NOTCH1基因3＇-UTR段双荧光素酶报告载体的构建及其活性鉴定  

Construction 3′-UTR of NOTCH1 dual luciferase recombintant vector and evaluation of its activity

作　　者：邵新宏[1] 韩渊[2] 于游[3] 张才全[3] 

机构地区：[1]新疆医科大学第五附属医院普外科,乌鲁木齐8300002 [2]新疆医科大学第二附属医院肿瘤外科,乌鲁木齐830000 [3]重庆医科大学附属第一医院胃肠外科,重庆430016

出　　处：《中国免疫学杂志》2013年第3期312-316,共5页Chinese Journal of Immunology

摘　　要：目的:构建含NOTCH1基因3'-UTR段双荧光素酶报告载体,并验证其活性。方法:使用PCR方法扩增含NOTCH1基因3'-UTR区序列,插入到双酶切的双荧光素酶报告载体中;使用生物信息学方法预测可能与NOTCH1基因3'-UTR相互作用的miRNA;使用lipofectamine 2000转染试剂将重组质粒或空质粒和miR-34a inhibitor或control真核表达载体共转染HEK293T细胞,双荧光素酶检测试剂盒测定荧光素酶活性。结果:得到含NOTCH1基因3'-UTR(1 648 bp)序列的双荧光素酶报告重组质粒,并用凝胶电泳和基因测序的方法验证。NOTCH1基因3'-UTR上可能有miR-34a的调控作用靶点;用重组质粒和miR-34a inhibitor共转染的HEK293T组的荧光素酶活性比空质粒组高45%。结论:含NOTCH1基因3'-UTR双荧光素酶报告载体构建成功,miR-34a对NOTCH1基因有调控作用。Objective：To construct the dual-luciferase recombinant vector which contain 3＇-UTR of NOTCH1 gene and verify its activity. Methods：3＇-UTR of NOTCH1 gene was amplified through PCR method, and was inserted in the dual luciferase reporter vector which was digested by enzyme. 3＇-UTR of NOTCH1 which targeting effect miRNA was predicted by bioinformation. HEK293T cells was treated with recombinant vector or empty vector and miR-34a inhibitor or control by transfection reagent. The dual Luciferase Reporter Assay System was used to evaluate the activity of luciferase. Results：3＇-UTR of NOTCH1 gene was successfully cloned into the pmiR-RB-REPORTTM vector, which was vertified by Gel electrophoresis and DNA sequencing methods. 3＇-UTR of NOTCH1 gene may has a binding site of miR-34a. Comparing with HEK293T cells was treated with the empty vector , the luciferase activity of HEK293T cells which was cotransfected with recombinant vector and miRNA-34a inhibitor was increased to 45%. Conclusion：3＇-UTR of NOTCH1 gene dual luciferase combintant vector was successfully constructed, preliminary evidence show that miR-34a could regu- late expression of NOTCH1.

关 键 词：NOTCH1 微小RNA 3'非编码区 双荧光素酶报告载体 基因调控 基因转染 

分 类 号：R392.4[医药卫生—免疫学]