摘要
目的构建肝细胞生长因子(hepatocyte growth factor,HGF)真核表达载体,并观察其在人正常肝细胞中的表达.方法利用基因重组技术将 HGF 与绿色荧光蛋白(greenfluorescence protein,GFP)融合并构建真核表达质粒,通过脂质体介导法,导入正常人肝细胞系中.用荧光显微镜以及免疫组化方法观察 HGF 的表达.结果酶切鉴定证实 HGF 片断已克隆到 pEGFP-N3的 BamHⅠ和 Sal Ⅰ位点之间,在转染人正常肝细胞后,利用荧光显微镜观察可见到绿色荧光蛋白的表达,用免疫组化方法进一步证实了 HGF 蛋白在细胞中的表达.结论 pEGFP-HGF 表达载体便于观察转染细胞中 HGF-GFP融合蛋白的表达情况及蛋白定位,适用于对 HGF 分子生物学特性及治疗应用的研究.
AIM To construct pEGFP-hepatocyte growth factor(HGF) expression vector and to detect its transient expression in transfected human hepatocytes. METHODS Techniques of gone recombination and gene transfection were employed.Human HGF cDNA was ligated to EGFP gone of pEGFP-N3.The recombinant plssmid was transfected into human hepatocyte line QZG with liposome.The expression of HGF protein was observed by fluorescence microscopy and immunohistochemistry method. RESULTS Identification of pEGFP-HGF by enzyme digestion showed that HGF fragment had been cloned into BamH Ⅰ and Sal Ⅰ sites of pEGFP-N3,and the expression of GFP in transfected hepatocytes was observed.Under fluorescence microscopy,QZG cells which highly expressed GFP protein showed green fluorescence.Using immunohistochemistry method,expression of HGF was observed in QZG cells transfected with pEGFP-HGF. CONCLUSIONS pEGFP-HGF expression vector makes it easy to assess the expression of HGF-GFP fusion protein and the protein localization in transfected cells.
出处
《世界华人消化杂志》
CAS
2001年第10期1143-1146,共4页
World Chinese Journal of Digestology
关键词
肝细胞生长因子
表达载体
基因转染
免疫组化
绿色荧光蛋白
hepatocyte growth factor
express ion vector/construction
gene transfection
immunohist ochemistry
green fluorescence protein
