人Notch受体胞内区基因N2ICD和N3ICD克隆及真核表达体系的构建  

Cloning and Construction of Human Notch Receptor Gene N2ICD and N3ICD Eukaryotic Expression System

作　　者：孙芳[1,2] 李玉霞[1] 毛艳[1,3] 凌焱[1] 张小男[2] 李伟东[1] 刘刚[1] 梁龙[1] 陈珊[2] 陈惠鹏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]东北师范大学,长春130024 [3]安徽中医学院,合肥230031

出　　处：《生物技术通报》2012年第7期108-113,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(81000763)

摘　　要：Notch信号通路与多种肿瘤的发生发展密切相关。对该通路中各受体功能的深入研究能够为揭示其致癌作用奠定基础。本研究采用RT-PCR的方法从人宫颈癌细胞系HeLa细胞的cDNA中克隆人Notch2和Notch3受体胞内区基因(N2ICD和N3ICD基因),并构建携带增强型绿色荧光蛋白(EGFP)的真核表达载体N2ICD/pEGFP和N3ICD/pEGFP,将序列正确的重组质粒转染HeLa细胞,显微镜下可见明显的绿色荧光,并定位在细胞核内。Western blotting检测目的蛋白成功融合表达,并能够提高其下游靶基因Hes1的转录活性。Notch signaling has been associated with a number of human cancers. The study on the receptor of Notch could provide a foundation for further investigation of Notch signaling. Total RNA was extracted from the HeLa cells and reverse transcription polymerase chain reaction （ RT-PCR ） was performed to obtain the cDNA fragment encoding N2ICD and N3ICD, which was inserted into the pEGFP-N3 vector in frame. The new constructs was confirmed by restriction enzyme digestion and DNA sequencing. HeLa cells were transfected with the N2ICD/ pEGFP and N3ICD/pEGFP vectors, respectively, and the expression of N2ICD and N3ICD were detected by Western blotting. The expression of recombinant proteins was located in the nucleus detected by fluorescent microscopy, and significant enhanced the transcriptional activity of the target gene Hesl

关 键 词：NOTCH信号通路 N2ICD/pEGFP N3ICD/pEGFP 真核表达 

分 类 号：Q78[生物学—分子生物学]