小鼠高迁移率族蛋白B1启动子的构建与转录活性检测

Construction of Luciferase Reporter Vector Containing High-mobility Group Box 1 Promoter in Mice and Characterization of Its Transcriptional Activity

【目的】构建小鼠高迁移率族蛋白B1(high-mobility group box 1 protein,HMGB1)启动子全长序列的荧光素酶报告基因用于药物筛选。【方法】采用PCR技术扩增小鼠HMGB1基因启动子全长序列,用GV238载体构建报告质粒GV238-HMGB1-P-Luc,以pGL3-basic作为阴性对照质粒,与内参质粒pRL共转染Hela细胞,以内毒素(LPS,0.2μg/mL)作为激活剂,以正丁酸钠(SB,10 mmol/L)作为抑制剂,分组处理24 h后检测荧光素酶活性。【结果】经酶切、PCR及测序鉴定证实克隆的HMGB1基因启动子全长2 140 bp,DNA序列正确无突变。与pGL3-basic组比较,GV238-HMGB1-P-Luc组荧光素酶活性显著增强(P<0.05);与GV238-HMGB1-P-Luc组比较,LPS组荧光素酶活性显著增强(P<0.01);与LPS组比较,SB组荧光素酶活性显著降低(P<0.01)。【结论】HMGB1基因启动子报告基因构建成功,LPS可以增强其表达,SB则可以抑制LPS刺激下的HMGB1启动子表达增强,可为下一步筛选具有调控HMGB1启动子活性的药物奠定基础。

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 ceils （pGL3-basic group, which served as negative control group） . Additionally, lipopolysaccharides （LPS, 0.2 pbg/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-I-IMGB1-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 inhibited by SB. The model can be used for further screening of medicine with the activities of regulating HMGB1 promoter.