Preparation Of Monoclonal Antibodies Against PEDV S1 Truncated Protein And Establishment Of Indirect Elisa Method

Porcine Epidemic Diarrhea（PED）is a contagious intestinal disease caused by the Porcine Epidemic Diarrhea Virus（PEDV）.Its main symptoms include diarrhea,anorexia,and dehydration.After infection,it causes a large number of deaths in newborn piglets,causing huge economic losses to the pig industry.PEDV’s entry into cells is mediated by the binding of its spike protein（S protein）to host cell receptors.The S1 domain contains multiple receptor binding sites and is often used as the preferred target for vaccine development and diagnostic reagents.In this study,the truncated S1 protein was expressed by baculovirus,and the recombinant protein was used to immunize mice to prepare monoclonal antibody,providing an effective detection tool for the in-depth study of PEDV S1 protein.In addition,the recombinant protein S1_401-769 containing more antigen epitopes was used as the coated antigen to establish an indirect ELISA method.To provide technical means for the prevention and control of PEDV disease and clinical diagnosis.1.Construction of recombinant baculovirus expression system with truncated protein PEDV S1In this study,the optimized S1 protein was sequentially truncated to obtain 1～400 aa and 401～769 aa fragments.The amplified fragments were connected with p Fast Bac1 vector to obtain the recombinant plasmids p Fast Bac1-S1_1-400 and p Fast Bac1-S1_401-769.The recombinant plasmid was translocated into DH10Bac receptive cells,the recombinant baculovirus shuttle plasmid was constructed,and then transfected into Sf9 insect cells for expression.The plasmid was verified by IFA and Western Blot.The results showed that two proteins,S1_1-400 and S1_401-769,were successfully expressed in the insect cells,and both were expressed in the crushed cell supernatant.SDS-PAGE and Western Blot results showed that the expressed proteins could react specifically with PEDV positive sera.2.Preparation of monoclonal antibodies against PEDV S1truncated proteinThe recombinant proteins S1_1-400 and S1_401-769 expressed by baculovirus were used to immunize mice,and the serum titer of mice was measured four times after immunization.When the titer reached 1:5120,the enhanced immunization was carried out.Spleen cells and myeloma cells of immunized mice were taken for cell fusion.Hybridoma cells were screened by indirect ELISA method,and three positive cell lines with stable antibody secretion were obtained after three subclones,which were verified by Western Blot and IFA,and two monoclonal antibodies against PEDV S1_1-400,named 400C5 and 400G8,were obtained.And one monoclonal antibody against PEDV S1_401-769 was named 769H4.3.Establishment of an indirect ELISA method for PEDV S1_401-769proteinUsing recombinant protein S1_401-769 as antigen for protein coating,the optimal coating concentration was determined by square titration as 0.25μg/m L,and the optimal serum dilution ratio was 1:40.According to these conditions,the optimal sealing solution was also found and the sealing time was 5%BSA for 2 hours,and the serum was incubated at 37℃for 1.5 h.HRP-the sheep anti-swine IgG antibody was incubated at 37℃for 30 min at a dilution ratio of 1:5000.According to the optimized indirect ELISA conditions,64 clinical serum samples were detected and compared with the results of domestic commercial kit.The results showed that the total coincidence rate of PEDV IgG antibody indirect ELISA assay was 81.25%（52/64）,which confirmed the sensitivity of the established indirect ELISA assay.In summary,this experiment constructed recombinant rod-shaped viruses of PEDV S1_1-400 and S1_401-769,and utilized this recombinant rod-shaped virus to stably express S1truncated protein in insect cells.After immunizing mice with the expressed protein,three monoclonal antibody cell lines targeting the truncated protein of PEDV S1 were successfully screened,which laid the foundation for further study of PEDV S1 protein in the future.An indirect ELISA method for detecting the truncated protein of PEDV S1 was established by encapsulating the recombinant protein S1_401-769 expressed by a rod-shaped virus on an enzyme-labeled plate,providing an effective tool for the prevention and control of PEDV.