Construction of human vascular endothelial growth factor 165 gene recombinant eukaryotic expression vector and its effect on the proliferation of vascular endothelial cells

Abstract

Objective To construct a recombinant eukaryotic expression vector pIRES-VEGF165-tPA containing functional region of human vascular endothelial growth factor 165 (VEGF165) gene, and investigate its in vitro expression and effect on the proliferation of vascular endothelial cells in transfected human umbilical vein endothelial cell lines (EA. hy926). Methods The VEGF165 gene was inserted into the eukaryotic expression vector plRES-EGFP to construct the recombinant plasmid, which was transfected into human umbilical vein endothelial cell lines (EA. hy926) at 1.2 μg/well subsequently. The transcription and expression of VEGF165 gene were detected by reverse transcription-polymerase chain reaction ( RT-PCR, n = 3 ) and Western blotting ( n = 5 ) respectively. And the effect on the proliferation of vascular endothelial cells was evaluated by MTT assay (n =5). Results The sequence of pIRES-EGFP-VEGF165 approved that the gene of VEGF165 was inserted into the eukaryotic expression vector correctly. The transfection efficiency was about ( 11.2 ± 2. 5) %, detected by counting the positive cells of green fluorescence.The mRNA and protein levels of VEGF165 in the pIRES-EGFP-VEGF165 transfected group were significantly higher than those in the control groups respectively (P ＜ 0. 01 ). Also, MTT assay indicated the proliferation of endothelia cells was greatly improved in the pIRES-EGFP-VEGF165 transfected group. Conclusion The recombinant eukaryotic expression vector pIRES-EGFP-VEGF165 is successfully constructed and can be expressed in transfected EA. hy926 cells, and it is proved that transfection with this vector could improve the proliferation of vascular endothelial cells. Key words: Vascular endothelial growth factor; Eukaryotic expression vector; Cellular proliferation; Endothelialization