摘要
目的克隆烟酰胺腺嘌呤二核苷酸磷酸氧化酶(Nox)上游启动子序列,为研究Nox1基因的转录调控奠定基础。方法以人基因组DNA为模板,通过PCR扩增获得1415bp(-1415～0)、1276bp(-1415～-140)、997bp(-1415～-419)、839bp(-1415～-577)、470bp(-1415～-946)大小的Nox1启动子片段,将其定向克隆入pGL3-Basic荧光素酶表达载体,构建荧光素酶报告基因载体,进行真核细胞转染后鉴定目的片段启动子活性。结果在A549细胞中,各Nox1启动子片段均有活性;-140～-419bp和-577～-946bp区域可能存在Nox1启动子核心区域。结论成功克隆了在肺泡上皮样细胞中具有活性的Nox1启动子序列,为进一步研究Nox1基因的转录调控机制奠定了基础。
Objective To construct the pGL3-Basic luciferase expression vector containing Noxl promoter, and to identify the promoter activity of Noxl in eukaryotic ceils. Methods The 1 415 bp fragment in the upstream of translation initiation site of the human Noxl gene was obtained by PCR.Human genomic DNA was used as the template. Five segments of Noxl promoter, namely the 1 415 bp （-1415~0）,1 276 bp （-1415^-140）,997 bp （-1415^-419）,839 bp （-1415^-577）, and 470 bp （-1415--946）, were obtained and cloned into a lueiferase expression vector. The recombinant plasmid was then used for transient transfection and the promoter activity was determined by measuring the relative luciferase activity in the transfected cells. Results When transiently transfected into A549 cells, all the fragments were found to have the promoter activity. Regions of -140--419 bp and -577--946 bp might contained the core element of Noxl promoter. Conclusion We have successfully cloned the functionally active Noxl promoter.It provides the foundation for the further study on the mechanism of transcriptional regulation of Noxl gene.
出处
《热带医学杂志》
CAS
2013年第5期528-530,545,共4页
Journal of Tropical Medicine
基金
国家自然科学基金(81070003)
