抗体Fc片段的离子交换法制备和印渍法鉴定

PREPAPATION OF ANTIBODY Fc FRAGMENT BY ION EXCHANGE AND ITS BLOTTING

免疫球蛋白经羧甲基纤维素(CM22)柱交换,并用增加盐浓度分段洗脱,可分得4个蛋白峰。其中,用0.1M醋酸钠缓冲液洗脱出的3个蛋白峰具Fc性质,而用0.225M醋酸钠缓冲液洗脱的蛋白质不与葡萄球菌A蛋白(SPA)相结合。将具Fc性质的组分合并,由木瓜蛋白酶消化后,各蛋白质片段再经CM22柱分段洗脱,得到5个蛋白峰。分离出的蛋白质经聚丙烯酰胺凝胶电泳后转移至滤膜上。膜上各蛋白质用SPA-HRP探针作酶分析,从而鉴定IgG的Fc片段。实验提示,0.225M醋酸钠缓冲液洗脱出的蛋白蜂是抗体Fc片段。

Four pcaks of immunoglobulin were scparated through stepwise elution from carboxymethl ccllulose (CM22) colum and by increasing salt concentration. Among them, the three protcin pcaks which were elutcd with 0.1M sodium acetate buffer have the Fc characteristics and the fourth one which was clutcd with 0.225M sodium acetate buffer did not bind with Staphylococcal protein A(SPA). The fractions, which have Fc quality, were digested into fragmerits by papain and were then isolated by stcpwise elution from CM22 column. Five protein peaks can be obtained from mixture fragments. Subsequently the isolated proteins were fractionated by polyacrylamide gcl elcctrophoresis and blotted to the filter, and the Fc fragments of IgG on the shects wcre probed with SPA-HRP and identified by enzyme assay. The experiment suggested that the protein pcak cluted by 0.225 M sodium acetate buffer is antibody Fc fragment.