Objective： To construct a recombinant adenovirus (pAdxsi-GFP-HIF) encoding human hypoxia inducible factor 1αgene (HIF-1α) and to express it in endothelial cells.
Methods： HIF-1αgene was obtained from human lung cancer cell line A549，which was cultured in hypoxia condition，by RT-PCR.The HIF-1αgene was subcloned into shuttle vector pShuttle-CMV-EGFP at KpnI and BamHI sites.After identified with restriction enzymes，plasmid pShuttle-GFP-HIF was linearized by digestion with restriction endonuclease I-CeuI and I-SceI，and subsequently cotransformed into E.coli DH5a with adenoviral backbone plasmid pAdxsi to make homologous recombination.After linearized by PacI，the homologous recombinant adenovirus plasmid was transfected into 293 cells to package and amplify.The recombinant adenovirus was infected with human umbilical vein endothelial cells (ECV304)，and the expression level of HIF-1αprotein was evaluated by ELISA.
Results： The recombinant adenovirus vector containing HIF-1αgene (pAdxsi-GFP-HIF) was successfully constructed and amplified with titer of 3.38×1010 pfu/mL.The green fluorescence protein was detected under fluorescent microscope in ECV304 at 24h after transfection and with a stronger degree after 48h.The concentration of HIF-1 protein was (48.93±3.86)ng/mL in supernatant at 48 h after transfection.
Conclusion： A recombinant adenovirus vector pAdxsi-GFP-HIF，encoding human hypoxia inducible factor 1αgene，has been constructed in vitro and expressed successfully in ECV304 cells.