Abstract: African Swine fever (ASF), caused by infection with the ASF virus (ASFV), is one of the most serious viral diseases affecting pigs. It is featured by acute, febrile, hemorrhagic, high morbidity and mortality, etc. The aim of this paper is to develop an efficient and clinically convenient indirect ELISA antibody detection method for ASFV. Here, the ASFV-CP204L gene was amplified from positive samples, and the p30 protein was expressed through the pET-30a prokaryotic expression system. The expressed product was further purified with Ni-NTA, and the His protein was removed by enterokinase. Thus, an indirect ELISA method was established based on the purified recombinant tag-free p30 protein. An approximate 30 ku fusion p30 tag-free protein was observed. The results showed that the protein expressed in this study has good reactogenicity with ASF-positive pig serum. It was finally determined that the ELISA antigen coating concentration was 1 μg·mL^-1. The positive value (S/P) was more than 0.398 by the area under the ROC curve. The coefficients of inter-and intra-batches were less than 10%, indicating the good repeatability of the method. This method can detect at least 512-fold diluted standard positive serum samples. In the detection of experimentally infected sera, 80% of the samples were seroconversion on the 10th day. In this study, a total of 644 pig serum samples were collected. The results showed that the positive rates were 7.61%. Among them, the positive rates of antibodies in sows, gilts, piglets, nursery pigs and fattening pigs were 3.03%, 0%, 4.94%, 7.55% and 28.7%, respectively. The indirect ELISA method based on the tag-free ASFV-p30 protein has good specificity, sensitivity and reproducibility, and can be initially applied to the detection of antibodies of ASFV. It provides a technical tool for epidemiologically serological investigation of ASF.

Key words: African swine fever, CP204L gene, p30 protein, prokaryotic expression, ELISA antibody detection