FGF21-Fc融合蛋白体外活性试验的建立和优化  

Establishment and Optimization of FGF21-Fc Fusion Protein Activity Assay in vitro

作　　者：徐金华 赵文鹏 赵丽丽 曹宇 彭伟 刘忠 XU Jinhua;ZHAO Wenpeng;ZHAO Lili;CAO Yu;PENG Wei;LIU Zhong(Shandong New Time Pharmaceutical Co.,Ltd.,Linyi 273400;State Engineering Lab.of High Expression of Mammalian Cells,Linyi 273400;Shenyang Pharmaceutical University,Shenyang 110016;Lunan Pharmaceutical Group Co.,Ltd.,Linyi 276000)

机构地区：[1]山东新时代药业有限公司,山东临沂273400 [2]哺乳动物细胞高效表达国家工程实验室,山东临沂273400 [3]沈阳药科大学,辽宁沈阳110016 [4]鲁南制药集团股份有限公司,山东临沂276000

出　　处：《中国医药工业杂志》2023年第4期545-553,共9页Chinese Journal of Pharmaceuticals

基　　金：山东省重点研发计划(泰山产业领军人才工程)(2018TSCYCX-30、tscy20200329)。

摘　　要：成纤维细胞生长因子-21(FGF21)-Fc融合蛋白体外活性试验的建立分为蛋白水平的结合活性试验和细胞水平的生物学活性试验2种。该研究建立了均相时间分辨荧光(HTRF)法用于检测FGF21-Fc融合蛋白与β-Klotho配体蛋白的结合活性;并自主构建了报告基因细胞,当FGF21-Fc融合蛋白与β-Klotho的结合,激活其FGF受体和诱导胞内信号转导所必需的共同受体,可通过检测萤光素酶活性,间接定量测定生物学活性。在建立过程中,分别对结合活性试验中样品和配体的浓度、供体和配体的稀释比例以及孵育时间进行优化,并进行了方法学验证,证明该HTRF技术具有良好的重复性和特异性,可用于FGF21-Fc融合蛋白和β-Klotho的结合活性检测。随后,对生物学活性试验中胎牛血清的浓度、报告基因细胞的密度、试验板的类型以及细胞种板形式进行优化,并进行了方法学验证,证明该报告基因法具有良好的重复性和特异性,可用于FGF21-Fc融合蛋白的生物学活性检测。本研究建立了FGF21-Fc融合蛋白的质控方法,为有同样活性检测难点的药物提供了一种分析技术参考,填补了国内外空白。The establishment of the fibroblast growth factor 21(FGF21)-Fc fusion protein in vitro activity test can be divided into binding assay at the protein level and biological assay at the cell level.This study established a homogeneous time-resolved fluorescence(HTRF)method to detect the binding activity of FGF21-Fc fusion protein andβ-Klotho ligand protein.And the reporter gene cells were constructed independently to test biological activity.When the FGF21-Fc fusion protein binded withβ-Klotho,the FGF receptor and the intracellular signal transduction were activated to further affect luciferase activity,which could be used to monitor and quantitatively measure biological activity indirectly.During the establishment process of the binding assay,the sample and ligand concentrations,dilution ratios of donor and acceptor,and incubation time were optimized comprehensively.And methodological verification was carried out to prove that the HTRF technology had good repeatability and specificity and this method could be used to detect the binding activity of FGF21-Fc fusion protein andβ-Klotho.Later,the fetal bovine serum concentration,density of report gene cells,plate type,and cells in the plate form in bioactivity assay were optimized.And methodological verification was carried out to prove that the reporter gene method had good repeatability and specificity and this method could be used to test the biological activity of FGF21-Fc fusion protein.In this study,a quality control method for FGF21-Fc fusion protein was established,which provided a reference for analysis technology of drugs with the same activity detection difficulties,filling the gap at home and abroad.

关 键 词：成纤维细胞生长因子-21 均相时间分辨荧光 结合活性 报告基因法 生物学活性 

分 类 号：R917[医药卫生—药物分析学]