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

描述（由申请人提供）：这是一份建立载体系统原理证明的提案，通过酵母和大肠杆菌的选择性轮分离高亲和力抗体。抗体已迅速成为非常有用的治疗药物，用于治疗各种各样的疾病。我们建议开发一种方法，使免疫疗法的发现更快，更便宜。在这个提议中，我们描述了一个系统，从酵母双杂交中分离抗原特异性抗体，然后利用噬菌体展示快速进化它们具有更高的亲和力和特异性。这种新的载体系统将消除对蛋白质表达、Y2H和噬菌体展示所需的任何亚克隆的需要。直接从大肠杆菌或酵母中表达和纯化抗体的选择是所描述系统的直接应用。所描述的系统将允许在酵母和大肠杆菌为基础的系统中同时检测cDNA和抗体-抗原相互作用。该项目的完成将为一个可扩展的系统奠定基础，该系统可以快速、廉价地对在Y2H中筛选的重组抗体进行亲和成熟，并增加我们对蛋白质-蛋白质相互作用的理解。

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.