从免疫鲨鱼中生成 VNAR 文库和选择靶标特异性克隆。

Generation of VNAR Libraries from Immunized Sharks and Selection of Target-Specific Clones.

机构信息

Department of Microbiology & Immunology, University of Maryland School of Medicine, Institute of Marine & Environmental Technology, Baltimore, MD, USA.

出版信息

Methods Mol Biol. 2022;2421:57-72. doi: 10.1007/978-1-0716-1944-5_4.

DOI:10.1007/978-1-0716-1944-5_4

PMID:34870811

Abstract

Cartilaginous fishes (sharks, skates, rays, and chimeras) are the most phylogenetically distant lineage relative to mammals in which somatically rearranging immunoglobulins (Igs or antibodies) have also been found. Alongside their conventional (heavy-light chain) isotypes, IgM and IgW, sharks produce the novel isotype, IgNAR, a heavy-chain homodimer. Naturally lacking light chains, antigen binding is mediated by two highly soluble and independently functioning variable domains, or VNARs, each having a molecular weight of approximately 12 kDa. The small size, high affinity for antigen, and extreme structural stability of single-domain VNARs make them an emerging prospect for use in therapeutic, diagnostic, and research applications. In this chapter, we detail the immunization protocol we use to raise an antigen-specific IgNAR response in the nurse shark (Ginglymostoma cirratum), the subsequent cloning of the variable domains from this isotype, and the selection of antigen-specific VNARs by phage display.

摘要

软骨鱼类（鲨鱼、鳐鱼、魟鱼和嵌合体鱼）是与哺乳动物亲缘关系最远的谱系，在其中也发现了体细胞重排免疫球蛋白（Ig 或抗体）。除了它们的常规（重轻链）同种型 IgM 和 IgW 外，鲨鱼还产生了新型同种型 IgNAR，即重链同源二聚体。由于缺乏轻链，抗原结合由两个高度可溶性且独立发挥作用的可变结构域（VNAR）介导，每个 VNAR 的分子量约为 12 kDa。单域 VNAR 的体积小、抗原亲和力高、结构稳定性极强，这使得它们成为治疗、诊断和研究应用中的新兴前景。在本章中，我们详细介绍了我们在护士鲨（Ginglymostoma cirratum）中用于产生抗原特异性 IgNAR 反应的免疫方案，随后从这种同种型中克隆可变结构域，以及通过噬菌体展示选择抗原特异性 VNAR。