人源性Notch 1胞内段真核表达载体的构建及鉴定  

Construction and identification of the eukaryotic expression vector of human Notch 1 intracellular domain

作　　者：李斌[1] 梁亮[2] 甄海宁[1] 林伟[1] 晁晓东[1] 董文鹏[1] 李娟[1] 章翔[1] 

机构地区：[1]第四军医大学西京脑科医院神经外科 [2]第四军医大学基础部医学遗传学与发育生物学教研室,陕西西安710032

出　　处：《中华神经外科疾病研究杂志》2008年第1期32-36,共5页Chinese Journal of Neurosurgical Disease Research

基　　金：国家自然科学基金资助项目(30672371)

摘　　要：目的构建人源性Notch 1胞内段(NICD)真核表达载体pIRES2-EGFP-NICD,并观察其在真核细胞中的表达。方法通过反转录-聚合酶链反应(RT-PCR)从人脑胶质母细胞瘤细胞系U251细胞中获得编码NICD的cDNA,定向克隆至真核表达载体pIRES2-EGFP;经酶切和测序鉴定正确后,应用脂质体法转染HeLa细胞,并通过蛋白免疫印迹法检测细胞内NICD的表达。结果构建了真核表达载体pIRES2-EGFP-NICD,将其转染HeLa细胞72h后,蛋白免疫印迹法检测到细胞内NICD的表达显著增高。结论成功构建了真核表达载体pIRES2-EGFP-NICD,为研究Notch信号通路在人脑胶质细胞瘤发生发展中的作用机制奠定了基础。Objective To construct the eukaryotic expression vector of human Notch 1 intracellular domain （ NICD）, pIRES2-EGFP-NICD, and to examine its expression in vitro. Methods Total RNA was extracted from the U251MG cells and reverse transcription-polymerase chain reaction （RT-PCR） was performed to obtain the cDNA fragment encoding NICD, which was inserted into the pIRES2-EGFP vector in-frame. And the new construct was confirmed by restriction enzyme digestion and DNA sequencing. HeLa cells were transfected with the pIRES2-EGFP-NICD vector and pIRES2-EGFP vector, respectively. The expression of NICD was detected by Western blotting. Results The eukaryotic expression vector pIRES2-EGFP-NICD was constructed, and significant increase of NICD expression was detected in the HeLa cells 72 hours after transfection. Conclusion The eukaryotic expression vector pIRES2-EGFP-NICD is constructed successfully. This study lay a foundation for the research on the mechanism of Notch signaling in tumorigenesis and development of glioma.

关 键 词：NOTCH信号 胶质细胞瘤 真核表达 

分 类 号：R739.4[医药卫生—肿瘤]