Antibody Validation Vector Core

抗体验证载体核心

基本信息

批准号：
10058978
负责人：
Feng Qian
金额：
$ 17.6万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-24 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10058978
关键词：
3-Dimensional Affinity Animal Model Animals Antibodies Antibody Affinity Antigens Area Basic Science Binding Biochemistry Biotechnology Cell Culture System Cell surface Cells Clinical Trials Cloning Communities Complementary DNA Complex Critical Pathways Development Epitopes Gene Expression Gene Proteins Genes Goals Immunization Immunize Immunoprecipitation Length Libraries Lipids Mammalian Cell Maryland Membrane Proteins Monoclonal Antibodies Mus Mutate PKD2 protein Pathway interactions Phage Display Polycystic Kidney Diseases Positioning Attribute Principal Investigator Property Proprotein Convertase 1 Proprotein Convertase 2 Protein Fragment Proteins Protocols documentation Reagent Research Research Personnel Role Science Silent Mutation Site Specificity Surface Technical Expertise Translations Validation Western Blotting Work anti-PD1 antibodies experience expression vector in vivo innovation mutant nanobodies nanodisk next generation novel novel strategies polycystic kidney disease 1 protein programmed cell death protein 1 protein complex repository restriction enzyme tool vector

项目摘要

Project Summary A major obstacle to the development of protective therapies for polycystic kidney disease (PKD) has been our limited understanding of the cellular pathways that are altered when PKD genes are mutated. In general researchers have identified critical pathways in cell culture systems and then proceeded to validate them in animal models with the goal of moving on to clinical trials. This pipeline requires a robust tool kit of basic science reagents including high quality antibodies and expression constructs. This has been particularly challenging for PKD because polycystins in particular are multi-domain membrane proteins that are difficult to work with. Many of the available antibodies lack sufficient affinity and specificity and large cDNA constructs are difficult to manipulate in cloning applications. The overarching goal of the Antibody and Vector Core is not only to supply investigators with our existing reagents, but to apply recent advances in biotechnology to develop the most innovative tools that will advance the next generation of discoveries in PKD research. This application builds on the Principal Investigator’s decades long experience in the study of polycystin biochemistry that will allow him to provide technical expertise so that investigators can fully leverage these novel reagents. In order to accomplish this goal we propose the following specific aims: 1) Maintain and provide validated PKD antibody kits that detect full length endogenous proteins 2) Use a novel strategy of whole cell immunization with cell surface PC1/PC2 to develop mouse monoclonal antibodies directed against the polycystin complex in its native configuration 3) Develop the first PC1 and PC2 Camelid VHH nanobodies using purified and stabilized PC1/PC2 complex antigen 4) Develop the first PC2 VHH nanobodies using purified and functional PC2-tetramer embedded in lipid nanodiscs and 5) Provide and develop easy-to-use vectors for PKD gene expression, inactivation and manipulation. In summary the Antibody and Vector Core will provide the research community with a comprehensive array of validated reagents and unique expertise that will facilitate discoveries in the PKD field.

项目概要开发多囊肾病（PKD）保护性疗法的一个主要障碍是我们的一般来说，对 PKD 基因突变时发生的细胞通路的了解有限。研究人员已经确定了细胞培养系统中的关键途径，然后在以进入临床试验为目标的动物模型需要强大的基础科学工具包。包括高质量抗体和表达构建体的试剂，这对于我们来说尤其具有挑战性。 PKD 是因为多囊蛋白尤其是难以使用的多结构域膜蛋白。的可用抗体缺乏足够的亲和力和特异性，并且大型 cDNA 构建体难以构建抗体和载体核心的总体目标不仅仅是提供克隆应用中的操作。研究人员使用我们现有的试剂，但应用生物技术的最新进展来开发最将推动 PKD 研究的下一代发现的创新工具。首席研究员在多囊蛋白生物化学研究方面数十年的经验将使他能够提供研究人员可以充分利用这些新颖试剂来完成的技术专业知识。为实现这一目标，我们提出以下具体目标： 1) 维护并提供经过验证的 PKD 抗体试剂盒，用于检测全长内源蛋白 2) 使用细胞表面 PC1/PC2 进行全细胞免疫的新策略开发针对天然构型多囊蛋白复合物的小鼠单克隆抗体 3) 使用纯化且稳定的 PC1/PC2 复合抗原开发第一个 PC1 和 PC2 Camelid VHH 纳米抗体 4) 使用嵌入脂质的纯化和功能性 PC2 四聚体开发第一个 PC2 VHH 纳米抗体 nanodiscs 和 5) 提供和开发易于使用的载体，用于 PKD 基因表达、失活和总之，抗体和载体核心将为研究界提供一种操作方法。全面的经过验证的试剂和独特的专业知识将促进 PKD 领域的发现。