小鼠干扰素调节因子3启动子质粒的构建及其启动子活性分析  

Construction of plasmid containing promoter of mouse interferon regulatory factor 3 and analysis of its promoter activity

作　　者：刘立飞 杨婷婷 徐华国 周国平 

机构地区：[1]南京医科大学第一附属医院儿科,江苏省210029

出　　处：《江苏医药》2012年第2期134-136,共3页Jiangsu Medical Journal

基　　金：国家自然科学基金(30872804);江苏省医学重点人才项目资助(RC2007050)

摘　　要：目的构建包含小鼠干扰素调节因子3(mIRF-3)基因启动子的质粒,并评价其启动子活性。方法以小鼠全血细胞总DNA为模板,PCR扩增mIRF-3目的片段,亚克隆此片段至pGL3-basic荧光素酶报告基因的多克隆位点,构建含mIRF-3启动子的重组报告质粒pmIRF-3。将pmIRF-3、pGL3-basic和pGL3-control质粒分别转染小鼠胚胎成纤维细胞NIH3T3后,行荧光素酶活性检测,计算相对活性单位(RLU)。生物信息学分析转录因子结合位点。结果成功构建了重组报告质粒pmIRF-3。与pGL3-basic组相比,pmIRF-3、pGL3-control组RLU明显增加(0.443±0.113vs.18.907±3.335、25.704±5.850)(P<0.01)。mIRF-3启动子区域内存在AML-1a、E2F、MZF1、CdxA、OCT-x等多个转录因子结合位点。结论 mIRF-3转录起始位点附近1479bp序列在NIH3T3细胞中具有较强的启动子活性。Objective To construct a plasmid containing the promoter of mouse interferon regulatory factor 3（mIRF-3） gene and evaluate its promoter activity.Methods The mIRF-3 fragment was amplified by PCR with mouse genomic DNA from total blood cells as a template and was directionally subcloned into the multiple cloning sites of pGL3-basic luciferase reporter gene to construct recombinant pmIRF-3 reporter plasmid containing mIRF-3 promoter.Transfection of mouse fibroblast cells NIH3T3 with the pmIRF-3,pGL3-basic and pGL3-control plasmids was performed to induce luciferase gene expression and calculate the relative luciferase activity unit（RLU）.The binding sites of transcription factor were analyzed by bioinformatics.Results It was successful to construct the recombinant pmIRF-3 reporter plasmid.Compared with group pGL3-basic,the RLU was significantly increased in groups of pmIRF-3 and pGL3-control（0.443±0.113 vs.18.907±3.335 and 25.704±5.850）（P〈0.01）.Sequence analysis revealed that AML-1α,E2F,MZF1,CdxA and OCT-x transcription factors might be involved in the mIRF-3 promoter region.Conclusion The onset site of mIRF-3 transcription with a sequence of 1479 bp has a strong promoter activity in NIH3T3 cells.

关 键 词：干扰素调节因子3 启动子活性 

分 类 号：R34[医药卫生—基础医学]