麦胚凝集素的分离纯化及其在亲和层析中的应用

Isolation and purification of WGA and its application to affinity chromatography

目的 :将分离纯化麦胚凝集素 (WGA)的方法和固定化WGA亲和层析柱的制备方法进行改良 ,从而得到能够有效结合细胞膜受体的亲和层析柱。方法 :依次用去脂抽提、分级盐析、离子交换层析、甲壳素亲和层析的方法分离纯化WGA ;将制备的WGA与用溴化氰活化的琼脂糖 4B(Sepharose 4B)耦联固定。结果 :得到的纯化倍数为 15 8 5倍的WGA单体相对分子质量为 160 0 0 ,表观相对分子质量为 3 2 0 0 0 ,血凝活力为 8 3mg·L-1。WGA Sepharose4B可以与红细胞表面糖基特异性结合。结论 :改良的WGA分离纯化方法和固定化WGA亲和层析柱的制备方法简便、有效 。

Objective To innovate the methods of purification of wheat germ agglutinin(WGA)and preparation of WGA?Sepharose 4B chromatographic column,and to obtain affinity chromatographic column which can be bound efficiently with cell membrane receptor.Methods Purified WGA is attained in turn of acetone extraction,salt precipitation with ammonium sulfate,ion exchange chromatography and chinin affinity chromatography,then the purified WGA is coupled by the BrCN activated Sepharose 4B.Results The purity of WGA,whose molecular weight is 16?000,increased by 158.5 times.Its relative apparent moleculuar weight is 32?000.The hemoagglutination activity is 8.3?mg·L -1 .WGA?Sepharose 4B bound with the specific sugar group on erythrocytes surface.Conclusion The improved methods of purification of WGA and preparation of WGA?Sepharose 4B column is convenient and available for laboratory research of membrane receptor.