一种增强型酵母双杂交系统的设计及其应用的研究

The yeast two-hybrid (Y2H) is an important molecular genetic tool for the study of protein-protein interaction, especially for its wide application on the screening of interacting partners of proteins. The yeast surface display (YSD) presents foreign proteins on its surface and it is widely used for protein directed evolution. On the basis of YSD, the applicant previously has designed a system dubbed yeast surface two-hybrid (YS2H), which anchors the protein-protein interaction on the yeast surface for the sake of quantitative measurement of the affinity. In this study, an enhanced yeast two hybrid system (eY2H) will be built by grafting of the advantages of the above three systems. This new eY2H uses the concepts of traditional Y2H, of which a certain transcription factor can be divided into two structural independent domains but these two have to spatially complement onto the operon to activate the downstream transcription. This concept is fulfilled by a single plasmid originated from the distinguished novel design of the YS2H. Meanwhile, the reporter gene is displayed on the yeast surface based on the YSD technology for better examination and screening. Compared to the classic Y2H, the eY2H is much easier in examination and verificationa. It saves labor, expenditure, and time, and thus it is especially suitable for the screening libraries of super large sizes.Using the eY2H strategy of library against library, the interaction between influenza virus and different hosts will be investigated. The research may help to disclose the mechanism underlying the viral corss-species transmission.

酵母双杂交是研究蛋白-蛋白相互作用的重要分子遗传学工具，特别是在筛选蛋白的未知受体上应用非常广泛。酵母表面展示将外源蛋白展示于细胞表面，它是研究蛋白定向进化的有力武器。申请者前期在酵母表面展示的基础上创立了酵母表面双杂交，将相互作用的蛋白展示于细胞表面，便于定量检测。在本研究中，申请者拟利用上述三个系统各自的优势，创建一个增强型的酵母双杂交系统。该系统依赖传统双杂交关于转录因子结构上可分为独立的结合结构域与激活结构域，但在功能上彼此依赖的基本原理；利用酵母表面双杂交独特而简便的载体设计，将上述双杂交体系置入单个质粒系统中。同时，借助于酵母表面展示，将报告基因展示于酵母表面，以利于检测和筛选。增强型双杂交系统与原有系统比较，检测与验证都十分容易，省时省力，非常适用于超大文库的高通量筛选。本项目拟以文库对文库筛选策略，研究流感病毒与不同宿主蛋白组的相互作用，以帮助揭示病毒跨种族传播机理。

酵母双杂交是研究蛋白-蛋白相互作用的重要分子遗传学工具，特别是在筛选蛋白的未知受体上应用非常广泛。酵母表面展示将外源蛋白展示于细胞表面，它是研究蛋白定向进化的有力武器。申请者前期在酵母表面展示的基础上创立了酵母表面双杂交，将相互作用的蛋白展示于细胞表面，便于定量检测。在本研究中，申请者拟利用上述三个系统各自的优势，创建一个增强型的酵母双杂交系统。该系统依赖传统双杂交关于转录因子结构上可分为独立的结合结构域与激活结构域，但在功能上彼此依赖的基本原理；利用酵母表面双杂交独特而简便的载体设计，将上述双杂交体系置入单个质粒系统中。同时，借助于酵母表面展示，将报告基因展示于酵母表面，以利于检测和筛选。增强型双杂交系统与原有系统比较，检测与验证都十分容易，省时省力，非常适用于超大文库的高通量筛选。