人致病性呼肠孤病毒M3编码蛋白纯化及多克隆抗体制备

Purification of human pathogenic reovirus M3 gene encoded protein and preparation of its polyclonal antibody

摘要:

目的  根据构建的人致病性纳尔逊海湾病毒（NBVs）M3基因片段质粒，纯化融合蛋白制备NBVs M3多克隆抗体。方法  利用构建的纳尔逊海湾病毒（NBVs）M3基因质粒Pris His MB-M3，转化至大肠埃希菌（E.coil）BL21（DE3）感受态细胞中，异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白表达，产物经过镍柱亲和层析法获得Pris His MB-M3融合蛋白；将纯化的融合蛋白作为抗原免疫家兔，获得NBVs M3多克隆抗体；蛋白免疫印迹检测（Western blot）蛋白准确性以及多克隆抗体抗性。结果  SDS-PAGE电泳考马斯亮蓝染色检测显示：25℃、IPTG浓度为0.3 mmol/L，诱导12 h，Pris His MB-M3融合蛋白表达量最高；在咪唑浓度为MCAC-20、MCAC-40条件时洗脱下目的蛋白。用纯化的融合蛋白免疫家兔制备抗体，Western blot验证成功制备出NBVs M3多克隆抗体。结论  成功获得了灵敏性及特异性较高的纳尔逊海湾病毒（NBVs）M3多克隆抗体，为进一步研究该病毒的致病性提供了很高的应用价值。

Abstract:

Objective To prepare an anti-NBVs (Nelson Bay orthoreoviruses) M3 polyclonal antibody according to the human pathogenic NBVs M3 gene. Methods NBVs M3 plasmid-Pris His MB-M3 was built and transformed into E. coli BL21 (DE3) competent cells. Isopropyl-β-D-thiogalactoside (IPTG) was used to induce protein expression, the NBVs M3 fusion protein was obtained by the method of Ni-NTA agarose; at the same time, the purified fusion protein was utilized as an antigen to immunize rabbits to obtain anti-NBVs M3 polyclonal antibody. The accuracy of the protein and polyclonal antibody resistance were detected by Western blot. Results SDS-PAGE electrophoresis and Coomassie blue staining showed that when the induction was carried out with 0.3 mmol/L IPTG at 25℃ for 12 h, the fusion protein expression was the highest. When the imidazole concentration was MCAC-20 and MCAC-40, the fusion protein could be obtained. Western blot showed anti-NBVS M3 polyclonal antibody was successfully prepared with strong specificity when the purified fusion protein was used to immune rabbits. Conclusions A highly sensitive and specific anti-NBVs M3 polyclonal antibody has been successfully obtained. It has a high application value for further study of the pathogenicity of the viruses.