First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate
First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate
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DOI：
10.1073/pnas.0508341103
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发表时间：
2006-02-07
影响因子：
11.1
通讯作者：
Flajnik, MF
中科院分区：
文献类型：
--
作者：
Dooley, H;Stanfield, RL;Flajnik, MF
软骨鱼类是发现免疫球蛋白的最古老的系统类群，鲨鱼产生独特的IG同种型IgINAR，一种不与轻链结合的重链同源二聚体。相反，IgNAR的可变（V）区作为可溶性单结构域结合抗原。我们的小组已经表明，IgNAR在抗原攻击后护士鲨（Ginglymostoma cirratum）的体液反应中起着不可或缺的作用。在这里，我们从免疫动物中产生IgNAR V区的噬菌体展示文库，并发现一个家族的克隆来源于相同的重排事件，但在扩增过程中差异突变。由于鲨鱼IG基因的集群组织和IgNAR的缺乏性质，我们能够构建该家族的推定祖先。通过研究克隆亲和力背景下的突变，我们发现了这种同种型亲和力成熟的证据。随后，我们能够鉴定在该家族亲和力改善中重要的突变。因为家族克隆都是在免疫后获得的，它们提供了对体内成熟机制的了解，一般来说，以及对单结构域抗体片段的了解。
The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgINAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.