Construction and identification of recombinant plasmid pmCherry-C1-Argonaute1
CUI Xiaoteng
GAO Xingjie
ZHANG Chunyan
FU Xue
SU Chao
REN Yuanyuan
YANG Jie
Abstract：Objective To construct and identify the recombinant plasmid pmCherry-C1-Argonaute1 and to provide the basis for further research on the biological role of Argonaute1 protein in the field of cell stress and its related diseases. Methods The total RNA was extracted from Hela cells and cDNA containing Argonaute1 was inverse transcribed.Argo-naute1 fragment containing EcoRI and BamHI site was amplified by PCR from purified tamplate from HeLa cell line and in-serted into pmCherry-C1 fluorescent expressing vector.The recombinant pmCherry-C1-Argonaute1 plasmid was transfected into HeLa cells and the expression of red fluorescent fusion proteins was examined by Western blotting and the co-localiza-tion in stress condition of Argonaute1 and G3BP ( biomarker in stress granules) and DCP1α( biomarker in processing bodies) was detected by fluorescence microscope.Results The recombinant plasmid was sequenced correctly which was i-dentified by EcoRI, BamHI, the restriction single/double enzyme digestion and gene sequencing.The red fluorescent fu-sion protein was also detected in transfected HeLa cell by Western blotting.The result of three-color immunofluorescence co-localization indicated that Cherry-red labeled Argonaute1 was co-localized with G3BP and DCP1αin cells under stress condition.Conclusions The recombinant eukaryotic plasmid of pmCherry-C1-Argonaute1 is constructed successfully and expresses Cherry-red labeled Argonaute1 fusion protein effectively .These results will help to study the biological role of Argonaute1 in cell stress and related diseases.
Keywords：human Argonaute1 proteinpmCherry-C1recombinant plasmidfusion proteinstress granules
Publication Date：2016-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 9-12 )