本文選題：Nlrp　切入點(diǎn)：過表達(dá)　出處：《基因組學(xué)與應(yīng)用生物學(xué)》2017年09期 　論文類型：期刊論文

【摘要】：本研究旨在構(gòu)建攜帶小鼠nlrp3蛋白的過表達(dá)真核載體。首先從高脂飲食喂養(yǎng)3個(gè)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(zhuǎn)錄獲得c DNA,然后PCR擴(kuò)增nlrp3基因的編碼區(qū)。PCR產(chǎn)物電泳回收,經(jīng)過NotⅠ及NheⅠ雙酶切后,將其克隆到真核表達(dá)載體p CDH-CMV-MCS-EF1-cop GFP-T2A-Puro中。重組質(zhì)粒電轉(zhuǎn)進(jìn)入感受態(tài)DH5α,過夜培養(yǎng)后挑取單個(gè)菌落過夜擴(kuò)大培養(yǎng)。然后提取質(zhì)粒,酶切鑒定后測序。將克隆成功的重組質(zhì)粒轉(zhuǎn)染HEK293T細(xì)胞,培養(yǎng)3 d后收集細(xì)胞蛋白,用Western blotting檢測nlrp3的表達(dá)水平,研究顯示,重組質(zhì)粒p CDH-NLRP3較空白質(zhì)粒p CDH-NULL的nlrp3蛋白表達(dá)水平明顯增加(p0.01)。本研究成功構(gòu)建了nlrp3過表達(dá)載體,為進(jìn)一步研究其作用機(jī)制提供了基礎(chǔ)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