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目的检测锌指蛋白转录抑制因子14(positive regulatory domain zinc finger protein 14,PRDM14)5′非翻译区(5′untranslated region,5′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′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 RLPRDM14 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′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′UTR分别转染HCT-8/WT、Hep-2、Bel7402/WT和NIH3T3细胞,检测各细胞中PRDM14 5′UTR IRES元件的活性。结果 PRDM14 5′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′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′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 detect the internal ribosome entry site (IRES) of 5 ’untranslated region (5’UTR) of the positive regulatory domain zinc finger protein 14 (PRDM14) active. Methods The gene and full-length gene of each truncated sequence of PRDM14 5’UTR were amplified by polymerase chain reaction (PCR) and inserted into dual luciferase and red and green fluorescence reporter gene respectively to construct recombinant plasmid p RLPRDM14 S1-FL and p RL-PRDM14 S2-FL , PRL-PRDM14 S3-FL and p G-PRDM 14-R. The recombinant plasmids were transfected into HEK293 cells respectively to detect the activity of 5’UTR IRES element of PRDM14, the internal cleavage site and its own promoter and the sequence of IRES activity. The recombinant plasmid p RL-FL / 5’UTR was transfected into HCT-8 / WT, Hep-2, Bel7402 / WT and NIH3T3 cells respectively to detect the activity of 5’UTR IRES elements in each cell. Results PRDM14 5’UTR had internal ribosome entry site activity, no internal cleavage site and self promoter activity. PRDM14 5 ’UTR activity was located at 25 ~ 60 nt and activity inhibition center was located at 60 ~ 95 nt ; Except NIH3T3 cells, other cells have IRES activity. Conclusion PRDM14 5’UTR has typical IRES activity, which lays the foundation for further study on the regulatory mechanism of PRDM14 expression in the future.