MRC Prion Unit and Department of Neurodegenerative Disease, UCL Institute of Neurology, National Hospital for Neurology and Neurosurgery, London, United Kingdom.
PLoS One. 2010 Dec 20;5(12):e15679. doi: 10.1371/journal.pone.0015679.
Disease-related prion protein, PrP(Sc), is classically distinguished from its normal cellular precursor, PrP(C), by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrP(Sc) using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrP(C), while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrP(C).
与疾病相关的朊病毒蛋白 PrP(Sc) 经典的特征是去污剂不溶性和部分抗蛋白酶水解。朊病毒病的分子诊断通常依赖于使用蛋白酶 K 检测 PrP(Sc) 的蛋白酶抗性片段，然而现在很明显，大多数与疾病相关的 PrP 实际上可能会被这种处理破坏。在这里，我们报告说，用糜蛋白酶 E 消化 RML 朊病毒感染的小鼠脑组织，然后用磷钨酸钠沉淀，可以消除大部分脑蛋白，包括 PrP(C)，同时保持 >70%的感染性朊病毒滴度。该方法现在允许对蛋白酶 K 敏感的朊病毒进行表征，并研究其在人和动物朊病毒病中的临床相关性，而不会受到污染的 PrP(C)的干扰。
