CORE--PEPTIDE SYNTHESIS AND ANTIBODY PRODUCTION

核心--多肽合成与抗体生产

• 批准号: 6218850
• 负责人: JEAN E RIVIER
• 金额: $ 27.95万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6218850
• 关键词: antibody; biomedical facility; capillary electrophoresis; high performance liquid chromatography; ion exchange chromatography; mass spectrometry; peptide chemical synthesis; protein purification

The purpose of this Core is to provide synthetic peptides and polyclonal antisera required by the Projects of this Program to carry out structural, functional and mechanistic studies. The two PI's of this Core have collaborated for 25 years to design peptide immunogens and produce high affinity, high specificity antibodies towards peptides and proteins. Using the Merrifield automated solid phase approach to peptide synthesis, this Core will synthesize at the request of each PI of this Program Project a total of approximately 30 peptides (including phosphopeptides) per year. From among these 30 synthetic peptides or from expressed proteins, approximately seven will be selected every year as immunogens for the production of polyclonal antisera. Using the latest techniques for peptide/protein isolation and analysis, we would provide relatively large quantities (25 to 100 mg) of purified, fully characterized peptides. Preparative peptide purification will be achieved by reversed phase high performance liquid chromatography (RP- HPLC) in two solvent systems. Determination of purity will be accomplished using RP-HPLC, ion exchange chromatography on FPLC and capillary zone electrophoresis; characterization will use amino acid analysis, mass spectroscopy (MS) and Edman degradation when necessary. We will use conjugated synthetic peptides as immunogens to develop antibodies directed towards gene products predicted from the cDNA sequences or from amino acid sequencing of tryptic fragments of purified natural products. Peptides will be carefully designed in such a way that the same peptide can be coupled through a unique bond to a protein carrier or a chromatographic support to raise antibodies and facilitate their affinity purification. Alternatively, fusion proteins produced in bacteria or recombinant proteins using the baculovirus/insect cell system and provided by individual projects will be injected directly in rabbits for production of antisera. To reduce cross-reactivity with antigens other than the protein of interest, selected antisera will be immunoaffinity purified using the cognate peptide, recombinant protein, or fusion protein covalently bound to an agarose-based gel.

本核心的目的是提供合成肽和多克隆 本计划项目所需的抗血清， 结构、功能和机制研究。 这两个PI 核心已经合作了25年，设计肽免疫原， 产生针对肽的高亲和力、高特异性抗体， proteins. 使用梅里菲尔德自动化固相方法 综合，本核心将根据本核心的每个PI的要求进行综合。 计划项目共约30肽（包括 磷酸肽）。 从这30种合成肽或 从表达的蛋白质中，每年将选择大约七种 作为免疫原用于生产多克隆抗血清。 使用 肽/蛋白质分离和分析的最新技术，我们将 提供相对大量（25至100 mg）的纯化的、完全 特征肽。 制备性肽纯化将是 通过反相高效液相色谱法（RP- HPLC）。 纯度测定将 使用RP-HPLC、FPLC上的离子交换色谱和 毛细管区带电泳;表征将使用氨基酸 分析、质谱（MS）和Edman降解（必要时）。 我们将使用结合的合成肽作为免疫原， 针对从cDNA预测的基因产物的抗体 序列或来自纯化的胰蛋白酶片段的氨基酸测序。 天然产品。 肽将被精心设计， 相同的肽可以通过独特的键与蛋白质偶联 载体或色谱支持物，以产生抗体并促进 其亲和纯化。 可替代地，可以使用在大肠杆菌中产生的融合蛋白。 使用杆状病毒/昆虫细胞系统 并由个别项目提供将直接注射在兔子 用于生产抗血清。 降低与抗原的交叉反应性 除了感兴趣的蛋白质， 使用同源肽，重组蛋白， 或共价结合于琼脂糖基凝胶的融合蛋白。