Establishment Of Indirect ELISA For Antibody Detection Based On The P22 Protein Of African Swine Fever Virus

African swine fever(ASF)is a highly lethal viral infectious disease of pigs caused by African swine fever virus(ASFV),which is a legally reported animal disease by the World Organization for Animal Health(OIE)and belongs to a category of animal diseases in China.The disease was introduced into China in August 2018,and due to its very high mortality rate,it has caused a rapid decline in the stock of sows,leading to an increase in pork prices and posing a serious challenge to the development of the pig industry and socioeconomic stability.At present,there is no effective treatment for ASF and no effective vaccine that can be used for prevention and control,and the means of prevention and control mainly rely on biosecurity and culling.However,with the gradual prevalence and occurrence of the disease,some animals exhibit subacute infection and survive.recovered animals with ASF infection have high antibody levels and the virus is usually undetectable in the blood,so antibody detection is important for ASF screening and decontamination.In this study,we prepared ASFV p22 protein by prokaryotic expression and used it to establish an ELISA method suitable for ASFV antibody detection.In this study,the amino acid sequence of p22 gene in ASFV was analyzed by DNASTAR software based on the sequence of p22 gene published by NCBI(Gen Bank:MK128995.1),and the amino acid sequence of positions 24-145 with good hydrophilicity and antigenicity was screened,codon optimized,and 6×His tag gene was added downstream of the fragment to synthesize the fusion protein expressed by the above-mentioned fusion protein.The gene fragment of the above fusion protein was synthesized and ligated into the prokaryotic expression vector p ET-32 a plasmid.The recombinant plasmid p ET32a-p22 was transformed into the receptor E.coli BL21(DE3)expression host,and the target protein was expressed after IPTG induction,then purified by Ni column affinity chromatography,and the concentration of recombinant protein was determined by BCA at 2.5 ?g/?L.The results of Western Blot assay showed that the p22 protein expressed in this study was able to reacted specifically with inactivated ASFV-positive sera,but not with positive sera of other common pathogens such as PRRSV,PCV2,CSFV,PRV,and sera of healthy pigs.The purified His-p22 recombinant protein was used as the antigen for encapsulating ELISA reaction plates to establish and optimize an indirect ELISA method for the detection of ASFV antibodies in pig sera,and standard ASFV inactivated positive and negative sera were kindly provided by the China Animal Health and Epidemiology Center.After the exploration and optimization of the assay conditions,the optimal antigen coating concentration was finally determined to be 0.125 ?g/m L,the dilution of the examined sera was 1:800,the antigen was coated overnight at 4?,the closure solution was selected from5% skim milk powder(200 ?L per well),the closure time was 60 min,the serum was acted at37?for 60 min,the secondary antibody was diluted at 1:10,000(The optimized ELISA method was applied to detect 60 negative sera from pigs without ASFV infection,and the critical values of the ELISA method were calculated based on statistical principles.When the OD450 value of the serum samples was ? 0.384,they were positive;when the OD450 value was < 0.384,they were negative.The positive sera of PCV2,PRRSV,PRV and CSFV were tested using this assay and the results were negative,indicating that the specificity of the assay is relatively good.The results of the repeatability test showed that the values of the same serum samples in the same batch of tests and different batches of tests were 4.5%?7.5%and 1.7%?9.08%,respectively,which were less than 10%,indicating that the indirect ELISA method has good reproducibility.The results showed that the indirect ELISA method established in our laboratory and the commercialized kits met 97.7% with no significant difference;it indicated that the indirect ELISA method established in this paper for the detection of p22 antibody can be used for the detection of ASFV p22 antibody detection.In conclusion,this experiment was based on the expression of p22 protein in the prokaryotic expression system,and an indirect ELISA method for the detection of ASFV antibodies based on ASFV p22 protein was established,which has good reproducibility and specificity.It laid the foundation for the development of ASFV vaccine in China.