【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。更多还原

【Abstract】 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.更多还原