Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein
Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein
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DOI：
10.1038/nmeth1066
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发表时间：
2007-08-01
期刊：
影响因子：
48
通讯作者：
Caughey, Byron
中科院分区：
文献类型：
--
作者：
Atarashi, Ryuichiro;Moore, Roger A.;Caughey, Byron
PrPSc是PrPSc蛋白的异构体，是一种与PrP蛋白相关的标志物，可诱导正常蛋白酶敏感的PrP-Sen蛋白的构象转化和聚合。这种种子活性允许使用循环超声放大(PMCA)反应和脑匀浆作为PrP-Sen的来源来超灵敏地检测PrPSc。在这里，我们描述了一种更快的种子聚合方法(rPrP-pMCA)，它可以在2-3天内检测到~gt；=50ag的仓鼠PrPSc(约0.003致死剂量)。该技术使用重组仓鼠PrP-sen，与脑源性PrP-sen不同，它可以很容易地浓缩、突变和合成标记。我们产生了抗蛋白酶的重组PrP纤维，这种纤维在蛋白分解敏感性和红外光谱上与自发启动的纤维不同。使用2亩L等份脑脊液，该方法可以区分感染和未感染瘙痒病的仓鼠。这种方法除了有助于PrPSc结构和形成机制的基础研究外，还将促进快速、超灵敏的Prion分析和诊断测试的发展。
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 >= 50 ag of hamster PrPSc (approximate to 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-mu l 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.