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

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摘要目的：研究细胞因子信号抑制蛋白3（suppressors of cytokine signaling3，SOCS3）基因3′端非翻译区（3′untranslated region，3′UTR）微小RNA（microRNA，miRNA）的调控作用，构建重组SOCS3基因3′UTR荧光素酶报告载体，并通过荧光素酶活性测定，初步分析可能调控SOCS3基因表达的miRNAs。方法：采用PCR方法从原代培养的小鼠星形胶质细胞基因组DNA中扩增SOCS3基因3′UTR序列，插入荧光素酶报告载体pGL3Promoter，获得重组载体pGL3Promoter/SOCS3；通过Target Scan5.2、RNAhybrid 和FINDTAR3软件预测可能与SOCS3基因3′UTR结合的miRNAs；将pGL3Promoter/SOCS3重组质粒和miRNAs共转染HEK 293T细胞，测定SOCS3 3′UTR荧光素酶的活性。结果：酶切及核酸测序证实，成功构建了SOCS3基因3′UTR序列的荧光素酶报告重组子；miRNA靶位点预测显示，SOCS3基因可能是miR203、miR291a5p、miR9、miR140和miR130b的作用靶标；与对照组相比，miR203、miR291a5p、miR140能使SOCS3 3′UTR荧光素酶的活性显著降低。结论：成功构建了SOCS3 基因3′UTR荧光素酶报告载体，且miR203、miR291a5p、miR140可显著降低其荧光素酶的活性。

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	刘晓梅1
	庞蓉蓉2
	赵聃2
	李妍2
	单锴2
	王迎伟2

关键词 ：细胞因子信号抑制蛋白-3基因； 3&prime, 端非翻译区；微小RNA；荧光素酶报告载体

Abstract：[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 constructed 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 recombinant pGL3Promoter/SOCS3 was identified. The miRNAs targeting SOCS3 3′UTR was predicted by Target Scan5.2, RNAhybrid and FINDTAR3 softwares. Thereafter, pGL3Promoter/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 verified by Xba I digestion and DNA sequencing. The predicted miRNAs targeting SOCS3 3′UTR included miR203, miR291a5p, miR9, miR140 and miR130b. Compared with the control group, the luciferase activity of pGL3Promoter/SOCS3 treated with miR203, miR291a5p, or miR140 was remarkably decreased, respectively. Conclusion: The SOCS3 3′UTR luciferase reporter vector was constructed successfully, and the luciferase activity of the recombinant vector can be suppressed significantly by miR20, miR291a5p or miR140.

引用本文:

刘晓梅1, 庞蓉蓉2, 赵聃2, 李妍2, 单锴2, 王迎伟2. 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. LIU Xiao-Mei-1, Pang-Rong-Rong-2, Zhao-Dan-2, Li-Yan-2, Dan-Kai-2, Wang-Ying-Wei-2. Construction of SOCS3 3′-UTR luciferase reporter vector and identification of its activity. Journal of Jiangsu University(Medicine Edition), 2012, 22(4): 307-311.

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http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2012/V22/I4/307