A bifunctional antibody screening system for both phage display and yeast two-hyb

用于噬菌体展示和酵母双杂交的双功能抗体筛选系统

• 批准号: 7802030
• 负责人: MICHAEL P WEINER
• 金额: $ 20万
• 依托单位: AFFOMIX, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2012-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7802030
• 关键词: Affinity; Antibodies; Antibody Affinity; Binding; Bispecific Antibodies; Cell Nucleus; Chimeric Proteins; Cloning; Cloning Vectors; Complementarity Determining Regions; Complementary DNA; DNA Binding Domain; Detection; Disease; Epitopes; Escherichia coli; Gene Rearrangement; Genes; Human; Immunoglobulin Variable Region; Immunoglobulins; Immunotherapeutic agent; In Vitro; Libraries; Light; Maintenance; Medicine; Messenger RNA; Methods; Monoclonal Antibodies; Nature; Nuclear; Phage Display; Plasmids; Proteins; Proteomics; Reagent; Recombinant Antibody; Reporter Genes; Research; Research Personnel; Saccharomyces; Screening procedure; Signal Transduction; Simian virus 40; Single-Stranded DNA; Site; Specificity; System; Testing; Therapeutic; Time; Transcriptional Activation Domain; Yeasts; base; combinatorial; cost; design; direct application; gene cloning; promoter; protein expression; protein protein interaction; public health relevance; transcription termination; vector; yeast two hybrid system

DESCRIPTION (provided by applicant): This is a proposal to establish a proof-of-principle for a vector system to isolate high-affinty antibodies through selective rounds in both yeast and Escherichia coli. Antibodies have quickly become extremely useful as therapeutic medicines to treat a wide-variety of disorders. We propose to develop a means of making the discovery of immunotherapeutics faster and cheaper. In this proposal we describe a system to isolate antigen-specific antibodies in yeast two-hybrid and then quickly evolve them for both higher affinity and specificity using phage display. This new vector system will eliminate the need for any subcloning needed for protein expression, Y2H and phage display. The option for expressing and purifying antibodies directly from E. coli or yeast is a direct application of the described system. The described system will allow for simultaneous testing of cDNA and antibody-antigen interactions in both yeast and E.coi-based systems. Completion of this project will form the basis of a scalable system for a rapid and inexpensive means of affinity-maturation of recombinant antibodies screened in Y2H and increasing our understanding of protein- protein interactions. PUBLIC HEALTH RELEVANCE: Researchers will benefit from an increased understanding of the function of human proteins. Methods are needed that can facilitate this understanding. The immediate objective of our research is to generate a method that will enable researchers to multiplex the screening of affinity reagents. We anticipate using our high-throughput pipeline for producing recombinant antibodies as a means to generate the reagents needed to make this method possible.

描述（由申请方提供）：这是一项建立载体系统原理验证的提案，通过酵母和大肠杆菌中的选择性轮次分离高亲和力抗体。抗体作为治疗多种疾病的治疗药物已迅速变得非常有用。我们建议开发一种方法，使免疫治疗的发现更快，更便宜。在这个建议中，我们描述了一个系统，以分离抗原特异性抗体在酵母双杂交，然后快速进化他们更高的亲和力和特异性使用噬菌体展示。这种新的载体系统将消除对蛋白质表达、Y2 H和噬菌体展示所需的任何亚克隆的需要。直接从大肠杆菌表达和纯化抗体的选择。大肠杆菌或酵母是所述系统的直接应用。所述系统将允许在基于酵母和大肠杆菌的系统中同时测试cDNA和抗体-抗原相互作用。该项目的完成将形成可扩展系统的基础，该系统用于在Y2 H中筛选的重组抗体的亲和力成熟的快速且廉价的手段，并增加我们对蛋白质-蛋白质相互作用的理解。 公共卫生相关性：研究人员将受益于对人类蛋白质功能的进一步了解。需要有方法来促进这种理解。我们研究的直接目标是产生一种方法，使研究人员能够多重筛选亲和试剂。我们预计使用我们的高通量生产线生产重组抗体，作为一种手段，以产生所需的试剂，使这种方法成为可能。