猪丁型冠状病毒截短S蛋白的原核表达及间接ELISA检测方法的建立与应用OACSTPCD

In order to establish a rapid and efficient method for serological detection of porcine deltacoronavirus(PDCoV),the recombinant plasmid pET-32a-S-S1b was constructed,and the recombinant S1b protein was obtained by prokaryotic expression.An indirect ELISA method for detecting PDCoV IgA an-tibody in serum was established.After optimizing the reaction conditions of the method,its specifici-ty,sensitivity and repeatability were verified.The results showed that the recombinant S1b protein was expressed in inclusion bodies,the size was 34.7 ku and the reactivity was good,and the best reaction conditions of indirect ELISA were as follows:the antigen was coated at a concentration of 250 ng/well and incubated overnight at 4 ℃,10 g/L BSA was incubated at 37 ℃ for 2 h as a blocking step,the tested serum was diluted at a ratio of 1:200 and incubated at 37 ℃ for 45 min,and the secondary antibody was diluted at a ratio of 1∶10 000 and incubated at 37 ℃ for 1 h,with a coloration time of 10 min.The cutoff value was determined to be 0.388 after testing 24 negative sera.The sensitivity of detecting positive serum was 1∶1 600,and the coefficient of variation of 10 PDCoV-positive sera inter-and intra-batch assay of ELISA were both less than 10％.This method demonstrated high specificity and did not show cross reaction with standard sera against porcine epidemic diarrhea virus,pseudorabies virus,classi-cal swine fever virus,porcine reproductive and respiratory syndrome virus,African swine fever virus,porcine circovirus and porcine enterovirus G.Compared to the results obtained from 30 serum samples using Western-blot,the coincidence rate was 96.66％.A total of 486 clinical serum samples were detect-ed by this method,with a positive rate of 32.72％.The method established in this study provides techni-cal support for the prevention and control of PDCoV.