报告基因法检测促胰岛素分泌肽融合蛋白生物学活性

目的:建立报告基因法检测促胰岛素分泌肽融合蛋白(Exendin-4-HSA,Byetalog)生物学活性。方法:将胰高血糖素样肽-1受体(glucagon-likepeptide 1 receptor,GLP-1R)表达载体和cAMP反应元件(cAMP response element,CRE)报告基因载体共转染CHO-K1细胞,经加压筛选和单克隆分离培养获得稳定的单克隆细胞株;使用药物反应强的单克隆细胞株建立报告基因方法;对新建方法进行条件优化,并用优化的方法对促胰岛素分泌肽融合蛋白原液和成品进行生物学活性测定。结果:经加压筛选和单克隆分离培养后获得对Byetalog强反应性的细胞株CHOglpar/crec14;新建方法剂量反应曲线符合四参数方程,R²大于0.99;对促胰岛素分泌肽融合蛋白的原液和成品分别测定3次,RSD值均低于5.0%,回收率在70%~130%之间。结论:报告基因法操作简便,重复性和准确性都较高,有望作为替代方法应用于促胰岛素分泌肽融合蛋白的生物学活性测定。

Objective:To establish a reporter gene assay for biological activity determination of Exendin-4-HSA.Methods:Glucagon-likepeptide 1 receptor(GLP-1R) and cAMP response element(CRE) reporter gene vectors were transfected into CHO-K1 cells.After being cultured in selective media, the cell clone with the strongest responsiveness to Exendin-4-HSA was subcloned and used to establish a reporter gene assay for Exendin-4-HSA.Then the method was optimized and applied for bioactivity determination of Exendin-4-HSA bulk and final products.Results:The cell strain CHOglpar/crec14 strongly responsive to Byetalog was obtained after pressure selection and separate monoclonal cultivation.The dose-response curve of the new assay fitted 4-PL model, demonstrating good linearity(R²>0.99).The RSD was below 5.0%, and the recovery was between 70% and 130% according to respective tests of the Exendin-4-HSA bulk and final products for three times.Conclusion:The new reporter gene assay is convenient, with good repeatability and accuracy, and can be an available alternative to biological activity determination of Exendin-4-HSA.