Antibody Validation Vector Core

抗体验证载体核心

• 批准号: 10693926
• 负责人: Feng Qian
• 金额: $ 18.19万
• 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/10693926
• 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; Educational process of instructing; Epitopes; Gene Expression; 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; Transfection; 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.

项目摘要 多囊肾病(PKD)保护性疗法发展的主要障碍是我们的 对PKD基因突变时改变的细胞通路的了解有限。总体而言 研究人员已经确定了细胞培养系统中的关键途径，然后开始在 动物模型，目标是进入临床试验。这条管道需要一个强大的基础科学工具包 试剂包括高质量的抗体和表达构建体。这尤其具有挑战性， PKD，因为多囊蛋白是很难处理的多结构域膜蛋白。许多 现有的抗体缺乏足够的亲和力和特异性，大的cdna构建物很难 在克隆应用程序中进行操作。抗体和载体核心的首要目标不仅是提供 研究人员使用我们现有的试剂，但要应用生物技术的最新进展来开发最 将推动下一代PKD研究发现的创新工具。此应用程序构建在 首席研究员在多囊藻毒素生物化学研究方面的数十年经验使他能够 提供技术专业知识，以便研究人员能够充分利用这些新试剂。为了完成 为此，我们提出了以下具体目标：1)维护和提供有效的PKD抗体试剂盒，以检测 全长内源性蛋白2)使用细胞表面PC1/PC2全细胞免疫的新策略 研制针对多囊藻毒素复合体天然构型的鼠单抗3) 利用纯化和稳定的PC1/PC2复合抗原制备首个PC1和PC2骆驼VHH纳米体 4)利用纯化的功能性PC2-四聚体包埋在脂质中，制备出第一个PC2 VHH纳米体 纳米盘和5)提供和开发易于使用的载体，用于PKD基因的表达、失活和 操纵。总之，抗体和载体核心将为研究界提供一个 全面的经验证的试剂和独特的专业知识，将促进在PKD领域的发现。