猪瘟病毒E2蛋白主要抗原域的高效表达及间接ELISA方法的初步建立

The High Expression of the Main Antigenic Domains of CSFV E2 Protein in E. coli and the Establishment of the Indirect ELISA Assay for Detection CSF Sera Antibody

重组质粒pET-2e转化宿主菌BL21(DE3),以IPTG进行诱导表达,比较不同诱导条件下目的蛋白的表达量,确定其最佳表达条件,使外源基因获得了较高水平的表达,可达菌体蛋白总量的36.6％。Western-blot检测表明表达产物具有良好的免疫反应原性。将收集的纯培养物进行超声波裂解后以镍亲和层析柱纯化回收目的蛋白,再以纯化后的目的蛋白作为包被抗原进行方阵滴定试验,确定了目的蛋白作为包被抗原的最佳稀释度,初步建立了检测CSF血清抗体水平的间接ELISA方法。以此方法建立的间接ELISA方法对收集的约120头份血清样品进行检测,结果显示:使用囊素与疫苗共同免疫猪的血清抗体水平明显高于未加囊素组猪的血清抗体水平,与以往的报道一致,为以CSFV重组蛋白作为抗原建立的间接ELISA方法的标准化奠定基础。

The recombinant expressing plasmid pET-2e, which include the foreign gene fragment coding for the main antigenic domains of classical swine fever virus (CSFV), was transformed into the host cell BL21 (DE3) and induced with isopropyl-β-D-thiogalactopyranoside (IPTG). After the expressing, level under different inducing conditions has been compared, the optimal expressing condition was confirmed and the target protein can be expressed in a very high level and up to 36.6% of the total protein expressed by the host cell under this condition. Western-blot assay showed the good immunological reactivity and specificity of the target protein. Then, the target protein, after having being purified with the His band kit, was used as antigen in the chess titration test and the optimal circumstance for the ELISA (antigen concentration and sera dilutions) was confirmed.Then, 120 swine sera samples were detected with the indirect ELISA (I-ELISA) assay established here. The results showed that the sera samples got from the pigs immunized with Hog cholera haplized virus (HCLV) using bursin as adjuvant have higher antibody titers than those without using adjuvant according to the criterion established here, which conforms to the previous study. So the foundation for standardizing the I-ELISA assay was established here.