Antibody Validation Vector Core

抗体验证载体核心

• 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
• 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

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 对 PKD 基因突变时改变的细胞通路的了解有限。一般来说 研究人员已经确定了细胞培养系统中的关键途径，然后在 动物模型，目标是进入临床试验。该管道需要强大的基础科学工具包 试剂包括高质量抗体和表达构建体。这对于 PKD 是因为多囊蛋白尤其是难以使用的多结构域膜蛋白。许多 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 在克隆应用程序中进行操作。抗体和载体核心的总体目标不仅是提供 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 创新工具将推动 PKD 研究的下一代发现。该应用程序构建于 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 提供技术专业知识，以便研究人员能够充分利用这些新型试剂。为了实现 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 操纵。总之，抗体和载体核心将为研究界提供 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。