Analysis of diphtheria toxin receptor by molecular cloning.

通过分子克隆分析白喉毒素受体。

• 批准号: 62570189
• 负责人: MEKADA Eisuke
• 金额: $ 1.41万
• 依托单位: Kurume University (1988)Osaka University (1987)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570189/
• 关键词: Diphtheria toxin; Receptor; Monoclonal Antibody; エンドサイドーシス; エンドサイトーシス

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.

该项目的目的是了解白喉毒素进入细胞质的机制。本课题的研究策略如下：（1）培养细胞膜组分中白喉毒素受体的鉴定。（2）受体的纯化。（3）白喉毒素受体单克隆抗体的制备。（4）用单克隆抗体克隆白喉毒素受体的cDNA：1987年，我们用白喉毒素突变蛋白CRM 197在Vero细胞膜裂解液中鉴定出白喉毒素受体为14.5KD蛋白。然后通过连续层析将受体纯化10，000倍。在这一年中，我们试图分离产生针对白喉毒素受体的抗体的杂交瘤。分离了几个克隆。其中一个克隆产生的抗体抑制白喉毒素与受体的结合。该抗体特异性地沉淀部分纯化的受体部分中的14.5Kd蛋白。现在，我们尝试使用这些单克隆抗体分离受体的cDNA。

项目成果

Eisuke Mekada: "Hybrid Molecules in Handbook of Natural Toxin,vol.IV.," Mercel Dekker Inc., 36 (1988)

Eisuke Mekada：“天然毒素手册中的混合分子，第 IV 卷”，Mercel Dekker Inc.，36 (1988)

目加田英輔: 微生物. 3. 66-72 (1987)

Eisuke Mekada：微生物。3. 66-72 (1987)

Kenji Kohno: Exptl.Cell Res.172. 54-64 (1987)

Kenji Kohno：Exptl.Cell Res.172。

Kenji Kohno;H. Hayes;E. Mekada;T. Uchida: Exptl. Cell Res.172. 54-64 (1987)

小野健二；H.

Eisuke Mekada: "Identification of diphtheria toxin receptor and a nonproteinous diphtheria toxin-binding molecule in Vero cell membrane." J. Cell Biol.107. 511-519 (1988)

Eisuke Mekada：“鉴定 Vero 细胞膜中的白喉毒素受体和非蛋白白喉毒素结合分子。”