聚乙二醇化胰高血糖素样肽1类似物生物学活性的报告基因法测定

 目的  建立检测聚乙二醇化胰高血糖素样肽1类似物（polyethylene glycolylated  glucagon like peptide -1 analogue，PEG-GLP-1a）生物活性的报告基因法，并对该方法进行验证。 方法 将人胰高血糖素样肽1受体表达载体和分泌型碱性磷酸酶（secreted alkaline phosphatase, SEAP）报告基因载体瞬时共转染HEK293T细胞，培养后加入PEG-GLP-1a刺激细胞分泌SEAP，使用SEAP报告基因检测试剂盒进行测定，并验证其专属性、准确性、精密度、稳定性等指标。 结果  人胰高血糖素样肽1受体和SEAP双载体瞬时共转染的HEK293T细胞对PEG-GLP-1a具有强反应性。使用该方法测定PEG-GLP-1a具有良好的专属性。加标回收率为97.6%~102.23%，准确性较好。不同测定日期的日内半数效应浓度的变异系数都小于10.22%，不同测定日期的日间半数效应浓度的变异系数都小于13.02%。 结论  报告基因法可以用于PEG-GLP-1a的生物活性检测。

Reporter gene assay for the determination ofpolyethylene glycolylated glucagon like peptide -1 analoguebioactivity#br#

Objective  To establish a reporter gene assay to detect the biological activity of polyethylene glycolylated glucagon like peptide-1 analogue (PEG-GLP-1a) and validate the assay. Methods  The human glucagon like peptide-1 receptor (hGLP-1R) expression vector and secretory alkaline phosphatase (SEAP) reporter gene vector were co-transfected into HEK293T cells. PEG-GLP-1a was added to the cells to stimulate the secretion of SEAP and SEAP reporter gene kit was used to measure the activity. Method specificity, accuracy, precision, and stability were verified. Results  HEK293T cells transiently co-transfected with hGLP-1R and SEAP showed strong reactivity to PEG-GLP-1a. The method had a good specificity to PEG-GLP-1a. The recovery rate was 97.6%-102.23%. The  intraday coefficients of variation (CV) values of concentration for 50% of maximal effect (EC₅₀) in different days were all no more than 10.22%, and the interday CV values of EC₅₀ in different days was no more than 13.02%. Conclusion  The reporter gene assay is applicable for the method of bioactivity determination of PEG-GLP-1a.