HMGB1启动子荧光素酶报告基因的构建及鉴定
Construction and identification of luciferase reporter gene vector containing HMGB1 promoter
丁宁 1肖慧 2高巨 3许立新 1佘守章1
作者信息
- 1. 广州市第一人民医院,麻醉科及麻醉学实验室,广东,广州,510180
- 2. 广州市第一人民医院,门诊部,广东,广州,510180
- 3. 广州中医药大学第二附属医院麻醉科,广东,广州,510120
- 折叠
摘要
目的:构建高迁移率族蛋白B1(HMGB1)启动子驱动的荧光素酶报告基因的真核表达载体pGL3-HMGB1 P,转染肺泡上皮细胞(A549)后检测报告基因在机械牵张刺激中的转录活性.方法:以A549细胞基因组DNA为模板,应用PCR技术扩增HMGB1启动子片段,测序正确后克隆入荧光素酶报告基凶表达载体pGL3,将重组质粒pGL3-HMGB1 P导入A549细胞,检测不同强度机械牵张(分别为5%和20%)刺激下荧光素酶的活性变化.结果:PCR和测序结果表明扩增的HMGB1启动子序列正确,酶切榆测证实重组真核表达载体pGL3-HMGB1P构建成功.在5%牵张应变作用下,转染pGL3-HMGB1P的A549细胞荧光素酶活性是转染空载体pGL3-Basic的2.9倍(P<0.05);而在20%牵张应变作用下,转染pGL3-HMGB1 P的A549细胞荧光素酶活性是转染空载体pGL3-Basic的6.2倍(P<0.01).结论:利用荧光素酶报告基因系统证实机械牵张可诱导HMGB1转录表达增加,为深入研究HMGB1转录表达的调控机制提供了基础.
Abstract
AIM: To construct luciferase reporter gene vectors containing high mobility group box 1 protein ( HMGB1 ) promoter and to assay the transcriptional activity of HMGB1 promoter induced by mechanical stretch in alveolar epithelial ceils (A549). METHODS: HMGB1 promoter were amplified from the genomic DNA of A549 ceils by PCR and cloned into luciferase reporter gene vector, pGl3. The reeombined vector was transfeeted into A549 cells, and the activity of the luciferase was determined after the cells were stimulated by mechanical stretch at 5% and 20% elongation. RESULTS: PCR and sequencing results indicated that the amplified sequence was correct. The results of restriction enzyme digestion indicated that the recombinant eukaryotic vector pGL3HMGB1P was successfully constructed. The iuciferase reporter system showed that the transcription activation of pGL3-HMGB1 P was 2.9 times higher than pGL3-Basic when subject to 5% stretch ( P < 0.05 ), while the transcription activation of pGL3-HMGB1 P was 6. 2 times higher than pGL3-Basic when subject to 20% stretch( P < 0.01 ). CONCLUSION: Mechanical stretch induced high transcriptional activity of HMGB1 promoter in A549 cells was confirmed by luciferase reporter gcne system, and supplied an experimental base for further study of the transcriptional regulation of HMGB1.
关键词
机械牵张/高迁移率族蛋白1/基因/报告Key words
mechanical stretch/high mobility group box 1 protein/gene/reporter引用本文复制引用
基金项目
广州市医药卫生科技项目(2008-yB-012)
重点项目(2008-zDi-14)
广东省科技计划项目(2007831509007)
出版年
2009
NETL
NSTL
维普
万方数据