丙型肝炎NS5A基因多克隆抗体制备.docVIP

丙型肝炎NS5A基因多克隆抗体制备[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。 [关键词] HCV-NS5A基因;原核表达;蛋白纯化 [中图分类号] R37[文献标识码]A [文章编号]1673-7210(2010)05(c)-020-03 Preparation of polyclonal antibody of HCV-NS5A gene LIU Xiucai1, LI Genghui2, GUO Weihua3 (1.Department of Biochemistry, Qiqihar Medical College, Qiqihar161006, China; 2.Department of Veteran Ward, the First People’s Hospital of Qiqihar City, Qiqihar161006, China; 3.Department of Emergency, PLA 324 Hospital, Chongqing 400023, China) [Abstract] Objective: To construct proeukaryotic cell expressive vector of HCV-NS5A gene, abundantly express and purify 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). 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.