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

脊椎动物(鲨鱼、溜冰鱼和放射鱼)是系统发育中最早拥有哺乳动物中也观察到的所有适应性免疫分子(MHC、TCR、免疫球蛋白)的类群。出于这个原因，人们对这个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。到目前为止，工作已经在鲨鱼身上发现了三种免疫球蛋白同种类型：所谓的传统的重轻链同种类型IgM(以五聚体和单体的形式存在)和IGW(鲨鱼的IGD同源基因)。第三种非常规鲨鱼特有的同型异型被称为IgNAR(新型抗原受体)，由于它是一种天然缺乏轻链的重链同源二聚体，因此特别令人感兴趣。EM研究表明，每个重链都有一个可变的(V)结构域，通过一个柔性的铰链状区域与5(C)恒定结构域相连，但V区的二聚化不是高亲和力抗原结合所必需的。选定的IgNAR V区与其抗原的复合体的晶体结构表明，胚线编码的非规范半胱氨酸的存在和CDR2/框架区(Fr)2的截断使V‘s具有非常小的尺寸(12 KDa)和非常紧密的结构域结构。与抗原的结合主要由高度多样化的CDR3介导，CDR3是通过3D区域的重排而产生的。长的(9-24个氨基酸)CDR3环从V结构域的顶部伸出与抗原相互作用，在某些情况下，已被证明插入到抗原表面的裂隙和活性部位以提供高亲和力结合。在某些情况下，这具有阻断目标分子的酶功能的效果。由于它们的高亲和力结合、靶向活性部位、非常小的尺寸以及在恶劣环境中的一般稳定性，惠氏公司对将这些分子作为人类治疗药物的开发非常感兴趣。鲨鱼基础生物学小组成立在惠氏研究阿伯丁，以支持这一目标，通过研究棘角鱼(Squalus Acanthias)的B细胞发育和同型选择，特别是寻找在体内或体外诱导抗原特异性IgNAR V区产生的最佳方法。虽然分子方法使我们能够开始解决这一目标，但确实迫切需要建立鲨鱼B细胞体外长期培养的方法。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，在这项工作中，我们建议从刺角鱼中定位并测序一些细胞因子基因，从它们在其他物种中的作用来看，这些基因应该会驱动B细胞的生长和分化。然后，我们将把注意力集中在几个这样的分子上，以产生重组蛋白，并在体外研究它们对鲨鱼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.