CAJ | 학술논문

目的：利用 pmirGLO Dual-Luciferase miRNA Target Expression Vector(简称为pmirGLO 报告基因载体)构建并鉴定含 miR-497野生及突变结合位点的胰岛素受体 mRNA 3′UTR 区报告基因载体(pmir-Insr-3′UTR 及 pmir-mutant-Insr-3′UTR)。方法以大鼠肝脏 cDNA为模板,PCR 获取目的片段(即含 miR-497野生及突变结合位点的胰岛素受体 mRNA 3′UTR区)；用PmeI、XbaI双酶切 pmirGLO 报告基因载体和含 miR-497野生及突变结合位点的胰岛素受体 mRNA 3′UTR区,用T4 DNA连接酶连接纯化后的酶切产物；连接产物转化DH5α大肠杆菌感受态细胞并挑选阳性克隆,并通过PCR、双酶切、DNA测序鉴定构建的重组质粒。结果 PCR和双酶切证实pmir-Insr-3′UTR及 pmir-mutant-Insr-3′UTR重组载体中均插入目的片段；DNA测序结果进一步证实 pmir-Insr-3′UTR重组载体中成功插入了胰岛素受体mRNA 3′UTR区,pmir-mutant-Insr-3′UTR重组载体中成功插入了在miR-497结合位点含有3个突变碱基的胰岛素受体 mRNA 3′UTR 片段。结论成功构建了含 miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR报告基因载体 pmir-Insr-3′UTR及 pmir-mutant-Insr-3′UTR。
목적：이용 pmirGLO Dual-Luciferase miRNA Target Expression Vector(간칭위pmirGLO 보고기인재체)구건병감정함 miR-497야생급돌변결합위점적이도소수체 mRNA 3′UTR 구보고기인재체(pmir-Insr-3′UTR 급 pmir-mutant-Insr-3′UTR)。방법이대서간장 cDNA위모판,PCR 획취목적편단(즉함 miR-497야생급돌변결합위점적이도소수체 mRNA 3′UTR구)；용PmeI、XbaI쌍매절 pmirGLO 보고기인재체화함 miR-497야생급돌변결합위점적이도소수체 mRNA 3′UTR구,용T4 DNA련접매련접순화후적매절산물；련접산물전화DH5α대장간균감수태세포병도선양성극륭,병통과PCR、쌍매절、DNA측서감정구건적중조질립。결과 PCR화쌍매절증실pmir-Insr-3′UTR급 pmir-mutant-Insr-3′UTR중조재체중균삽입목적편단；DNA측서결과진일보증실 pmir-Insr-3′UTR중조재체중성공삽입료이도소수체mRNA 3′UTR구,pmir-mutant-Insr-3′UTR중조재체중성공삽입료재miR-497결합위점함유3개돌변감기적이도소수체 mRNA 3′UTR 편단。결론성공구건료함 miR-497야생급돌변결합위점적이도소수체mRNA 3′UTR보고기인재체 pmir-Insr-3′UTR급 pmir-mutant-Insr-3′UTR。
Objective To construct and identify the reporter gene vectors of the 3′UTR region of insulin re-ceptor mRNA containing the wild or mutant miR-497 binding site (pmir-Insr-3′UTR and pmir-mutant-Insr-3′UTR) using pmirGLO dual-Luciferase miRNA target expression vector (pmirGLO report gene vector).Methods The target fragments,which were the 3′UTR region of rat insulin receptor mRNA,including wild or mutant miR-497 binding site,were obtained by PCR using rat liver cDNA as template.The target fragments and pmirGLO report gene vector were cut by PmeI and XbaI restriction endonucleases and then the purified enzyme-digested products were ligated together with a T4 DNA ligase.Subsequently,the ligation productions were used to transform DH5αcompetent cells,and the positive clones were picked up from the agarose plate with ampicillin.Finally,the recombi-nant plasmids were identified through PCR,double-restrict-enzyme digestion of PmeI and XbaI and DNA sequence. Results PCR and double-restrict-enzyme digestion of PmeI and XbaI confirmed that the recombinant vectors of pmir-Insr-3′UTR and pmir-mutant-Insr-3′UTR successfully inserted into the target fragment respectively.The anal-ysis of DNA sequence further confirmed that pmir-Insr-3′UTR successfully inserted the 3′UTR region of rat insulin receptor mRNA,and pmir-mutant-Insr-3′UTR successfully inserted the 3’UTR region of rat insulin receptor mRNA containing three base mutation in miR-497 binding site.Conclusion We successfully constructed pmir-Insr-3′UTR and pmir-mutant-Insr-3′UTR,the reporter gene vectors of the 3′UTR region of insulin receptor mRNA containing wild or mutant miR-497 binding site.