长效GLP-1-IgG2σ Fc融合蛋白的真核表达与生物活性鉴定  

Eukaryotic Expression and Activity Detection of GLP-1-IgG2σ Fc Fusion Protein

作　　者：杨懿[1,2] 万德友[2] 刘蕴慧[2] 冯红茹[2] 杨利[2] 高新[2] 宋海峰[1,2] 

机构地区：[1]安徽医科大学,安徽合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《生物技术通讯》2016年第2期173-177,共5页Letters in Biotechnology

基　　金：国家自然科学基金(81272701)

摘　　要：目的:在真核细胞中融合表达胰高血糖素样肽1(GLP-1),纯化后探究产物的生物学活性。方法:通过预留酶切位点将合成的GLP-1-Ig G2σFc融合蛋白表达序列连入表达载体质粒,用电转方法将线性化质粒稳定转染CHO-K1细胞,通过亚克隆筛选出蛋白高表达细胞株;收集高表达细胞株培养上清并初步纯化,采用口服葡萄糖耐量试验(OGTT)探究GLP-1融合蛋白降低血糖的生物学活性。结果:经过一轮亚克隆,筛选得到的细胞株的GLP-1融合蛋白产量达1 g/L,OGTT实验结果表明GLP-1融合蛋白具有降血糖生物活性。结论:GLP-1变体和Ig G2σ的Fc段连接是一种可行的融合蛋白形式,融合蛋白具有GLP-1受体激动剂的降糖作用,并且细胞株的筛选方式和初步纯化方法也具有可行性,为后续的体内、外活性研究和纯化方法确定提供了基础。Objective： To eukaryotic express glucagon like peptide-1（GLP-1） fusion protein, and detect the bio-logical activities of GLP-1 fusion protein after purification. Methods： The GLP-1-Ig G2σ Fc fusion protein genewas digested by restriction enzymes HindⅢ and Eco RⅠ and inserted into a mammalian expression vector. CHO-K1 cell was stable transfected with expression plasmid. Stable expressing clones were chosen according to the re-sult of SDS-PAGE. The expression medium which continuous cultured cell line for 12 d was taken, filtered andpurified by using Hi-Trap Protein A column. Finally oral glucose tolerance test（OGTT） experiment was performedto detect the biological activities of GLP-1 fusion protein. Results： After the subcloning, the GLP-1 fusion pro-tein yield of stable expression cell line has reached 1 g/L. The results of the OGTT experiment showed that thepreliminary purification methods do not affect blood glucose lowing effect of GLP-1 fusion protein. Conclusion： Itis a feasible form of fusion proteins which link GLP-1 variant with Ig G2σ Fc fragment. The GLP-1 fusion proteineukaryotic expressed lays the foundation for further research on biological activity in vitro and in vivo.

关 键 词：胰高血糖素样肽1 受体激动剂 融合蛋白 真核表达 口服葡萄糖耐量试验 

分 类 号：Q78[生物学—分子生物学] Q25