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

Prokaryotic expression of truncated S protein of porcine deltacoronavirus and establishment and application of indirect ELISA detection method

为建立快速、高效检测猪丁型冠状病毒(Porcine deltacoronavirus,PDCoV)的血清学方法,构建了pET-32a-S-S1b重组质粒,通过原核表达获得重组S1b蛋白,以此为包被抗原建立了检测血清中PDCoV IgA抗体的间接ELISA方法,对该检测方法的反应条件进行优化,验证了其特异性、敏感性和重复性等。结果显示:重组S1b蛋白呈包涵体表达,大小为34.7 ku,反应原性良好;建立的间接ELISA检测方法的最佳反应条件为:250 ng/孔抗原4℃过夜包被,10 g/L BSA 37℃封闭2 h,待检血清1∶200稀释37℃孵育45 min,二抗1∶10000稀释37℃孵育1 h,显色时间为10 min。测定24份阴性血清,确定临界值为0.388。检测阳性血清敏感度为1∶1600;批间和批内重复试验的变异系数均小于10%;本方法特异性高,与猪流行性腹泻病毒、伪狂犬病病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、非洲猪瘟病毒、猪圆环病毒和猪肠病毒标准血清无交叉反应。选取30份血清,与Western-blot检测结果比较,该间接ELISA的符合率为96.66%。应用该方法对486份临床血清样品进行检测,阳性率为32.72%。本研究中建立的方法为PDCoV的防控提供了技术支持。

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 antibody in serum was established.After optimizing the reaction conditions of the method,its specificity,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∶10000 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∶1600,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,classical 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 detected by this method,with a positive rate of 32.72%.The method established in this study provides technical support for the prevention and control of PDCoV.