Construction and identification of eukaryotic expression plasmid of human hypoxia-inducible factor-1α

Objective:To construct the eukaryotic expression plasmid of human hypoxia-inducible factor-1α and study its expression in HepG2 cells.Methods:Total RNA was isolated from HepG2 cells and cDNA library was constructed by reverse transcriptional PCR method.The cDNA prepared was inserted into pcDNA3.1(+) vector.All sequences amplified by PCR were confirmed by complete sequencing.After lipofectamine-mediated transient transfection of HepG2 with pcDNA3.1(+)-HIF-1α plasmid,the expression levels of HIF-1α protein were determined by immunofluorescent staining and Western bloting.Results:DNA sequence analysis demonstrated the pcDNA3.1(+)-HIF-1α plasmid was obtained,which could express HIF-1α protein in HepG2 in normixa.Conclusion:The pcDNA3.1(+)-HIF-1α plasmid has been successfully constructed with efficient expressions in HepG2.