真核表达分泌型簇集素蛋白的纯化及其功能的初步研究  

Purification and characterization of secretory clusterin proteins from an eukaryotic expression system and the preliminary exploration of its functions

作　　者：马晓[1] 罗晓莹[2] 朱晓丽[3] 李宗海[2] 李鹤成[1] 

机构地区：[1]复旦大学附属肿瘤医院胸外科,复旦大学上海医学院肿瘤学系,上海200032 [2]上海交通大学医学院附属仁济医院上海市肿瘤研究所癌基因及相关基因重点实验室,上海200032 [3]复旦大学附属肿瘤医院病理科,复旦大学上海医学院肿瘤学系,上海200032

出　　处：《肿瘤》2013年第7期597-603,共7页Tumor

基　　金：上海市科技启明星跟踪计划资助项目(编号:11QH1400600);国家自然科学基金资助项目(编号:81272608);国家自然科学基金资助项目(编号:81102044)

摘　　要：目的:本研究旨在通过纯化分泌型簇集素(secretory clusterin,sCLU)蛋白,并对其生物学功能进行初步探索。方法:以人肺癌NCI-H460细胞cDNA为模板,构建真核表达质粒pRAG5-ag-sCLU,并转染HEK-293F细胞表达目的蛋白;采用亲和层析柱纯化目的蛋白后,分别采用Flag抗体及clusterin抗体行蛋白质印迹法检测鉴定纯化的sCLU蛋白。采用不同浓度纯化的sCLU蛋白处理NCI-H460细胞,观察其对细胞形态的影响。采用Boyden小室法鉴定sCLU蛋白对肺癌LETP-a-2细胞迁移能力的影响。结果:成功纯化获得具有二级结构的sCLU蛋白;sCLU蛋白浓度为5.4μmol/L时可使NCI-H460细胞的形态发生改变,并促进LETP-a-2细胞的迁移(P=0.0021)。结论:sCLU蛋白可能通过上皮间质转化促进肺癌细胞的迁移,sCLU蛋白的纯化将为其进一步应用于临床研究奠定了良好的实验基础。Objective： To purify sCLU （secretory clusterin） proteins from an eukaryotic expression system, and to explore their preliminary functions. Methods： Clusterin fragment was amplified from human lung cancer cDNA. The eukaryotic expression vector of pRAG5-flag-sCLU was constructed and then transfected into HEK-293F cells. The sCLU proteins were purified by affinity chromatography and then they were detected using Flag antibody and anti-clusterin antibody. Finally, the purified sCLU proteins were used to treat lung cancer cells and the biological functions of the sCLU proteins were observed. Results： The secondary structure of sCLU protein was successfully gained and the morphological changes of NCI-H460 cells were observed. After treatment with sCLU of 5.4 μmol/L, the morphology of NCI-H460 cells was changed, and the migration ability of LETP-a-2 cells was enhanced （P = 0.002 1）. Conclusion： The purified sCLU proteins may promote tumor metastasis through epithelial-mesenchymal transition, and these results will be the basis of further study.

关 键 词：分泌型簇集素 真核表达载体 纯化蛋白 上皮间质转化 

分 类 号：R734.2[医药卫生—肿瘤]