pmCherry-C1-Argonaute1重组质粒的构建及鉴定

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He La细胞总RNA,反转录出含Argonaute1的c DNA;以Argonaute1 c DNA为模板,采用PCR法扩增出带有EcoRⅠ和Bam HⅠ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He La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...

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Bibliographic Details
Published in山东医药 Vol. 56; no. 12; pp. 9 - 12
Main Author 崔晓腾 高星杰 张春燕 付雪 苏超 任媛媛 杨洁
Format Magazine Article
LanguageChinese
Published 天津医科大学,天津,300070 2016
Subjects
pmCherry-C1
人类Argonaute1蛋白
应激颗粒
融合蛋白
重组质粒
human Argonaute1 protein
fusion protein
应激颗粒
人类Argonaute1蛋白
重组质粒
pmCherry-C1
融合蛋白
stress granules
recombinant plasmid
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Summary: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He La细胞总RNA,反转录出含Argonaute1的c DNA;以Argonaute1 c DNA为模板,采用PCR法扩增出带有EcoRⅠ和Bam HⅠ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He La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、Bam HⅠ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。
Bibliography:37-1156/R
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 c DNA containing Argonaute1 was inverse transcribed. Argonaute1 fragment containing EcoRI and Bam HI site was amplified by PCR from purified tamplate from He La cell line and inserted into pmCherry-C1 fluorescent expressing vector. The recombinant pmCherry-C1-Argonaute1 plasmid was transfected into He La cells and the expression of red fluorescent fusion proteins was examined by Western blotting and the co-localization 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 identified by EcoRI,Bam HI,the restriction single / double enzyme digestion and gene
ISSN:1002-266X
DOI:10.3969/j.issn.1002-266X.2016.12.003