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视黄酸刺激RARE调控荧光素酶报告基因表达系统的构建与应用

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.06.033, PP. 209-215

Keywords: 视黄酸,RARE,荧光素酶报告基因,节律,代谢

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Abstract:

类维生素A,通过视黄酸(retinoicacid,RA)代谢旺盛组织的靶基因调控,与生物节律通路相互作用,在代谢性疾病发展过程中发挥着重要作用。在293T及小鼠肝原代细胞中,构建视黄酸反应元件(RARE)调控的荧光素酶报告基因表达系统,为研究类维生素A与节律和代谢研究中靶基因上游调控信号分子提供可能。用定点突变PCR方法在pGL3-Basic载体中插入RARE片段,检测报告基因表达及RARE对视黄酸响应的半数有效浓度(EC50),初步筛选可能调控RARE的靶基因,通过酶切连接将荧光素酶报告基因Luciferase和启动子RARE片段构入穿梭载体pShuttle,转入感受态细胞BJ518获得重组腺病毒载体Ad-Basic-RARE-Luc,转染MGH细胞并进行扩增,在小鼠肝原代细胞检测腺病毒活性。结果显示,构建的RARE调控的荧光素酶报告基因表达载体在293T细胞可被RA刺激,RARβ促进RA刺激RARE表达,而CRY1抑制RARE-Luc对RA的响应,并成功构建了在小鼠肝原代细胞响应RA刺激的Adeasy-Basic-RARE-Luc腺病毒载体。

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