Camelid antibodies as sensitive proteomics tools for developmental studies

描述(申请人提供)：非洲爪哇是研究脊椎动物发育的重要模式生物。这些研究由于缺乏跟踪胚胎发育过程中蛋白质表达、定位和功能所必需的分子工具而受到限制。这一应用将探索来自骆驼的单域抗体(或纳米体)在非洲爪哇胚胎蛋白质组鉴定和功能探索中的应用。与制造和维护费用昂贵的传统单抗相比，纳米抗体非常稳定，可以很容易地在大肠杆菌中表达，而不会失去它们的结合活性。在我们的初步实验中，通过同胞选择从用复杂的原肠胚胚胎裂解物免疫的大羊驼中，成功地分离出了针对非洲爪哇抗原的特异性纳米抗体。在这些初步研究的基础上，我们建议开发一类新的抗体工具来分析非洲爪哇蛋白质组。为了实现这一目标，将通过表达克隆的方法制备和筛选相关的纳米体cDNA文库，以分离针对非洲爪哇发育抗原的纳米体。这些实验应该会产生一些有用的分子标记，并将评估这种方法是否可以扩展到非洲爪哇蛋白质组和其他脊椎动物蛋白质组。这些研究还将使我们能够评估纳米体表达克隆用于功能胚胎学研究的可行性。通过将纳米体技术应用于胚胎学研究，这些实验将对未来非洲爪哇胚胎发育的系统水平表征和功能分析产生重大影响，并将对非洲爪哇群落构成重大帮助。生成的纳米体将用于分析不同发育过程中的蛋白质-蛋白质相互作用和基因调控网络。对这些过程的先进知识对于未来理解人类出生缺陷的潜在机制和癌症的分子原因至关重要。

DESCRIPTION (provided by applicant): Xenopus laevis is an important model organism for studies of vertebrate development. These studies are limited by lack of molecular tools that are necessary for following protein expression, localization and function during embryogenesis. This application will explore the utility of single domain antibodies (or nanobodies) from camelids for characterization and functional exploration of the Xenopus embryo proteome. In contrast to conventional monoclonal antibodies, which are expensive to generate and maintain, nanobodies are exceptionally stable and can be easily expressed in E. coli without loss of their binding activity. In our preliminary experiments, specific nanobodies against Xenopus antigens have been successfully isolated by sib-selection of a pilot cDNA library from llamas immunized with a complex gastrula embryo lysate. Based on these preliminary studies, we propose to develop a new class of antibody tools for the analysis of the Xenopus proteome. To achieve this goal, relevant nanobody cDNA libraries will be prepared and screened by expression cloning approaches to isolate nanobodies against specific Xenopus developmental antigens. These experiments should generate a number of useful molecular markers and will assess whether this approach can be extended to the Xenopus proteome and other vertebrate proteomes. These studies will also enable us to evaluate feasibility of nanobody expression cloning for functional embryological studies. By applying the nanobody technology to embryological studies, these experiments will have a significant impact on the future systems- level characterization and functional analysis of Xenopus embryonic development and will constitute a major help to the Xenopus community. The generated nanobodies will be used for the analysis of protein-protein interactions and gene regulatory networks in diverse developmental processes. The advanced knowledge of these processes is critical for the future understanding of mechanisms underlying human birth defects and the molecular causes of cancer.