目的 真核表达杜拉鲁肽类似药胰高糖素样肽l（GLP-1）-Fc融合蛋白（27C7），并与杜拉鲁肽进行理化性质比较。方法 构建真核表达载体pCHO1.0-GLP-1-Fc，并用脂质体转染CHO细胞，经甲氨蝶呤和嘌呤霉素加压筛选，再单克隆筛选，得到1株高表达的细胞模型。采用Protein A亲和层析法纯化目的蛋白，SDS-PAGE检测蛋白大小，质谱仪进行高分辨分子量检测，高效液相色谱法检测蛋白纯度，毛细管等点聚焦电泳（CIEF）检测蛋白等电点，细胞活性功能评价杜拉鲁肽与27C7生物活性的一致性。结果 通过SDS-PAGE、样品高分辨分子量检测，27C7与杜拉鲁肽分子量一致；分子排阻色谱检测27C7与杜拉鲁肽单体纯度接近，CIEF显示27C7与杜拉鲁肽的等电点一致；体外活性检测结果表明，27C7与杜拉鲁肽的生物学活性一致。结论 类似药27C7与杜拉鲁肽具有一定的一致性，可进行下一步的类似药研发工作。

Objective To express dulaglutide similar drugs Glp-1-fc fusion protein (27C7) with eukaryotic expression system, and compared the physicochemical properties with the dulaglutide. Methods Construct Eukaryotic expression vector of pCHO1.0-GLP-1-Fc, and transfect CHO cells by Liposomes, after MTX and puromycin pressure screening, then the monoclonal screening was carried out, a high expression cell model was obtained. Purified the fusion protein by Protein A affinity chromatography. Detect protein size by SDS-PAGE, detect high resolution molecular weight detection by mass spectrometer, detect protein purity by High Performance Liquid Chromatography (HPLC). And detect protein isoelectric point by Capillary isocenter focusing electrophoresis (CIEF), cell biology function to assess whether the cytological function were consistent between the biological similar drugs and dulaglutide. Results By SDS-PAGE and high-resolution molecular weight detection, the molecular weight of 27C7 was the same as that of Dularutin; the ratio of 27C7 to Dularutin was similar by molecular exclusion chromatography; and the isoelectric point of 27C7 was the same by CIEF; the biological activity of 27C7 was the same as that of Dularutin in vitro. Conclusion The Similar drug 27C7 has a certain degree of consistency with the dulaglutide, and can be used for further research.

R962.2