| 虢灿杰卞兆连盛黎马雄△.miR-126报告基因载体的构建及其功能鉴定*[J].,2014,14(5):801-804 |
| miR-126报告基因载体的构建及其功能鉴定* |
| Construction of miR-126 Reporter Gene Vector andDetection of Its Function |
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| DOI： |
| 中文关键词: miR-126 荧光素酶报告基因 靶基因 |
| 英文关键词: miR-126 Luciferase reporter gene Target gene |
| 基金项目:国家自然科学基金青年科学基金项目(81100296）；上海市青年科技启明星
计划项目（13QA1402500） |
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| 中文摘要: |
| 摘要目的：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DH5琢，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。 |
| 英文摘要: |
| ABSTRACR Objective：To construct miR-126 luciferase reporter gene vector according to miR-126 predicted target sequences and
todetect 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 plasmid 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 of miR-126. |
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