抗重组蛋白rVVC多克隆抗体制备及其保护作用鉴定.doc

抗重组蛋白rVVC多克隆抗体制备及其保护作用鉴定 　 作者：姚蔚，周建娟，李桂军，谢旦立，楼永良堃 【摘要】 目的:纯化rVVC免疫家兔，制备并纯化多克隆抗体。观察复性rVVC、纯化多克隆抗体、rVVC和纯化多克隆抗体混合作用肝癌细胞SMMC7721后，肝癌细胞SMMC7721形态学和活性变化，评估抗重组rVVC蛋白多克隆抗体对肝癌细胞SMMC772的保护作用。方法:纯化重组蛋白rVVC免疫日本大耳兔获得多克隆抗体，多克隆抗体经过硫酸铵盐析、Sephadex G25除盐和DEAE-Sephadex G100层析纯化并进行 Westerm Blot鉴定和抗体效价分析。复性rVVC、纯化多克隆抗体、rVVC和纯化多克隆抗体混合物作用肝癌细胞SMMC7721后，显微镜观察并以MTT法确定抗重组rVVC蛋白多克隆抗体对肝癌细胞SMMC7721的保护作用。结果:兔抗重组rVVC多克隆抗体经盐析法、分子筛和阴离子交换层析法纯化后，得到较纯的IgG分子。免疫印迹结果显示，抗重组rVVC蛋白多克隆抗体与纯化抗原呈现特异性反应条带。显微镜观察和MTT结果表明，纯化多克隆抗体能够阻断重组rVVC活性蛋白对肝癌细胞SMMC772的损伤。结论:成功制备了具有保护和阻断作用的兔抗重组rVVC多克隆抗体，为以后rVVC促炎症、促应激作用机制和信号传导机制研究提供一个重要的阻断剂。 【关键词】 创伤弧菌溶细胞素；多克隆抗体；保护作用；阻断作用 Abstract: Objective: To prepare and purify rabbit polyclonal antibody against rVVC，to evaluate the protection of polyclonal antibody by observing the morphology and activity changes of SMMC7721 cells acted by purified rVVC，purified polyclonal antibody，the mixture of rVVC and polyclonal antibody. Method: Rabbit polyclonal antibody against rVVC prepared by immunning rabbit was purified by persulfate，Sephadex G25 and DEAE-Sephadex G100 followed by analysis of Western Blot hybridization and antibody titer. After SMMC7721 cells were acted by purified rVVC， purified polyclonal antibody and the mixture of VVC and polyclonal antibody， rabbit polyclonal antibody protection were observed under microscope and with MTT method. Result: Rabbit polyclonal antibody against rVVC was purified successfully by methods of Persulfate， Sephadex G25 and DEAE-Sephadex G100. Polyclonal antibody showed high immunoreactivity. The results of MTT and microscope observatien indicated that polyclonal antibody could block up the damage of SMMC7721 cells acted by rVVC. Conclusion: Rabbit polyclonal antibody against rVVC with functions of protection and blockade is prepared successfully， thus to provide a significant blocking agent for further study on mechanism of inflammation，stress and signal transduction of rVVC. Key words: vibrio vulnificus cytolysin；polyclonal antibody；protect