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

基本信息

批准号：
9208974
负责人：
DOMINIQUE R ALFANDARI
金额：
$ 29.52万
依托单位：
UNIVERSITY OF MASSACHUSETTS AMHERST
依托单位国家：
美国
项目类别：
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/9208974
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 Disease 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&apos s Organizer Spleen Testing Tissues Vertebral column Work Xenopus Xenopus Proteins Xenopus laevis antibody libraries base cell type chromatin immunoprecipitation cross reactivity design 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

项目摘要

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.

项目摘要：两栖动物胚胎，尤其是青蛙Xenopus laevis是一种出色的模型全球胚胎学家和细胞生物学家。该模型系统领导了轴形成的发现（前/后和腹侧/腹侧），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该模型恢复活力。在 Xenopus社区最近的白皮书已经确定了缺乏足够的资源研究爪蟾蛋白质并提出这种资源的生产优先级的抗体。我们建议至少产生一百个完全表征转录的单克隆抗体因素，细胞表面蛋白，包括信号受体和配体以及特定于组织标记。为了实现这一目标，我们将用两种选择的重组蛋白免疫小鼠免疫在哺乳动物细胞和复杂蛋白提取物中表达，从爪蟾胚胎分离出来。重组蛋白靶标将由由Xenopus组成的指导委员会选择研究人员基于蛋白质在发育和疾病过程中的重要性以及缺乏交叉反应性商业抗体。通过这些免疫接种，我们将产生，并档案超过10,000多个表达IgG的克隆人，将通过免疫荧光筛选 Escence，整个免疫染色和免疫沉淀以识别单克隆抗体到天然蛋白质。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法进行，然后在重组推定目标。这些单克隆抗体以及表征数据将通过发展性杂交瘤研究银行和Xenbase自由分发给社区。