hnRNP A1的增强型绿色荧光标记及细胞应激定位分析

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

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,并对增强型绿色荧光蛋白(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(应激颗粒的标记成分)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-hnRNPA1质粒成功构建并表达,应激状态下EGFP标记的hnRNPA1参与应激颗粒的构成。

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 ribonucleo-protein A1）, and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly （A）＋mRNA （the marker of the stress granules）, or DCP1a （the marker of processome） were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly（A）＋mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.