关键词: 猪流行性腹泻; S2蛋白; IgA抗体; ELISA

Abstract: 【Objective】 The purpose of this study was to establish an ELISA method for detection of IgA antibody against Porcine epidemic diarrhea virus (PEDV).【Method】 In this study,PEDV epidemic strain CH/HNPJ/2017(MF152604.1)was used as the biological material,and the S2 gene (2 368—4 170 bp) was amplified by RT-PCR and inserted into prokaryotic expression vector pET-24a to transform the competent cells of Escherichia coli BL21 (DE3).It was induced by IPTG and purified by nickel column.Western blotting was used to verify the reactivity and specificity of recombinant protein S2 with PEDV positive serum.Using it as the coating antigen,an IgA antibody ELISA method based on recombinant full-length S2 protein of PEDV variant strain was established.The chessboard method was used to determine the optimal detection conditions,and its sensitivity,specificity and repeatability were determined.It was preliminarily applied to the detection of 130 porcine serum and 40 porcine oral mucus.【Result】 The S2 gene was amplified by RT-PCR,and the recombinant expression vector pET-24a-S2 was successfully constructed.The recombinant S2 protein was obtained after being transformed into Escherichia coli and expressed and purified by induction.SDS-PAGE results showed that the purified S2 protein had specific bands and no heterobands.Western blotting test showed that the protein had good reactivity with PEDV positive pig serum.The optimal coating conditions for recombinant S2 protein were 0.5 μg per well at 4 ℃ overnight,the optimal serum reaction conditions were 1∶160 dilution at 37 ℃ for 60 min,the best reaction conditions for HRP-conjugated antibody were 1∶5 000 dilution at 37 ℃ for 30 min,and the optimal color rendering time of TMB was 10 min under ambient temperatures.The sample was positive when the S/P value ≥ 0.04,it was negative when the S/P value ≤ 0.02,and it was suspicious when the S/P value was between 0.02 and 0.04.The ELISA method established in this study was used to detect the standard positive serum of swine fever virus,porcine circovirus,porcine reproductive and respiratory syndrome virus,pseudorabies virus and porcine deltacoronavirus.The S/P values were all less than 0.02,indicating that the method had good specificity.When the PEDV positive serum was diluted by 640 times,it was still positive by this method,indicating that this method had good sensitivity.This method was used for inter assay and intra assay,and the coefficient of variation were all less than 8% (within 10%),which shows that this method had good repeatability.130 pig sera were detected by this method,and the coincidence rate between the results and IDEXX IgA antibody detection kit was 90.77%.The detection results of 40 samples of pig oral mucus showed that the positive detection rate was 90.0% and the negative detection rate was 93.3%.【Conclusion】 In this study,the recombinant plasmid pET-24a-S2 was successfully constructed,and the recombinant S2 protein with high purity was obtained,which was used as coating protein to establish an ELISA method for IgA antibody based on full-length S2 protein.The method could effectively detect both serum and pig oral mucus type samples,and had strong sensitivity,high specificity,stability,and repeatability,which was of great clinical practical significance to the clinical diagnosis and prevention and control of PEDV.

Key words: porcine epidemic diarrhea; S2 protein; IgA antibody; ELISA