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.