miR-126报告基因载体的构建及其功能鉴定

目的:根据miR-126的预测靶点构建荧光素酶报告基因重组质粒,并进行功能鉴定。方法:利用sanger数据库提供的miR-126靶序列设计引物,PCR扩增目的微小RNA(microRNAs,miRNAs)靶基因3'非编码区(three-prime untranslated regions,3'UTRs)序列,PCR产物双酶切,后连入经过同样双酶切的pGL3-control载体中,连接产物转化大肠杆菌DH5α,进行阳性克隆鉴定。同样,将候选靶基因3'UTRs突变,突变型3'UTR克隆入pGL3-control报告载体,构建野生型和突变型的报告基因重组质粒。将野生型和突变型的报告基因载体分别和化学合成的microRNA以及内参质粒共转染293TN细胞,进行双荧光素酶检测。结果:成功构建miR-126报告基因野生型和突变型重组质粒pGL3-VEGF-A-3'UTR和pGL3-VEGF-A-3'UTR,质粒测序及酶切结果完全正确。瞬时转染实验显示,过表达miR-126能直接抑制VEGF-A-3'UTRs报告基因活性。结论:miR-126对VEGF-A具有靶向调节功能。

Construction of miR-126 Reporter Gene Vector and Detection of Its Function

Objective： To construct miR-126 luciferase reporter gene vector according to miR-126 predicted target sequences and to detect its function. Methods： The primers were designed and amplified by using the putative target site for miR-126 predicted by Sanger database and inserted at the XbaI site, and immediately downstream of the luciferase gene in the pGL3-control vector. A mutant version with a deletion the site of perfect complementarity was also generated. Wild type and mutant recombinant plasmid for pGL3-vegfa -2-3＇UTR and pGL3-mutvegfa -2-3＇UTR were constructed and confirmed by sequencing. Twenty four hours before transfection, pGL3-vegfa-3＇UTR or pGL3-mutvegfa-3＇UTR plus pRL-TK were transfect alone or in combination with miR-126 mimics. Luciferase activity was measured 24hr after transfection. Results： pGL3-vegfa-3＇UTR and pGL3-mut vegfa-3＇UTR recombined luciferase reporter gene vector were constructed successfully, and the result of sequencing and double digesting of recombined plasrnid were completely correct. The experiment showed that luciferase activity of pGL3-vegfa-3＇UTR reporter gene decreased after miR-126 overexpression. Conclusion： VEGF-A is the target ofmiR-126.