Production and characterization of monoclonal antibodies to Xenopus Proteins

非洲爪蟾蛋白单克隆抗体的生产和表征

• 批准号: 9897195
• 负责人: DOMINIQUE R ALFANDARI
• 金额: $ 3.98万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-07 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9897195
• 关键词: Affinity Chromatography; Amphibia; Animals; Antibodies; Antibody Affinity; Antigens; Archives; Biological Assay; Biological Models; Birds; Cell Line; Cell Surface Proteins; Cell physiology; Cell surface; Cells; Charge; Chimeric Proteins; Communities; Competence; Complex; Data; Development; Developmental Process; Dorsal; Embryo; Epitopes; Female; Freezing; Gene Silencing; Goals; HE protein; Hybridomas; Immunization; Immunize; Immunofluorescence Immunologic; Immunoglobulin G; Immunoprecipitation; Individual; Ion Exchange; Libraries; Ligands; Mammalian Cell; Mammals; Masks; Mass Spectrum Analysis; Membrane Proteins; Mesoderm; Modeling; Monoclonal Antibodies; Mus; Nature; Neural Crest Cell; Nuclear; Paper; Pattern; Peptides; Post-Translational Protein Processing; Process; Production; Protein Family; Proteins; Proteomics; Rana; Receptor Signaling; Recombinant Proteins; Recombinants; Research Personnel; Resolution; Resources; Screening Result; Serum; Signaling Protein; Spemann' s Organizer; Spleen; Testing; Tissues; Vertebral column; Work; Xenopus; Xenopus Proteins; Xenopus laevis; antibody libraries; base; cell type; chromatin immunoprecipitation; cross reactivity; design; developmental disease; embryo membrane; embryonic protein; genome sequencing; glycosylation; in vivo Model; liquid chromatography mass spectrometry; meetings; nuclear transfer; polyclonal antibody; protein complex; relating to nervous system; success; tool; transcription factor

PROJECT SUMMARY: The amphibian embryo and in particular the frog Xenopus laevis is an outstanding model used by embryologist and cell biologist worldwide. This model system has led the discovery of axis formation (antero/posterior and dorso/ventral), tissue induction and nuclear reprograming among others. Advances in genome sequencing, proteomics and gene silencing have rejuvenated this model. In its most recent White Paper, the Xenopus community has identified a lack of adequate resource of antibody to study Xenopus proteins and proposed the production of such resources a priority. We propose to produce at least a hundred fully characterized monoclonal antibodies to transcription factors, cell surface proteins including signaling receptors and ligands as well as tissue specific markers. To achieve this goal we will immunize mice with both selected recombinant proteins expressed in mammalian cells and complex protein extracts isolated from Xenopus embryos. The recombinant protein targets will be selected by a steering committee composed of Xenopus investigators based on the importance of the proteins in developmental and diseases processes and the absence of cross-reactive commercial antibodies. From these immunizations we will produce and archive more than 10,000 clonal IgG-expressing hyrbidomas that will be screened by immunofluor- escence, whole mount immunostaining and immunoprecipitation to identify monoclonal anti-bodies to native proteins. Final characterization of monoclonal antibodies produced against the complex embryonic extracts will be performed by mass spectrometry followed by immuno-fluorescence on the recombinant putative target(s). These monoclonal antibodies, together with the characterization data will be freely distributed to the community via the developmental hybridoma study bank and Xenbase.

项目概要： 两栖动物胚胎，特别是非洲爪蟾 (Xenopus laevis) 是一个杰出的模型。 世界各地的胚胎学家和细胞生物学家。该模型系统导致了轴形成的发现 （前/后和背/腹）、组织诱导和核重编程等。 基因组测序、蛋白质组学和基因沉默的进步使这一模型焕发了活力。在 在其最新的白皮书中，Xenopus 社区发现缺乏足够的资源 研究爪蟾蛋白的抗体并提出优先生产此类资源。我们 提议生产至少一百种完全表征的转录单克隆抗体 因子、细胞表面蛋白（包括信号受体和配体）以及组织特异性 标记。为了实现这一目标，我们将用两种选定的重组蛋白对小鼠进行免疫接种 在哺乳动物细胞和从爪蟾胚胎中分离的复杂蛋白质提取物中表达。 重组蛋白靶点将由 Xenopus 组成的指导委员会选出 研究人员基于蛋白质在发育和疾病过程中的重要性， 不存在交叉反应的商业抗体。通过这些免疫接种，我们将生产和 归档超过 10,000 个表达 IgG 的克隆性杂交瘤，将通过免疫荧光进行筛选 荧光、整体免疫染色和免疫沉淀来鉴定单克隆抗体 到天然蛋白质。针对复合物产生的单克隆抗体的最终表征 胚胎提取物将通过质谱分析进行，然后对胚胎进行免疫荧光分析 重组假定目标。这些单克隆抗体以及表征数据 将通过发育杂交瘤研究库和 Xenbase 免费分发给社区。