Establishment And Preliminary Application Of Diagnostic Methods For ASF Based On P30 And P54 Antigens

African swine fever(ASF)is an acute,haemorrhagic infectious disease caused by African swine fever virus(ASFV),which can infect both domestic and wild pigs.Mortality of the infected pigs may be up to 100%caused by the virulent strains.The disease was first reported in Kenya in 1921,then gradually spread around the world,and entered China in 2018.Due to the lack of effective vaccines and therapeutic drugs,hundreds of millions of pigs were slaughtered or died in China.Therefore,rapid and effective diagnostics combined with effective control measures are now effective methods to prevent the transmission and epidemic of ASFV.To establish a simple preparation and sensitive method for the rapid detection of African swine fever virus(ASFV)antigen,the recombinant protein P30 with His tag and Flag tag were co-expressed in 293A cells using the expression vector pIRES.After lysing the cells,P30 was found to be a polymer by immunoprecipitation.The relative molecular mass of purified P30 was further determined by mass spectrometry,the results confirmed that ASFV P30 could form dimers spontaneously.With that,a colloidal gold immunochromatographic method was established for the detection of P30 dimers by a single monoclonal antibody 6H9A10 against P30,and the ASFV p30 recombinant antigen at the concentration of 2.1ng/mL could be detected out instead of the common porcine viral antigens,such as antigens of PCV-2,CSFV,PRRSV,PRV,PEDV,PPV and SVA.100 clinical samples including 20 ASFV negative sera,20 ASFV negative tissues,10 ASFV negative plasma,21 ASFV positive sera,21 ASFV positive tissues,and 8 ASFV positive plasma,identified by the real-time PCR were tested by the immunochromatographic method,the results showed that the 48 negative samples and 49 positive samples were consistent with that by the fluorescent PCR method,compliance rate for testing was 97%.Which indicates that the established colloidal gold immunochromatographic method for the detection of P30 by single monoclonal antibody sandwich has good specificity and sensitivity.Analysis of the antigenicity of ASFV P30,and in order to improve the immunoreactivity of the antigen,the gene encoding the carboxyl terminus of P30 was fused with the ASFV p54 gene,then was cloned into the prokaryotic expression vectors pET-30a(+)and pGEX-6P-1 to construct the recombinant plasmids pET-30a-5430 and pGEX-5430.The expressed recombinant proteins were purified after the E.coli BL21(DE3)containing recombinant plasmids being induced with IPTG,and the recombinant proteins could react with monoclonal antibodies against ASFV P30 and P54 specifically by Western blot,which indicates that the recombinant proteins P5430His and GST-P5430 were successfully expressed and purified.The recombinant protein P5430-His was used as the encapsulated antigen and GST-P5430 as the detection antigen.By optimizing the reaction conditions,a double-antigen sandwich ELISA for the detection of ASFV antibodies was established,and the cutoff value of 0.084 was determined by testing 200 ASFV-negative sera.The results of 456 clinical serum samples tested and compared with the ID Vet indirect ELISA diagnostic kit showed that the double antigen sandwich ELISA and ID Vet indirect ELISA assay results were 95.8%.The results showed that the inter-batch CV was ≤6.86%and the intra-batch CV was ≤5.02%,indicating that the ELISA method has good reproducibility.In conclusion,the colloidal gold immunochromatographic method for the detection of ASFV P30 antigen and the double-antigen sandwich ELISA method for the detection of ASFV P30 and P54 antibodies established in this study both have good specificity and detection compliance,and are expected to be used for the clinical detection of ASF.