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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590410/
• 关键词: Clostridium perfringens; epsilon-toxin; monoclonal antibody; MDCK cell; MDCK細胞; ε毒素; モノクローナル抗体

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.准备乳梭状芽胞杆菌蛋白酶毒素的单克抗体准备好确定埃氏蛋白石毒素的毒性活性与其分子结构之间的关系。 Epsilon toxin非常有毒。因此，用Folmarin固定Epsilon原始毒素，将毒素用作抗原。杂交瘤细胞的十二个克隆，产生了与依克隆毒素的单克隆抗体。通过诱惑骨髓瘤细胞（SP2/0-AGL4）和免疫化BALB/C小鼠的脾细胞获得。七个单克隆抗体的土地中和伊氏抗毒素的毒性活性。但是，其表位的氨基酸序列未通过噬菌体肽显示克隆系统确定，因为单克隆抗体识别出依克隆 - 毒素的高阶结构，而不是主要结构。为了解决此问题的重组毒素片段，由LYS-14到Glys-139组成，或从Met 77到Lys 296用作抗原。产生单克隆抗体的杂交瘤细胞反应反应重组毒素片段，由从Ser-12到Met-47组成的重组毒素片段，从Met-47到77，从Met-47到77，从Gly-139到ASN 109，从Gly-139到ALE 173，或从IIE-206到ALE-206到Lys-296，均获得了Moncocies and Monocipies of Monocipies的纯度，并具有当前的purecipies and saterals puripie of Monocipies usoclipies usoclipies usoclipies peribipe of puripies usoclipies usoclipies usocl。 进步。 nitones中的11种确定。2。Epsilon毒素与MDCK细胞的膜微域结合并形成七聚体孔，称为MDCK细胞的膜（称为清洁剂耐热膜（DRMS））。用富莫尼菌素B1和PDMP，鞘脂和葡萄糖糖脂合成的抑制剂治疗MDCK细胞，增加了易感性，而D609（鞘氨质抑制剂抑制剂）具有相反的作用。神经节苷脂GM1的外源添加大大降低了乳蛋白毒素的结合，七聚体和细胞毒性，这些结果表明，DRMS中的糖磷脂脂会干扰依氏蛋白酶毒素的细胞毒性。

项目成果

Changes in ganglioside content affect the binding of clostridium perfringens epsilon-toxin to detergent-resistant membranes of Madin-Darby canine kidney cells

• DOI: 10.1111/j.1348-0421.2005.tb03726.x
• 发表时间: 2005-01-01
• 期刊: MICROBIOLOGY AND IMMUNOLOGY
• 影响因子: 2.6
• 作者: Shimamoto, S;Tamai, E;Miyata, S
• 通讯作者: Miyata, S