Construction and identification of eukaryotic expression plasmid of human Iron regulation protein1 and function in HepG2 cells

Objective:To construct the eukaryotic expression plasmid of human Iron regulation protein1 and study its expression and function 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 FuGENEHD-mediated transient transfection of HepG2 with pcDNA3.1(+)-IRP1 plasmid,the expression levels of IRP1 and its regulated genes were determined by QRT-PCR.Results:DNA sequence analysis demonstrated that pcDNA3.1(+)-IRP1 plasmid was obtained,which express IRP1 in HepG2.The mRNA expressions of TfR1 and DMT1 were up-regulated significantly when pcDNA3.1(+)-IRP1 was transfected into HepG2 cells.Conclusion:The pcDNA3.1(+)-IRP1 plasmid has been successfully constructed with efficient expressions in HepG2.The mRNA expressions of TfR1 and DMT1 were increased significantly by approximately 3-fold and 1.5-fold compared with the control.