基于胞内cAMP浓度测定融合蛋白GGH活性的改良方法

An Improved Method to Measure Bioactivity of the Fusion Protein GGH Based on the Intracellular cAMP Level

目的:改良一种基于胞内cAMP浓度变化,用于融合蛋白GGH生物学活性测定的方法。方法:根据融合蛋白GGH是GLP-1类似物,能促进胰岛β细胞增殖,产生胞内第二信使cAMP的原理,利用酶联免疫法测定胞内cAMP的含量。结果:Exenatide或融合蛋白GGH刺激大鼠β胰岛素瘤细胞RIN m5f后,选用100nmol/L IBMX作为cAMP保护剂,用DPBS为药物稀释剂,采用液氮—100℃反复冻融2次裂解细胞,最后用酶联免疫法可稳定的测定上清液中cAMP含量。结论:成功改良了融合蛋白GGH体外高通量生物学活性测定方法。运用此方法可快速鉴定融合蛋白GGH及其它GLP-1类似物的生物活性。

Objective： To improve a method to determine bioactivity of the fusion protein GGH based on the intraeellular cAMP level. Methods： According to GGH is the analog of GLP-1 which can promote islet 13 cells to produce second messenger cAMP, detecting intracellular cAMP levels by enzyme-linked immunosorbent assay （ELISA） can be used to characterize GGH biological activity. Results： Exenatide or GGH was used to stimulate rat insulinoma cell line RIN mSf to produce cAMP. The optimal cAMP protective agent and drug diluent were 100nmol/L IBMX and DPBS. We found that freezing and thawing cells in liquid nitrogen 2 times led to maximum and stable production of cAMP in the supernatant measured by ELISA. Conclusion： An in vitro high-throughput screening method for detecting the bioactivities of the fusion protein GGH was successfully modified. Using this method, the biological activity of GGH and GLP-1 analogs can be rapidly identified.