人Notch受体胞内区基因N2ICD克隆及真核表达  

Cloning and eukaryotic expression of human Notch2 intracellular domain

作　　者：陈中标[1] 叶健斌 梁来妹 杨宜[1] 宁立军[1] 宁云山[1,2] 李妍[1] 

机构地区：[1]南方医科大学生物技术学院生物治疗研究所,广东广州510515 [2]珠海南医大生物医药公共服务平台有限公司

出　　处：《中国公共卫生》2016年第3期310-314,共5页Chinese Journal of Public Health

基　　金：国家高技术研究发展技术(863计划)(2014AA020909);国家自然科学基金-广东省联合基金(U1401223);国家自然科学基金面上项目(81470831);广东省创新载体建设项目(2013B090800036)

摘　　要：目的构建真核重组质粒N2ICD/p CMV-Tag4并转染HEK 293T细胞进行表达。方法根据GenBank上Notch2的NICD序列设计引物,采用逆转录聚合酶链反应(RT-PCR)的方法从人宫颈癌细胞Hela细胞扩增人Notch2受体胞内区基因(N2ICD),酶切和测序鉴定后克隆至携带FLAG标签的真核表达载体pCMV-Tag4并进行瞬时和稳定转染HEK 293T细胞,荧光定量PCR(Q-PCR)和蛋白印迹(WB)检测目的蛋白的表达。结果通过RT-PCR克隆获得人N2ICD基因并成功构建重组质粒N2ICD/pCMV-Tag4,目的蛋白N2ICD在HEK 293T细胞中获得表达。结论成功构建稳定表达N2ICD的HEK 293T细胞株,为进一步探讨Notch2受体的功能奠定基础。Objective To construct a recombinant eukaryotic expression plasmid N2ICD / pCMV-Tag4 and transfect it into HEK 293 T cells. Methods Human Notch2 intracellular domain gene（ N2ICD） from Hela cells was amplified by reverse transcription-PCR（ RT-PCR） and identified by enzyme restriction,sequencing and blasting and then cloned into eukaryotic expression vector pCMV-Tag4 to construct N2ICD / pCMV-Tag4,which was then transiently and stably transfected into HEK 293 T cells. Quantitative-PCR（ Q-PCR） and Western blot was used to detect target protein expression.Results N2ICD was obtained by RT-PCR. The recombinant eukaryotic expression vector Notch2 intracellular domain（ N2ICD） /p CMV-Tag4 was successfully constructed and N2ICD was expressed in HEK 293 T cells. Conclusion N2ICD was expressed in HEK 293 T cells,which provides a foundation for further investigating the function of Notch2 receptor.

关 键 词：Notch2信号通路 N2ICD 真核表达 pCMV-Tag4 

分 类 号：R512.62[医药卫生—内科学]