Establishment Of Indirect ELISA And Preparation Of Monoclonal Antibody Based On African Swine Fever Virus P35 Protein

African classical swine fever(ASF)is a highly lethal contagious disease caused by African classical swine fever virus(ASFV)in pigs.The disease is transmitted by soft ticks and the African wild boar is used as the storage host.Since the epidemic of classical swine fever in Africa was introduced into China,it has brought severe challenges to the development of pig industry and social and economic stability in China.In addition,ASF has complex mechanisms of infection and immune escape,so commercial vaccines have not been developed to prevent and control the disease in China.Therefore,the establishment of routine serological diagnosis method which is rapid,accurate,low-cost,relatively convenient and suitable for large-scale screening is particularly important for the regular surveillance of ASF epidemic situation.The antigenicity of diagnostic antigens and the development of effective monoclonal antibodies are very important for the establishment of reliable serological diagnostic methods.The indirect ELISA method using ASFV pp62 protein as diagnostic antigen is significantly better than the indirect ELISA method based on p30 or p54 protein for poorly preserved field serum samples.ASFV p35 protein is a mature protein originated from pp62 polyprotein cleaved by ASFV S273R protease.It is mainly involved in the assembly of ASFV nucleocapsid and the stabilization of virions.Therefore,the diagnostic antigenicity of p35 protein was firstly confirmed by using ASFV p35 mature protein as the research target,and the monoclonal antibody against p35 protein was prepared in the study.The contents and results of the study are as follows:1.In order to verify the potential of p35 protein as a diagnostic antigen,the antigenicity and potential of ASFV p35 protein expressed in prokaryotic expression system as diagnostic antigen were discussed for the first time by establishing enzyme-linked immunosorbent assay(Enzyme linked immunosorbent assay,ELISA)with reference to ASFV p30 protein expressed in baculovirus insect cell expression system.The results of indirect immunofluorescence and immunoblotting showed that the recombinant p35 protein of 40 k Da and the p30 protein of 30 k Da were obtained.Both proteins and ASFV positive serum had good immunogenicity.ELISA methods were established by using recombinant p30 and p35 proteins as diagnostic antigens,and their sensitivity,stability and coincidence with imported kits were verified.The results showed that although the sensitivity of p35-ELISA method was slightly lower than that of p30-ELISA method,the sensitivity was still 95.8%,and the intra-and inter-assay coefficients of variation of p35-ELISA method and p30-ELISA method were less than 10%.Compared with the imported kit,the coincidence rate of p35-ELISA method was 97.2%.The results showed that the established p35-ELISA method was stable and sensitive,and could be used to detect ASFV infection in the field.2.Mice were immunized with the purified p35 protein,when antibody titer against p35 protein achieved 1?204800,the collected splenocytes are fused with SP2/0 myeloma cells.Hybridoma cells were screened by the established p35-ELISA method and were subcloned,and 15 strains of hybridoma cells stably secreted monoclonal antibodies were obtained.The results of Western blot and indirect immunofluorescence indicted that 2-D7E5 and 3-F7E8 named monoclonal antibody can specifically recognize p35 antigen;The obtained monoclonal antibodies can specifically recognize the p35 antigen;the antibody subclass identification results showed that the two monoclonal antibodies are both Ig G1subtype,and the light chain is Kappa;The ascites titers measured by indirect ELISA method are all greater than 2¹⁴.This study provides important biological raw materials for the analysis of p35 protein functional epitopes and the establishment of ASF serological diagnostic methods.