von Eichel-Streiber C, Harperath U, Bosse D, Hadding U
Institut für Medizinische Mikrobiologie, Johannes-Gutenberg-Universität Mainz, F.R.G.
Microb Pathog. 1987 May;2(5):307-18. doi: 10.1016/0882-4010(87)90073-8.
Two Cl. difficile toxins were isolated from cultures of Cl. difficile strain VPI 10463. A purification procedure to prepare homogenous Cl. difficile toxins is given. This procedure allows purification of high molecular weight toxins A and B without using immunaffinity chromatography. The main step of the purification is the separation of a partially purified toxin preparation over a FPLC-Mono Q column by anion exchange chromatography. The experimental conditions for a rechromatography were determined to prepare the two major toxic activities as homogenous high molecular weight proteins. Our toxin A has a molecular weight (Mr) of ca. 300 kDa and an IP of 4.7. The Mr of our toxin B is ca. 250 kDa, the isoelectric focusing gives rise to two bands one at 4.7 and the other at 4.8. The two bands represent charge isomers as have been described for other bacterial toxins. Both toxins differ in cytotoxicity testing by a factor of 1000 but have the same activity when tested in vivo. Toxin specific monoclonal antibodies (mabs) were elicited by separate immunization of mice either with toxin A or toxin B, respectively. All of our mabs cross react with pure toxin A and toxin B when tested by ELISA or Western Blotting. Some mabs strongly cross react indicating that both toxins have major epitopes in common. A hypothesis for the structural and possible functional relatedness between the two toxins is discussed.
从艰难梭菌菌株VPI 10463的培养物中分离出两种艰难梭菌毒素。给出了一种制备纯艰难梭菌毒素的纯化程序。该程序无需使用免疫亲和色谱法即可纯化高分子量毒素A和B。纯化的主要步骤是通过阴离子交换色谱法在FPLC-Mono Q柱上分离部分纯化的毒素制剂。确定了再色谱法的实验条件，以制备两种主要的具有毒性活性的高分子量纯蛋白。我们的毒素A分子量（Mr）约为300 kDa，等电点为4.7。我们的毒素B的Mr约为250 kDa，等电聚焦产生两条带，一条在4.7，另一条在4.8。这两条带代表电荷异构体，正如其他细菌毒素所描述的那样。两种毒素在细胞毒性测试中的差异为1000倍，但在体内测试时具有相同的活性。分别用毒素A或毒素B单独免疫小鼠，诱导产生毒素特异性单克隆抗体（mab）。当通过ELISA或Western印迹法检测时，我们所有的mab都与纯毒素A和毒素B发生交叉反应。一些mab强烈交叉反应，表明两种毒素具有共同的主要表位。讨论了两种毒素之间结构和可能的功能相关性的假设。
