法尼醇X受体激动剂细胞筛选体系的建立与优化

Development and optimization of a cell screening system for farnesoid X receptor agonist

摘要:

本研究旨在建立一种基于双荧光素酶报告基因检测体系的法尼醇X受体(farnesoid X receptor, FXR)激动剂细胞筛选体系，以满足对FXR激动剂先导化合物的高通量筛选。通过在报告基因质粒pGL4-luc2P-Hygro中的萤火虫荧光素酶(firefly luciferase, Luc)基因上游克隆并插入来自FXR靶基因的FXR反应元件(FXR response element, FXRE)片段，构建用于筛选FXR激动剂的报告基因质粒，并结合海肾荧光素酶内参质粒，建立能够有效反映药物对FXR激动效应的双荧光素酶报告基因细胞检测体系。通过一系列优化实验，比较了过表达RXR、鼠源和人源FXR、不同的FXRE片段、FXR过表达质粒与报告基因质粒的混合比对筛选体系诱导效率和灵敏度的影响。根据上述结果，最终确定了优化条件，优化后体系Z因子达到0.83。本研究建立了一种用于FXR激动剂筛选的改良的基于双荧光素酶报告基因检测体系的细胞筛选体系，其主要特征在于，使用多段FXR靶基因上的FXRE片段叠加组成一种新型的增强型FXRE元件，而非传统的反向重复序列-1 (inverted repeats-1, IR-1)片段的叠加，为FXR激动剂的发现提供了更好的手段和工具。

Abstract:

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.