MOLECULAR-CLONING OF THE 3' HALF OF THE CLOSTRIDIUM-PERFRINGENS ENTERO-TOXIN GENE AND DEMONSTRATION THAT THIS REGION ENCODES RECEPTOR-BINDING ACTIVITY
MOLECULAR-CLONING OF THE 3' HALF OF THE CLOSTRIDIUM-PERFRINGENS ENTERO-TOXIN GENE AND DEMONSTRATION THAT THIS REGION ENCODES RECEPTOR-BINDING ACTIVITY
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DOI：
10.1128/jb.171.12.6815-6820.1989
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发表时间：
1989-12-01
影响因子：
3.2
通讯作者：
MCCLANE, BA
中科院分区：
文献类型：
--
作者：
HANNA, PC;WNEK, AP;MCCLANE, BA
产气荚膜梭菌A型肠毒素（CPE）引起与产气荚膜梭菌食物中毒相关的症状。为了确定CPE的c端半段是否具有受体结合活性，我们用大肠杆菌表达载体系统克隆了CPE结构基因的3”半段。然后检测含有表达的c端CPE片段（cperag）的大肠杆菌裂解物的CPE样血清学、受体结合和细胞毒性活性。CPEfrag被证明含有一个位于CPE分子的受体结合域或附近的表位。竞争结合研究表明，CPE和cpefragment裂解物对CPE受体具有特异性竞争。CPEfrag裂解物对Vero（非洲绿猴肾）细胞不产生细胞毒性。然而，用cpefragment裂解物预孵育可以特异性地保护Vero细胞免受随后的CPE攻击。这表明CPEfrag识别介导CPE细胞毒性的生理受体。总的来说，这些研究表明CPE的c端一半包含一个受体结合域，但CPE的细胞毒性似乎需要额外的氨基酸序列。
Clostridium perfringens type A enterotoxin (CPE) causes the symptoms associated with C. perfringens food poisioning. To determine whether the C-terminal half of CPE contains receptor-binding activity, the 3'' half of the cpe structural gene was cloned with an Escherichia coli expression vector system. E. coli lysates containing the expressed C-terminal CPE fragment (CPEfrag) were then assayed for CPE-like serologic, receptor-binding, and cytotoxic activities. CPEfrag was shown to contain an epitope located at or near the receptor-binding domain of the CPE molecule. Competitive-binding studies showed specific competition for CPE receptors between CPE and CPEfrag lysates. CPEfrag lysates did not cause cytotoxicity in Vero (African green monkey kidney) cells. However, preincubation with CPEfrag lysates specifically protected Vero cells from subsequent CPE challenge. This indicates that CPEfrag recognizes the physiologic receptor which mediates CPE cytotoxicity. Collectively, these studies indicate that the C-terminal half of CPE contains a receptor-binding domain but additional amino acid sequences appear to be required for CPE cytotoxicity.