摘要
目的:构建丙型肝炎病毒(HCV)NS5A蛋白原核表达载体,获得高产量的纯化目的蛋白,并制备其多克隆抗体。方法:应用聚合酶链反应技术,PCR扩增获得NS5A基因,将NS5A基因插入至原核表达载体pET-32a(+)中,转化大肠埃希菌DH5α提取质粒,验证正确后,转化大肠埃希菌BL21中,以IPTG诱导,获得NS5A融合蛋白的可诱导性表达,通过SDS-PAGE电泳、Western blot免疫印迹分析证实融合蛋白表达的特异性。利用Ni+亲和柱对表达蛋白进行纯化及柱上复性。纯化蛋白免疫新西兰兔,利用Western blot和ELISA法对多克隆抗体进行特异性和效价检测。结果:NS5A融合蛋白表达成功,成功获得了融合蛋白及兔抗NS5A多克隆抗体。结论:成功表达NS5A基因融合蛋白,获得高特异性、兔抗NS5A多克隆抗体,为研究NS5A基因的生物学功能提供了重要制剂。
Objective： To construct proeukaryotic cell expressive vector of HCV-NS5A gene, abundantly express and pu- rify NS5A protein, and prepare its polyclonal antibody. Methods： The DNA fragment of NS5A was amplified by PCR. The correct DNA fragment was inserted into inducible proeukaryotic expressive vector pET-32a（＋） and transformed into the competent E.coli BL21. E.coli was induced with IPTG. Expressed bacteria were quassationed by supersound and analyzed with sodium dodecylsulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The expressed product was purified and renatured by Ni^＋ affinity column chromatography. Then the purified pET-32a（＋）-NS5A fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA. Results： The NS5A fusion protein was highly expressed. The purified protein and polyclonal antibody were obtained successfully. Conclusion： The successful expression and purification of NS5A fusion protein and the preparation of NS5A specific polyclonal antibody will be valuable for the study of the biological function of NS5A.
出处
《中国医药导报》
CAS
2010年第15期20-22,共3页
China Medical Herald
