【摘要】 [目的]构建FBXO47基因3’-UTR双荧光素酶报告载体,并验证miR-33b-5p与FBXO47的靶向关系。[方法]采用生物信息学软件预测miR-33b-5p与FBXO47结合位点;利用PCR扩增FBXO47基因3’-UTR序列,将其克隆到GV272载体中,构建FBXO47野生型及突变型双荧光素酶报告质粒;将miR-33b-5p或阴性对照分别与野生型GV272-FBXO47-wt 3’-UTR或突变型GV272-FBXO47-mut 3’-UTR双荧光素酶报告质粒共转染至293T细胞中,双荧光素酶报告系统检测各组荧光素酶活性。[结果]酶切及测序结果表明,野生型GV272-FBXO47-wt 3’-UTR及突变型GV272-FBXO47-mut 3’-UTR双荧光素酶报告载体构建成功;荧光素酶活性结果表明,相较于对照组,miR-33b-5p可使野生型GV272-FBXO47-wt 3’-UTR的荧光素酶活性降低66%左右;而定点突变GV272-FBXO47-mut 3’-UTR的荧光素酶荧光素酶活性没有显著变化。[结论]成功构建了FBXO47基因3’-UTR的荧光素酶报告基因载体,miR-33b-5p与FBXO47存在靶向性。更多还原

【Abstract】 [Objective]To construct FBXO47 3’-UTR dual luciferase reporter vector,and verify the targeted relationship between miR-33 b-5 p and FBXO47. [Methods] Bioinformatics software was used to predict the binding sites between miR-33 b-5 p and FBXO47. PCR was used to amplify FBXO47 gene 3’-UTR sequence,then cloned it into GV272 vector to construct FBXO47 wild type and mutant dual luciferase report plasmid. Cotransfection of miR-33 b-5 p or negative control with wild type GV272-FBXO47-wt 3’-UTR or mutant GV272-FBXO47-mut 3’-UTR dual luciferase report plasmid to 293 T cells,respectively. Dual luciferase reporter system was used to detect the luciferase activity. [Results] The enzyme digestion and sequencing data showed that the wild type GV272-FBXO47-wt 3’-UTR and mutant GV272-FBXO47-mut 3’-UTR dual luciferase report vector were constructed successfully. Luciferase activity results showed that compared with the control group,miR-33 b-5 p could descrease wild type GV272-FBXO47-wt 3’-UTR luciferase activity about 66%,and site-directed mutagenesis of GV272-FBXO47-mut 3’-UTR luciferase activity do not change significantly compared with control.[Conclusion]The FBXO47 3’-UTR dual luciferase reporter vector was constructed successfully and FBXO47 was a target of miR-33 b-5 p.更多还原