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

破伤风和白喉毒素通过包括HPLC在内的步骤得到高度纯化。使用FPLC系统中的Mono Q柱，从经二硫苏糖醇(DTT)还原和4M-2M尿素处理的解离的毒素中分离并高度纯化两个互补的多肽片段。用CDI[1-乙基-3-(3-L-二甲氨基丙基)碳二亚胺-盐酸盐]或SPDP[N-琥珀酰亚胺3-(2-吡啶二硫)丙酸酯]或通过氧化和透析法从解离的毒素混合物中去除尿素，制备了由不同毒素的N端酶活性片段和C端细胞结合片段组成的复合体。测定了它们对Vero和PC12细胞的细胞毒性、皮肤坏死活性和对豚鼠的致死性、神经性毒性。与原始毒素和合成毒素相比，这些由不同来源的酶活性片段和细胞结合片段组成的配合物只有轻微的毒性。而用CDI法制备的白喉毒素片段A或敌百虫毒素与神经生长因子形成的复合体具有胆碱能神经元特异性的细胞毒作用，表明CDI制备的复合体中的片段A仍具有酶活性。DTT还原的尿素解离毒素混合物的重组复合体对Vero细胞的细胞毒性似乎增加。因此，重组和重组材料的分离和纯化制剂的细胞毒性正在进行研究。正在研究纯化的重组毒素对神经分泌PC12细胞释放去甲肾上腺素的影响，以及这些材料对豚鼠和白喉毒素抗性大鼠和小鼠的致死和神经毒性作用，以及针对每个毒素片段的抗体的中和作用。

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.