Objective     To construct a bicistronic euk

aryotic expression plasmid carrying vascular endothelial growth factor (VEGF) 165 and human beta defensins (HBD) 3 genes

and to detect the expression and activity of VEGF165 and HBD3 in human embryonic kidney 293FT cells.     Methods     VEGF165 and HBD3 cDNA were amplified using total RNA from leukemic cell HL60 and gingival tissue through RT-PCR

respectively

and inserted into eukaryotic expression plasmid vector pVIVO1-mcs to construct bicistronic eukaryotic expression plasmid pVIVO1-VEGF165-HBD3. The plasmid was verified through restriction enzyme analysis and DNA sequencing and then transferred into 293FT cells through lipofection. VEGF165 and HBD3 expression in the transfected cells was detected through RT-PCR and Western blotting. Serum-free culture media supernatant of transfected 293FT cells was used to stimulate endothelial cell EA.hy926 proliferation

which was detected through the cell counting kit-8 (CCK-8) method. Antibacterial effects of the concentrated serum-free supernatant were detected via the Kleihauer-Betke (K-B) test.     Results     Restriction enzyme analysis and DNA sequencing results verified the successful construction of eukaryotic expression plasmid pVIVO1-VEGF165-HBD3. RT-PCR and Western blotting results exhibited high expression levels of VEGF165 and HBD3 in the transfected 293FT cells. The serum-free culture media supernatant can promote EA.hy926 cell proliferation significantly

reflecting the activity of secreted VEGF165. The concentrated serum-free supernatant could produce obvious inhibition rings against

Escherichia coli

(ATCC No. 25922)

Staphylococcus aureus

(ATCC No. 25923)

Pseudomonas aeruginosa

(ATCC No. 27853) and

Bacillus subtilis

(ATCC No. 62037)

showing the activity of secreted HBD3.      Conclusion     Bicistronic eukaryotic expression plasmid pVIVO1-VEGF165-HBD3 can express active VEGF165 and HBD3 in 293FT cells.