枯草杆菌重组水蛭素的分离纯化工艺研究  

Studies on the Separation and Purification of Recombinant Hirudin Variant 3 from the Bacillus Subtilis

作　　者：陈华友[1,2] 齐向辉[1,2] 耿旭[3] 徐庆刚[1] 

机构地区：[1]江苏大学,江苏镇江212013 [2]南京工业大学,江苏南京210009 [3]河南大学医学院,河南开封475004

出　　处：《安徽农业科学》2009年第34期16757-16759,16768,共4页Journal of Anhui Agricultural Sciences

基　　金：国家"863"项目(2006AA03Z0453);江苏省高校自然科学研究项目(09KJB230001);国家"973"项目(2009CB724700);江苏-大学校基金项目(08JDG009)

摘　　要：[目的]优化枯草杆菌重组水蛭素的分离纯化工艺。[方法]采用系列预处理、初步层析和精细纯化试验。[结果]优化的分离纯化工艺为:发酵液经离心、三氯乙酸处理、超滤浓缩脱盐,再上阴离子交换柱,或用乙醇处理后贮存,为离子交换备用。初步层析介质用阴离子交换QSepharoseF.F.,缓冲体系为Tris-HCl(pH值8.0),体系电导率为6.0mS/cm,上样量为240.0ATU/ml介质,产品纯度为70.2%,回收率达90.0%。用SephacrylSi-100凝胶过滤柱精细纯化水蛭素,在一定流速范围内,流速对纯化效果影响不大,上样量可为10.0ml,得到的纯品纯度可达95.1%,得率为93.0%,SDS-PAGE电泳为单一条带。[结论]可为进一步工业化分离纯化水蛭素研究提供借鉴。[ Objective] The research aimed to get the optimized separation and purification conditions of the hirudin produced from Bacillus subtilis DB403 （pUBH5）. [ Method] Through the systemic pretreatment ,preliminary chromatography and fine chromatography. [ Result] The op- timized separation and purification conditions were that： Supernatant was treated by trichloroacetic acid,then by ultrafihration desalt and anion exchange chromatography. Strong anion Q F. F. was better than weak anion DEAE F.F. The proper balanced solution was Tris-HCl（ pH 8.0）. The proper conductivity was 6.0 mS/cm. The maximum applied sample was 240.0 ATU/ml to matrix of strong anion Q F. F. This opti- mized procedure was magnified in strong anion exchange HiPrep 16/IOQ with the 90.0% recovery and 70.2% purity. The purification of gel filtration of Sephacryl S-IO0 to hirudin was not relative to flow rate within certain scope. The application size of sample was lOml. The purity checked by HPLC was 95.1% , and the recovery was 93.0% , and the band of SDS-PAGE was single. [ Conclusion ] The research provided the reference of the further industrialization separation and purification of hiruin.

关 键 词：水蛭素 枯草杆菌 离子交换 分离纯化 

分 类 号：Q813.1[生物学—生物工程]