Isolation and functional characterisation of cyokines from the spiny dogfish Squalus acanthias.

板鳃类（鲨鱼、鳐和鳐鱼）是哺乳动物中最早拥有所有适应性免疫分子（MHC、TCR、免疫球蛋白）的物种。出于这个原因，人们对这一群体进行了广泛的研究，试图了解适应性免疫系统是如何进化的。迄今为止，在鲨鱼中发现了3种免疫球蛋白同种型;所谓的“常规”重轻链同种型IgM（以五聚体和单体形式出现）和IgW（鲨鱼IgD直系同源物）。第三种非常规的鲨鱼特异性同种型称为IgNAR（新型抗原受体），并且特别令人感兴趣，因为它是天然缺乏轻链的重链同源二聚体。EM研究表明，每条重链具有一个可变（V）结构域，其通过柔性铰链样区与5个（C）恒定结构域连接，然而，高亲和力抗原结合不需要V区的二聚化。与其抗原复合的所选IgNAR V区的晶体结构显示，种系编码的非典型半胱氨酸的存在和通过CDR 2/框架区（Fr）2的截短赋予V非常小的尺寸（12 kDa）和极其紧凑的结构域结构。与抗原的结合主要由高度多样性的CDR 3介导，其通过3D区域的重排产生。长（9-24 aa）CDR 3环从V结构域的顶部突出以与抗原相互作用，并且在一些情况下，已显示将其自身插入抗原表面上的裂缝和活性位点中以提供高亲和力结合。在某些情况下，这具有阻断靶分子的酶功能的作用。由于它们的高亲和力结合、活性位点的靶向、非常小的尺寸和在恶劣环境中的一般稳健性，惠氏公司对开发这些分子作为人类治疗剂具有重大兴趣。鲨鱼基础生物学小组在Wyeth Research Aberdeen内成立，通过研究多刺角鲨（Squalus acanthias）中的B细胞发育和同种型选择来支持这一目标，特别是寻找诱导体内或体外抗原特异性IgNAR V区域产生的最佳方法。虽然分子方法使我们能够开始解决这一目标，但仍有真实的迫切需要建立体外长期培养鲨鱼B细胞的方法。为此，我们需要分离和研究可能参与鲨鱼淋巴细胞生长和发育的细胞因子。因此，对于这项工作，我们建议定位（通过基因同线性）和序列的细胞因子基因从刺角鲨，从他们在其他物种中的作用，应该驱动B细胞的生长和分化。然后，我们将把注意力集中在这些分子中的一对上，以产生重组蛋白，并研究它们对体外鲨鱼B细胞的影响。

Elasmobranchs (sharks, skates and rays) are the earliest group in phylogeny to have all of the molecules of adaptive immunity also observed in mammals (MHC, TCR, Immunoglobulin). For this reason this group has been extensively studied to try to understand how the adaptive immune system evolved. Work to date has found 3 immunoglobulin isotypes in sharks; the so-called 'conventional' heavy-light chain isotypes IgM (which occurs in a pentameric and monomeric form) and IgW (the shark IgD orthologue). The third, non-conventional, shark specific isotype is called IgNAR (novel antigen receptor) and is of special interest as it is a heavy chain homodimer that naturally lacks light chain. EM studies have shown that each heavy chain has one variable (V) domain tethered to 5 (C) constant domains via a flexible, hinge-like region however dimerization of the V regions is not required for high-affinity antigen binding. Crystal structures of selected IgNAR V regions in complex with their antigen showed that the presence of germline-encoded non-canonical cysteines and a truncation through CDR2/framework region (Fr)2 give the V's a very small size (12 kDa) and extremely compact domain structure. Binding to antigen is mediated mainly by the highly diverse CDR3, which is generated through the rearrangement of 3 D regions. The long (9-24 aa) CDR3 loop protrudes from the top of the V domain to interact with antigen, and in some instances, has been shown to insert itself into clefts and active sites on the antigen surface to provide high affinity binding. In some instances this has the effect of blocking the enzymatic function of the target molecule. Due to their high affinity binding, targeting of active sites, very small size and general robustness in harsh environments there is significant interest within Wyeth towards the development of these molecules as human therapeutics. The shark basic biology group was established within Wyeth Research Aberdeen to support this aim by studying B-cell development and isotype choice in the spiny dogfish (Squalus acanthias), specifically looking at the best way to induce the production of antigen-specific IgNAR V regions, either in vivo or in vitro. Whilst molecular approaches allow us to begin to address this aim there is a real and pressing need to establish methods for the long-term culture of shark B-cells in vitro. For this we need to isolate and study the cytokines which may be involved in lymphocyte growth and development in sharks. Thus, for this work we propose to locate (via gene synteny) and sequence a number of cytokine genes from spiny dogfish which, from their role in other species, should drive B-cell growth and differentiation. We will then focus our attention on a couple of these molecules to produce as recombinant proteins and study their effects on shark B-cells in vitro.