【摘要】 为了研制乙型脑炎病毒EⅢ-间接ELISA抗体检测试剂盒,将乙型脑炎病毒EⅢ基因克隆至原核表达载体p ET-32a(+),重组质粒转化大肠杆菌BL21进行诱导表达。以纯化后的重组蛋白EⅢ包被酶标板,优化ELISA反应条件,组装试剂盒。SDS-PAGE和Western bloting分析表明,重组蛋白分子质量约为30ku,经IPTG诱导后高效表达,且具有来良好的免疫原性。优化试验确定重组抗原最佳包被浓度为4.7μg/m L,血清样本的阳性临界值为0.255。特异性试验结果表明,重组蛋白与猪瘟、猪伪狂犬、猪圆环病等多种病原体的阳性血清无交叉反应。敏感性试验结果表明,血清稀释至1∶6400时仍检测为阳性。该试剂盒的批内、批间变异系数均小于10%。用该试剂盒检测224份临床猪血清样本,并与商品化ELISA试剂盒检测结果比较,两者的符合率为90.7%。研究结果表明,原核表达的猪流行性乙型脑炎病毒EⅢ蛋白具有良好的免疫原性,研制的猪流行性乙型脑炎病毒EⅢ-间接ELISA抗体检测试剂盒敏感性、特异性和重复性较好,为猪流行性乙型脑炎的抗体水平检测、流行病学调查、类症鉴别等方面提供了重要方法。更多还原

【Abstract】 In order to develop an indirect ELISA method for detecting antibody against Japanese encephalitis virus, the EⅢ gene of Japanese encephalitis virus was inserted into p ET-32a(+) vector and the recombinant plasmid was transformed into E.coli BL21 and expressed. Using the purified recombinant protein E Ⅲ as coating antigen to establish indirect ELISA method, and the reaction conditions was optimized and kit was assembled. The recombinant protein was expressed in high level in BL21 as confirmed by SDS-PAGE, and showed strong immunological reaction with Japanese encephalitis virus positive serum detected by Western blot. The test confirmed that the best coating concentration of the recombinant antigen was 4.7 μg/m L, and the critical value of positive serum samples was 0.255. The specificity test showed that there were no cross-reactivity between the recombinant protein and CF,PRS, PC and other pathogens positive serum. The sensitivity tests indicated that the diluted serum ratio was 1 ∶6400.The coefficient of variation of intra-assay and inter-assay were less than 10%. There was 90.7% coincidence rate between the indirect ELISA kit and the commercial indirect ELISA kit based on detection of 224 clinical swine serum samples. Conclusion: The prokaryotic expression of recombinant protein E Ⅲ has good immunogenicity, and the established the indirect ELISA method has good sensitivity, specificity and reproducibility, which can provide an important method for antibody levels detected, epidemiological investigation, syndrome identification of Japanese encephalitis virus.更多还原