蛋白A的固定及在抗体亲和纯化中的应用

摘要

目的 以蛋白A作配基偶联活化的Sepharose 4FF后纯化兔免疫蛋白IgG.方法 以环氧氯丙烷(ECH)活化凝胶,正交试验确定最佳活化条件;以纯化数据确定抗体兔免疫蛋白IgG纯化最佳缓冲液盐浓度.结果 Sepharose 4FF最佳活化条件:ECH浓度20％(v/v),0.8 mol/L NaOH添加量为1 mL/1 g Sepharose 4FF湿胶,活化时间2.5 h,反应温度40℃.结论 建立将活化琼脂糖凝胶Sepharose 4FF与蛋白A偶联的条件,可应用于纯化兔免疫蛋白IgG.%Objective To isolate the IgG from rabbit using Sepharose 4FF coupled with protein A.Methods The optimum conditions of Sepharose 4FF activated by epichlorohydrin were determined by an orthogonal experiment with four factors and three levels,and the optimum salt concentration for IgG purification was determined by changing the buffer concentration.Results The optimum conditions of Sepharose 4FF activated by epichlorohydrin were as follows:20 ％ (v/v) epichlorohydrin,1 mL 0.8 mol/L sodium hydroxide for incubation of 1 g Sepharose 4FF at 40℃ for 2.5 h.Conclusion Proper condition for Sepharose 4FF coupled with protein A can be established for the purification of IgG from rabbit.