【摘要】 目的　构建携带人血管内皮生长因子 (VEGF) 16 5基因的重组腺病毒载体 ,用于治疗重症冠心病。方法　将人VEGF16 5cDNA正向插入到穿梭质粒 pHCMVSP1A的CMV启动子之下 ,构建重组质粒 ,即pAd hVEGF16 5 ,通过脂质体与pJM17共转染 2 93细胞 ,经同源重组获得携带人VEGF16 5基因的重组腺病毒载体Ad hVEGF16 5。通过PCR扩增法鉴别Ad hVEGF16 5 ,根据A2 60nm计算病毒滴度。结果　人VEGF16 5cDNA成功地正向插入到 pHCMVSP1A载体中 ,以重组病毒基因组DNA为模板 ,同时扩增出了 5 76bp的VEGF16 5cDNA基因片段和 86 0bp的腺病毒骨架基因片段 ,证实了Ad hVEGF16 5的正确性。测病毒滴度为 1.94× 10 12 pfu/ml。结论　携带人VEGF16 5的腺病毒载体的成功构建为用VEGF16 5基因治疗冠心病奠定了基础。更多还原

【Abstract】 Objective To construct recombinant adenovirus vector carrying the gene of human vascular endothelial growth factor 165 for study of therapeutic angiogenesis in severe coronary artery disease.Methods The VEGF165 cDNA was extracted from pUCCAGGS/hVEGF165 with EcoRI and inserted into the E 1-deleted expression plasmid pHCMVSP1A shuttle vector,hamed Ad-hVEGF165.Ad-hVEGF165 was co-transfected with the plasmid pJM17 into 293 cells by liposome-maliated mehtad Ad-hVEGF165 expressing human VEGF165 cDNA was generated by homolgous recombination and confirmed by PCR.Ad-hVEGF165 was propagated in 293 cells and purified by CsC1 density pruification.The titer of each viral stock was determined by measuring the absorbance at 260 nm.Results VEGF165 cDNA was successfully inserted into the shuttle vector pHCMVSP1A.pAd-hVEGF165 was confirmed by NcoI and XhoI digestion.Ad-hVEGF165 was characterized by PCR.The virus titer was 1.94×10 12pfu/ml?Conclusions The recombinant replication-deficient adenovirus vector coding for human vascular endothelial growth factor 165 is successfully constructd.This investigation provides the basis for study of therapeutic angiogenesis of coronary artery disease.更多还原