RECOMBINANT ANTIBODIES FOR MAPPING PRP STRUCTURES

蛋白质病是一种蛋白质构象疾病。确定了 不同形态的朊病毒蛋白的构象差异 (PRP)是了解这些疾病的性质的核心。然而， PrP的物理形式，例如作为膜相关蛋白或在 不可溶的聚集体，使这一测定复杂化。我们的方法是 用单抗作为蛋白质的灵敏特异探针 构象，可在广泛的环境中使用。这个 使用这些探头，结合详细的结构数据 可从核磁共振和有限数量的PrP的结晶学获得 分子和片段，允许PrP以多种形式和 有待探索的环境。产生PrP抗体的早期问题 已经通过免疫PrP基因消融的小鼠和拯救 通过噬菌体展示文库获得抗体。这种方法产生了一笔财富 或明确定义表位差异的重组抗体 正常细胞表面PrP分子与蛋白酶的相互作用 具有感染性的普恩粒子的抗性核心。 在这篇文章中描述了新形式感染性PrP分子的产生 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 普恩病毒的传染性。应特别关注PrP 具有三重A-V突变和PrP缩短的分子(PrP106)。 这两个分子似乎都采用了刮痕构象- 例如，它们与传染性或蛋白酶抵抗力有关。 我们建议在体外和原位使用 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。产生的新PrP分子的构象 随着项目1的发展，将同样通过抗体方法进行探测。

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.