Construction and identification of recombinant adeno- associated virus vector co-expressing human vascular endothelial growth factor and green fluorescent protein*☆

Huang Xiang-hui1, Shi Zhi-bin1, Wang Kun-zheng1, Dang Xiao-qian1, Yang Pei1, Yu Peng-bo2

1Department of Orthopaedics, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an   710004, Shaanxi Province, China; 2Laboratory of Virus, Shanxi Provincial Center for Disease Control and Prevention, Xi’an   710054, Shanxi Province, China

Huang Xiang-hui☆, Studying for doctorate, Attending Physician, Department of Orthopaedics, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an   710004, Shaanxi Province, China
drhxh@163.com

Correspondence to: Shi Zhi-bin, Doctor, Lecturer, Attending Physician, Department of Orthopaedics, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an   710004, Shaanxi Province, China
jackky9999@ sohu.com

Supported by: the National Natural Science Foundation of China, No. 30600624*

Received:2008-01-08
Accepted:2008-03-11
(08-50-1-185/ZS)

Huang XH, Shi ZB, Wang KZ, Dang XQ, Yang P, Yu PB. Construction and identification of recombinant adeno-associated virus vector co-expressing human vascular endothelial growth factor and green fluorescent protein.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(29):
5755-5758(China)
[www.zglckf.com/ zglckf/ ejournal/ upfiles/08-29/ 29k-5755(ps).pdf]

Abstract
BACKGROUND: Vascular endothelial growth factor (VEGF) can specifically promote the division and proliferation of endothelial cells and the revascularization, finally induce angiopoiesis. Recently, VEGF-based gene therapy has been gradually used in clinical trials, but some limits on usually used vectors deserve further studies, including the low transfection efficiency of plasmid vector, the immunogenicity of adnovirus vector to host cells and the potential risk of infection.
OBJECTIVE: To construct the non-pathogenic recombinant adeno-associated virus (AAV) co-expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP) label, and measure the virus titer and assess its biological activity.
DESIGN, TIME AND SETTING: The open experiment was performed at the Virus Laboratory of Shanxi Provincial Center for Disease Control and Prevention from March to September 2007.
MATERIALS: AAV-293 virus packaging cell line, AAV HT-1080 cells were purchased from Stratagene, USA. E.coli DH5α was a stocked strain from Shanxi Provincial Center for Disease Control and Prevention. AAV Helper Free System (pAAV-IRES-GFP vector containing GFP label) was purchased from Stratagene, USA. Plasmid pUC18-hVEGF165 was constructed previously by Dr.Shi from Department of Orthopaedics of Second Affiliated Hospital of Xi’an Jiaotong University.
METHODS: The hVEGF165 gene from plasmid pUC18-hVEGF165 was amplified and inserted into plasmid pAAV-IRES-hrGFP. Then recombinant plasmid pAAV-hVEGF165-IRES-hrGFP, pAAV-RC and pAAV-Helper 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. The recombinant virus infected the AAV-HT1080 cells, the virus titer was measured by fluorescence counting, and the recombinant AAV-hVEGF165-IRES-hrGFP was verified by the amplification of the exogenous gene of hVEGF165 from virus genome.
MAIN OUTCOME MEASURES: Virus packaging efficiency was monitored under the fluorescence microscope. Virus titer was measured by fluorescent counting. The packaging of recombinant virus was determined by the amplification of the exogenous hVEGF165 gene.
RESULTS: The amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-hrGFP was confirmed by double digestion. The system provided a high packaging ratio of over 95% and the purified recombinant virus had a high titer of 5.5×1011 vp/mL. The recombinant virus was confirmed by exogenous human VEGF165 gene from virus genome.
CONCLUSION: The non-pathogenic rAAV-hVEGF165-GFP simultaneously carrying hVEGF165 and GFP label is successfully constructed, with a high titer and satisfying biological activity.