牛病毒腹泻病毒(BVDV)E2重组蛋白ELISA检测方法的初步建立

Establishment of Bovine Viral Diarrhea Virus Recombinant Protein ELISA Assay

【目的】为牛病毒性腹泻病毒抗体快速检测提供敏感、特异检测方法。【方法】重组质粒pET-(1-345)转化宿主菌BL21(DE),以IPTG进行诱导表达,使外源基因获得了较高水平的表达;Westem一blot检测表达产物反应原性,同时进行特异性检测;将收集的纯培养物进行超声波裂解后以His bind kit蛋白质纯化试剂盒纯化回收目的蛋白,再以纯化后的目的蛋白作为包被抗原进行方阵滴定试验,确定目的蛋白作为包被抗原的最佳稀释度。【结果】重组蛋白表达可达菌体蛋白总量的19.7%,且具有较好的反应原性和特异性,以此方法建立的间接ELISA方法对收集的约43头份血清样品进行检测,检测阳性结果与进口试剂盒检测结果符合率达到93.55%。【结论】以BVDV E2重组蛋白作为抗原建立的间接ELISA检测方法是可行的,为其进一步的标准化莫定基础。

[ Objective and Method ] The recombinant expressing plasmid PET - （ 1 - 345 ）, which include the foreign gene fragment coding the main antigenic of bovine viral diarrhea virus （ BVDV }, was transformed into host cell BL21 （DE3） and induced with IPTG.. West- Blot assay reactivity and specificity of the target protein . Then, the target protein, after being purified with the his band kit, was used as an antigen in the square titration test and optimal dilution rate for the ELISA was confirmed . [Result]The recombinant protein expression can reach 19.7% in total amount of somatic protein. West - Blot assay showed the good immunological reactivity and specificity of the target protein, 43 bovine sera samples were detected with the indirect ELISA assay. The result showed that the consistancy rate between the result detected the bovine sera sample and the result detected with imported agent is 93.55 %. [ Conclusion] Bovine viral diarrhea virus recombinant protein E2 ELISA assay is a practicable method.