中南大学学报(医学版) J Cent South Univ (Med Sci) 2014, 39(8) hp://www.csumed.org 764 IPO8 基因启动子的克隆及转录活性分析 熊建军 1 ,龚帧 1 ,周小鸥 1 ,王庭 1 ,刘建云 2 ,李卫东 2 (1. 九江学院基础医学院药理教研室,江西 九江 332000;2. 江西省系统生物医学重点实验室,江西 九江 332000) [摘要] 目的:克隆IPO8 基因5' 端非编码序列,探讨其转录活性。方法:应用cDNA 5' 末端快速扩增(rapid amplification of cDNA ends,5'CE)方法定位IPO8基因转录起始点;在此基础上针对其5'端非编码区包括转录起始点在 内的–3302 ~ +134区域分段进行PCR扩增,将产物克隆入荧光素酶表达载体pGL3-Basic。经酶切鉴定后,将重组质粒与 内参质粒pRL-TK共转染人骨肉瘤细胞Saos-2,用双荧光素酶活性检测以确定其转录活性。结果:成功确定IPO8基因 转录起始位点,酶切结果证实IPO8基因5'端非编码区6条续减片段正确插入到荧光素酶报告基因载体pGL3-Basic。重组 质粒转染Saos-2细胞后检测到荧光素酶的高表达(P<0.05)。结论:成功构建具有转录活性的IPO8启动子片段,为进一 步研究IPO8表达调控机制奠定了基础。 [关键词] IPO8;启动子;报告基因 Cloning of IPO8 promoter and analysis of its transcription activity XIONG Jianjun 1 , GONG Zhen 1 , ZHOU Xiao’ou 1 , WANG Ting 1 , LIU Jianyun 2 , LI Weidong 2 (1. Department of Pharmacology, College of Basic Medical Science, Jiujiang University, Jiujiang Jiangxi 332000; 2. Key Laboratory of Jiangxi Province for the Systems Bio-medicine, Jiujiang Jiangxi 332000, China) ABSTRACT Objective: To clone 5' untranslated region of human IPO8 gene and determine its transcription activity. Methods: We used 5' rapid amplification of cDNA ends (RACE) analysis to identify the IPO8 transcription start site (TSS), and amplified series truncated 5' UTR fragment containing transcription start site. The PCR productions were inserted into luciferase report vector pGL3- Basic. After confirmation by restriction enzyme digestion, the recombinant plasmids were co- transfected into Saos-2 cells with plasmid pRL-TK. e luciferase activities were measured by dual luciferase reporter system. Results: The IPO8 gene transcription start site was established. The electrophoresis analysis of restriction enzyme digestion and DNA sequencing verified the fragments were successfully 收稿日期(Date of reception):2014-03-10 作者简介(Biography):熊建军,博士,副教授,主要从事基因表达调控方面的研究。 通信作者(Corresponding author):李卫东,Email: [email protected]基金项目(Foundation item):国家自然科学基金(81060075,81260140);江西省教育厅科学研究项目(GJJ12683)。This work was supported by the National Nature Science Foundation of China (81060075, 81260140), and the Key Projects of Jiangxi Provincial Education Department, P. R. China (GJJ12683). DOI:10.11817/j.issn.1672-7347.2014.08.002 www.csumed.org/xbwk/fileup/PDF/201408764.pdf
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中南大学学报(医学版)
J Cent South Univ (Med Sci) 2014, 39(8) http://www.csumed.org764
Cloning of IPO8 promoter and analysis of its transcription activity
XIONG Jianjun1, GONG Zhen1, ZHOU Xiao’ou1, WANG Ting1, LIU Jianyun2, LI Weidong2
(1. Department of Pharmacology, College of Basic Medical Science, Jiujiang University, Jiujiang Jiangxi 332000; 2. Key Laboratory of Jiangxi Province for the Systems Bio-medicine, Jiujiang Jiangxi 332000, China)
ABSTRACT Objective: To clone 5' untranslated region of human IPO8 gene and determine its transcription activity.
Methods: We used 5' rapid amplification of cDNA ends (RACE) analysis to identify the IPO8 transcription start site (TSS), and amplified series truncated 5' UTR fragment containing transcription start site. The PCR productions were inserted into luciferase report vector pGL3-Basic. After confirmation by restriction enzyme digestion, the recombinant plasmids were co-transfected into Saos-2 cells with plasmid pRL-TK. The luciferase activities were measured by dual luciferase reporter system.
Results: The IPO8 gene transcription start site was established. The electrophoresis analysis of restriction enzyme digestion and DNA sequencing verified the fragments were successfully
基金项目(Foundation item):国家自然科学基金(81060075,81260140);江西省教育厅科学研究项目(GJJ12683)。This work was supported by the
National Nature Science Foundation of China (81060075, 81260140), and the Key Projects of Jiangxi Provincial Education Department, P. R. China (GJJ12683).
DOI:10.11817/j.issn.1672-7347.2014.08.002
www.csumed.org/xbwk/fileup/PDF/201408764.pdf
IPO8 基因启动子的克隆及转录活性分析 熊建军,等 765
amplified and inserted into pGL3-Basic. After the recombinant plasmids transfected, the high-expressions of luciferase were detected in Saos-2 cells.
Conclusion: The recombinant vector containing IPO8 promoter is constructed successfully, which provides a foundation for determining expressional regulation of IPO8 in the further study.
KEY WORDS IPO8; promoter; report gene
核 转 运 受 体 包 括 Importin α 和 Importin β 两
个家族,在蛋白的主动运输过程中发挥了决定性
的 作 用。Importin α 和 β 分 别 识 别 不 同 的 蛋 白 核
定位信号,介导不同类型核蛋白入核 [1]。IPO8 是
Importin β 家 族 成 员 之 一, 定 位 于 人 类 常 染 色 体
的 12p11.21,其编码蛋白质的 N 端含有 Ran-GTP结合域,能够与核孔蛋白相互作用介导核蛋白的
约 3.4 kb 片段。正向引物,5'-TTCCACGCGTAATC-AAAGGGTTAGGAATGT-3'(–3 302),反向引物,5'- A A G G A G A T C T C G A C C C C T G G A T TA C C T C -AC-3'(+134)。PCR 产物经琼脂糖凝胶电泳鉴定,
引 物 序 列:5'-TTCCACGCGTGTGGGTCAAGGC-TAGAGTTA-3'(−2 193);5'-TTCCACGCGTTGTGG-CTCGCTTCTTCAGTG-3'(−1 337);5'-TTCCACGC-G T G T G T C A G T C C T C A C C TA G G T- 3 ' ( − 7 3 2 ) ;
5 ' -T TC C A C G C G TC G ATG C C C ATA C A G T TC T-CG-3'(−330);5'-TTCCACGCGTGACGGGAGGCG-GTCATAGC-3'(−97)。 以 上 PCR 产 物 经 琼 脂 糖 凝