江苏大学学报（医学版）2012，Vol.22Issue(4)：307-311,5.
SOCS3基因3′端非翻译区荧光素酶报告载体的构建及活性鉴定
Construction of SOCS3 3′-UTR luciferase reporter vector and identification of its activity
刘晓梅 1庞蓉蓉 2赵聃 2李妍 2单锴 2王迎伟2
作者信息
- 1. 徐州医学院微生物与免疫学教研室,江苏,徐州,221002
- 2. 南京医科大学微生物与免疫学系,江苏,南京,210029
- 折叠
摘要
Abstract
Objective: To study the microRNA(miRNA) targeting to 3'-untranslated region(3'-UTR) of suppressors of cytokine signaling -3 ( SOCS3 ) gene , a SOCS3 3 '-UTR luciferase reporter vector was construe -ted and to analyse the miRNAs regulated SOCS3 expression through detecting the luciferase activity of SOCS3 3'-UTR. Methods: The 3'-UTR fragment of SOCS3 gene was amplified by PCR from genomic DNA of primary astrocytes of mouse and cloned into luciferase reporter vector (pGL3-Promoter) , then the recom-binant pGL3-Promoter/SOCS3 was identified. The miRNAs targeting SOCS3 3'-UTR was predicted by Tar-get Scan5.2, RNAhybrid and FINDTAR3 softwares. Thereafter, pGL34>romoter/SOCS3 and miRNAs were co-transfected into HEK 293T cells and the luciferase activity of SOCS3 3'-UTR was measured. Results; 3'-UTR fragment of SOCS3 gene was successfully cloned into the pGL3-Promoter reporter vector, which ver-ified by Xba I digestion and DNA sequencing. The predicted miRNAs targeting SOCS3 3'-UTR included miR-203 , miR-291a-5p, miR-9 , miR440 and miR430b. Compared with the control group , the luciferase activity of pGL3-Promoter/SOCS3 treated with miR-203, miR-291a-5p, or miR440 was remarkably de-creased, respectively. Conclusion; The SOCS3 3'-UTR luciferase reporter vector was constructed success -fully , and the luciferase activity of the recombinant vector can be suppressed significantly by miR -20 , miR-291a-5pormiR440.关键词
细胞因子信号抑制蛋白-3基因/3′端非翻译区/微小RNA/荧光素酶报告载体Key words
suppressors of cytokine signaling-3/ 3'-untranslated region/ miRNA/ luciferase reporter vector分类
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刘晓梅,庞蓉蓉,赵聃,李妍,单锴,王迎伟..SOCS3基因3′端非翻译区荧光素酶报告载体的构建及活性鉴定[J].江苏大学学报（医学版）,2012,22(4):307-311,5.
