一种基于连续PMCA的PrPSc体外扩增方法的建立

为建立一种基于蛋白质错误折叠循环扩增(PMCA)的体外稳定扩增方法以观察PrPSc是否能在体外连续传代,我们分别制备了正常仓鼠和羊瘙痒病因子263K感染的发病仓鼠的全脑匀浆,将两种脑匀浆以不同体积比混合后,分别进行144个循环的直接PMCA和每轮48个循环、共8轮的连续PMCA,用Western blot对PrPSc的扩增情况进行检测.结果显示,与常规的直接PMCA方法相比,连续PMCA能更有效地使低浓度的PrPSc扩增到可检出的水平,表明连续PMCA可以支持羊瘙痒因子263K在体外长期稳定的复制.连续PMCA方法是一种体外高效地扩增PrPSc的方法,有潜力成为一种Prion体外培养方法,用于研究Prion错误折叠和复制机制,以及检测脑组织、外周组织和体液样品中的微量PrPSc.

Establishment of PrPSc Conversion Based on serial PMCA In Vitro

In order to establish an amplification system in vitro with which the PrPSc is able to convert PrPC into proteinase K-resistant isoform infinitely and whether this system is more efficient than conventional protein misfolding cyclic amplification(PMCA),scrapie strain 263K-infected hamster's brain homogenate and homologous normal brain homogenate were prepared,respectively.A new methodology, namely serial PMCA,was utilized to reveal the continuous propagation ability of PrPSc.Totally 8 rounds of serial PMCA were proceeded and each round contained 48 cycles of alternative sonication and incubation.Simultaneously 144 cycles of conventional PMCA was used as a control.The results showed the PrPSc from hamsters' brain tissues of scrapie agent 263K could replicate efficiently and infinitely with serial PMCA compared with finite propagation of PrPSc with conventional PMCA system.The study of PrPSc continuous propagation in brain homogenate with serial PMCA may further provide insight into the unsettled mechanism of prions misfolding and replication and apply to detect trace amount of PrPSc.