摘要
目的利用GFP—MS2系统在活细胞内对胰岛素样生长因子结合蛋白2（insulin-hke growth factor，bindin gprotein2，IGFBP2）mRNA3’非翻译区（3’untranslated region，3’UTR）进行绿色荧光标记，构建pT-／-IGFBP2-3’UTR-24×MS2重组质粒。方法提取HeLa细胞总RNA，以针对IGFBP2-3’非翻译区的特异性片段为反转录引物，反转录出cDNA，并以其为模板，降落PCR法扩增出带有EcoRI和BamHI双酶切位点的1GFBP2—3’UTR目的基因，再利用双酶切法将目的片段连接到psG5载体（启动子为T7）上，构建重组质粒pT-／．IGFBP2-3’UTR；同时。以BglII和BamHI双酶切法从pCR4-24xMS2SL．stable质粒上切下24×MS2结合位点片段并将其插入pT7-IGFBP2-3’UTR，构建重组质粒pT7-IGFBP2-3’UTR-24×MS2；然后将构建的重组质粒pT7-IGFBP2-3’UTR-24×MS2、pMS2．GFP质粒与pERFP-Tudor-SN质粒共同转染人HeLa细胞内，以激光共聚焦荧光显微镜检测IGFBP2—3’UTR的荧光标记情况及应激共定位。结果以酶切质粒法及基因测序法鉴定构建的重组质粒均无误．激光共聚焦结果显示在胞浆中检测到IGFBP2．3’UTR的绿色荧光信号，在细胞受到氧化应激时IGFBP2—3’UTR信号呈颗粒状聚集，且与应激颗粒（stressgranules，SGs）的标志蛋白Tudor-SN呈共定位关系。结论针对IGFBP2—3’UTR的GFP-MS2荧光标记系统质粒构建成功；细胞应激时，ICFBP2-3’UTR被招募至sGs结构中。
Objective To construct the eukaryotic recombinant plasmid pT7-IGFBP2-3＇UTR-24 × MS2, tagging the 3＇ untranslated region （3＇UTR） of insulin-like growth factor-binding protein 2 （IGFBP2） mRNA with green fluorescent protein （GFP） via GFP-MS2 system. Methods The total RNA was isolated from HeLa cells and used for the first-strand cDNA synthesis with reverse prim- ers specific for the 3＇UTR of IGFBP2. IGFBP2-3＇UTR fragment was then amplified by touch-down PCR from the cDNA and inserted into empty pSG5 vector （T7 promoter） at Eco RI and BamHI sites to generate the pTT-IGFBP2-3＇ UTR. 24 × MS2 stem loop repeats derived from the pCR4-24 x MS2SL-stable plasmid were subsequently inserted into the BglII and BamH I sites of pTT-IGFBP2- 3＇ UTR to generate pTT-IGFBP2-3＇ UTR-24 x MS2 plasmid, which was then co-transfected with pMS2-GFP and pERFP-Tudor-SN plasmids into HeLa cells. Image data were then collected by the confocal microscopy. Results The pT7-IGFBP2-3＇UTR-24 × MS2 plasmid was sequenced and correctly digested with the restriction enzyme. The GFP tagged IGFBP2-3＇UTR was located in the cytoplasm of HeLa cells under normal condition and aggregated to form a granule structure under oxidative stress, which colocalized with the stress granules （SGs） marker protein Tudor-SN. Conclusion The recombinant eukaryotic plasmid pT7-IGFBP2-3＇ UTR-MS2 was successfully constructed. IGFBP2-3＇UTR was recruited into the SGs structure under oxidative stress condition.
出处
《医学分子生物学杂志》
CAS
2014年第1期7-11,16,共6页
Journal of Medical Molecular Biology
基金
国家杰出青年基金项目（No.31125012）,国家自然科学基金（No.31100967,31170830,81202102,21305103,31370749）,天津市高等学校科技发展基金计划项目（No.20110104）,中国博士后科学基金（No.2013T60258）
