Hyperefficient PrPSc amplification of mouse-adapted BSE and scrapie strain by protein misfolding cyclic amplification technique
Hyperefficient PrPSc amplification of mouse-adapted BSE and scrapie strain by protein misfolding cyclic amplification technique
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DOI：
10.1111/j.1742-4658.2009.07007.x
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发表时间：
2009-05-01
期刊：
影响因子：
5.4
通讯作者：
Nishida, Noriyuki
中科院分区：
文献类型：
--
作者：
Fujihara, Aiko;Atarashi, Ryuichiro;Nishida, Noriyuki
在传染性海绵状脑病的进展过程中，异常形式的朊病毒蛋白 (PrPSc) 通过正常 PrP (PrPC) 的结构转变而积累。在无细胞条件下，可以使用体外 PrPSc 扩增方法（包括蛋白质错误折叠循环扩增）有效复制该过程。这些方法能够实现 PrPSc 的超灵敏检测；然而，在实践中使用它们仍然存在困难。例如，迄今为止，需要进行几轮蛋白质错误折叠循环扩增才能达到最大灵敏度，这不仅需要数周时间，而且还会导致污染风险增加。在这项研究中，我们试图使用感染小鼠适应牛海绵状脑病或小鼠适应羊瘙痒病、钱德勒株的小鼠传染性海绵状脑病模型，进一步提高蛋白质错误折叠循环扩增技术中PrPSc的扩增率。在这里，我们证明，适当的超声处理调节可显着加速两种菌株的 PrPSc 扩增。事实上，仅经过一到两轮反应，我们就达到了最大灵敏度，可以超灵敏地检测稀释的脑匀浆中 < 1 LD50 的 PrPSc，此外，我们还在适应小鼠的牛海绵状脑病感染小鼠的血浆中检测到了 PrPSc。我们相信，这些结果将推动建立一种快速、超灵敏的传染性海绵状脑病诊断测试。
Abnormal forms of prion protein (PrPSc) accumulate via structural conversion of normal PrP (PrPC) in the progression of transmissible spongiform encephalopathy. Under cell-free conditions, the process can be efficiently replicated using in vitro PrPSc amplification methods, including protein misfolding cyclic amplification. These methods enable ultrasensitive detection of PrPSc; however, there remain difficulties in utilizing them in practice. For example, to date, several rounds of protein misfolding cyclic amplification have been necessary to reach maximal sensitivity, which not only take several weeks, but also result in an increased risk of contamination. In this study, we sought to further promote the rate of PrPSc amplification in the protein misfolding cyclic amplification technique using mouse transmissible spongiform encephalopathy models infected with either mouse-adapted bovine spongiform encephalopathy or mouse-adapted scrapie, Chandler strain. Here, we demonstrate that appropriate regulation of sonication dramatically accelerates PrPSc amplification in both strains. In fact, we reached maximum sensitivity, allowing the ultrasensitive detection of < 1 LD50 of PrPSc in the diluted brain homogenates, after only one or two reaction rounds, and in addition, we detected PrPSc in the plasma of mouse-adapted bovine spongiform encephalopathy-infected mice. We believe that these results will advance the establishment of a fast, ultrasensitive diagnostic test for transmissible spongiform encephalopathies.