Atarashi Ryuichiro, Moore Roger A, Sim Valerie L, Hughson Andrew G, Dorward David W, Onwubiko Henry A, Priola Suzette A, Caughey Byron
Laboratory of Persistent Viral Diseases, National Institutes of Health, 903 S. 4th Street, Hamilton, Montana 59840, USA.
Nat Methods. 2007 Aug;4(8):645-50. doi: 10.1038/nmeth1066. Epub 2007 Jul 22.
The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects >or=50 ag of hamster PrPSc (approximately 0.003 lethal dose) within 2-3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-microl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.
瘙痒病朊病毒蛋白异构体PrPSc是一种与朊病毒相关的标志物，它能引发正常蛋白酶敏感型朊病毒蛋白（PrP-sen）的构象转变和聚合反应。这种引发活性使得利用循环超声放大（PMCA）反应和脑匀浆作为PrP-sen来源对PrPSc进行超灵敏检测成为可能。在此，我们描述了一种更快的引发聚合方法（rPrP-PMCA），该方法能在2至3天内检测出≥50 纳克的仓鼠PrPSc（约0.003个致死剂量）。这项技术使用重组仓鼠PrP-sen，与源自脑的PrP-sen不同，它能够轻松浓缩、突变并进行合成标记。我们生成了蛋白酶抗性重组PrP纤维，其在蛋白水解敏感性和红外光谱方面与自发形成的纤维有所不同。该检测方法能够使用2微升脑脊液区分感染瘙痒病和未感染的仓鼠。除了有助于对PrPSc形成的结构和机制进行基础研究外，这种方法还应能推动快速、超灵敏朊病毒检测和诊断测试的发展。
