多梳蛋白Nspc1对微管基因Tubulin βⅢ启动子活性的影响  

Effect of polycomb protein Nspc1 on the promoter activity of microtubule gene Tubulin βIII

作　　者：李辉[1] 樊嵘[2] 正午 孙奕[3] 龚燕华[2,4] 

机构地区：[1]武警后勤学院人体解剖与组织胚胎学教研室,天津300309 [2]武警后勤学院生物化学与分子生物学教研室,天津300309 [3]武警后勤学院病原生物学与免疫学教研室,天津300309 [4]天津市心血管重塑与靶器官损伤重点实验室,天津300162

出　　处：《武警后勤学院学报（医学版）》2015年第4期253-256,共4页Journal of Logistics University of PAP（Medical Sciences）

基　　金：国家自然科学基金项目(31070929;81201757);天津市自然科学基金青年项目(13JCQNJC09600);武警后勤部项目(WJHQ2012-13);天津市心血管重塑与靶器官损伤重点实验室开放基金项目(TJC1406)

摘　　要：【目的】克隆TubulinβIII基因的启动子,插入荧光素酶报告基因载体中并检测多梳蛋白Nspc1对其活性的影响。【方法】提取小鼠畸胎瘤细胞系P19的DNA,PCR扩增TubulinβIII近端启动子片段,构建2个不同长度的p GL3basic-TubulinβIII荧光素酶报告基因载体,双酶切及测序确定所扩增的DNA序列,将重组的报告基因载体与转录调控因子多梳蛋白Nspc1瞬时共转染小鼠畸胎瘤细胞系P19细胞,使用双荧光素酶报告基因检测系统检测Nspc1对TubulinβIII启动子活性的影响。【结果】鉴定结果表明构建的TubulinβIII启动子序列正确,P19细胞中双荧光素酶活性实验表明构建的报告基因具有启动子活性,并对转录调控因子Nspc1有应答。【结论】成功构建了TubulinβIII荧光素酶报告基因载体,为进一步在多能干细胞分化模型中研究多梳蛋白Nspc1对TubulinβIII基因的表达调控机制奠定了重要基础。【Objective】To clone TubulinβIII promoter and insert it into a luciferase reporter gene vector, and to characterize the promoter activity.【Methods】The promoter of Tubulin βIII was amplified from mouse embryonic stem cell P19 genomic DNA by PCR and cloned into p GL3 basic. The p GL3basic- Tubulin βIII plasmid was confirmed by double digestion and DNA sequencing. The luciferase activity of P19 cells transiently co-transfected by p GL3basic-Tubulin βIII and a transcriptional regulator polycomb protein Nspc1 was measured.【Results】The sequence of the cloned Tubulin βIII promoter was correct. The promoter activity of the p GL3 basicTubulin βIII was confirmed by Dual-luciferase assay, and its luciferase activity responded to the transcriptional regulator Nspc1 in P19 cells.【Conclusions】Tubulin βIII promoter luciferase reporter gene vector was cloned successfully, and this will be an important basis for researching the regulation mechanism of Tubulin βIII by polycomb protein Nspc1 in pluripotent stem cells.

关 键 词：多梳蛋白 TubulinβIII启动子 荧光素酶报告基因 转录活性 

分 类 号：Q78[生物学—分子生物学]