Cellular Localization Analysis of Enhanced Green Fluorescent Protein Tagged hnRNP A1 Under Stress
10.3969/j.issn.0253-9896.2014.06.003
- VernacularTitle:hnRNP A1的增强型绿色荧光标记及细胞应激定位分析
- Author:
Xingjie GAO
;
Juan SONG
;
Lin GE
;
Xue FU
;
Xiaoming SUN
;
Wei ZHANG
;
Jinyan HE
;
Zhi YAO
;
Jie YANG
- Publication Type:Journal Article
- Keywords:
heterogeneous-nuclear ribonucleoproteins;
green fluorescent proteins;
membrane fusion proteins;
human hnRNP A1 protein;
pEGFP-C1;
fusion protein;
stress granules
- From:
Tianjin Medical Journal
2014;(6):522-525
- CountryChina
- Language:Chinese
-
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 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.
