首页|期刊导航|南京医科大学学报（英文版）|Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor
南京医科大学学报（英文版）2008，Vol.22Issue(1)：52-56,5.
Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor
Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor
摘要
Abstract
Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.关键词
vascular endothelial growth factor/ adenovirus vector/ genetic transfectionKey words
vascular endothelial growth factor/ adenovirus vector/ genetic transfection分类
医药卫生引用本文复制引用
Hongyu Yang,Hong Qi,Junjie Zou,Xiwei Zhang..Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor[J].南京医科大学学报（英文版）,2008,22(1):52-56,5.基金项目
This study is supported by Natural Science Foundation of Jiangsu Province(BK2005158) （BK2005158）
