天津医药

• 细胞与分子生物学 • 上一篇    下一篇

针对人SND1基因两个AUG的细胞应激分析

高星杰1,何津岩2,葛林3,张毅4,付雪1,尹洁1,张纬5,史雪彬1,苏征6,姚智7,杨洁8   

  1. 1. 天津医科大学基础医学研究中心
    2. 天津医科大学生理教研室
    3. 天津医科大学基础医学院免疫学教研室
    4. 天津医科大学免疫教研室
    5. 生化教研室
    6. 天津医科大学基础医学院
    7.
    8. 天津医科大学
  • 收稿日期:2014-02-11 修回日期:2014-03-19 出版日期:2014-07-15 发布日期:2014-07-15
  • 通讯作者: 高星杰

Analysis of Cellular Stress Response in Two AUG of Human SND1 Gene

  • Received:2014-02-11 Revised:2014-03-19 Published:2014-07-15 Online:2014-07-15
  • Contact: Xing-Jie Gao

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摘要： 【摘要】目的 针对人SND1基因2个蛋白翻译起始密码子AUG构建真核表达质粒pCMV-N-Flag-SND1-No1/ 2，并分析2个AUG在SND1应激颗粒形成中的作用。方法 以SND1全长转录本为模板，PCR法扩增含BamHⅠ和 EcoRⅠ酶切位点的目的基因SND1-No1/2，双酶切法分别酶切目的基因片段和线性pCMV-N-Flag，以T4-DNA连接酶将两者连接成pCMV-N-Flag-SND1-No1/2重组质粒，然后将构建的重组质粒转染入HeLa细胞内，以Western印迹法检测Flag标签（DYKDDDDK）与SND1-No1/2的融合表达，最后以细胞免疫荧光实验检测在氧化应激状态下Flag SND1-No1/2融合蛋白与内源性SND1应激颗粒的胞内共定位情况。结果以单/双酶切及基因测序法鉴定构建的重组质粒无误，Western印迹结果检测到融合蛋白Flag-SND1-No1/2的表达；细胞免疫荧光结果显示Flag-SND1-No1/2 均可与内源性SND1应激颗粒共定位。结论重组pCMV-N-Flag-SND1-No1/2质粒构建成功，SND1基因第1个 AUG的缺失并不影响SND1应激颗粒的形成。

关键词: SND1, AUG, 融合蛋白, 应激颗粒

Abstract:

[Abstract] Objective To construct eukaryotic Flag (DYKDDDDK) expressing recombinant plasmids, pCMV- N Flag-SND1-No1/2, which contain the coding sequence of human SND1-No1（from 1 st AUG）orSND1-No2(from 2 nd AUG), and perform the cellular localization analysis of Flag-tagged SND1-No1/2under stress condition to study the function of the two AUG in the SND1containing stress granules formation.Methods The gene fragments ofSND1-No1/2were amplified by PCR from the wholeSND1transcript and inserted into pCMV-N-Flag expressing vector through BamHI/EcoRI double en? zyme digestion and T4DNA Ligase connection. The recombinant pCMV-N-Flag-SND1-No1/2plasmids were transfected in? to HeLa cells and the expression of Flag-SND1-No1/2fusion proteins was examined by Western blotting assay. Immunofluo? rescence assay was performed to detect the co-localization of Flag-SND1-No1/2with endogenous SND1granule.Results The pCMV-N-Flag-SND1-No1/2were sequenced and digested correctly by restriction single/double enzyme. The Flag tagged SND1- No1/2fusion proteins were also detected in transfected HeLa cell by Western blotting assay. Both of them showed the co- localization with endogenous SND1 granule. Conclusion The recombinant eukaryotic plasmids of pCMV-N-Flag-SND1-No1/2were constructed successfully and expressed effectively. The depletion of1 st AUG failed to af? fect the formation of SND1containing stress granules.

Key words: SND1, AUG, fusion protein, stress granules