摘要
产肠毒素性大肠杆菌工程菌株pSLM004在限定性培养基上培养后,上清液经硫酸铵沉淀、丙酮抽提、Sephadex G-25凝胶过滤、DEAE-Sephadex A-25离子交换层析、Bio-gel P-4凝胶及反相高压液相层析,获得的STa纯化倍数为302.7倍,有效剂量达10.72ng/鼠单位,回收率为9.9%。纯化的STa在Sephadex G-25凝胶过滤时出现1个蛋白峰,聚丙烯酰胺凝胶电泳(PAGE)呈现1条蛋白带;在100℃30min不失活,121℃15min则失活;在pH 1～10范围内保持活性;胰蛋白酶、链霉蛋白酶对其活性无影响;2-巯基乙醇、二硫苏糖醇(DTT)可使其迅速失活,表明在STa分子中有生物活性所必需的二硫键存在。
A E. coli heat-stable enterotoxin (STa) was produced by cultivating a gene engineering strain pLSM 004 in a defined medium and purified by ammonium sulfate precipi-tation, acetone fractionation, Sephadex G-25 gel filtration and a series of chromatography. The STa was purified 302.7 fold and had a minimal effective dose of 10.27ng, its recovery rate was 9.9%. The purified STa showed only a single peak on SephadexG-25 gel filtration, and only a single band on polyacrylamide gel electrophoresis. After heating by 100℃ in 30 min the STa was still biologically active but inactivated at 121℃ for 15 min. It was stable at pH 1-10 and could not be hydrolyzed by trypsase and pronase. Its biological activity was promptly lost on treatment with 2-mercaptoethanol or dithiothreitol(TDD).
关键词
大肠杆菌
肠毒素
纯化
耐热性
性质
enterotoxigenic E. coli
STa
purification
property
