锌指蛋白转录抑制因子145′非翻译区内部核糖体进入位点的活性  

Activity of internal ribosome entry site in 5′untranslated region of positive regulatory domain zinc finger protein 14

作　　者：丁鹏鹏[1] 马文囡 朱瑞宇[1] 陈蕴[1] 金坚[1] 

机构地区：[1]江南大学药学院药物设计与分子药理学实验室,江苏无锡214122

出　　处：《中国生物制品学杂志》2016年第1期23-27,30,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81101667);江苏省基础研究计划(自然科学基金)资助项目(BK2009-071)

摘　　要：目的 检测锌指蛋白转录抑制因子14（positive regulatory domain zinc finger protein 14,PRDM14）5忆非翻译区（5忆untranslated region,5忆UTR）内部核糖体进入位点（internal ribosome entry site,IRES）的活性。方法 PCR扩增PRDM14 5忆UTR各截短序列的基因及全长基因,分别插入至双荧光素酶和红绿荧光报告基因中,构建重组质粒p RL-PRDM14 S1-FL、p RL-PRDM14 S2-FL、p RL-PRDM14 S3-FL及p G-PRDM 14-R。将各重组质粒分别转染HEK293细胞,检测PRDM14 5忆UTR IRES元件的活性、内部剪切位点和自身启动子的活性及影响IRES活性的序列。将重组质粒p RL-FL/5忆UTR分别转染HCT-8/WT、Hep-2、Bel7402/WT和NIH3T3细胞,检测各细胞中PRDM14 5忆UTR IRES元件的活性。结果 PRDM14 5忆UTR具有内部核糖体进入位点元件活性;不具有内部剪切位点和自身启动子活性;PRDM14 5忆UTR的活性激活中心位于25~60 nt,活性抑制中心位于60~95 nt;除NIH3T3细胞外,其他细胞均具有IRES活性。结论 PRDM14 5忆UTR具有典型的IRES活性,为今后深入研究PRDM14表达的调控机制奠定了基础。Objective To determine the activity of internal ribosome entry site（IRES）in 5′ untranslated region（UTR）of positive regulatory domain zinc finger protein 14（PRDM 14）. Methods The truncated and full-length genes of PRDM14 5′UTR were amplified by PCR and inserted to vector p RL-FL containing dual-luciferase reporter gene. The constructed recombinant plasmids p RL-PRDM14 S1-FL, p RL-PRDM14 S2-FL, p RL-PRDM14 S3-FL and p G-PRDM 14-R were transfected to HEK293 cells respectively, and the activities of PRDM14 5′ UTR IRES, internal cleavage site and self promoter, as well as the sequence influencing IRES activity, were determined. HCT-8 / WT, Hep-2, Bel7402 / WT and NIH3T3 cells were transfected with recombinant plasmid p RL-FL / 5′ UTR respectively, in which the activity of PRDM145′ UTR IRES was determined. Results PRDM 14 5′UTR showed IRES activity while no internal cleavage site activity or self promoter activity. The activation and inhibition centers were located at 25 ~ 60 and 60 ~ 95 nt respectively. All the selected tumor cell lines showed IRES activity. Conclusion PRDM 14 5′ UTR showed typical IRES activity, which laid a foundation of furthers study on mechanism in expression and regulation of PRDM14.

关 键 词：锌指蛋白转录抑制因子14 内部核糖体进入位点 萤火虫荧光素酶 海肾荧光素酶 

分 类 号：Q74[生物学—分子生物学]