Antibody Validation Vector Core

抗体验证载体核心

• 批准号: 10231257
• 负责人: Feng Qian
• 金额: $ 18.2万
• 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/10231257
• 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.

项目摘要 多囊肾（PKD）保护性治疗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理解有限。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，然后继续验证它们， 动物模型，目的是进行临床试验。这条管道需要一个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。这一点尤其具有挑战性， PKD是因为多囊蛋白特别是难以处理的多结构域膜蛋白。许多 的可用抗体缺乏足够的亲和力和特异性，并且大的cDNA构建体很难 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 研究人员与我们现有的试剂，但应用生物技术的最新进展，以开发最 创新的工具，将推动下一代的发现在PKD研究。此应用程序基于 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 提供技术专长，使研究人员能够充分利用这些新试剂。为了实现 为此，我们提出以下具体目标：1）维护和提供经验证的PKD抗体试剂盒， 全长内源性蛋白2）使用用细胞表面PC 1/PC 2进行全细胞免疫的新策略， 开发针对天然构型的多囊蛋白复合物的小鼠单克隆抗体3） 使用纯化和稳定的PC 1/PC 2复合抗原开发第一个PC 1和PC 2骆驼科VHH纳米抗体 4)使用包埋在脂质中的纯化的和功能性的PC 2-四聚体开发第一个PC 2 VHH纳米抗体 5）提供和开发用于PKD基因表达、失活和表达的易于使用的载体。 操纵总之，抗体和载体核心将为研究界提供一个 全面的经过验证的试剂和独特的专业知识，将有助于在PKD领域的发现。