Takeda Y, Takeda T, Yano T, Yamamoto K, Miwatani T
Infect Immun. 1979 Sep;25(3):978-85. doi: 10.1128/iai.25.3.978-985.1979.
Heat-stable enterotoxin was purified from a strain of enterotoxigenic Escherichia coli 53402 A-1 from human intestine. The cells were cultured in Casamino Acids-yeast extract-salts medium, and the purification procedure consisted of protamine sulfate treatment of the culture supernatant, ultrafiltration with an Amicon PM-10 membrane, diethylaminoethyl-cellulose column chromatography, hydroxyapatite column chromatography, Bio-Gel P-10 gel filtration, 90% ethanol extraction, and preparative polyacrylamide gel disc electrophoresis. About 300-fold purification was achieved, with a yield of about 12%. However, the homogeneity of the purified heat-stable enterotoxin was not rigorously demonstrated. The purified heat-stable enterotoxin had an absorption maximum at about 275 nm, and its isoelectric point was about 3.90. The molecular weight of the purified heat-stable enterotoxin was ca. 4,000 by Sephadex G-100 gel filtration. The minimum effective dose of purified heat-stable enterotoxin was about 2.5 ng in the suckling mouse assay. The purified heat-stable enterotoxin gave a positive reaction in not only the suckling mouse assay but also the mouse intestinal loop test and the guinea pig skin permeability test.
热稳定肠毒素是从一株源自人类肠道的产肠毒素大肠杆菌53402 A-1中纯化得到的。细胞在酪蛋白氨基酸-酵母提取物-盐培养基中培养，纯化步骤包括用硫酸鱼精蛋白处理培养上清液、用Amicon PM-10膜进行超滤、二乙氨基乙基纤维素柱色谱、羟基磷灰石柱色谱、Bio-Gel P-10凝胶过滤、90%乙醇提取以及制备性聚丙烯酰胺凝胶圆盘电泳。实现了约300倍的纯化，产率约为12%。然而，纯化后的热稳定肠毒素的均一性并未得到严格证明。纯化后的热稳定肠毒素在约275 nm处有最大吸收峰，其等电点约为3.90。通过Sephadex G-100凝胶过滤法测得纯化后的热稳定肠毒素的分子量约为4000。在乳鼠试验中，纯化后的热稳定肠毒素的最小有效剂量约为2.5 ng。纯化后的热稳定肠毒素不仅在乳鼠试验中呈阳性反应，在小鼠肠襻试验和豚鼠皮肤通透性试验中也呈阳性反应。
