CORE--TECHNICAL CORE

核心--技术核心

• 批准号: 6849108
• 负责人: WOLFGANG H FISCHER
• 金额: $ 25.7万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6849108
• 关键词: SDS polyacrylamide gel electrophoresis; activins; biological signal transduction; biomedical facility; cell surface receptors; gonadotropins; high performance liquid chromatography; inhibin; mass spectrometry; nuclear magnetic resonance spectroscopy; proteoglycan; radioimmunoassay; recombinant proteins; synthetic peptide

This Program Project focuses on the role of activins and inhibins and their membrane-bound receptors, soluble binding proteins and modulators in the regulation of reproductive function, cellular growth and development. The primary focus of this Program is to characterize the physical interactions and molecular events that lead to signal propagation by activin and inhibin. The second focus is to explore the physiologic and pathophysiologic significance of these molecules at the cellular and system levels and to study the control of protein expression and secretion as well as modes of action. The Technical Core (Core Component B) provides vital services, including protein expression, peptide synthesis, peptide/protein characterization, and generation of antibodies, needed by the Projects of this Program to carry out structural, functional and mechanistic studies. Large quantities of recombinant activin, inhibin and betaglycan fragments, essential for the structural and biological studies, will be expressed by the Core. Peptides for generation of antisera and for specific blockers of antibodies will be synthesized and purified by the Core. Synthetic peptides and recombinant proteins generated by the Core, additional recombinant proteins produced by individual Projects, and novel proteins isolated by this Program will be characterized. Posttranslational modifications of ligands and/or receptors will be identified. The analysis of polypeptides will be achieved using a number of techniques including HPLC, mass spectroscopy, NMR and automated Edman degradation. Synthetic peptides conjugated to carrier proteins will be used as immunogens to develop antibodies directed towards gene products predicted from cDNA sequences. Alternatively, proteins produced using in vitro cell expression systems will be injected directly into host animals for production of antisera. To reduce cross-reactivity with antigens other than the protein of interest, selected antisera will be immunoaffinity purified when necessary. Core B will additionally provide immunoassays of pituitary gonadotropic hormones and sex steroids for physiologic and pharmalogic studies proposed. The reagents and services provided require specialized equipment and highly trained personnel which can be most efficiently consolidated in a Core. In addition, the integrated nature of the Core assures cost efficiency, standardization of protocols and facilitates quality control.

本计划项目的重点是激活素和激活素及其膜结合受体，可溶性结合蛋白和调节剂在调节生殖功能，细胞生长和发育中的作用。该计划的主要重点是表征物理相互作用和分子事件，导致信号传播的激活素和tubin。第二个重点是探索这些分子在细胞和系统水平上的生理和病理生理意义，并研究蛋白质表达和分泌的控制以及作用模式。技术核心（核心组件B）提供重要服务，包括蛋白质表达、肽合成、肽/蛋白质表征和抗体生成，这些都是本计划项目进行结构、功能和机制研究所需的。Core将表达大量重组激活素、β-葡聚糖和β-葡聚糖片段，这些片段对于结构和生物学研究至关重要。用于产生抗血清和抗体特异性阻断剂的肽将由核心合成和纯化。将对核心产生的合成肽和重组蛋白、个别项目产生的其他重组蛋白以及本项目分离的新型蛋白进行表征。将鉴定配体和/或受体的翻译后修饰。多肽的分析将使用多种技术实现，包括HPLC、质谱、NMR和自动Edman降解。与载体蛋白缀合的合成肽将用作免疫原以开发针对从cDNA序列预测的基因产物的抗体。可选择地， 将使用体外细胞表达系统产生的蛋白质直接注射到宿主动物中以产生抗血清。为了减少与目标蛋白以外的抗原的交叉反应性，必要时将对所选抗血清进行免疫亲和纯化。核心B将额外提供用于拟定生理学和药理学研究的垂体促性腺激素和性类固醇的免疫测定。所提供的试剂和服务需要专门的设备和训练有素的人员，这些设备和人员可以最有效地整合在一个核心中。此外，核心的集成性确保了成本效益、协议标准化和便于质量控制。