抗癌胚抗原纳米抗体与人Fc融合蛋白在毕赤酵母和HEK293细胞中的表达、纯化和性质比较  

A Comparative Study on the Expression,Purification and Functional Characterization of Anti-serum Carcinoembryonic Antigen Nanobody-fusion Fc-fragment in Pichia Pastoris and HEK293 Cell

作　　者：郭万美 陈玉娟[1] 周宇杭 陈泉[3] 李飞 咸漠[3] 冯东晓 年锐 宋海鹏 GUOWan-mei;CHEN Yu-juan;ZHOU Yu-hang;CHEN Quan;LI Fei;XIANMo;FENG Dong-xiao;NIAN Rui;SONG Hai-peng(College of Life Science and Technology,Changchun University of Science and Technology1,Changchun 130022,China;Shenzhen Innova Nanobodi Co.,Ltd^2,Guangzliou 518000,China;CAS Key Laboratory of Biobased Materials,Qingdao Institute of Bioenergy and Bioprocess Technology,Chinese Academy of Sciences3,Jinan 266101,China;Binzhou Medical University4,Jinan 264003,China)

机构地区：[1]长春理工大学生命科学技术学院,长春130022 [2]深圳市国创纳米抗体技术有限公司,广州518000 [3]中国科学院青岛生物能源与过程研究所,济南266101 [4]滨州医学院,济南264003

出　　处：《科学技术与工程》2018年第21期30-35,共6页Science Technology and Engineering

摘　　要：通过对不同系统表达的肿瘤标记物癌胚抗原(CEA)纳米抗体与人Fc融合蛋白(11C12-Fc)的表达、纯化及抗体性质等的比较分析,比较不同表达系统的特点及其对融合抗体性质的影响;从而为CEA融合纳米抗体在体内检测方面的应用提供理论依据。构建两种11C12-Fc表达系统(Pichia pastoris和HEK293)相应的表达载体和菌株,分别进行诱导表达、分离纯化;并对其纯化产物的生物学活性等进行初步研究。成功构建了毕赤酵母、HEK293细胞两种CEA纳米抗体融合蛋白(11C12-Fc)表达系统并实现11C12-Fc的胞外分泌表达;通过protein A柱及阴离子交换层析纯化,获得了较高纯度和浓度的11C12-Fc样品;通过突变糖基化位点的方式有效去除了毕赤酵母表达11C12的过度糖基化作用;并且其抗体亲和力较突变前未发生明显改变,与HEK293细胞表达的11C12-Fc都表现出较高的亲和力。毕赤酵母和HEK293系统均能够表达具有较高生物活性的11C12-Fc,后续的科研或生产可根据实际需要和条件来合理选择表达系统。为后续的纳米抗体融合蛋白规模化生产、体内诊断与靶向治疗的应用提供了理论和技术参考。Expression, purification and functional characterization of anti-CEA nanobody-Fc fusion protein（11C12-Fc ） in Pichia pastoris and HEK293 Cell were studied,and their effects on the properties on the properties of fusion antibodies were compared and sunmarized. These results provided theoretical basis of CEA fusion nanobodtion .Two expression vectors and strains were constucted corresponding to expressHEK 293 ） of 11C12-Fc. Subsequently,11C12-Fc of different expression system were expressed,purified respec-tively,and their biological activities of the purified products were studied preliminarily. Expression systemspastoris and HEK 293 ） of anti-CEA nanobody fusion proteins （ 11C12-Fc ） were successfully constructed,and 11C12-Fc samples of high purity and concentration were obtained with purified by protein a column and anion ex-change chromatography ; The hyperglycosylation of 11C12 in Pichii pastoris was removed efectively by glycosylation sites mutation,and its antibody affinity did not change significantly compared to original antibody. In addition,these 11C12-Fc expressed by Pichia pastoris and HEK293 cell showed higher affinity . Both Pichia pastoris and HEK293 system are able to express 11C12-Fc with high biological activity so that follow-up research or production can be based on actual needs and conditions to choose a reasonable expression system. This study provides a theo-retical and technical reference for the subsequent mass production,in vivo diagnosis,and targeted therapy of nanobody fusion protein.

关 键 词：癌胚抗原 纳米抗体 Fc片段 融合蛋白 毕赤酵母 HEK293 

分 类 号：Q816[生物学—生物工程]