Arkadia和UCH37表达载体的构建及其对TGF-beta;1_Smads信号通路的影响.pdfVIP

中国农业科学 2012,45(9): 1848-1856 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2012.09.021 Arkadia 和UCH37表达载体的构建及其对TGF-β1/Smads信号通 路的影响 廖芳芳, 苑嗣纯, 张中文, 吴国娟 （北京农学院动物科技学院，北京102206） 摘要：【目的】构建泛素启动酶Arkadia 和泛素羧基末端水解酶-5 （亦称UCH37）的表达载体，并探讨Smad7 蛋白的泛素化修饰对马兜铃酸肾病中 TGF- β1/Smads 信号通路的影响。【方法】通过观察绿色荧光的表达量及 real-time PCR 反应优化转染条件，转染Arkadia 和UCH37 siRNA，real-time PCR 检测mRNA 表达抑制率。分别 构建Arkadia 和UCH37 的表达载体pGPU6/GFP/Neo，经鉴定后转染细胞，real-time PCR 和Western blotting 检 测Arkadia、UCH37 和Smad7 的表达变化。【结果】①30 pmol siRNA 和 1.5 μL LipofectamineTM 2000 为最佳转 染条件；②Arkadia-1490和UCH37-721 的干扰效果最明显；③载体的插入序列完全正确，且pGPU6/GFP/Neo-Arkadia 能上调马兜铃酸肾病模型中Smad7 的表达，而pGPU6/GFP/Neo-UCH37 使Smad7 表达下降。【结论】成功构建了表 达载体pGPU6/GFP/Neo-Arkadia 和pGPU6/GFP/Neo-UCH37，且Arkadia 和UCH37 可分别放大、抑制TGF- β1/Smads 信号通路。 关键词：Arkadia 基因; UCH37 基因; 转染; 表达载体; TGF- β1/Smads 信号通路 Constructions of Arkadia and UCH37 Expression Vectors and Effects on TGF-β1/Smad7 Signal Pathway LIAO Fang-fang, YUAN Si-chun, ZHANG Zhong-wen, WU Guo-juan (College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206) Abstract: 【Objective 】The objective of the study is to construct expression vectors of ubiquitination starting enzyme Arkadia and ubiquitin COOH-terminal hydmlase-L5 (also known as UCH37), and to investigate the effect of Smad7 modified by ubiquitination on TGF-β1/Smad7 signal pathway in aristolochic acid nephropathy (AAN). 【Method 】The transfection condition was optimized by observing expressions of green fluorescences and undertaking real-time PCR reactions. After transferring the siRNA of Arkadia and UCH37, mRNA inhibition ratios were