Establishment of a recombinant adeno-associated virus expressing hVEGF165*☆
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Publisher：Quzwzb Publish Time：Friday, November 21, 2008
Source：Neural Regen Res,2008,3(6),610-3 |
Xianghui Huang1, Zhibin Shi1, Xiaoqian Dang1, Chen Zhang1, Pengbo Yu2, Kunzheng Wang1
1Department of Orthopedics, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
2Laboratory of Shaanxi Provincial Center for Disease Control and Prevention, Xi’an 710054, Shaanxi Province, China
Xianghui Huang☆, Studying for doctorate, Attending physician, Department of Orthopedics, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
drhxh@163.com
Supported by: the National Natural Science Foundation of China, No. 30600624*
Huang XH, Shi ZB, Dang XQ, Zhang C, Yu PB, Wang KZ. Establishment of a recombinant adeno-associated virus expressing hVEGF165. Neural Regen Res 2008;3(6):610-3
| Abstract
BACKGROUND: Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration.
OBJECTIVE: To construct a non-pathogenic, recombinant adeno-associated virus (AAV) simultaneously expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP).
DESIGN, TIME AND SETTING: A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007.
MATERIALS: AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi.
METHODS: The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC (the rep/cap-gene containing plasmid), and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP.
MAIN OUTCOME MEASURES: Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter (FACS) upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR.
RESULTS: The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system provided a high packaging ratio of 95%, and the purified recombinant virus had a high titer of 5.5×1011 virus particles/mL. The AAV-HT1080 cells were infected at a ratio of 90.4%. The recombinant virus was confirmed by PCR to contain the exogenous hVEGF165 gene.
CONCLUSION: The non-pathogenic rAAV-hVEGF165-GFP vector, carrying hVEGF165 and GFP reporter gene, was successfully constructed with a high titer and infection efficiency.
Key Words: vascular endothelial factor; adeno-associated virus; nerve regeneration
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