天津医药

• 细胞与分子生物学 • 上一篇    下一篇

hnRNP A1的增强型绿色荧光标记及细胞应激定位分析

高星杰1,宋娟2,葛林3,付雪1,孙晓明4,张纬5,何津岩2,姚智6,杨洁7   

  1. 1. 天津医科大学基础医学研究中心
    2. 天津医科大学免疫教研室
    3. 天津医科大学基础医学院免疫学教研室
    4. 天津医科大学生物化学教研室
    5. 生化教研室
    6.
    7. 天津医科大学
  • 收稿日期:2013-11-14 修回日期:2014-02-10 出版日期:2014-06-15 发布日期:2014-06-15
  • 通讯作者: 宋娟

Cellular Localization Analysis of Enhanced Green Fluorescent Protein Tagged hnRNP A1 Under Stress Condition

  • Received:2013-11-14 Revised:2014-02-10 Published:2014-06-15 Online:2014-06-15

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摘要： 目的 构建包含有核不均一核糖核蛋白（hnRNP）A1蛋白编码区序列的真核表达质粒pEGFP-C1-hnRNP A1，并对EGFP标记的hnRNP A1蛋白进行细胞应激共定位分析。方法 提取HeLa细胞总RNA，以针对hnRNPA1-3'非翻译区的特异性片段为反转录引物，反转录出包含hnRNP A1编码区序列的cDNA，并以其为模板，降落PCR法扩增出带EcoRI和BamHI双酶切位点的目的基因，利用双酶切法分别酶切目的基因片段和线性pEGFP-C1，在T4-DNA连接酶的催化下将两者连接构建成pEGFP-C1-hnRNP A1重组质粒，然后将重组质粒转染入HeLa细胞内，以激光共聚焦荧光显微镜观察EGFP-hnRNP A1的荧光表达情况，Western印迹法检测EGFP与hnRNP A1的融合表达情况，最后进行细胞原位杂交及细胞免疫荧光实验检测在氧化应激状态下EGFP-hnRNP A1蛋白与poly(A)+ mRNA（应激颗粒的标记蛋白）及DPC1a（加工体的标记蛋白）的应激共定位。结果 以单/双酶切及基因测序法鉴定构建的重组质粒无误，激光共聚焦荧光显微镜观察和Western印迹结果检测到绿色荧光融合蛋白的表达；EGFP-hnRNP A1蛋白与poly(A)+ mRNA呈现共定位，但与DPC1a无共定位关系。结论 重组pEGFP-C1-hnRNP A1质粒成功构建并表达，应激状态下EGFP标记的hnRNP A1参与应激颗粒的构成

关键词: hnRNP A1蛋白, pEGFP-C1, 融合蛋白, 应激颗粒

Abstract: Objective:To construct eukaryotic enhanced green fluorescent protein(EGFP) expressing recombinant plasmid,pEGFP-C1-hnRNP A1,which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1),and perform the cellular localization analysis of EGFP tagged hnRNP A1 under stress condition. Methods:The total RNA was isolated from HeLa cell and used for the first-strand cDNA synthesis with reverse primers specific for the 3'-untranslated region of hnRNP A1.The gene of hnRNP A1 fragment was then amplified by touch-down PCR from the cDNA and inserted into pEGFP-C1 fluorescent expressing vector through EcoRI/BamHI double enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and the expression of green fluorescent fusion proteins was examined by Western blotting assay and confocal fluorescence microscopy. RNA Fluorescence in situ Hybridization and Immunofluorescence assays were also performed to detect the co-localization of EGFP-hnRNP A1 with poly(A)+ mRNA (the marker of the stress granules),or DCP1a (the marker of processing bodies). Results: The pEGFP-C1-hnRNP A1 was sequenced and digested correctly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blotting assay and confocal fluorescence microscopy. EGFP-hnRNP A1 localizes with poly(A)+ mRNA, but not DCP1a. Conclusion:The recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effectively.EGFP tagged hnRNP A1 takes part in the formation of stress granules.

Key words: Human hnRNP A1 protein, pEGFP-C1, fusion protein, stress granules