表达荧光素酶报告基因稳定细胞系的建立及对CBLB502活性的检测  

Establishment of stable cell line expressing NF-κB luciferase reporter gene and activity detection of CBLB502 protein

作　　者：汤立宣[1] 王治东[2] 许望翔[2] 葛常辉[2] 杨晓明[2] 

机构地区：[1]安徽医科大学病理生理学教研室,合肥230032 [2]军事医学科学院放射与辐射研究所,北京100850

出　　处：《安徽医科大学学报》2011年第4期310-313,共4页Acta Universitatis Medicinalis Anhui

摘　　要：目的建立表达NF-κB-RE-luc2P荧光素酶报告基因的稳定细胞系以检测CBLB502的活性。方法利用脂质体转染试剂Lipofectin2000将含有NF-κB报告基因的质粒NF-κB-RE-luc2P转染入人胚肾细胞(HEK293细胞),并通过潮霉素B进行克隆筛选,最后挑取单克隆进行放大并应用荧光素酶检测方法进行鉴定。结果经200μg/ml的潮霉素B筛选,得到所需要的单克隆株,鉴定并保存。应用该潮霉素抗性单克隆细胞系对基于NF-κB信号通路的新型抗辐射蛋白药物CBLB502活性的检测显示出良好的量效关系。结论本实验通过Lipofectin2000转染法并在潮霉B筛选成功培育出稳定表达NF-κB报告基因活性的HEK293细胞系。Objective To establish a cell line which stably express NF-κB luciferase reporter and detect the activity of radiation protection protein CBLB502.Methods Lipofectin 2 000 transfection reagent was used to transfer plasmid carrying NF-κB receptor gene（NF-κB-RE-luc2P） into HEK293 cells.Hygromycin B resistant clones were selected and characterizated by luciferase assay.Results After selection with 200 μg/ml hygromycin B,the positive clones were obtained.The examination of CBLB502 activity used this cell line which showed a good dose-dependant relationship.Conclusion This experiment successfully obtained stable cell line that expressing NF-κB reporter gene by Lipofectin 2 000 transfection followed by hygromycin B selection.

关 键 词：荧光素酶类 NF-ΚB 潮霉素B 

分 类 号：R345[医药卫生—基础医学] R392.11