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Abstract：
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.
摘要：
诸如鲨鱼的软骨鱼类具有与哺乳动物类似的基于免疫球蛋白的适应性免疫系统。在他们的进化过程中,软骨鱼个体获得了称为免疫球蛋白新抗原受体（IgNAR）的适应性免疫系统。IgNAR在鲨鱼血清的恶劣环境中保持其功能，其中含有高浓度的尿素以防止海水中的水分流失。因此,IgNARs具有较高的结构稳定性,并有望在不同于常规IgG抗体的应用中用作下一代抗体。然而,没有IgNAR的重组表达系统，作为二聚体和多个N-糖基化位点，其分子量约为147kDa，目前已建成。这阻碍了对IgNAR开发的研究。这里,我们利用中国仓鼠卵巢(CHO)细胞构建了IgNAR的重组表达系统,广泛用作IgG抗体生产的宿主。使用这个系统，IgNAR成功表达并纯化为人IgGFc融合蛋白，并显示出抗原结合能力。蛋白A亲和纯化后，然后特异性切割和去除人Fc区，IgNAR的最终产量为1.07mg/L-培养基。此外，这种CHO细胞表达系统修饰了具有各种N-聚糖的IgNAR，包括高甘露糖和复杂类型。这个表达系统将允许我们分析结构，物理化学性质，和IgNAR的生物学功能。这些基本信息将促进用于工业和生物技术应用的IgNAR的开发。
