Nanobodies as markers and tools for studies of Xenopus embryonic development

 描述（由申请人提供）： 脊椎动物发育生物学在很大程度上依赖于使用低等脊椎动物模型，例如非洲爪蟾，然而，由于缺乏蛋白质表达、定位和功能所必需的分子工具，其使用受到限制。该提案将评估骆驼衍生的纳米抗体（单域抗体）用于爪蟾胚胎蛋白质组的表征和功能探索的效用。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，噬菌体展示库已从免疫美洲驼淋巴细胞构建，并显示出适合的来源，针对青蛙抗原的纳米抗体。此外，已从这些文库中分离出对非洲爪蟾抗原具有特异性的纳米抗体，并优化了基于质谱的鉴定相应抗原的方法。基于这些初步研究，我们计划开发一类新的抗体工具，用于非洲爪蟾蛋白质组的分析。为了实现这一目标，构建的cDNA文库将用于几种表达克隆方法，以建立Xenopus特异性纳米抗体库，并评估纳米抗体在功能胚胎学研究中的效用。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水平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。生成的纳米抗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。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): Vertebrate developmental biology heavily relies on the use of lower vertebrate models, such as Xenopus laevis, however, its use has been limited by lack of molecular tools that are necessary for following protein expression, localization and function. This proposal will evaluate the utilit of llama-derived nanobodies (single domain antibodies) 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, phage display libraries have been constructed from immune llama lymphocytes and shown to be suitable sources for nanobodies against frog antigens. Moreover, nanobodies specific for Xenopus antigens have been isolated from these libraries and a mass spectrometry-based approach to identify the corresponding antigens has been optimized. Based on these preliminary studies, we plan to develop a new class of antibody tools for the analysis of the Xenopus proteome. To achieve this goal, the constructed cDNA libraries will be used in several expression cloning approaches to establish a bank of Xenopus-specific nanobodies and to evaluate the utility of nanobodies in 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.