Isolation and Identification of Promoter Sequence of Interferon Regulatory Factor-3 and Detection of Its Promotor Activity in Human Embryonic Kidney-293 Cells
- VernacularTitle:干扰素调节因子-3启动子区序列分离、鉴定及在人类胚胎肾-293细胞中启动活性分析
- Author:
wei, REN
;
hua-gao, XU
;
chao, LU
;
guo-ping, ZHOU
- Publication Type:Journal Article
- Keywords:
interferon regulatory factor 3;
promoter;
promoter activity
- From:Journal of Applied Clinical Pediatrics
2006;0(17):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a luciferase reporter plasmid containing interferon regulatory factor 3(IRF-3)human gene promoter and to evaluate promoter activity in human embryonic kidney(HEK)-293 cells.Methods The 1 000 bp fragment was amplified by PCR with human genomic DNA as a template and was directionally cloned into pGL3-basic multiple cloning sites to construct the luciferase repor-ter plasmid pGL3-pIRF-3.Transfection of HEK-293 cells with the promoter-driven lucife-rase construct was performed to induce lucife-rase gene expression and calculate the relative luciferase activity unit(RLU).Promoter sequence of 1 000 bp upstream of transcription initiation site of IRF-3 was analyzed by using Promoter 2.0 Prediction software.Results DNA sequencing and restriction endonuclease analysis verified the successful construction of the plasmid pGL3-pIRF-3.This IRF-3 promoter exhibited a strong promoter activity with an increase of 42.2-fold of RLU in HEK-293 cells when compared with pGL-3 basic vector.The transfection experiment confirmed that the levels of its activation were significantly higher than that in controls in HEK-293 cells.Function analysis of IRF-3 promoter disclosed seve-ral GATA-1 and specific protein 1(Sp1) sites and E2F in minimal promoter region.Conclusion The plasmid pGL3-pIRF-3 promoter is successfully constructed and has a strong basal promoter activity in HEK-293 cells.
