RECOMBINANT ANTIBODIES FOR MAPPING PRP STRUCTURES

用于绘制 PRP 结构的重组抗体

• 批准号: 6565182
• 负责人: Dennis R. Burton
• 金额: $ 25.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6565182
• 关键词: genetically modified animals; hamsters; laboratory mouse; monoclonal antibody; mutant; prions; protein structure; recombinant proteins; scrapie

Prion diseases are disease of protein conformation. Determination of the conformation differences between different forms of the prion protein (PrP) is central to understanding the nature of these diseases. However the physical forms of PrP, e.g. as a membrane-associated protein or in insoluble aggregates, complicate this determination. Our approach is to use monoclonal antibodies as sensitive specific probes of protein conformation which can be employed in a wide range of environments. The use of these probes, in conjunction with the detailed structural data available from NMR and crystallography of a limited number of PrP molecules and fragments, allows the conformations of PrP in many forms and milieu to be explored. Earlier problems in generating antibodies to PrP have been circumvented by immunizing PrP-gene ablated mice and rescuing antibodies via phage display libraries. This approach has yielded a wealth or recombinant antibodies that clearly define differences in epitope exposure between the normal cell surface PrP molecule and the protease resistant core of infectious prion particles. The generation of new forms of infective PrP molecules described in this program provides new opportunities, using the antibodies and approaches developed, to elucidate the critical changes involved in the acquisition of prion infectivity. Particular attention is to be focussed on a PrP molecule with a triple A-V mutation and a shortened PrP molecule (PrP106). Both of these molecules appear to adopt conformation which are scrapie- like in that they are associated with infectivity or protease resistance. We propose to investigate these molecules in vitro and in situ using the panel of existing antibodies and to use the molecules as immunogens to generate new antibodies. The conformations of new PrP molecules generated as Project 1 develops will similarly be probed by the antibody approach.

朊病毒疾病是蛋白质构象的疾病。测定 不同形式朊病毒蛋白之间的构象差异 (PrP)是理解这些疾病本质的关键然而 PrP的物理形式，例如作为膜结合蛋白或 不溶性聚集物使该测定复杂化。我们的做法是 用单克隆抗体作为敏感的蛋白质特异性探针 这是一种可以在广泛的环境中使用的构造。的 使用这些探针，结合详细的结构数据， 可从有限数量的PrP的NMR和晶体学获得 分子和片段，允许PrP以多种形式构象， 环境有待探索。产生PrP抗体的早期问题 通过免疫PrP基因消融的小鼠和拯救 抗体通过噬菌体展示文库。这种方法产生了大量的 或重组抗体， 正常细胞表面PrP分子和蛋白酶之间的暴露 感染性朊病毒颗粒的抵抗核心。 本文中描述的感染性PrP分子的新形式的产生 计划提供了新的机会，使用抗体和方法， 开发，以阐明收购中涉及的关键变化 朊病毒的传染性应特别注意PrP 具有三重A-V突变和缩短的PrP分子（PrP 106）。 这两种分子似乎都采用了羊瘙痒症的构象- 比如它们与感染性或蛋白酶抗性有关。 我们建议在体外和原位研究这些分子， 一组现有的抗体，并使用这些分子作为免疫原， 产生新的抗体生成的新PrP分子的构象 随着项目1的发展，将类似地通过抗体方法进行探测。