pmCherry-C1-Argonaute1重组质粒的构建及鉴定

摘要

目的 构建并鉴定pmCherry-C1-Argonaute1重组质粒,为深入研究Argonaute1蛋白在细胞应激及相关疾病的生物学作用奠定基础.方法 提取HeLa细胞总RNA,反转录出含Argonaute1的cDNA;以Argonaute1 cDNA为模板,采用PCR法扩增出带有EcoRⅠ和BamHⅠ双酶切的目的基因;采用双酶切法将目的片段连接到pmCherry-C1载体上;将构建成功的pmCherry-C1-Argonaute1重组质粒转染入HeLa细胞内,以Western blotting法检测Argonaute1与樱桃红(Cherry)蛋白的融合表达情况;以激光共聚焦显微镜观察细胞内Argonaute1蛋白与细胞应激颗粒的标记蛋白(G3BP)及加工体的标记蛋白(DCP1α)的应激共定位关系.结果 以EcoRⅠ、BamHⅠ单、双酶切及基因测序法鉴定构建的重组质粒无误;融合蛋白表达阳性;当细胞受到应激时,Argonaute1蛋白与G3BP、DCP1α蛋白共定位.结论 本研究成功构建重组pmCherry-C1-Argonaute1质粒.该质粒可有效表达Cherry标记的Argonaute1融合蛋白,有助于深入研究Argonaute1蛋白在细胞应激及相关疾病领域的生物学作用.%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.