Yutsudo T, Nakabayashi N, Hirayama T, Takeda Y
Department of Bacterial Infection, University of Tokyo, Japan.
Microb Pathog. 1987 Jul;3(1):21-30. doi: 10.1016/0882-4010(87)90034-9.
A cytotoxin to Vero cells (Vero toxin) was purified from Escherichia coli O157:H7 isolated from a patient with hemorrhagic colitis by ammonium sulfate fractionation, DEAE-cellulose column chromatography, repeated chromatofocusing column chromatography and repeated high performance liquid chromatography. About 440 micrograms of purified Vero toxin was obtained from 12 liters of culture with a yield of about 22%. The purified Vero toxin showed similar cytotoxic activity to that of Shiga toxin to Vero cells and killed about 50% of the Vero cells at 1 pg. The activity was lost on heating the toxin at 80 degrees C for 10 minutes, but not at 60 degrees C for 10 minutes. The toxin also showed lethal toxicity to mice when injected intraperitoneally, the LD50 being 1 ng per mouse. The purified Vero toxin consisted of A and B subunits with molecular weights of about 35,000 and 10,700, respectively, which were slightly larger than those of Shiga toxin. On polyacrylamide gel disc electrophoresis, the mobility of the purified Vero toxin differed from that of Shiga toxin. The isoelectric point of the toxin was 4.1, which was also different from that of Shiga toxin (pI = 7.0). Furthermore, Vero toxin and Shiga toxin were found to be immunologically unrelated; anti-Vero toxin did not react with Shiga toxin, and similarly anti-Shiga toxin did not react with the Vero toxin in either the Ouchterlony double gel diffusion test or enzyme-linked immunosorbent assay. The Vero toxin purified in this work was found to be immunologically identical to VT2 and Shiga-like toxin II reported previously.
通过硫酸铵分级沉淀、DEAE - 纤维素柱色谱、反复的色谱聚焦柱色谱和反复的高效液相色谱法，从一名出血性结肠炎患者分离出的大肠杆菌O157:H7中纯化出一种对Vero细胞的细胞毒素（Vero毒素）。从12升培养物中获得了约440微克纯化的Vero毒素，产率约为22%。纯化的Vero毒素对Vero细胞显示出与志贺毒素相似的细胞毒性活性，在1皮克时可杀死约50%的Vero细胞。在80℃加热毒素10分钟后活性丧失，但在60℃加热10分钟则不会。当腹腔注射时，该毒素对小鼠也显示出致死毒性，半数致死量为每只小鼠1纳克。纯化的Vero毒素由A和B亚基组成，分子量分别约为35000和10700，略大于志贺毒素的分子量。在聚丙烯酰胺凝胶圆盘电泳中，纯化的Vero毒素的迁移率与志贺毒素不同。该毒素的等电点为4.1，也与志贺毒素的等电点（pI = 7.0）不同。此外，发现Vero毒素和志贺毒素在免疫上不相关；在免疫双扩散试验或酶联免疫吸附试验中，抗Vero毒素与志贺毒素不发生反应，同样，抗志贺毒素与Vero毒素也不发生反应。在这项工作中纯化的Vero毒素在免疫上与先前报道的VT2和类志贺毒素II相同。
