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Analysis of physiological functions of target proteins of tetanus and diphtheria toxins using complexes of homologous fragments of the toxins

使用毒素同源片段复合物分析破伤风和白喉毒素靶蛋白的生理功能

基本信息

批准号：
08457087
负责人：
MATSUDA Morihiro
金额：
$ 4.22万
依托单位：
Koshien University (1997)Osaka University (1996)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Tetanus and diphtheria toxins were highly purified by procedures includind HPLC.Two complementary polypeptide fragments from each toxin were separated and highly purified, using a Mono Q column in an FPLC system, from toxins dissociated by reduction with dithiothreitol (DTT) and treatment with 4M-2M urea. Complexes composed of an N-terminal enzymatic, active frament and a C-terminal cell-binding fragment from the different toxins were prepared by using CDI [1-ethyl-3- (3-L-dimethyl aminopropyl) carbodiimide-HCl] or SPDP [N-succinimidyl 3- (2-pyridyldithio) propionate] or by oxidation and removing urea by dialysis from a mixture of the dissociated toxins. Cytotoxicity on Vero and PC12 cells, dermonecrotizing activity and lethal, neurotoxic activities on guinea pigs of the complexes were examined, In contrast to the oridinal toxins and the respectivereconstituted toxins, these complexes composed of an enzymatic active fragment and a cell-binding fragment from different origins elicited only slight toxicity. But the complexes composed of the diphtheria toxin fragment A or diphrheria toxin and a nerve growth factor prepared by using CDI elicited cholinergic neuron-specfic cytotoxicity, indicating that the fragment A in the complex pepeared using CDI retained its enzymatic activity. The mixture of reconstituted complexes from the mixture of DTT-reduced, urea-dissociated toxins appeared to show an increase in cytotoxicity on Vero cells. So the cytotoxicity of the isolated and purified preparations of reconstituted and recombined materials are now being investigaed. The effects of the purified recombined toxins on noradrenaline release from neurosecretoy PC12 cell and lethal, neurotoxic activity of these materials on guinea pigs and diphtheria toxin-resistant rats and mice are being investigated together with the neutralizing effects of antibodies against each toxin-fragment.

采用高效液相色谱等方法对破伤风和白喉毒素进行了高度纯化。通过双硫苏糖醇（DTT）还原和4M-2M尿素处理，从每种毒素中分离出两个互补的多肽片段，并使用FPLC系统中的Mono Q柱进行高度纯化。用CDI[1-乙基-3- （3- l-二甲基氨基丙基）碳二酰亚胺- hcl]或SPDP [n -琥珀酰亚基3-（2-吡啶二硫）丙酸盐]或从解离毒素混合物中氧化和透析去除尿素，制备了由不同毒素组成的n端酶促、活性片段和c端细胞结合片段组成的配合物。研究了复合物对Vero和PC12细胞的细胞毒性、皮肤坏死活性以及对豚鼠的致死性神经毒性。与原始毒素及其各自构成的毒素相比，这些复合物由不同来源的酶活性片段和细胞结合片段组成，仅引起轻微的毒性。但用CDI制备的白喉毒素片段A或白喉毒素与神经生长因子组成的复合物可引起胆碱能神经元特异性细胞毒性，说明用CDI制备的复合物中的片段A保留了其酶活性。从dtt还原的尿素解离毒素混合物中重组复合物的混合物似乎显示出对Vero细胞的细胞毒性增加。因此，目前正在研究重组和重组材料的分离和纯化制剂的细胞毒性。纯化的重组毒素对神经分泌腺PC12细胞释放去甲肾上腺素的影响，以及这些物质对豚鼠和白喉毒素抗性大鼠和小鼠的致死性神经毒性活性，以及针对每种毒素片段的抗体的中和作用，正在进行研究。