南方医科大学学报2016，Vol.36Issue(3)：332-338,7.DOI:10.3969/j.issn.1673-4254.2016.03.07
人细胞表面黏附分子P选择素启动子报告基因的构建及鉴定
Construction and identification of human p-selectin promotor luciferase reporter gene vector
摘要
Abstract
Objective To construct a luciferase reporter gene vector of p-selectin gene promoter and determine its transcriptional activity for screening the effect of drugs on the transcriptional activity of p-selectin promoter. Methods Primers were designed based on human p-selectin promoter sequence from UCSC software. The p-selectin promoter from human genome DNA was then amplified. After digestion of pGL3-Basic vector and p-selectin promoter with KpnⅠand XhoⅠ, p-selectin promoter was inserted into pGL3-basic vector. The recombinant plasmid, namely pGL3-p-selectin-promoter, was transiently cotransfected into 293F cells with pRL-SV40 as the control vector, and the activity of the dual luciferase was detected. The transcription activity of serially truncated segments of the p-selectin promoter reporter gene was quantified by luciferase expression. 293F cells transfected with pGL3-p-selectin-promoter reporter gene and dual luciferase were stimulated with LPS, TNF-αand As2O3, and the transcriptional activity of p-selectin promoter were assessed. Results pGL3-p-selectin-promoter was constructed successfully as verified by restriction digestion and sequence analysis. The luciferase activity was higher in pGL3-p-selectin-promoter/pRL-SV40 group than in pGL3-basic/pRL-SV40 group (0.8573 ± 0.4703 vs 0.03955 ± 0.05894). pGL3-1826 bp was actively transcribed compared with pGL3-1092 bp and pGL3-3738 bp. LPS, TNF-α and As2O3 significantly enhanced the transcriptional activity of p-selectin promoter. Conclusion pGL3-p-selectin-promoter can be transcribed and activated in 293F cells. This study provided an important basis for acquiring transcriptional factors and screening inflammatory factors and drugs.关键词
细胞黏附分子类/P选择素/炎症反应/荧光素酶Key words
adhesion molecules/p-selectin/inflammatory response/luciferase引用本文复制引用
徐若霆,周辉,刘玮璐,吴炜,刘鲜艳,张文强,谭洁,赵明..人细胞表面黏附分子P选择素启动子报告基因的构建及鉴定[J].南方医科大学学报,2016,36(3):332-338,7.基金项目
国家自然科学基金（81272095）；大学生创新创业训练计划项目（201512121268）Supported by National Natural Science Foundation of China (81272095) （81272095）
