Prokaryotic Expression Of P22 Protein Of African Swine Fever And Establishment Of An Indirect ELISA Method For Detection

African classical swine fever（African Swine Fever,ASF）is an acute hemorrhagic and infectious viral animal disease caused by African classical swine fever virus（African Swine Fever Virus,ASFV）infection.The World Organization for Animal Health（WOAH）requires countries with ASF outbreaks to notify them,and China classifies it as a kind of animal infectious disease.However,so far,there are no commercial vaccines and drugs that can effectively prevent or treat ASF.Therefore,timely and accurate detection and cutting off the route of transmission is an effective way to reduce the loss caused by the ASF epidemic.In this study,a high purity recombinant ASFVp22 protein was prepared by E.coli expression system and used as a coating protein to establish an indirect ELISA method for the detection of ASFV antibodies.In this study,42-189amino acids with good antigenicity in the KP177R gene sequence corresponding to p22 protein of ASFV strain SY-18（Gen Bank:MH766894）were ligated into pET-30a（+）prokaryotic expression vector to construct pET30a-p22recombinant plasmid.The recombinant plasmid was expressed in prokaryotic cells,and the target protein was expressed in the supernatant of the bacterial liquid by optimizing the induction conditions.The induced protein was purified and concentrated,and the concentration was 670μg/m L.The results of Western Bolt test showed that the recombinant protein pET30a-p22 expressed in E.coli was correct and had good antigenicity and could bind strongly to ASFV antibody positive serum.Using pET30a-p22 as antigen protein,the ELISA test conditions were explored and optimized.The final optimum test conditions are as follows:the best coating solution is PBS,the best antigen dilution concentration is 0.25μg/m L（100μL per well）,the best serum dilution ratio is 1PBS 1600（100μL per well）,the best sealing solution is 5%skimmed milk powder（200μL per well）,and the best sealing condition is 12-16h at 4℃.The serum to be tested was incubated at 37℃for 0.5 h.The best dilution ratio of secondary antibody is 10 000（100μL per well）,incubated at 37℃for 1.5 h,and the best reaction time of chromogenic solution（100μL per well）is 15min.The criterion of this indirect ELISA method is that the OD₆₃₀value of the sample to be tested≥0.261 is ASFV antibody positive,0.261>the sample OD₆₃₀value≥0.224 is suspicious,and the OD₆₃₀value<0.224 of the sample to be tested is ASFV antibody negative.The indirect ELISA method was tested and evaluated:this method was used to detect the positive sera of 7 different epidemic swine diseases,such as pseudorabies,and the results of each sample were negative,which indicated that this method had no cross-reaction with other epidemic swine disease antibodies and had good specificity.Different ASFV positive sera can still be judged to be positive when the dilution is as high as 3200,indicating that this method has high sensitivity.Intra-and inter-batch repeatability tests show that the intra-batch coefficient of variation is 0.30%color 3.23%,and the inter-batch coefficient of variation is 1.04%color 7.33%,both of which are less than 10%,indicating that the detection method has good repeatability and stability.The indirect ELISA method was used to detect 226 clinical samples with two different commercial ELISA kits.The results showed that the coincidence rate of the two commercial ELISA antibody detection kits was 100%.Compared with the commercial kit,the coincidence rate of this method is 98.6%（223）,indicating that the indirect ELISA detection method can be used to detect a large number of ASFV antibodies.In this study,an indirect ELISA detection method was successfully optimized and established based on the recombinant protein pET30a-p22 expressed in E.coli prokaryotic expression system.The experimental results show that this method has good sensitivity,specificity,repeatability and stability for the detection of ASFV antibody,can detect ASF antibody in field samples quickly,efficiently and in large quantities,and can be used for large-scale screening of ASFV epidemic situation.The results of this study provide a reliable and effective technical means for clinical detection and epidemiological investigation of ASFV,and are of great significance for the prevention and control of ASF epidemic.