Lallier R, Lariviere S, St-Pierre S
Infect Immun. 1980 May;28(2):469-74. doi: 10.1128/iai.28.2.469-474.1980.
In this study we used Escherichia coli strain F11(P155) of porcine origin. The heat-stable enterotoxin (ST) was produced with a batch fermentor under agitation (500 rpm) and forced aeration (5 liters/min) in Casamino Acids yeast extract medium containing 0.2% glucose. The pH varied from 7.2 to 7.8. The maximum amount of ST was obtained after 7 h of growth. ST was purified by ammonium sulfate precipitation, ultrafiltration on Amicon membranes, and chromatography in Bio-Gel P-4. The enterotoxin, which was purified approximately 1,000 times, was active at nanogram levels. On 20% polyacrylamide gel electrophoresis, ST exhibited an Rf of 0.6 ST was recovered from the gel slices by eluting in buffer and testing the activity in suckling mice, since no band appeared on the gel after staining with Coomasie brilliant blue R, Schiff reagent, or red oil. ST was resistant at pH 2 to 10 and at 100 degrees C for 15 min, but it was inactivated at 121 degrees C; it did not lose biological activity after treatment with pronase, lipase, or amylase. In suckling mice antiserum obtained from rabbits or goats immunized with ST neutralized the enterotoxin activity of a cell-free supernatant of purified ST.
在本研究中，我们使用了源自猪的大肠杆菌F11(P155)菌株。在含有0.2%葡萄糖的酪蛋白氨基酸酵母提取物培养基中，使用分批发酵罐在搅拌（500转/分钟）和强制通气（5升/分钟）的条件下生产热稳定肠毒素（ST）。pH值在7.2至7.8之间变化。生长7小时后获得了最大量的ST。通过硫酸铵沉淀、在Amicon膜上进行超滤以及在Bio-Gel P-4上进行色谱法对ST进行纯化。纯化后的肠毒素活性在纳克水平，其纯化倍数约为1000倍。在20%聚丙烯酰胺凝胶电泳中，ST的Rf值为0.6。由于用考马斯亮蓝R、席夫试剂或红油染色后凝胶上未出现条带，因此通过在缓冲液中洗脱并在乳鼠中测试活性，从凝胶切片中回收了ST。ST在pH 2至10以及100℃下15分钟时具有抗性，但在121℃时失活；用链霉蛋白酶、脂肪酶或淀粉酶处理后它不会丧失生物活性。在用ST免疫的兔子或山羊获得的抗血清中，乳鼠抗血清可中和纯化ST的无细胞上清液的肠毒素活性。
