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Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor
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    Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods:Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

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Hongyu Yang, Hong Qi, Junjie Zou, Xi-wei Zhang.[J].南京医科大学学报(自然科学版),2008,28(1):52-56

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  • 收稿日期:2007-11-23
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