In vitro analysis of prion proliferation : Modified PMCA method using PrP-gene deficient cell line

本研究利用朊蛋白（PrP）基因缺陷细胞系作为细胞PrP的来源，进行了蛋白错误折叠环扩增（PMCA）。以小鼠和仓鼠瘙痒病和牛海绵状脑病（BSE）朊病毒为种子。2008年，将朊蛋白（PrP）基因缺陷细胞重新引入小鼠、仓鼠和牛的PrP基因，制备了细胞裂解液。2009年，我们比较了仓鼠痒病263K、小鼠痒病Obihiro、小鼠Chandler痒病朊病毒PMCA的体外扩增效率。结果表明，在含有1% TritonX-100, 4mM EDTA（40循环）的PBS条件下，可以从263K和Chandler朊病毒感染的脑匀浆中扩增出PrPres，而Obihiro朊病毒不能扩增。此外，与脑匀浆相比，使用细胞裂解液的PMCA需要不同的条件进行PrPres扩增。因此，我们得出结论，根据朊病毒菌株和PrPC来源的不同，需要适应PMCA条件来实现PrPres的高效增殖。

In this study, protein misfolding cyclic amplification (PMCA) was performed using prion protein (PrP)-gene deficient cell line as the source of cellular PrP. Mouse and hamster scrapie and bovine spongiform encephalopathy (BSE) prion were used for seed. In 2008, cell lysate was prepared from prion protein (PrP)-gene deficient cell re-introduced with mouse, hamster, and bovine PrP-gene. In 2009, we compared the in vitro amplification efficiency in PMCA among hamster scrapie 263K, mouse scrapie Obihiro, mouse Chandler scrapie prions. As the results, PrPres could be amplified from brain homogenates infected with 263K and Chandler prion under the condition of PBS containing 1% TritonX-100, 4mM EDTA (40 cycles), whereas Obihiro prion could not be amplified. In addition, PMCA using cell lysate required different conditions for PrPres amplification compared to brain homogenate. Therefore, we concluded that the adaptation of PMCA conditions were required for efficient PrPres proliferation depending on prion strains and PrPC sources.