Construction and identification of recombinant adenovirus vector containing the hVEGF165 gene.

Abstract

To construct the recombinant adenovirus vector containing the cDNA for human vascular endothelial growth factor (hVEGF165), the cDNA for hVEGF165 was subcloned into pACCMV.pLpA. Subsequently, this recombinant pACCMV.hVEGF was co-transfected into 293 cells together with pJM17 to obtain the replication-deficient recombinant adenovirus containing hVEGF gene--AdCMV.hVEGF. The VEGF gene expression was detected by using RT-PCR and Western blot in rabbit aorta vascular smooth muscle cells (VSMC) infected with AdCMV.hVEGF. Cultured human umbilical vein endothelial cells (HUVEC) were incubated with the conditioned medium (CM) from above mentioned VSMC infected with AdCMV.hVEGF to observe the effect of VEGF on proliferation of HUVEC. 48 h after the infection with AdCMV.hVEGF, VSMC demonstrated VEGF expression, and the expressed VEGF could stimulate the proliferation of HUVEC in vitro. Successfully prepared AdCMV.hVEGF165 could express biologically active VEGF in infected VSMC, and stimulate proliferation of HUVEC.