郧阳医学院学报2009，Vol.28Issue(3)：214-217,封2,5.
携带绿色荧光蛋白基因的重组腺病毒的构建及其在人血管平滑肌细胞中的表达
Construction of Recombinant Adenovirus Carrying gfp Gene and Adenovirus-mediated GFP Expression in Human Vascular Smooth Muscle Cells
摘要
Abstract
Obieaive To construct recombinant edvenovirus phsmid containing GFP gene and prepare recombinant adenovirus pAd-GFP and GFP will be efficiently expressed in human vascular smooth muscle cells(hVSMC).Methods pRNATH1.1/Adeno was linearized with Pme I,and transformed into ultracompletent BJ5183 containing pAdeasy-1,then reeombinant advenovirus pAd-GFP was constructed by homologous recombination in bacteria BJ5 183.The recombinant adenoviral plasmid DAd-GFP Was identified bv Pac I dizestion.Linearized DAd-GFP was transfeeted into AD293 cells with llposome to generate recombinant adenovirus particles which were purified and concentrated by Cesium cMoride(CsCl)density gradient eentrifugation.Adenovirus particles were used to infect hVSMC,and the infection efficiency and GFP expression were observed under inveaed phase-contrast microscope.Results The adenovirus vector WaS constructed successfully and adenoviruff encoding GFP gene Wag prepared with titers of about 4.5 × 1012pfu/ml.The infection efficiency of aden06rus for hVSMC Was 100%.GFP could be efficiently expressed in hVSMC.Conclusion The recombinant adenovims expressing GFP Was packaged and prepared successfully.The adenovirus could infect hVSMC efficiently and GFP could express very well.This investigation provides a good control vector for study on GFP as a reporter gene in gene therapy of RNA interference.关键词
报告基因/绿色荧光蛋白/细菌内同源重组/人血管平滑肌细胞/RNA干扰Key words
Reporter gene/Green fluorescent protein/Homologous recombination in bacteria/Human vascular smooth muscle ceils/RNA interferenee分类
生物学引用本文复制引用
张蕾,王家宁,郭凌郧,孔霞,杨建业,唐俊明,黄永章,郑飞..携带绿色荧光蛋白基因的重组腺病毒的构建及其在人血管平滑肌细胞中的表达[J].郧阳医学院学报,2009,28(3):214-217,封2,5.基金项目
湖北省教育厅青年人才基金(No.200524003). （No.200524003）
