猪流行性腹泻病毒S1蛋白IgG抗体间接ELISA检测方法的建立

Establishment of indirect ELISA IgG antibody detection method based on S1 protein of porcine epidemic diarrhea virus

根据猪流行性腹泻病毒(PEDV)变异株的S1基因序列,与pcDNA3.1载体构建重组表达质粒pcDNA-12S1,利用瞬时转染方法将pcDNA-12S1转染至293F细胞,每隔24 h取样,进行Western-blot检测,确定最佳收样时间,并采用镍离子亲和层析技术进行蛋白纯化。SDS-PAGE和Western-blot结果显示,该S1蛋白的纯度高、免疫原性好。以S1蛋白作为包被抗原,建立并优化了IgG抗体间接ELISA方法。结果显示,S1蛋白的最佳包被浓度为0.25μg/m L;最佳封闭剂和最佳封闭时间为5%脱脂奶粉溶液37℃作用3 h;待检血清和酶标二抗的最佳稀释度分别为1∶100和1∶20000,最佳孵育时间均为37℃30 min。采用该方法检测猪非典型瘟病毒(APPV)、猪传染性胃肠炎病毒(TGEV)、猪德尔塔冠状病毒(PDCoV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪轮状病毒(PoRV)的阳性血清,结果均为阴性,表明该方法特异性好;该方法的批内和批间变异系数(CV)均<9%,表明该方法重复性较好;该方法与间接免疫荧光(IFA)相比较,阳性符合率为93.54%,阴性符合率为94.74%,总符合率为94%。上述结果表明,该方法可用于检测临床猪血清中的PEDV抗体,为PEDV的感染与接种疫苗后的免疫保护状态监测提供有效依据。

Based on the Sl gene sequence of porcine epidemic diarrhea virus(PEDV)variant,a recombinant expression plasmid pcDNA-12S1 was constructed with pcDNA3.1 vector.The pcDNA-12S1 was transfected into 293F cells using transient transfection method.Samples were taken every 24 h and the optimal sample collection time was determined by Western-blot assay.The protein was purified by Nickel ion affinity chromatography.The results of SDS-PAGE and Western-blot showed that the obtained S1 protein was of high purity and immunogenicity.An indirect ELISA method was established and optimized by using the purified Sl protein as the coating antigen.The results showed that the optimal antigen coating concentration was 0.25μg/mL.The optimal blocking solution and blocking time was 5%skimmed milk at 37℃C for 3 h.The working concentrations of tested serum and HRP-labelled secondary antibody were 1:100 and 1:20000,respectively,and the optimal incubation time was 37 C for 30 min.Moreover,atypical porcine pestivirus(APPV),transmissible gastroenteritis virus(TGEV),porcine deltacoronavirus(PDCoV),porcine reproductive and respiratory syndrome virus(PRRSV),and porcine rotavirus(PoRv)positive serum samples were detected with the negative results by this method,which showed high specificity.The coefficients of variation(CV)of intra and inter batch repetitive tests of the ELISA were less than 9%,indicating good repeatability of the method.Compared with the indirect immunofluorescence assay(IFA),the positive coincidence rate was 93.54%,the negative coincidence rate was 94.74%and the total coincidence rate was 94%.The above results indicate that the method can be used in clinical serum antibody screening and provide an effective basis for PEDV infection and immune protection status aftervaccination.