首页|期刊导航|南京医科大学学报（英文版）|Construction of recombinant adeno-associated virus vector co-expressing hVEGF165 and hBMP7 gene
南京医科大学学报（英文版）2008，Vol.22Issue(4)：205-210,6.
Construction of recombinant adeno-associated virus vector co-expressing hVEGF165 and hBMP7 gene
Construction of recombinant adeno-associated virus vector co-expressing hVEGF165 and hBMP7 gene
摘要
Abstract
Objective:To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165(hVEGF165) and bone morphogenetic protein 7(hBMPT), measure the virus titer and verify the recombination. Methods:The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid pIRES was cut down and subcloned into ITR/ MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then, recombinant plasmid pAAV- hVEGF165-IRES-hBMPT, pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells, and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results:Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5×1011vp/ml, and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion:Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer, which may offer foundation for/n vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.关键词
adeno-associated virus/human vascular endothelial factor/human bone morphogenetic protein/internal ribosome entry siteKey words
adeno-associated virus/human vascular endothelial factor/human bone morphogenetic protein/internal ribosome entry site分类
医药卫生引用本文复制引用
Zhibin Shi,Xianghui Huang,Kunzheng Wang,Xiaoqian Dang,Pei Yang,Pengbo Yu..Construction of recombinant adeno-associated virus vector co-expressing hVEGF165 and hBMP7 gene[J].南京医科大学学报（英文版）,2008,22(4):205-210,6.基金项目
This work was supported by National Natural Science Foundation ofChina(No.30600624) （No.30600624）
