Preparation Of Monoclonal Antibody Against Clostridium Perfringens Beta1 Toxin And Establishment Of Indirect ELISA

Clostridium perfringens,as an important opportunistic pathogen,widely exists in the intestines of healthy people and animals,causing a series of diseases in hosts,including necrotic enteritis,gas gangrene,enterotoxemia and food poisoning,which poses a serious threat to the development of animal husbandry and human health.Betal toxin,as a kind of pore-form protein which produced by Clostridium perfringens type B and type C strains.The Betal toxin is deadly poisonous to the cells,and nerve toxicity also.Betal toxin is the key factor of the Clostridium perfringens cause human and animal necrotic enteritis.However,the lack of specificity and sensitivity monoclonal antibodies for Clostridium perfringens Betal toxin.This restraint the research on pathogenic mechanism of CPB1 toxin and the research and development of immunological detection products.In this study,the potential antigen peptide segment of Clostridium perfringens Betal toxin was analyzed and predicted.Hybridioma cell lines targeting CPB1 toxin were prepared by using monoclonal antibody technology.Two specificity and sensitivity monoclonal antibody cell lines were obtained by screening the monoclonal antibodies through Western-blot.An indirect ELISA method was established based on a recombinant Betal toxin and monoclonal antibodies,to detect the level of Clostridium perfringens Betal toxin antibody in animal serum.The main research results are as follows:(1)Recombinant strain BL21(DE3(pET32-cpbl)expressing Clostridium perfringens Betal toxin was obtained by genetic engineering technology;Recombinant Escherichia coli expressing Clostridium perfringens Alpha,Beta2 and Episilon toxins were obtained.After IPTG induction,the corresponding toxin proteins were successfully expressed.The antigens were prepared that could be used for the Western-blot screening and identification of monoclonal antibodies and the establishment of indirect ELISA detection method.(2)The selected epitopes of CPB1 toxin for antigen production Based on software analysis and Blast with other Clostridium perfringens toxin sequences in database,three potential epitopes are selected form the target protein.The selected peptides will be synthesized to generate antigens for further immunization in mice.Two monoclonal antibody cell lines with high specificity and sensitivity were screened by Western-blot from 17 peptide-positive hybridioma cell lines.Named McAb 1H11 and 1K19.The optimal detection limits of Betal toxin in Clostridium perfringens B strain(C58-2)and C strain(C59-2)by McAb 1K19 and McAb 1H11 were 0.5×LD100 and 0.25xLD100.The heavy chain of antibody secreted by McAb 1K19 cell line was identified as IgG2b and light chain was identified as ?.These monoclonal antibodies provide good materials for the detected the natural Clostridium perfringens Beta1 toxin.(3)Based on McAb 1K19,an indirect ELISA method for detecting CPB1 toxin antibody was established and optimized with Clostridium perfringens Beta1 recombinant toxin protein as the coated antigen.Finally,the optimal coating concentration of the antigen was determined to be 10?g/mL,and the dilution ratio of the McAb 1K19,was 1:1024.The results of repeated test between batches showed that the method had good specificity,sensitivity and stability.The establishment of this detection method provides technical support and detection means for the clinical diagnosis of diseases caused by Clostridium perfringens Betal toxin at antibody level,and has a wide application prospect in the evaluation of the immune effect of Clostridium perfringens vaccine.In conclusion the prokaryotic expression strain of Clostridium perfringens Betal toxin recombinant protein was successfully constructed in this study to prepare CPB1 toxin recombinant protein.Monoclonal antibodies with high specificity and sensitivity against CPB1 toxin were prepared and screened by the fusion technique of immunizing mice with antigen peptide,which can be used to detect natural CPB1 toxin.The indirect ELISA method of CPB1 toxin antibody was established by using CPB1 recombinant toxin as the coating antigen and McAb 1K19 as the primary antibody.These results indicated the monoclonal antibodies generated in this study are useful tools for studies of biological functions and pathogenic mechanisms of CPB1 in future,which.warrant for further investigations,and also provide a good detection product for better prevention and detection of Clostridium perfringens Betal toxin.