CORE--PEPTIDE SYNTHESIS AND ANTIBODY PRODUCTION

核心--多肽合成与抗体生产

• 批准号: 6311528
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6311528
• 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.

这个核心的目的是提供合成肽和多克隆 本计划项目实施所需的抗血清 结构、功能和力学研究。这两个圆周率 CORE已经合作了25年来设计多肽免疫原和 产生针对多肽的高亲和力、高特异性抗体和 蛋白质。用Merrifield自动固相法测定多肽 合成，此核心将在此的每个PI的请求下合成 计划项目总共约30个多肽(包括 磷酸多肽)每年。从这30个合成肽中或 每年将从表达的蛋白质中挑选出大约七个 作为免疫原用于生产多克隆抗血清。使用 最新的多肽/蛋白质分离和分析技术，我们将 提供相对较大数量(25至100毫克)的纯化、完全 具有特征的多肽。将进行制备性多肽纯化 通过反相高效液相色谱(RP- 在两种溶剂体系中)。纯度的测定将是 用反相高效液相色谱、FPLC离子交换层析和 毛细管区带电泳法；将使用氨基酸进行表征 必要时进行分析、质谱分析和埃德曼降解。 我们将使用结合的合成肽作为免疫原来开发 针对从cDNA预测的基因产物的抗体 纯化的胰酶片段的氨基酸序列或测序 天然产品。多肽将以这样一种方式精心设计 相同的多肽可以通过一个独特的键与蛋白质偶联 载体或层析载体以提高抗体和促进 它们的亲和纯化。或者，融合蛋白在体内产生 利用杆状病毒/昆虫细胞系统的细菌或重组蛋白 并由个别项目提供，将直接注射到兔子体内 用于生产抗血清。减少与抗原的交叉反应 除了感兴趣的蛋白质，选定的抗血清将是 使用同源肽、重组蛋白、 或融合蛋白共价结合到琼脂糖凝胶上。