Expression And Immunogenicity Of Recombinant CctA Gene Of Clostridium Chauvoei In E.coli

Object:Clostridium chauvoei is one of the most pathogenic Clostridium bacteria,mainly causing emphysema,also known as black leg disease,which has a high fatality rate and causes significant losses in livestock production worldwide.Clostridium chauvoei cytotoxin?CctA?is the most typical pathogenic toxin among the five toxins it produces.It has cytotoxicity and hemolytic activity.It is the main protective antigen and plays a leading role in pathogenicity.In this study,bioinformatics analysis was conducted on the full-length CctA gene of Clostridium chauvoei,prokaryotic expression plasmid of CctA gene was constructed and protein expression was induced,and guinea pigs were immunized with subunit vaccine.Indirect ELISA method was established to detect antibody levels in guinea pigs,and the immune effect of subunit vaccine was preliminarily analyzed.Methods:1.Specific primers were designed and synthesized according to the published CctA gene sequence.Bioinformatics analysis software DNAStar,EXPASY,Signal P,NetPhos Server v.3.1 and other programs were used to predict the structure and function of CctA proteins,glycosylation sites,and dominant antigen epitopes.2.According to the results of bioinformatics analysis,the full-length CctA gene was synthesized to remove signal peptide and partial sequence fragments.The truncated CctA gene was cloned by pMD19-T and inserted into the prokaryotic expression vector pET-28a?+?,which was transformed into BL21?DE3?pLysS receptor cells.The protein expression was induced by IPTG and its reactivity was analyzed.3.The expressed protein was inactivated by formaldehyde and mixed with Al?OH?₃ adjuvant to prepare the vaccine.Guinea pigs were divided into the primary and secondary immunization groups with subunit vaccine,commercial inactivated vaccine primary immunization group and blank control group.Each guinea pig in vaccine immunization group was injected with 1ml intramuscular injection and 6 in each group.The liquid of Clostridium chauvoei was prepared and the half of the lethal dose was calculated.After immunization for 21 days,challenge protection test was conducted to record the immune protection of guinea pigs.An indirect ELISA method was established to detect antibody levels in immune guinea pigs.Results:1.The full-length CctA gene was about 970bp amplified by PCR,which was consistent with the expectation.Bioinformatics analysis showed that CctA protein is a hydrophilic protein.The structural domain analysis showed that the protein had no transmembrane region,there was signal peptide,and there was a leucocyte-killing superfamily protein.There are 4N-glycosylation sites,1 O-glycosylation site,and multiple phosphorylation sites,among which the secondary structure is mixed,with the most random curling and 22 dominant B-cell epitopes.2.The size of truncated CctA gene was 853bp by PCR amplified,which was consistent with the expectation.In this experiment,the recombinant expression plasmid was successfully constructed.After IPTG induced expression,SDS-PAGE analysis showed that the inclusion body protein with the size of 35KDa was obtained,which was consistent with the expected size.The results of Western blot showed that the reactivity of the protein was good.3.The lethal dose of the strong toadstool solution prepared was 0.5×10⁶CFU.The results of the challenge test showed that the protection rate of guinea pigs in the immune group with the inactivated vaccine of Clostridium chauvoei was 83%.The protective rate of primary and secondary immunization with subunit vaccine was 66.7%.By establishing an indirect ELISA method to detect the antibody level in guinea pigs after immunization,the data analysis showed that there was a very significant difference between the immunization group with inactivated vaccine of Clostridium chauvoei and the primary group with subunit vaccine?p<0.01?.Comparison with the secondary subunit vaccine immunization group showed significant difference?p<0.05?.There was no significant difference between the primary and secondary immunization groups?ns?.All three groups showed very significant differences compared with the control group?p<0.001?.Conclusion:In this study,the characteristics of CctA protein were analyzed,so that the truncated CctA gene was successfully expressed in the E.coli expression system,and the immunization of the prepared subunit vaccine could also achieve a certain protective effect,while the inactivated vaccine stimulated the body to produce significantly higher protective antibodies than the subunit vaccine.Further optimization of experimental scheme is needed to improve the immune effect of subunit vaccine.