一种在哺乳动物细胞中研究蛋白分子转录激活活性系统的建立  

A Novel System for Characterization of the Transcription Activating Proteins in Mammalian Cells

作　　者：张浩[1] 周建光[1] 李杰之[1] 王健[1] 孙玉龙[1] 陈珊[1] 黄翠芬[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《Acta Genetica Sinica》2002年第11期983-989,共7页

基　　金：国家自然科学基金资助项目 (编号 3 0 0 70 2 96)~~

摘　　要：为了在哺乳动物细胞中建立一套用于研究蛋白分子转录激活活性的系统 ,首先以质粒pTet Off和真核表达载体pCDNA3.1B( ) /myc his为基础 ,分别构建重组质粒pZHO1(用于插入待测基因并作为该系统的阴性对照 )、pZHO2(用于作阳性对照 ) ,此外 ,该系统还包括质粒pTRE luc(编码Firefly荧光素酶报道基因 )和质粒pRL TK(编码Renilla荧光素酶基因 ,用作内参对照 )。为验证该系统的可行性 ,分别将质粒pZHO1、pZHO2、pZHO3(编码p5 3分子N端转录激活区 73个氨基酸片段 ,作为实验组 )与质粒pTRE luc和pRL TK共转染至C4 2、MCF 7、COS73种不同的细胞株中 ,通过检测各转染组细胞中Firefly荧光素酶相对活性的大小来判断该系统的可行性。结果表明 ,所构建的系统可以在哺乳动物细胞中检测目的分子的转录激活活性。A system used for detecting the transcriptional activating activity of the function unknown gene products in mammalian cells was developed. Based on the plasmid pTet Off and the eukaryotic expressing vector pCDNA3.1B( )/myc his, firstly, we constructed a set of recombinant plasmids namely pZHO1 (for cloning into the foreign gene fragment and as a negtive control)?pZHO2 (as a positive control). The system also includes the plasmids pTRE luc(encoding the Firefly luciferase reporter gene) and pRL TK(encoding Renilla luciferase gene as blackground control). To confirm the feasibility of the system, the plasmids pZHO1,pZHO2 and pZHO3(encoding p53 transcriptional activating domain, containing 73 amino acids in its N terminal) was cotransfected into such mammalian cells as C4 2,MCF 7,COS7 respectively, each with pTRE luc and pRL TK plasmids, the feasibility of the system was determined by comparing the relative activity of Firefly luciferase activity ratio of and Renilla in different transfecting panels. Our research result showed that the system we constructed can be used for detecting the transcriptional activating activity of the target protein molecules in mammalian cells.

关 键 词：哺乳动物细胞 蛋白分子 转录激活活性 双荧光素酶报道系统 TET-OFF 

分 类 号：Q953[生物学—动物学]