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Analysis of the structure and mode of action of Clostridium perfringens ε-toxin with monoclonal antibody

单克隆抗体分析产气荚膜梭菌ε-毒素的结构和作用方式

基本信息

批准号：
15590410
负责人：
MINAMI Junzaburo
金额：
$ 1.98万
依托单位：
Kagawa Prefctural College of Health Sciences (2004)Kagawa Prefectural College of Health science (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

1.Monoclonal antibodies to epsilon toxin of Clostridum perfringens were prepared to determine the relation between the toxic activity of epsilon-toxin and its molecular structure. Epsilon-toxin was very toxic. Therefore, epsilon prototoxin was fixed with folmarin and the toxoid was used as antigen. Twelve clones of hybridoma cells producing monoclonal antibodies to epsilon toxin. were obtained by fuzing myeloma cells (SP2/0-Agl4) and spleen cells of the immunized BALB/c mouse. Seven lands of monoclonal antibodies neutralized the toxic activity of epsilon toxin. However, the amino acids sequences of their epitopes were not determined by phage peptide display cloning system because the higher-order structure of epsilon-toxin, but not the primary structure, was recognized by the monoclonal antibodies. In order to resolve this problem recombinant toxin fragments composedd of amino acids from Lys-14 to Glys-139, or from Met 77 to Lys 296 were used as antigen. Hybridoma cells producing monoclonal antibodies which react recombinant toxin fragments composed of amino acids from Ser-12 to Met-47, from Met-47 to Met 77, from Met 77 to Asn 109, from Gly-139 to Ale 173, or from IIe-206 to Lys-296 were obtained The purification of the monoclonal antibodies and analysis of the epitopes are presently in progress. Eleven of the nitones havebeen determined.2.Epsilon toxin binds to and forms a heptameric pore within the microdomain of membranes, known as detergent resistant membranes (DRMs), of MDCK cells. Treatment of MDCK cells with fumonisin B1 and PDMP, inhibitors of sphingolipid and glucosphingolipid synthesis, respectively, increased the susceptibility, while D609, a sphingomyelin synthesis inhibitor, had the opposite effect. The exogenous addition of ganglioside GM1 dramatically decreased the epsilon toxin binding, heptamerization and cytotoxicity These results suggest that glycosphingolipid in DRMs interferes the cytotoxicity of epsilon-toxin.

1.制备了抗产气荚膜梭菌ε毒素的单克隆抗体，以研究ε毒素的毒性与其分子结构的关系。ε毒素毒性很强。因此，用福尔马林固定化黄曲霉原毒素，并将该类毒素用作抗原。12株产生抗肉毒毒素单克隆抗体的杂交瘤细胞克隆。免疫BALB/c小鼠，取骨髓瘤细胞（SP 2/0-Agl 4）与脾细胞融合，获得重组质粒。七种单克隆抗体中和了该毒素的毒性。然而，它们的表位的氨基酸序列不能通过噬菌体肽展示克隆系统确定，因为ε毒素的高级结构，而不是一级结构，被单克隆抗体识别。为了解决这一问题，用由Lys-14至Glys-139或Met-77至Lys-296的氨基酸组成的重组毒素片段作为抗原。获得了产生单克隆抗体的杂交细胞，所述单克隆抗体与由Ser-12至Met-47、Met-47至Met-77、Met-77至Asn-109、Gly-139至Ale-173或IIe-206至Lys-296的氨基酸组成的重组毒素片段反应。2.Epileptic毒素与MDCK细胞膜的微区结合并形成一个七聚体孔，称为抗洗涤剂膜（DRMs）。MDCK细胞与伏马菌素B1和P450，鞘脂和葡萄糖鞘脂合成的抑制剂，分别治疗，增加了易感性，而D 609，鞘磷脂合成抑制剂，有相反的效果。外源性神经节苷脂GM 1的加入显著降低了ε毒素的结合、七聚化和细胞毒性。这些结果表明，DRM中的鞘糖脂干扰了ε毒素的细胞毒性。