Construction and expression of eukaryotic expression vector of pEGFP-N1/PDX1

Aim:To construct recombinant eukaryotic expression vector of Pdx1 transcription factor and detect its expression in eukaryocyte. Methods: We obtained the Pdx1 gene exon by using human embryo pancreas tissue mRNA as the template,and cloned it into multiple cloning sites of eukaryotic expression vector pEGFP-N1 to construct pEGFP-N1/Pdx1 eukaryotic expression plasmid and the constructed plasmid was transfected L02 cell.The expression in transfected cells was detected by RT-PCR immunocytochemistry,indirect fluorescence assay and Western Blot respectively.Results: Restriction endonucleas and sequence analysis verified that the fragment cloned in pEGFP-N1 vector was Pdx1 cDNA.In the supernatant of L02 cells transfected by the pEGFP-N1/Pdx1 plasmid.RT-PCR verified that Pdx1 mRNA was positive in L02 cells,the expression of PDX1 protein in the nuclears of L02 cells was detected by indirect fluorescence assay and immunohistochemistry,and about 48KDa protein was detected by Western blot in the cellular nucleus of L02 cells.Conclusion:Eukaryotic expression plasmid of Pdx1 gene(pEGFP-N1/Pdx1) is successfully constructed and expressed effectively in L02 cells.