Construction of promoter CD2AP and analysis of its promoter activity in human embryonic kidney-293T cells

Objective To construct a luciferase reporter plasmid containing CD2AP human gene promoter and evaluate its activity in human embryonic kidney(HEK)-293T cells.Methods The 2082 bp fragment was amplified by PCR with human genomic DNA as a template and directionally cloned into pGL3-basic multiple cloning sites to construct the luciferase reporter plasmid pGL3-2k.Transfection of HEK-293T cells with the promoter-driven luciferase construct was performed to induce luciferase gene expression and to calculate the relative luciferase activity unit(RLU).Promoter sequence of 2082 bp upstream of transcription initiation site of CD2AP was analyzed with TFSEARCH ver.1.3 software.Results DNA sequencing and restriction endonuclease analysis verified the successful construction of the plasmid pGL3-2k.This CD2AP promoter exhibited a strong promoter activity with an increase of 74.8-fold of RLU in HEK-293T cells when compared with pGL-3 basic vector.The transfection experiment confirmed that the levels of its activation were significantly higher than those in the controls in HEK-293T cells.Function analysis of CD2AP promoter disclosed several AP1,Sp1,CREB and GATA-1 sites in minimal promoter region.ConclusionThe plasmid pGL3-2k promoter has been successfully constructed and has a strong basal promoter activity in HEK-293T cells.