核转录因子红细胞系相关因子-2干扰质粒构建及鉴定

发布时间：2018-06-01 08:13

  本文选题：核转录因子红细胞系相关因子- + RNA干扰　； 参考：《临床神经病学杂志》2017年02期

【摘要】：目的设计及构建小鼠核转录因子红细胞系相关因子-2(Nrf2)基因的干扰质粒,并筛选出效果最好的干扰质粒。方法设计3组针对Nrf2基因的核糖核酸干扰(RNAi)序列,应用基因重组技术克隆入载体中构建短发夹RNA(shRNA),分别为shRNA1、shRNA2及shRNA3,通过基因测序鉴定,经Lipofectamine2000转染至BV2细胞,实时PCR检测Nrf2 mRNA的表达,Western Blot法检测Nrf2蛋白的表达。结果测序表明,克隆入载体中的Nrf2干扰序列及读码框完全正确,实时PCR及Western Blot显示shRNA3干扰效果最强。结论成功构建小鼠Nrf2的有效干扰质粒,为Nrf2信号通路在脑卒中领域的功能研究奠定了基础。
[Abstract]:Objective to design and construct the interference plasmid of mouse nuclear transcription factor RBC related factor -2nrf2, and to screen out the best interference plasmid. Methods three sets of RNA interference RNAi (RNAi) sequences targeting Nrf2 gene were designed and cloned into the vector by gene recombination technique to construct shRNA2 and shRNA3 for short hairpin RNAs, which were identified by gene sequencing and transfected into BV2 cells by Lipofectamine2000. The expression of Nrf2 mRNA was detected by real time PCR and the expression of Nrf2 protein was detected by Western Blot. Results sequencing showed that the Nrf2 interference sequence and the reading frame cloned into the vector were completely correct, and real-time PCR and Western Blot showed the best effect on shRNA3 interference. Conclusion the effective interference plasmid of mouse Nrf2 was successfully constructed, which laid a foundation for the study of the function of Nrf2 signaling pathway in the field of stroke.
【作者单位】： 江苏大学附属宜兴医院(扬州大学医学院宜兴临床学院)神经内科;
【基金】：江苏省科技厅自然科学基金面上项目(BK20141121) 无锡市卫生局青年基金(Q201301;Q201625)
【分类号】：R743.3
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本文编号：1963493