检测猪瘟病毒E0抗体ELISA方法的建立与应用

Establishment and application of an ELISA method for detecting CSFV E0 antibody

E0蛋白是猪瘟病毒(CSFV)重要的结构蛋白之一,能刺激机体产生中和性保护抗体,同时也是作为鉴别诊断的标识之一。本研究通过RT-PCR扩增得到石门株E0基因,构建了重组质粒pET-28a-E0,原核表达E0目的蛋白后鉴定了其免疫反应性。将纯化后的目的蛋白进行梯度稀释,建立了ELISA检测方法,优化反应条件如下:最佳蛋白包被浓度2μg/mL,最佳一抗和二抗的稀释倍数分别为1∶100及1∶40 000,一抗孵育时间为30 min,阴阳性临界值分别为0.293和0.418。结果显示组间与组内变异系数均小于10%,无交叉反应性,特异性良好。用建立的方法与商品化试剂盒同时检测206份临床免疫疫苗的血清样本,阳性和阴性及总体符合率分别为96.55%、84.21%及94.17%,表明ELISA方法具有良好的敏感性、稳定性和符合率,可用于临床样本的检测。用该方法检测了85份免疫了E2亚单位疫苗的临床猪血清样本,阳性率为10.59%,表明该方法可初步用于临床CSFV抗体的快速检测及鉴别诊断。

E0 is an important structural protein of CSFV, which can stimulate the host to produce neutral protective antibodies, and it is also one of the identifiers of differential diagnosis. In this study, the E0 gene of the Shimen strain was amplified by RT-PCR, and the recombinant plasmid pET-28 a-E0 was constructed. After prokaryotic expression of the E0 target protein, its immunogenicity was identified. The target protein was purified, subjected to gradient dilution and coated with an ELISA antigen plate to optimize its reaction conditions. The results showed that the optimal antigen coating concentration was 2 μg/mL;the dilution ratios of the optimal primary antibody and the secondary antibody were 1∶100 and 1∶40 000, respectively;the incubation time of the primary antibody was 30 min;and the critical values were 0.293 and 0.418. The coefficient of variation between and within the groups was less than 10%;there was no cross-reactivity, and the specificity was good. Detection of 206 clinical serum samples using the established methods and the commercial kits at the same time showed that the positive, negative and overall coincidence rates were 96.55%, 84.21% and 94.17%, respectively, which indicated that the ELISA method here had good sensitivity, stability and coincidence, and might be used for the detection of clinical samples. The test results of 85 pig serum samples immunized with the E2 subunit vaccine showed that the positive rate of the E2 commercial kit test was 91.76%, and the positive rate of this method was 15.29%. This proved that this present method might be used for rapid detection and differential diagnosis of clinical CSFV antibodies.