SOD2启动子的克隆及转录活性的检测

Cloning of SOD2 promoter and identification of its transcriptional activity

目的:克隆小鼠锰超氧化物歧化酶基因(MnSOD,SOD2)5非编码区启动子序列,通过报告基因技术检测在静息或脂多糖(LPS)、亚砷酸钠(NaAsO2)等刺激下该段启动子的转录活性。方法:提取小鼠肝组织基因组DNA,PCR扩增小鼠SOD2启动子序列(-1554～+48);采用基因重组技术构建由SOD2启动子驱动的红色荧光蛋白报告基因载体,将该载体瞬时转染小鼠胚胎成纤维细胞(MEF),荧光显微镜下观察静息或NaAsO2、LPS、佛波酯(PMA)刺激下红色荧光蛋白表达。结果:正确扩增出小鼠SOD2启动子(-1554～+48)片段;成功构建其红色荧光蛋白报告基因载体,证实该质粒转染MEF细胞后静息状态下仅可见少量而微弱的红色荧光,经NaAsO2、LPS、PMA刺激后,红色荧光强度和亮度明显增加。结论:小鼠SOD2启动子(-1554～+48)在静息状态下即具有转录活性,炎性、氧化应激刺激后SOD2表达增强;该启动子的成功克隆和其报告基因载体的构建,为研究SOD2的基因表达调控机制提供了重要基础和工具。

AIM： To clone the promoter sequence in 5＇ non -coding region （NCR） of murine manganum superoxide dismutase gene （ MnSOD, SOD2） , and to identify the transcription activity of SOD2 promoter at rest or induced by l ipopolysaccharide （LPS）, NaAsO2, etc in murine embryonic fibroblasts （MEF） by the reporter gene technology. METHODS ： Genomic DNA was extracted from mouse liver, and the promoter sequence of SOD2 （ - 1 554- ＋ 48 ） was amplified by polymerase chain reaction （PCR） method. The red fluorescent protein reporter gene vector driven by SOD2 promoter was constructed by gene recombination technique. The recombinant vector was transiently transfected into MEF cells. The expression of the red fluorescent protein was observed by fluorescent microscopy in MEF cells at rest or stimula- ted by NaAsO2, LPS or phorbol - 12 - myristate - 13 - acetate （PMA）. RESULTS： SOD2 promoter（ - 1 554 - ＋48） of mouse was correctly amplified and the red fluorescent protein reporter gene vector was successfully constructed. It was observed that the MEF cells transfected with reporter gene vector only expressed few and weak red fluorescent protein at rest. However, red fluorescent signals were evidently enhanced after the cells were stimulated by NaAsO2 , LPS or PMA. CONCLUSION： SOD2 promoter（ - 1 554- ＋ 48） of mouse has the transcriptional activity at rest, and the expression of SOD2 has enhanced after the cells are stimulated by inflammatory substance or oxidative stress. The reporter gene vector driven by SOD2 promoter will provide an experimental tool for the further study on the regulatory mechanism of the SOD2 expression.