真核表达载体pcDNA3.1(-)+KG的构建及其在HeLa细胞中的表达.pdfVIP

生物技术通报 BIO TE CHN OLOG Y B ULLETIN 2010 1 pcDNA31(- ) + KG HeLa 1 2 3 王洪振 王晓光 翟雷 1 2 3 ( , 136000; , 130032; , 130024) : 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 在 pcD N 31() + 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 pUCmT 克 载体中, 再将目的片段重组至 pcDN 31() 中, 对阳性克 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 pcDN 31() + KG真核表达载体 pcDN 31() + 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 : Kozak序列 核定位信号 绿色荧光蛋白 真核表达载体 Construction of Eukaryotic Expression Vector pcDNA31() + KG Containin Green Fluorescent Protein and Its Expression in HeLa Cell 1 2 3 W angHongzhen W angX iaoguang Zhai Lei 1 2 ( College of LfieScience, JilinNormal University, Spi ing 136000; College of LfieScience, ChangchunNormal 3 University, Changchun 130032; Institute of Cytology and Genetics, NortheastNormal University, Changchun 130024) Abstrac:t To introduce green fluorescent protein into nucleus, an eukaryotic expression vector pcDN 31() + KG containing green fluorescent protein genew ith kozak sequence andNLS( nuclear localization signal)was constructedGFP sequencewas amplified by two rounds ofPCR from pcDN 31() + GFP vectorwith smi ultaneously being attachedKozak sequence andNLS, and then cloned into pUCmT vector following the routine proceduresThe interest gene fragmentwas recombinated into eukaryotic expression vector pcDN 31( ) to construct pcDN 31()