摘要
以Ｆ３ＧＡ（Ｃｉｂａｃｒｏｎ Ｂｌｕｅ Ｆ３ＧＡ）为配基建立了一种可用于免疫毒素（ＩＴ）分离纯化的亲和层析方法。实验中用三种不同来源的核糖体灭活蛋白（ＲＩＰ），即蓖麻毒素Ａ链（ＲＴＡ），苦瓜毒素（ｍｏｍｏｒｄｉｎ，ＭＴ）和Ｓａｐｏｒｉｎ，以探讨ＲＩＰ与Ｆ３ＧＡ的相互作用。分析显示三种ＲＩＰ均能引起Ｆ３ＧＡ吸收光诸明显红移，提示ＲＩＰ均可与Ｆ３ＧＡ发生特异结合。将Ｆ３ＧＡ与Ｓｅｐｈａｄｅｘ交联可获得Ｂｌｕｅｄｅｘ。Ｂｌｕｅｄｅｘ亲和层析是一种经济有效，简单易行，便于在各类实验室中使用的蛋白质亲和层析技术。结果表明：在低盐溶液中ＲＴＡ和ＭＴ均可迅速地与Ｂｌｕｅｄｅｘ结合，而在高盐溶液中（０．６５ｍｏｌ／ＬＮａＣｌ）又极易被洗脱回收。这一技术用于免疫毒素的研究可有效地去除游离抗体，而不影响其杀伤活性。
With Cibacron Blue F3GA as a ligand,a method is described for separating free antibodies from immunotoxins by affinity chromatography. Three kinds of ribosome inactivating protein (RIP),ricin A chains (RTA), momrdin (MT) and saporin from different plant seeds,can induce changes in the absorbance of F3GA,shifting the maximum absorption from about 620 nm to 685 nm. This effect, called'red shift' of F3GA, suggests that RIPs can bind to F3GA specifically. Bluedex, a gel for affinity chromotography was obtained by coupling F3GA to Sephadex. RTA and MT were bound to the column of Bluedex under the condition of low-concentrated salt buffer, and then eluted with buffer containing 0. 65mol/L NaCl. With this method, free antibodies were removed completely from immunotoxins without loss of cytotoxicity of immunotoxins.The results indicate that this method is simple, efficent and suitable for affinity chromatorgraphy of RIPs and their derivatives.
关键词
CibacronBlue
亲和层析
免疫毒素
制备
Cibacron Blue
Affinity chromoatography
Ribosome inactivating proteins
Immunotoxins
