HEK 293细胞中重组型抗体表达和分泌的监测系统的构建  

Construction of a System to Monitor Recombinant Immunoglobulin Secretion in HEK 293 Cells

作　　者：何曾安 柳艺石 张慧杰 高晓冬[1] 藤田盛久 HE Zeng'an;LIU Yishi;ZHANG Huijie;GAO Xiaodong;Morihisa Fujita(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院

出　　处：《食品与生物技术学报》2019年第7期1-8,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目（31400693）;江苏省自然科学基金项目（BK20140141）;中央高校基本科研业务费项目（JUSRP51508）

摘　　要：免疫球蛋白G(Immunoglobulin G,IgG)作为一种十分重要的生物药物,已被广泛应用于多种医学诊断和治疗。为构建高效表达重组抗体的哺乳动物生产菌株,运用可以灵活检测抗体分泌运输的表达细胞株,从而充分理解抗体的折叠、转运及分泌尤为重要。作者在哺乳动物细胞株(HEK 293)中构建了一个稳定表达绿色荧光蛋白(GFP)融合型抗体E-F-HyHEL10的表达系统,可通过流式细胞术检测抗体的分泌运输。此哺乳动物细胞表达系统具有良好的抗体表达和分泌水平,且稳定维持了抗体的功能活性。使用布雷菲德菌素A(brefeldin A)和放线菌酮处理细胞后,来自胞内E-F-HyHEL10的GFP荧光信号与抗体分泌水平呈正相关。结果表明,本研究中构建的抗体表达系统可实现抗体表达、累积和分泌水平的灵活准确监测,未来可应用于哺乳动物细胞中抗体分泌相关因子的筛选。Immunoglobulin G(IgG)is one of the most important pharmaceuticals,which has been widely used in many kinds of diagnoses and therapies.To construct cell lines that can produce high amounts of recombinant antibodies,it is essential to understand factors required for the folding and secretion/transport of antibodies using a monitoring system.In this study,we established a mammalian cell line(HEK 293)to stably expressing a green fluorescent protein(GFP)-fused IgG E-F-HyHEL10,which could monitor the recombinant antibody secretion using the flow cytometry.The E-F-HyHEL10 was successfully expressed and secreted well into the medium,and could retain the antibody's biofunction.After treatment with either brefeldin A or cycloheximide,the GFP fluorescence derived from E-F-HyHEL10 in the cells was positively correlated with the level of secreted antibody.These results suggested that the modified HEK293 cells could be used flexibly to monitor the secretion of recombinant immunoglobulin in cells,and possibly can be applied for identification of factors required for antibody secretion in mammalian cells in the future.

关 键 词：抗体 流式细胞术 蛋白质分泌 EGFP 

分 类 号：Q786[生物学—分子生物学]