Analysis of diphtheria toxin receptor by molecular cloning.

本项目旨在了解白喉毒素进入细胞质的机制。本课题的策略如下：(1)培养细胞膜组分白喉毒素受体的鉴定。(2)受体纯化。(3)白喉毒素受体单克隆抗体的分离。(4)利用单克隆抗体克隆受体cDNA。1987年，我们利用白喉毒素突变蛋白CRM197蛋白在Vero细胞膜裂解液中鉴定出白喉毒素受体为14.5Kd蛋白。然后用序层析法对受体进行10000倍纯化。今年，我们尝试分离出能产生针对白喉毒素受体抗体的杂交瘤。几个克隆被分离出来。其中一个克隆体产生的抗体抑制了白喉毒素与受体的结合。该抗体在部分纯化的受体片段中特异性沉淀14.5Kd蛋白。现在我们尝试用这些单克隆抗体分离受体的cDNA。

The aim of this project is to understand the entry mechanism of diphtheria toxin into cytosol. The strategy of this project is as follows: (1)Identification of diphtheria toxin receptor in membrane fractions of cultured cells. (2)Purification of the receptor. (3)Isolation of monoclonal antibodies against diphtheria toxin receptor. (4)cDNA cloning of the receptor using monoclonal antobodies.In 1987, we identified diphtheria toxin receptor as 14.5Kd protein in lysate of Vero cell membrane using CRM197 protein, a mutant protein of diphtheria toxin. Then the receptor was purified by a 10,000-folds by sequential chromatography. In this year, we tried to isolate hybridomas producing antibodies directed to diph-theria toxin receptor. Several clones were isolated. An antibody produced from one of these clones inhibited the binding of diphtheria toxin to the receptor. This antibody specefically precipitates the 14.5Kd protein in the fraction of partially purified receptor. Now we tried to isolate cDNA of the receptor using these monoclonal antibodies.