Preparation Of Monoclonal Antibody To Porcine Circovirus Type 2 Cap Protein And Establishment Of A Double Antibody Sandwich ELISA Assay

Porcine circovirus type 2（PCV2）is the main pathogen causing porcine circovirus disease（PCVD）,which mainly includes diseases such as multisystem failure syndrome in weaned piglets,porcine dermatitis nephrotic syndrome,sow reproductive disorder and porcine respiratory syndrome.PCV2 ORF2 encodes a Cap protein that binds to host cell receptors and induces specific immunity,and is the major immunogenic protein.In this experiment,PCV2 Cap protein was expressed in the primary nucleus,and monoclonal antibodies were prepared using it as the immunogen and a double antibody sandwich ELISA assay was established to lay the foundation for the rapid clinical detection of PCV2.The Cap gene was synthesized according to the PCV2 Cap gene sequence published in Gen Bank,and the Cap gene was cloned into the p ET-32a vector to obtain a recombinant plasmid p ET32a-Cap carrying the Cap gene,which was induced to be expressed in E.coli Rosetta（DE3）.Soluble expressed Cap protein was obtained by optimizing the induction temperature,IPTG concentration,and induction time.Meanwhile,the Cap gene was cloned into p GEX-6P-1 vector,and the Cap recombinant protein was purified by massive induction expression in E.coli Rosetta（DE3）,which was used as a screening antigen for subsequent ELISA screening of monoclonal antibodies.In this study,p ET32a-Cap and p GEX-6P-1-Cap recombinant expression vectors were successfully constructed,and two recombinant Cap proteins carrying different tags（His and GST）with sizes of 45.7 k Da and 54.8 k Da,respectively,were mass induced and purified.BALB/c mice were immunized with prokaryotic expression of His-tagged Cap protein,and the potency was detected after immunization according to the immunization procedure.Mouse spleen cells with higher potency were fused with Sp2/0 cells using the hybridoma technique,and the hybridoma cells were subcloned using the limited dilution method.Western blot and indirect immunofluorescence were used to identify the reactogenicity of monoclonal antibodies,and typing kits were used to determine their subtypes.In this study,three murine monoclonal antibodies of Cap protein with good reactivity,named F8G8F7C4,F8G8F7C5 and F5H7E3D2,whose isoforms are Ig G2a,Ig G2a and Ig G1,were successfully prepared to lay the foundation for the establishment of a double antibody sandwich ELISA assay.The monoclonal antibody F5H7E3D2 was labeled with sodium periodate,and the best antibody pairing was selected by antibody pairing to establish a double antibody sandwich ELISA assay,and the conditions of the assay were optimized as well as the specificity,sensitivity and reproducibility were evaluated.The results showed that with F5H7E3D2 as the enzyme-labeled antibody and F8G8F7C5 as the capture antibody as the best antibody pairing combination,the specificity tests show that the method does not cross-react with other common swine virus disease antigens,and the PCV2 virus titer was still detected at10^2.5TCID₅₀/m L in the sensitivity test,and the intra-batch inter-batch variation coefficients of variation were less than 5%in the reproducibility test.In summary,in this study,we successfully constructed recombinant plasmids p ET32a-Cap and p GEX-6P-1-Cap carrying Cap gene,and expressed purified Cap protein to prepare three well-reactive PCV2 Cap monoclonal antibodies.A double antibody sandwich ELISA assay with good specificity,sensitivity and reproducibility was also established to provide an effective tool for the rapid diagnosis of PCV2.