摘要
传统的抗IgG-Fc单克隆抗体制作过程繁琐、耗时长且批间差异大,极大的限制了其应用。因此寻找一种抗IgG-Fc单克隆抗体的替代元件是十分必要的。本研究基于微孔板指数富集的配体系统进化技术(微孔板-selex),设计了针对抗IgG-Fc蛋白ssDNA适配体的筛选方案。交替包被5种IgG单克隆抗体于酶标孔作为筛选靶分子,将从靶分子上洗脱的核酸适配体作为模板进行PCR扩增,生物素-链霉亲和素ELISA测定第3,7,13,14,16,18轮筛选获得ssDNA文库与内酰胺酶单克隆抗体的结合能力,并将第19轮筛选获得的ssDNA文库进行TA克隆验证、高通量测序。经过19轮筛选,得到69796条抗IgG-Fc蛋白的适配体,选取出现频率最高的ap-1(178次)、ap-2(129次)、ap-3(58次)体外合成并在其5,端标记生物素,经ELISA鉴定3条适配体分别与5种IgG单克隆抗体有着较强的结合能力,并与3%BSA几乎不结合。Ap-1结合能力最强,被认为是本次筛选得到最佳抗IgG-Fc蛋白核酸适配体。实验结果表明本文筛选获得抗IgG-Fc蛋白的核酸适配体,具有成为抗IgG单克隆抗体替代元件的潜力。
The traditional production process of anti-IgG Fc protein is time-consuming and differs greatly among batches,which greatly limits its application.In order to obtain an alternative component of anti-IgG-Fc protein,a screening scheme for biopanning ssDNA aptamer against anti-IgG-Fc protein was performed based the evolution of ligand systems with exponential enrichment of microporous plates.In this study,five IgG monoclonal antibodies were coated in enzyme-labeled pores alternately and the nucleic acid aptamer against anti-IgG-Fc protein was eluted.After 19 rounds of biopanning,the binding ability of the ssDNA library in rounds 3,7,13,14,16,and 18 for anti-lactamase mAb was determined by biotin-streptavidin enzyme-linked immunosorbent assay(bs-ELISA),and the ssDNA library obtained from the 19th round screening was tested by TA cloning and high-throughput sequencing.Finaly,the 69796 aptamers against anti-IgG-Fc proteins were obtained.The aptamer(ap)-1(178 times),ap-2(129 times),and ap-3(58 times)labeled biotins were identified with strong binding ability to IgG mAb and without binding to other protein such as bovine serum albumin.Among these aptamers,ap-1 had the best binding ability with anti-IgG-Fc protein.These results showed that the obtained aptamer had the potential to be a substitute for anti-IgG monoclonal antibody.
作者
杨叶民
胡文进
何庆华
YANG Yemin;HU Wenjin;HE Qinghua(Shanghai Yemin Biotechnology Company,Shanghai,200000,China;State Key Lab of Food Science and Technology,Nanchang Universitya,Nanchang 330047,China)
出处
《南昌大学学报（理科版）》
CAS
北大核心
2020年第4期340-345,353,共7页
Journal of Nanchang University(Natural Science)
基金
江西省主要学科学术与技术带头人项目(20194BCJ22003)。
