Construction of Luciferase Reporter Vector Containing High-mobility Group Box 1 Promoter in Mice and Characterization of Its Transcriptional Activity
10.13359/j.cnki.gzxbtcm.2014.05.028
- VernacularTitle:小鼠高迁移率族蛋白B1启动子的构建与转录活性检测
- Author:
Quan WEN
;
Shaohui DU
;
Ruimin TIAN
;
Hui LI
;
Ye BAI
;
Fei LI
;
Hang LEI
;
Dongfeng CHEN
;
Yiwei LI
;
Jianhong ZHOU
;
Saixia ZHANG
;
Rudong DENG
;
Yao YU
- Publication Type:Journal Article
- Keywords:
High-mobility group box 1;
Promoter;
Luciferase;
Reporter genes
- From:
Journal of Guangzhou University of Traditional Chinese Medicine
2014;(5):810-813,820
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct luciferase reporter vector containing full-length high-mobility group box 1 ( HMGB1, GenBank NM-010439) promoter for the screening of medicine. Methods The full-length HMGB1 promoter was amplified by polymerase chain reaction ( PCR) , and then was inserted into GV238 vector to construct plasmid GV238-HMGB1-P-Luc. GV238-HMGB1-P-Luc combined with internal reference plasmid pRL was co-transfected into Hela cells ( GV238-HMGB1-P-Luc group, which served as positive control group) . Plasmid pGL3-basic combined with pRL was co-transfected into Hela cells (pGL3-basic group, which served as negative control group) . Additionally, lipopolysaccharides ( LPS, 0.2 μg/mL) was used as the activator for the positive control group (LPS group), and then sodium butyrate (SB, 10 mmol/L) was used as the inhibitor for LPS group ( SB group) . At the end of experiment hour 24, luciferase activity was detected. Results The results of digestion, amplification, sequencing and identification showed that the full length of HMGB1 promoter was 2 140 bp, and the DNA sequence was correct, without mutation. Luciferase activity in GV238-HMGB1-P-Luc group was increased as compared with that of the pGL3-basic group ( P<0.05) . Luciferase activity in the LPS group was increased ( P<0.01, compared with that of GV238-HMGB1-P-Luc group) , and then was decreased after the administration of SB ( P<0.01, compared with that of the LPS group) . Conclusion A model of luciferase reporter vector containing HMGB1 promoter has been successfully constructed. Its activity can be increased by LPS, and then is in hibited by SB. The model can be used for further screening of medicine with the activities of regulating HMGB1 promoter.
