猪繁殖与呼吸综合征病毒N蛋白间接ELISA方法的建立

Establishment of ELISA Method for Detection of Porcine Reproductive and Respiratory Syndrome Virus Antibodies

关键词:  猪繁殖与呼吸综合征  PRRSV  ELISA

Key words:  porcine reproductive and respiratory syndrome  PRRSV  ELISA

摘要:

为研制特异性敏感性较好的猪繁殖与呼吸综合征病毒（PRRSV）抗体检测方法，更好地实现PRRSV的流行病学监测和诊断，将PRRSV GDr180 毒株N蛋白基因序列克隆到pET32a（+）载体中，在大肠杆菌BL21（DE3）细胞内实现了高效表达，表达形式为可溶性表达，通过Western blot证明表达产物与PRRSV GDr180株阳性血清具有很好的反应原性和特异性。将大肠杆菌表达的N蛋白经过超声、离心、过柱纯化后，作为间接ELISA包被抗原检测血清中的PRRSV抗体，通过对各参数和试剂的优化建立了能检测PRRSV血清抗体的间接 ELISA检测方法；对方法的特异性和重复性以及与同类成品试剂盒间的应用效果对比进行了试验。结果表明，研究建立的 ELISA抗体检测方法可用于检测猪血清中PRRSV抗体、监测猪繁殖与呼吸综合征的流行情况和评价相关疫苗的免疫效果。

Abstract:

In order to develop a specific and sensitive porcine reproductive and respiratory syndrome virus (PRRSV) antibody detection method, to better realize the epidemiological monitoring and diagnosis of PRRSV, the N-protein gene sequence of PRRSV GDr180 strain was cloned into pET32a(+) vector, and it was highly expressed in E.coli BL21(DE3) cells in the form of soluble expression. Western blotting showed that the expression product had good reactivity and specificity with the positive serum of PRRSV GDr180 strain. The N protein expressed by E.coli was purified by ultrasound, centrifugation and column chromatography, and used as a coating antigen of indirect ELISA to detect the PRRSV antibody in the serum. By optimizing the parameters and reagents, an indirect ELISA method for detecting PRRSV serum antibody was established. The specificity and repeatability of the method and the comparison with the application effects of the similar finished kits were tested. The results showed that the ELISA antibody detection method established in this study can be used to detect porcine reproductive and respiratory syndrome virus antibodies in pig serum, monitor the prevalence of porcine reproductive and respiratory syndrome and evaluate the immune effect of related vaccines.