重组人蛋白C在HEK293细胞中的表达与鉴定  

Expression of Recombinant Human Protein C in HEK293 Cells and Identification of Expressed Product

作　　者：贾莉玮[1] 吕茂民[1] 徐斯日古楞 沈永才[1] 于群[1] 张亚东[1] 章金刚[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《中国生物制品学杂志》2005年第4期294-296,共3页Chinese Journal of Biologicals

基　　金：军事医学科学院创新基金资助项目(020901)

摘　　要：目的获得转染人蛋白CcDNA的工程细胞株,实现重组蛋白C的表达。方法将构建好的重组表达载体pIRES-hPC用脂质体介导的方法转染HEK293细胞,经G418加压筛选、细胞有限稀释法等获得克隆细胞株,收集无血清培养上清,浓缩后进行鉴定。结果经G418筛选得到了21株克隆化细胞,SDS-PAGE和Westernblot鉴定表明,表达产物含有人蛋白C,且具有抗凝活性。结论在哺乳动物细胞中已成功表达重组人蛋白C,为进一步深入研究及产业化奠定了基础。Objective To obtain a recombinant cell strain transfected with human protein C cDNA and e xpress the protein. Methods Transfect HEK293 cells with a constructed recombinant expression vector pIRES-h PC in the mediation of liposome.Select positive transfectants by G418 pressure s creening and clone the single cell colony by limiting dilution.Culture the clone d cells in a serum-free medium for 3 days,collect the culture supernatant and c oncentrate with 70% PEG8000.Identify the expressed product by SDS-PAGE and West ern blot. Results A total of 21 cloned cell strains were obtained by G418 pressure screening.S DS- PAGE and Western blot proved that the human protein C with anticoagulation activ ity was expressed. Conclusion Recombinant human protein C was successfully expressed in mammal animal cells.It laid a foundation of further study and industrialization of human protein C.

关 键 词：重组人蛋白C HEIQ93细胞中 表达 鉴定 抗凝活性 

分 类 号：TQ464.7[化学工程—制药化工] Q786[生物学—分子生物学]