目的 真核表達(dá)杜拉魯肽類似藥胰高糖素樣肽l（GLP-1）-Fc融合蛋白（27C7），并與杜拉魯肽進(jìn)行理化性質(zhì)比較。方法 構(gòu)建真核表達(dá)載體pCHO1.0-GLP-1-Fc，并用脂質(zhì)體轉(zhuǎn)染CHO細(xì)胞，經(jīng)甲氨蝶呤和嘌呤霉素加壓篩選，再單克隆篩選，得到1株高表達(dá)的細(xì)胞模型。采用Protein A親和層析法純化目的蛋白，SDS-PAGE檢測(cè)蛋白大小，質(zhì)譜儀進(jìn)行高分辨分子量檢測(cè)，高效液相色譜法檢測(cè)蛋白純度，毛細(xì)管等點(diǎn)聚焦電泳（CIEF）檢測(cè)蛋白等電點(diǎn)，細(xì)胞活性功能評(píng)價(jià)杜拉魯肽與27C7生物活性的一致性。結(jié)果 通過(guò)SDS-PAGE、樣品高分辨分子量檢測(cè)，27C7與杜拉魯肽分子量一致；分子排阻色譜檢測(cè)27C7與杜拉魯肽單體純度接近，CIEF顯示27C7與杜拉魯肽的等電點(diǎn)一致；體外活性檢測(cè)結(jié)果表明，27C7與杜拉魯肽的生物學(xué)活性一致。結(jié)論 類似藥27C7與杜拉魯肽具有一定的一致性，可進(jìn)行下一步的類似藥研發(fā)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