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...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 × 10^9 pfu/ml. Conclusion：The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a ufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.展开更多
基金Natural Science Foundation of Jiangsu Province(BK2005158)
文摘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 × 10^9 pfu/ml. Conclusion：The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a ufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.
