以GLP-1受体为靶点的药物筛选模型的建立及功能鉴定
发布时间：2018-12-30 22:32
【摘要】：目的以GLP-1受体为靶点,建立GLP-1类似物活性检测的细胞模型,为GLP-1类似物以及GLP-1受体激动剂的药物筛选提供一种简单可靠的评价方法。方法首先将人源GLP-1受体基因插入pEGFP-N3,构建真核表达载体pEGFP-GLP-1R,并转染至HEK293A细胞中,经G418压力筛选后获得稳定表达GLP-1R-GFP的293A细胞株,然后通过GFP荧光信号和Western blot检测GLP-1R-GFP融合蛋白在该细胞中的表达与分布;最后利用GLP-1类似物利拉鲁肽刺激细胞,均相时间分辨荧光法检测细胞内cAMP含量变化。结果成功构建了GLP-1R-GFP-293A稳转细胞株,GLP-1RGFP蛋白主要分布在细胞膜表面,该细胞对GLP-1类似物利拉鲁肽具有高灵敏度和产生cAMP的特异性反应。结论利用所构建的细胞模型,可对小分子和GLP-1类似物进行体外活性分析,为筛选GLP-1受体激动剂奠定了模型基础。
[Abstract]:Objective to establish a cell model for the detection of the activity of GLP-1 analogues using GLP-1 receptor as the target, and to provide a simple and reliable method for the screening of GLP-1 analogues and GLP-1 receptor agonists. Methods Human GLP-1 receptor gene was inserted into pEGFP-N3, to construct eukaryotic expression vector pEGFP-GLP-1R, and transfected into HEK293A cells. After G418 pressure screening, 293A cell line expressing GLP-1R-GFP stably was obtained. Then the expression and distribution of GLP-1R-GFP fusion protein in the cells were detected by GFP fluorescence signal and Western blot. Finally, GLP-1 analogues were used to stimulate the cells, and the changes of cAMP content in the cells were detected by homogeneous time resolved fluorescence method. Results the GLP-1R-GFP-293A stable cell line was successfully constructed. The GLP-1RGFP protein was mainly distributed on the surface of the cell membrane. The cell had a high sensitivity to the GLP-1 analogue and a specific response to the production of cAMP. Conclusion the cell model can be used to analyze the activity of small molecules and GLP-1 analogues in vitro, which lays a foundation for screening GLP-1 receptor agonists.
【作者单位】： 中山大学药学院新药筛选中心新药成药性评估与评价国家地方联合工程实验室;深圳翰宇药业股份有限公司;
【基金】：广东省自然科学基金资助项目(No2016A030313335)
【分类号】：R96
本文编号：2396257
[Abstract]:Objective to establish a cell model for the detection of the activity of GLP-1 analogues using GLP-1 receptor as the target, and to provide a simple and reliable method for the screening of GLP-1 analogues and GLP-1 receptor agonists. Methods Human GLP-1 receptor gene was inserted into pEGFP-N3, to construct eukaryotic expression vector pEGFP-GLP-1R, and transfected into HEK293A cells. After G418 pressure screening, 293A cell line expressing GLP-1R-GFP stably was obtained. Then the expression and distribution of GLP-1R-GFP fusion protein in the cells were detected by GFP fluorescence signal and Western blot. Finally, GLP-1 analogues were used to stimulate the cells, and the changes of cAMP content in the cells were detected by homogeneous time resolved fluorescence method. Results the GLP-1R-GFP-293A stable cell line was successfully constructed. The GLP-1RGFP protein was mainly distributed on the surface of the cell membrane. The cell had a high sensitivity to the GLP-1 analogue and a specific response to the production of cAMP. Conclusion the cell model can be used to analyze the activity of small molecules and GLP-1 analogues in vitro, which lays a foundation for screening GLP-1 receptor agonists.
【作者单位】： 中山大学药学院新药筛选中心新药成药性评估与评价国家地方联合工程实验室;深圳翰宇药业股份有限公司;
【基金】：广东省自然科学基金资助项目(No2016A030313335)
【分类号】：R96
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