摘要
目的 构建在哺乳动物细胞中表达人VEGF1 65(hVEGF1 65)红色荧光蛋白 (RFP)的融合表达载体。方法 根据已知的人VEGF1 65序列 ,用PCR方法 ,从质粒pUC1 8/VEGF1 65中扩增出去除终止密码子的VEGF1 65片段 ,定向克隆至pDsRed1 N1质粒中。重组质粒经限制性内切酶和DNA序列测定鉴定。以DOTAP为介导 ,将pDsVEGF1 65Red1 N1转染 2 93 T细胞 ,4 8小时后用RT PCR、激光共聚焦显微镜检测VEGF1 65在细胞内表达和分布情况。结果 重组的融合蛋白表达载体经酶切、PCR和DNA序列测定证明构建正确 ,并在2 93 T细胞中表达。报告基因 -红色荧光蛋白在细胞质、细胞核中都有一定的分布。结论 成功构建了pDsVEGF1 65Red1 N1红色荧光蛋白融合表达载体 ,该载体能在哺乳动物细胞中表达 。
Objective To construct the vector that expresses the fusion protein of VEGF 165 and red fluorescent protein (RFP) in mammalian cells.Methods According to the nucleotide sequence of hVEGF 165 ,a pair of oligonucleotides was designed as primer which contained nucleotide sequence of HindⅢ and SacⅡ restriction endonuclease at the two end respectively,and no stop code. The gene encoding for hVEGF 165 (573bp) was amplified using PCR technique. The PCR product was digested with HindⅢ and SacⅡ,and then cloned into the plasmid containing the reporter gene pDsRed1 N1. The recombinant plasmid was identified by restriction endonuclease enzyme analysis and DNA sequence analysis. The plasmid was transfected into 293 T cells,human fetal renal epithelial cells T,with DOTAP. The expression and distribution of the VEGF 165 were observed with RT PCR,Confocal Laser Scanning Microscopy (CLSM) after 48 hours. Results The recombinant fusion protein expression vector was verified correctly by enzyme digestion,PCR and sequence analysis. This vector was highly expressed in the 293 T cells. The red fluorescence was found over the cytoplasm and in the nuclei as well by CLSM. Conclusion A red fluorescent protein reporter gene vector containing human vascular endothelial growth factor sequence has been constructed successfully,thus providing an important and convenient tool to study intracellular localization of VEGF.
出处
《创伤外科杂志》
2004年第3期209-212,共4页
Journal of Traumatic Surgery
关键词
血管内皮细胞生长因子
红色荧光蛋白
真核细胞
基因表达
vascular endothelial growth factor
red fluorescent protein
eukaryotic cell
gene expression
