HMGB2启动子报告基因载体的构建、活性验证及与其结合的转录因子的预测

Construction and identification of the activity of HMGB2 promoter reporter vector and forecast of its binding transcription factors

目的:构建高迁移率族蛋白B2(high mobility group box2,HMGB2)启动子荧光素酶报告基因载体,并对其进行活性验证,预测可能与HMGB2启动子结合的转录因子。方法:在NCBI Genbank数据库中查询HMGB2基因启动子区域序列,运用PCR扩增HMGB2启动子序列片段,将其克隆至pGL3-Basic载体中,构建重组质粒pGL3-HMGB2-promoter,并通过双酶切和测定核酸序列鉴定。采用虫荧光素酶报告实验验证pGL3-HMGB2-promoter重组质粒的活性,进一步通过在线软件PROMO预测可以与HMGB2启动子序列结合的转录因子。结果:经限制性内切酶酶切鉴定和核酸序列比对,证实pGL3-HMGB2-promoter重组质粒构建成功。虫荧光素酶报告实验结果显示,构建的重组质粒具有启动子活性。PROMO在线软件分析结果表明,有72个转录因子可能与HMGB2启动子序列结合。结论:成功构建了含有HMGB2启动子序列的荧光素酶报告基因重组质粒,该质粒具有启动子活性,且有72个转录因子可能与HMGB2启动子序列结合,促进HMGB2的转录。

Objective ： To construct the recombinant luciferase reporter plasmid carrying high mobility group box2（HMGB2） promoter,to identify the activity of the recombinant luciferase reporter plasmid,and to forecast the target transcription factors which can bind to the promoter. Methods： The fragment sequence of HMGB2 promoter, searched from NCBI Genbank database and amplified through PCR was cloned into luciferase reporter plasmid, pGL3-Basic,by digestion with restriction endonucleases and insertion with ligase. The activity of HMGB2 promoter plasmid was detected by luciferase reporter assay.The transcription factors which could bind to the promoter was forecasted by an online software PROMO.Results： The recombinant luciferase reporter plasmid carrying pGL3-HMGB2-promoter was constructed successfully by examination with enzyme digestion and sequencing. The result of luciferase reporter assay indicated that the recombinant luciferase reporter plasmid had transcript activity. With the help of the online software PROMO, 72 transcription factors were forecasted to bind to the promoter. Conclusion： The pGL3-HMGB2-promoter plasmid has the promoter activity and 72 transcription factors may bind to the promoter and promote the expression of HMGB2.