NLRP3基因过表达载体的建立及鉴定

发布时间：2018-03-05 03:28

  本文选题：Nlrp　切入点：过表达　出处：《基因组学与应用生物学》2017年09期 　论文类型：期刊论文

【摘要】：本研究旨在构建携带小鼠nlrp3蛋白的过表达真核载体。首先从高脂饮食喂养3个月的C57BL/6J小鼠上剪取肝脏组织,液氮冻存。快速剪取适量肝脏组织,冰上研磨,Trizol法提取总RNA,逆转录获得c DNA,然后PCR扩增nlrp3基因的编码区。PCR产物电泳回收,经过NotⅠ及NheⅠ双酶切后,将其克隆到真核表达载体p CDH-CMV-MCS-EF1-cop GFP-T2A-Puro中。重组质粒电转进入感受态DH5α,过夜培养后挑取单个菌落过夜扩大培养。然后提取质粒,酶切鉴定后测序。将克隆成功的重组质粒转染HEK293T细胞,培养3 d后收集细胞蛋白,用Western blotting检测nlrp3的表达水平,研究显示,重组质粒p CDH-NLRP3较空白质粒p CDH-NULL的nlrp3蛋白表达水平明显增加(p0.01)。本研究成功构建了nlrp3过表达载体,为进一步研究其作用机制提供了基础工具。
[Abstract]:The purpose of this study was to construct a eukaryotic vector carrying mouse nlrp3 protein. Firstly, liver tissue was extracted from C57BL / 6J mice fed with high fat diet for 3 months, and then frozen with liquid nitrogen. The total RNAs were extracted by trizol method on ice, then the cDNA was obtained by reverse transcription. The coding region of nlrp3 gene was amplified by PCR. The product was recovered by electrophoresis. After Not 鈪，

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