Construction and identification of rat vascular endothelial growth factor recombinant adenovirus-mediated gene transfer system / 中国普通外科杂志

ABSTRACT

Objective To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF) as preparation for later use for genetic transfection.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,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 carrying rat VEGF by PCR,and its titration value determined based on TCID50 assay was 3?109pfu/ml.Conclusions 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.