本文選題：Nlrp　切入點：過表達　出處：《基因組學與應用生物學》2017年09期 　論文類型：期刊論文

【摘要】：本研究旨在構建攜帶小鼠nlrp3蛋白的過表達真核載體。首先從高脂飲食喂養(yǎng)3個月的C57BL/6J小鼠上剪取肝臟組織,液氮凍存�？焖偌羧∵m量肝臟組織,冰上研磨,Trizol法提取總RNA,逆轉錄獲得c DNA,然后PCR擴增nlrp3基因的編碼區(qū)。PCR產物電泳回收,經過NotⅠ及NheⅠ雙酶切后,將其克隆到真核表達載體p CDH-CMV-MCS-EF1-cop GFP-T2A-Puro中。重組質粒電轉進入感受態(tài)DH5α,過夜培養(yǎng)后挑取單個菌落過夜擴大培養(yǎng)。然后提取質粒,酶切鑒定后測序。將克隆成功的重組質粒轉染HEK293T細胞,培養(yǎng)3 d后收集細胞蛋白,用Western blotting檢測nlrp3的表達水平,研究顯示,重組質粒p CDH-NLRP3較空白質粒p CDH-NULL的nlrp3蛋白表達水平明顯增加(p0.01)。本研究成功構建了nlrp3過表達載體,為進一步研究其作用機制提供了基礎工具。
[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，

本文編號：1568563