泛素化调节因子2真核表达质粒的构建及其在原代肾小管上皮细胞的表达

›› 2013, Vol. 40 ›› Issue (6): 37-40.

泛素化调节因子2真核表达质粒的构建及其在原代肾小管上皮细胞的表达

高磊, 赵海焦, 吴金栋, 田娅慧, 张中文, 吴国娟

北京农学院动物科学技术学院, 北京 102206

收稿日期:2012-12-21 出版日期:2013-06-20 发布日期:2013-06-20
通讯作者: 吴国娟;张中文 E-mail:wgj9288@sina.com;zwzhang9288@sina.com
作者简介:高磊(1986-),男,山东人,硕士生,研究方向:中药药理和毒理。
基金资助:
国家自然科学基金(31172362、30972215);北京市自然科学基金(5102014);北京市人才强教深化计划(教学创新人才)。

Construction of Eukaryotic Expression Vector for Smurf2 Gene and its Expression in Renal Tubular Epithelial Cells

GAO Lei, ZHAO Hai-jiao, WU Jin-dong, TIAN Ya-hui, ZHANG Zhong-wen, WU Guo-juan

Department of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China

Received:2012-12-21 Online:2013-06-20 Published:2013-06-20

摘要/Abstract

摘要： 本试验旨在构建带有增强型绿色荧光蛋白报告基因EGFP及目的基因泛素化调节因子2(Smurf2)的真核表达载体pEGFP-N3-Smurf2,并转染小鼠肾小管上皮细胞检测其表达情况。用RT-PCR法扩增小鼠肾小管上皮细胞Smurf2的CDS区,构建克隆载体pEASY-Zero-Smurf2和真核表达载体pEGFP-N3-Smurf2,酶切并测序鉴定。运用脂质体转染法将pEGFP-N3-Smurf2真核表达载体转染小鼠肾小管上皮细胞,实时荧光定量PCR、免疫印迹检测Smurf2的表达。PCR结果显示扩增出Smurf2基因片段大小约为2247 bp;重组质粒pEGFP-N3-Smurf2的酶切鉴定及测序结果均表明Smurf2基因成功克隆到真核表达载体中;脂质体转染后检测结果显示,转染pEGFP-N3-Smurf2表达载体的肾小管上皮细胞中Smurf2基因mRNA表达量升高且EGFP-Smurf2重组蛋白有表达。本试验成功构建了含有增强型绿色荧光蛋白标记的Smurf2真核表达质粒,转染后能明显提高细胞内Smurf2的表达,为研究Smurf2基因的功能奠定了基础。

关键词: 泛素化调节因子2; pEGFP-N3; 小鼠肾小管上皮细胞; 质粒; 表达

Abstract: The assay was aimed to construct the eukaryotic expression vector pEGFP-N3-Smurf2 carrying Smad ubiquitination regulatory factor 2 (Smurf2) and detect its expression after transfecting to renal tubular epithelial cells (RTECs).The CDS of Smurf2 gene was amplified and cloned from total RNA of RTECs by RT-PCR.Both prokaryotic expression vector pEASY-Zero-Smurf2 and eukaryotic expression vector pEGFP-N3-Smurf2 were constructed and then detected by double digestion and sequencing.pEGFP-N3-Smurf2 was transfected into RTECs with Lipofectamine^TM2000 and the expression of Smurf2 was detected by Real-time PCR and Western blotting.The amplified Smurf2 gene was about 2247 bp in length.Restriction analysis and sequencing proved that Smurf2 was successfully inserted into recombinant plasmid pEGFP-N3.The mRNA and protein of Smurf2 were detected by Real-time PCR and Western blotting.Smurf2 expression vector using green fluorescent protein as a reporter gene was constructed and transfected into RTECs successfully,which provided a powerful tool for the further study of function of Smurf2 gene.

Key words: Smad ubiquitination regulatory factor 2; pEGFP-N3; renal tubular epithelial cell; plasmid; expression

中图分类号:

Q786

高磊, 赵海焦, 吴金栋, 田娅慧, 张中文, 吴国娟. 泛素化调节因子2真核表达质粒的构建及其在原代肾小管上皮细胞的表达[J]. , 2013, 40(6): 37-40.

GAO Lei, ZHAO Hai-jiao, WU Jin-dong, TIAN Ya-hui, ZHANG Zhong-wen, WU Guo-juan. Construction of Eukaryotic Expression Vector for Smurf2 Gene and its Expression in Renal Tubular Epithelial Cells[J]. , 2013, 40(6): 37-40.

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