Camelid antibodies as sensitive proteomics tools for developmental studies

描述（申请人提供）：非洲爪蟾是脊椎动物发育研究的重要模式生物。这些研究受到缺乏分子工具的限制，这些工具是胚胎发生过程中蛋白质表达、定位和功能所必需的。本申请将探索骆驼科动物的单域抗体（或纳米抗体）的效用，用于爪蟾胚胎蛋白质组的表征和功能探索。与传统的单克隆抗体相比，纳米抗体的产生和维持都很昂贵，纳米抗体非常稳定，并且可以很容易地在大肠杆菌中表达。大肠杆菌中，而不丧失其结合活性。在我们的初步实验中，针对非洲爪蟾抗原的特异性纳米抗体已经通过从用复杂原肠胚胚胎裂解物免疫的美洲驼的先导cDNA文库的同胞选择成功地分离。基于这些初步的研究，我们建议开发一类新的抗体工具的非洲爪蟾蛋白质组的分析。为了实现这一目标，将通过表达克隆方法制备和筛选相关的纳米抗体cDNA文库，以分离针对特定非洲爪蟾发育抗原的纳米抗体。这些实验将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蟾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用于功能胚胎学研究的纳米抗体表达克隆的可行性。通过将纳米抗体技术应用于胚胎学研究，这些实验将对未来非洲爪蟾胚胎发育的系统级表征和功能分析产生重大影响，并将对非洲爪蟾社区构成重大帮助。产生的纳米抗体将用于分析蛋白质-蛋白质相互作用和不同发育过程中的基因调控网络。这些过程的先进知识对于未来了解人类出生缺陷的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.