郧阳医学院学报2001，Vol.20Issue(1)：1-4,4.
反义VEGF165腺病毒重组体的构建及鉴定
Construction and characterization of replication-deficient adenoviral vector containing the cDNA for human VEGF165 in an antisense orientation
王家宁 1黄永章 1王俊峰 1王卫民 1李瑞明 1张群林1
作者信息
- 1. 郧阳医学院附属太和医院生命科学研究所，湖北十堰 442000
- 折叠
摘要
Abstract
objective To construct the recombinant adenovirus vectorcontaining the cDNA for hVEGF165 in an antisense orientation for future study of tumor treatment by antisense hVEGF165 RNA strategy.Methods The VEGF165 cDNA has been extracted from pUCCAGGS/hVEGF165 with EcoRI and then inserted in an antisense orientation into the E1-deleted expression plasmid pHCMVsp1A shuttle vector, called pAd-ahVEGF165. pAd-ahVEGF165 was cotransfected with the plasmid pJM17 into the transformed human embryonic kidney cell line 293 cells by liposome-mediated method. Homologous recombination of the pAd-ahVEGF165 and pJM17 in 293 cells replaced the E1 region with the expression cassette from pAd-ahVEGF165. Ad-ahVEGF165 was confirmed by PCR.Ad-ahVEGF165 was propagated in 293 cells and then underwent CsCl density purification. subsequently, the preparations were didalyzed in dialysis buffer. The titer of each viral stock was determined by measuring the absorbance at 260nm. Results VEGF165 cDNA was successfully inserted into the shuttle vector pHCMVSPIA. pAd-ahVEGF165 was confirmed by NcoI and XhoI digestion. Ad-ahVEGF165 was characterized by PCR coamplification. The virus titer was 5.6×1011pfu/ml. Conclusions Ad-ahVEGF165 containing the antisense VEGF165 sequence was successfully constructd.This investigation provides the basis for future study of tumor treatment based on antisense VEGF RNA strategy.关键词
内皮生长因子/腺病毒/RNA,反义/基因扩增Key words
endothelial growth factors/adenovirus/RNA,antisense/geneamplification分类
生物科学引用本文复制引用
王家宁,黄永章,王俊峰,王卫民,李瑞明,张群林..反义VEGF165腺病毒重组体的构建及鉴定[J].郧阳医学院学报,2001,20(1):1-4,4.
