Construction of Combination Gene Vector Expressing VEGF-siRNA and Fusion Suicide Gene yCDglyTK and Its Application*

Abstract

This research aimed to construct a new combination gene vector: pcDNA3.1 (-)VEGF-siRNA/ yCDglyTK, study its expression quality and lethal effet in human gastric cancer cell line SGC7901. First, RNA interference (RNAi) targeting vascular endothelial growth factor (VEGF) was applied to construct interfering plasmid pGenesil-VEGF-siRNA. Then, the siRNA expression cassette (including U6 promotor ) was amplified by PCR and subcloned into pcDNA3.1 (-)CV-yCDglyTK to build a new combination gene plasmid: pcDNA3.1 (-) VEGF-siRNA/yCDglyTK. The recombinant plasmid was identified by restriction enzyme digestion and gene sequencing. All of the three plasmids were delivered into SGC7901 cells using calcium phosphate nanoparticles (CPNPs). Expressions of yCDglyTK and VEGF were detected by RT-PCR and Western-blot. MTT assays were applied to determine the cytotoxic effect of plasmids in the presence of 5-FC. Restriction enzyme digestion and gene sequencing confirmed the combination gene vector pcDNA3.1(-)VEGF-siRNA/yCDglyTK was constructed successfully. RT-PCR, Western-blot showed expression of yCDglyTK and inhibition of VEGF in SGC7901 cells transfected with the combined gene plasmid, which were the most sensitive to 5-FC in the MTT assays. The combination gene vector pcDNA3.1(-)VEGF-siRNA/yCDglyTK was constructed successfully. It was tentatively confirmed that RNAi targeting VEGF could synergize with suicide gene therapy.