中国康复理论与实践2017，Vol.23Issue(2)：166-172,7.DOI:10.3969/j.issn.1006-9771.2017.02.010
大鼠细胞外信号调节激酶1基因3'非编码区双荧光素酶报告载体的构建及rno-miR-15b-5p对其活性的影响
Construction of Rat Extracellular Signal-regulated Kinase 1 Gene 3' Untranslated Regions Dual-luciferase Re-porter Plasmids and Effect of rno-miR-15b-5p on Its Activitiy
摘要
Abstract
Objective To construct dual-luciferase reporter plasmids containing the wild type and mutant rat extracellular signal-regulat-ed kinase 1 (ERK1) gene 3' untranslated regions (UTR) which were used to detect rno-miR-15b-5p's putative target gene. Methods The rat ERK1 gene 3' UTR fragment was amplified by polymerase chain reaction (PCR) from PC12 cell cDNA and cloned into pmiR-RB-ReportTM vector. The mutant rat ERK1 gene 3' UTR fragment was obtained by overlap PCR and inserted into pmiR-RB-ReportTM vector. Successful wild type and mutant recombinant plasmids were confirmed by DNA sequencing. PC12 cells were co-transfected with rno-miR-15b-5p mim-ic and pmiR-ERK13' UTR or pmiR-ERK1-mut 3' UTR and then analyzed by dual-luciferase reporter assay system. The achieved mutation sequence of the target site TGCTGCT was mutated to CGAACGT and GTACACG, respectively. Results The wild-type reporter vector pmiR-ERK13' UTR and the mutant reporter vector pmiR-ERK1-mut 3' UTR were successfully constructed. The rno-miR-15b-5p mimic de-creased the activity of pmiR-ERK13' UTR plasmid (P<0.001) but did not decrease the activity of pmiR-ERK1-mut 3' UTR plasmid. Conclu-sion The recombinant pmiR-ERK13' UTR and pmiR-ERK1-mut 3' UTR plasmids were constructed successfully, and luciferase activities demonstrated that the 3' UTR of ERK1 gene might be a potential target of rno-miR-15b-5p.关键词
rno-miR-15b-5p/细胞外信号调节激酶1/重叠延伸聚合酶链式反应/pmiR-ERK13'UTR/pmiR-ERK1-mut3'UTR/荧光素酶活性检测Key words
rno-miR-15b-5p/extracellular signal-regulated kinase 1/overlap polymerase chain reaction/pmiR-ERK13' UTR/pmiR-ERK1-mut 3' UTR/luciferase reporter assay分类
医药卫生引用本文复制引用
罗汉将,徐云峰,李晓晓,杨予涛,徐志卿..大鼠细胞外信号调节激酶1基因3'非编码区双荧光素酶报告载体的构建及rno-miR-15b-5p对其活性的影响[J].中国康复理论与实践,2017,23(2):166-172,7.基金项目
1.北京市自然科学基金面上项目(No.7162016) （No.7162016）
2.国家自然科学基金面上项目(No.31271154 （）
No.31171032). （）
