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