重组毒素DT389-IL13的复性与纯化工艺  

Refolding and Purification of Recombinant Toxin DT389-IL13

作　　者：代新宪[1] 郑妍鹏[1] 郭韶霞[1] 

机构地区：[1]北京交通大学理学院,北京100044

出　　处：《北京交通大学学报》2010年第6期118-121,127,共5页JOURNAL OF BEIJING JIAOTONG UNIVERSITY

基　　金：国家自然科学基金资助项目(30700696);北京交通大学"十五"重大科技基金项目资助(2004SZ010)

摘　　要：为了获得有较高生物活性的靶向重组毒素DT389-IL13,在大肠杆菌中高效表达DT389-IL13,并采用凝胶过滤的方法对DT389-IL13进行柱上复性及初步纯化,然后使用离子交换色谱方法进一步纯化,最后应用CCK-8细胞活力检测试剂盒检测DT389-IL13对肿瘤细胞的抑制作用.实验结果显示,经柱上复性和纯化后,DT389-IL13的纯度可达90%以上,且对脑胶质瘤细胞U251有明显的抑制作用,存在明确的剂量依赖关系,48h的半数抑制浓度为8.87×10-10mol/L.表明联合应用凝胶过滤和离子交换两种色谱方法可获得高纯度的复性蛋白,是可行的复性及纯化方案.In order to obtain the targeted recombinant toxin DT389-IL13 with a high biological activity,the DT389-IL13 gene was overexpressed in E.coli.The recombinant protein was refolded and initially purified using a size exclusive chromatograph column,then the resulting protein was further purified by ion exchange chromatography.In the end,we performed cytotoxicity assay to verify the inhibitive effective of DT389-IL13 on tumor cells.The results showed that the purity of the refolded and purified DT389-IL13 was higher than 90%.DT389-IL13 could significantly suppress the U251 cells in a dose dependent manner and the IC50 at 48 hours was 8.87×10-10 mol/L.Therefore,the combination of size exclusive chromatography and ion exchange chromatography were the feasible program to refold and purify recombinat toxin DT389-IL13.

关 键 词：生物化学 重组蛋白 蛋白纯化 柱上复性 

分 类 号：Q78[生物学—分子生物学]