CORE 2: Protein Expression and Purification Core

核心 2：蛋白质表达和纯化核心

• 批准号: 10625689
• 负责人: BENJAMIN W SPILLER
• 金额: $ 25.3万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-03 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625689
• 关键词: Antibodies; Antibody Response; Antigens; Bacillus megaterium; Cells; Clostridium difficile; Codon Nucleotides; Core Protein; Cryoelectron Microscopy; Electron Microscopy; Epitopes; Escherichia coli; Evaluation; Excision; Fab Immunoglobulins; Frequencies; Futility; Goals; Human; IgG1; Immune response; Immunoglobulin A; Immunoglobulin G; Length; Methods; Molecular; Patients; Proteins; Reagent; Sampling; Sorting; Specificity; Structure; Subunit Vaccines; Toxin; Vaccination; Vaccines; Validation; X-Ray Crystallography; antigen binding; experience; experimental study; holotoxins; improved; member; neutralizing antibody; novel; novel vaccines; primary endpoint; protein expression; protein purification; structural biology; vaccine candidate; vaccine development; vaccine trial

Summary – Core 2: Protein Expression and Purification Core To support the Vanderbilt Antibody and Antigen Discovery for Clostridioides difficile Vaccines (VANDy-CdV) goals, the Protein Expression and Purification Core (Protein Core) will improve and establish protein expression and purification strategies to enable delivery of both antigens and antibodies for both Projects 1 and 2 of this proposal. Overall, the Protein Core will have three goals. The first goal is to express and purify C. difficile toxins and other antigens for use as sorting reagents and vaccine candidates in Projects 1, aim 1 and 2, and Project 2, aim 1. The second goal will be expression and purification of antibodies identified in Projects 1, aim 1, and Project 2, aim 2. Antibody expression and purification will be done on the μg and mg scale allowing purification of hundreds of clones. This will support antibody and antigen validation and neutralization experiments in both aims of Project 1. The third goal will support structural biology efforts in Project 1. Structural Biology has become an essential component of vaccine development. In the present application antigen discovery and antigen optimization will be used to produce new vaccine candidates with a focus on developing subunits that lack immunodominant yet non-neutralizing epitopes. These subunit vaccine candidates will be optimized and used for structural studies. We will identify antibody antigen pairs and produce molecular Fab fragments and appropriate antigens for structural studies in Project 1, aim 1.

总结-核心2：蛋白表达和纯化核心 支持艰难梭菌疫苗的范德比尔特抗体和抗原发现（VANDy-CdV） 蛋白质表达和纯化核心（Protein Core）将改善和建立蛋白质 表达和纯化策略，从而能够递送项目1和项目2的抗原和抗体。 2、这个建议。总的来说，蛋白质核心将有三个目标。第一个目标是表达和纯化C. 艰难梭菌毒素和其他抗原，用作项目1，目标1和 2，项目2，目标1。第二个目标将是表达和纯化项目中鉴定的抗体。 1，目标1，项目2，目标2。抗体表达和纯化将以μg和mg规模进行， 数百个克隆的纯化。这将支持抗体和抗原验证和中和 在项目1的两个目标的实验。第三个目标将支持项目1中的结构生物学工作。结构 生物学已成为疫苗开发的重要组成部分。在本申请中，抗原 发现和抗原优化将用于生产新的候选疫苗，重点是开发 缺乏免疫显性但非中和表位的亚基。这些候选亚单位疫苗将 优化并用于结构研究。我们将鉴定抗体抗原对， 片段和适当的抗原，用于项目1的结构研究，目标1。