Immunological test in BALB/c mice showed that after three times of immunization,levels of IgG2a and IgGl antibodies in the protein group were significantly higher than that of the PBS group and the pcDNA3.1 group(P<0.05),and significantly lower than that of the pcDNA3.1-CctA group(P<0.05);the expression levels of cytokines IFN-y and IL-4 in the protein group were remarkably higher than that of the PBS group and the pcDNA3.1 group,and was significantly lower than that of the pcDNA3.1-CctA group(P<0.05),which somehow enhanced the immunity level in mice.In the present study,data of the Clostridium chauvoei strain from Yanbian area are predicted and analyzed,pcDNA3.1-CctA plasmid for Clostridium chauvoei is successfully constructed and expressed,and humoral and cellular immune response are successfully induced in mice.