Production of monoclonal shark-derived immunoglobulin new antigen receptor antibodies using Chinese hamster ovary cell expression system
Production of monoclonal shark-derived immunoglobulin new antigen receptor antibodies using Chinese hamster ovary cell expression system
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DOI：
10.1016/j.jbiosc.2021.04.015
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发表时间：
2021-08-26
影响因子：
2.8
通讯作者：
Omasa, Takeshi
中科院分区：
文献类型：
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作者：
Enatsu, Hajime;Okamoto, Nako;Omasa, Takeshi
像鲨鱼这样的软骨鱼有类似于哺乳动物的基于免疫球蛋白的适应性免疫系统。在其进化过程中，软骨鱼个体获得了一种适应性免疫系统——免疫球蛋白新抗原受体（IgNARs）。ignar在鲨鱼血清的恶劣环境中保持其功能，鲨鱼血清中含有高浓度尿素，以防止海水中的水分流失。因此，ignar具有较高的结构稳定性，有望在不同于传统IgG抗体的应用领域作为下一代抗体。然而，目前还没有构建IgNAR的重组表达系统，其分子量约为147 kDa，为二聚体，具有多个n -糖基化位点。这阻碍了对IgNAR开发的研究。本研究利用中国仓鼠卵巢（CHO）细胞构建了igar的重组表达体系，CHO细胞被广泛用作IgG抗体的宿主。利用该系统成功表达和纯化了igar作为人IgG Fc融合蛋白，并显示出抗原结合能力。经过Protein A亲和纯化，特异性切割和去除人fc区域后，最终的IgNAR产量为1.07 mg/ l培养基。此外，该CHO细胞表达系统用各种n -聚糖修饰IgNAR，包括高甘露糖和复杂类型。这个表达系统将使我们能够分析IgNAR的结构、理化性质和生物学功能。这些基本信息将促进用于工业和生物技术应用的ignar的发展。(c) 2021，日本生物技术学会。版权所有。
Cartilaginous fishes such as sharks have adaptive immune systems based on immunoglobulins similar to those in mammals. During their evolution, cartilaginous fishes individually have acquired their adaptive immune system called immunoglobulin new antigen receptor (IgNARs). IgNARs maintain their functions in the harsh environment of shark serum, which contains a high concentration of urea to prevent water loss in seawater. Therefore, IgNARs have high structural stability, and are expected to be used as next-generation antibodies in applications different from those of conventional IgG antibodies. However, no recombinant expression system for IgNAR, which has a molecular weight of approximately 147 kDa as a dimer and multiple N-glycosylation sites, has yet been constructed. This has stalled research into IgNAR development. Here, we constructed a recombinant expression system for IgNAR using Chinese hamster ovary (CHO) cells, widely used as hosts for IgG antibody production. Using this system, IgNAR was successfully expressed and purified as a human IgG Fc fusion protein and showed antigen-binding ability. After Protein A affinity purification, followed by specific cleavage and removal of the human Fc-region, the final yield of IgNAR was 1.07 mg/L-medium. Moreover, this CHO cell expression system modified IgNAR with various N-glycans, including high-mannose and complex types. This expression system will allow us to analyze the structure, physicochemical properties, and biological functions of IgNAR. This fundamental information will advance the development of IgNARs for industrial and biotechnological applications. (c) 2021, The Society for Biotechnology, Japan. All rights reserved.