Biochemical Detection of Prions in Blood

血液中朊病毒的生化检测

• 批准号: 6829511
• 负责人: JOAQUIN CASTILLA
• 金额: $ 6.19万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6829511
• 关键词: SDS polyacrylamide gel electrophoresis; biochemistry; blood chemistry; brain disorder diagnosis; communicable disease transmission; enzyme linked immunosorbent assay; hamsters; human tissue; laboratory mouse; noninvasive diagnosis; nucleic acid amplification techniques; prions; protein folding; protein quantitation /detection; spongiform encephalopathy; western blottings

DESCRIPTION (provided by applicant): This application is in response to the PAR-03-056 (NIA Pilot Research Grant Program) and focuses on the topic number four, Transmissible Spongiform Encephalopathies. Transmissible spongiform encephalopathies, also called prion diseases, are a group of fatal infectious neurodegenerative disorders affecting humans and animals. Although rare diseases, the recent outbreak of Bovine Spongiform Encephalopathy and Chronic Wasting disease and the transmission of the disease from cattle to humans have risen a great concern about a possible epidemic of Creutzfeldt-Jakob disease. This problem is aggravated by the lack of an early and sensitive diagnosis to identify individuals incubating the disease during the pre-symptomatic phase. The infectious agent (termed prion) is composed exclusively by a misfolded version of a normal protein and does not contain any nucleic acid. According to the prion hypothesis, the disease is transmitted by propagation of the misfolding from the disease associated isoform (termed PrPres) to the normal host protein (termed PrPc), which become converted into the pathological form. We have recently described a procedure to induce the conversion of PrPc into PrPres in vitro starting with minute quantities of brain PrPres. This procedure, named Protein Misfolding Cyclic Amplification (PMCA) mimics the process of prion replication in vivo, but at an accelerated speed resulting in an exponential amplification of the initial amount of PrPres. PMCA has tremendous promise to increase detection of prions in tissues and biological fluids during early stages of the disease and thus may be useful for pre-symptomatic diagnosis of prion disease. The major goal of this project is to take advantage of the PMCA technology to attempt developing a highly sensitive and non-invasive diagnosis of prion diseases in humans and animals. In specific aim 1 we will attempt the pre-symptomatic detection of PrPres in the brain of experimentally infected cattle sacrificed at different times after infection. In specific aim 2 we will attempt detection of PrPres in peripheral tissues of humans and animals. Specific aim 3 proposes the development of the conditions for amplification of PrPres from blood of experimental animals in order to reach reproducible detection of prions in blood. The results generated in this project may provide the basis for the development of a novel highly-sensitive pre-mortem and pre-symptomatic diagnosis of prion disease. Such test will have tremendous applications in public health to minimize the risk of further propagation of prion to humans.

描述（由申请人提供）：本申请是对 PAR-03-056（NIA 试点研究资助计划）的回应，重点关注第四个主题：传染性海绵状脑病。 传染性海绵状脑病，也称为朊病毒病，是一组影响人类和动物的致命传染性神经退行性疾病。尽管疾病罕见，但最近爆发的牛海绵状脑病和慢性消耗性疾病以及该疾病从牛到人类的传播引起了人们对克雅氏病可能流行的高度关注。由于缺乏早期和敏感的诊断来识别在症状前阶段潜伏该疾病的个体，这个问题变得更加严重。感染因子（称为朊病毒）完全由正常蛋白质的错误折叠形式组成，不含任何核酸。根据朊病毒假说，疾病是通过从疾病相关异构体（称为 PrPres）到正常宿主蛋白（称为 PrPc）的错误折叠传播而传播的，正常宿主蛋白被转化为病理形式。我们最近描述了一种从微量大脑 PrPres 开始在体外诱导 PrPc 转化为 PrPres 的程序。这个过程被称为蛋白质错误折叠循环扩增（PMCA），模仿体内朊病毒复制的过程，但速度加快，导致初始 PrPres 量呈指数扩增。 PMCA 有望在疾病早期阶段增加组织和生物体液中朊病毒的检测，因此可能有助于朊病毒疾病的症状前诊断。该项目的主要目标是利用 PMCA 技术尝试开发一种对人类和动物朊病毒疾病的高灵敏度和非侵入性诊断方法。在具体目标 1 中，我们将尝试对感染后不同时间处死的实验感染牛的大脑中的 PrPres 进行症状前检测。在具体目标 2 中，我们将尝试检测人类和动物外周组织中的 PrPres。具体目标 3 提出开发从实验动物血液中扩增 PrPres 的条件，以实现血液中朊病毒的可重复检测。该项目产生的结果可能为开发新型高度敏感的朊病毒病死前和症状前诊断提供基础。这种测试将在公共卫生领域具有巨大的应用，以最大限度地减少朊病毒进一步传播给人类的风险。