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

摘要/Abstract

摘要： 类维生素A，通过视黄酸(retinoic acid，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。

关键词: 视黄酸, RARE, 荧光素酶报告基因, 节律, 代谢

Abstract: Biostearin plays a crucial role in the development of metabolic diseases by target gene regulation of retinoic acid(RA)in metabolically active tissues and interaction with circadian pathway. The luciferase reporter gene system regulated by retinoic acid response element(RARE)was constructed and expressed in 293T and primary hepatocyte of a mouse, which provides the possibility for studying biostearin and circadian, as well as signaling molecules of upstream regulation of target genes in researching metabolism. By PCR-based site-specific mutagenesis, RARE promoter was inserted into multiple clone sites of pGL3-Basic vector. In 293T cells, we detected the gene expression and half maximal effective concentration(EC50)of RARE responding to RA and screened the possible target genes of regulating RARE. The pGL3-RARE-Luc plasmid and pShuttle vector were digested by restriction enzyme and ligated. Through transformation into competent cell BJ518 the recombinant adenovirus vector Ad-Basic-RARE-Luc was obtained and amplified after transfection to MGH cells. The activity of recombinant adenovirus was detected in primary hepatocyte of a mouse with Dual-luciferase Reporter Assay System. The results indicated that luciferase reporter gene vector regulated by RARE in 293T cells was stimulated by RA. RARβ promoted the stimulation of RA in RARE regulation, and CRY1 prohibited the response of RARE-Luc to RA. The adenovirus vector Adeasy-Basic-RARE-Luc responding to RA stimulation in primary hepatocyte of a mouse was constructed successfully.

Key words: retinoid acid, RARE, luciferase reporter gene, circadian, metabolism

袁源, 陈亚琼. 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[J]. 生物技术通报, 2015, 31(6): 209-215.

Yuan Yuan, Chen Yaqiong. Construction and Application of RARE-regulated Luciferase Reporter Gene System Stimulated by Retinoid Acid[J]. Biotechnology Bulletin, 2015, 31(6): 209-215.

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