真核表达载体pcDNA3.1(-)+KG的构建及在HeLa细胞中的表达  

Construction of Eukaryotic Expression Vector pcDNA3.1(-)+KG Containing Green Fluorescent Protein and Its Expression in HeLa Cell

作　　者：王洪振[1] 王晓光[2] 翟雷[3] 

机构地区：[1]吉林师范大学生命科学学院,四平136000 [2]长春师范学院生命科学学院,长春130032 [3]东北师范大学遗传与细胞研究所,长春130024

出　　处：《生物技术通报》2010年第1期188-192,共5页Biotechnology Bulletin

基　　金：国家自然科学资金资助项目(30040031);吉林师范大学博士科研启动项目(2004010)

摘　　要：为了将绿色荧光蛋白(green fluorescent protein,GFP)引入细胞核内,采用两轮PCR方法从原先克隆在pcD-NA3.1(-)+GFP载体中将GFP编码序列扩增出来并引入Kozak序列和核定位信号,使用常规酶切和连接方法将其重组至pUCm-T克隆载体中,再将目的片段重组至pcDNA3.1(-)中,对阳性克隆进行酶切、PCR和测序鉴定后,构建了带有Kozak序列和核定位信号的绿色荧光蛋白(GFP)真核表达载体pcDNA3.1(-)+KG。真核表达载体pcDNA3.1(-)+KG被转染试剂Su-perfect转染至HeLa细胞中,绿色荧光蛋白基因在HeLa细胞中得到表达而且在细胞核中观察到绿色荧光。该研究以绿色荧光蛋白为标记初步建立了活体观察真核细胞核动态变化的研究体系。To introduce green fluorescent protein into nucleus, an eukaryotic expression vector pcDNA3.1 （-） ＋ KG containing green fluorescent protein gene with kozak sequence and NLS（nuclear localization signal）was constructed. GFP sequence was amplified by two rounds of PCR from pcDNA3.1 （-） ＋ GFP vector with simultaneously being attached Kozak sequence and NLS ,and then cloned into pUCm-T vector following the routine procedures. The interest gene fragment was recombinated into eukaryotic expression vector pcDNA3, 1 （-）to construct pcDNA3.1 （-） ＋ KG. After identification by enzyme digestion, PCR and sequencing, the positive clones were transformed into Hela cells with transfect reagent Superfect. GFP gene was expressed in Hela cells and green fluorescent light was observed in nucleus. This paper preliminarily established a system for study on dynamic changes of nucleus in live cells with GFP as a reporter.

关 键 词：KOZAK序列 核定位信号 绿色荧光蛋白 真核表达载体 

分 类 号：Q78[生物学—分子生物学]