一种基于连续PMCA的PrP^Sc体外扩增方法的建立  

Establishment of PrP^(Sc) Conversion Based on serial PMCA In Vitro

作　　者：石崧[1] 董辰方[1] 张宝云[1] 石琦[1] 王桂荣[1] 王新[1] 韩俊[1] 董小平[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,传染病预防控制国家重点实验室,北京100052

出　　处：《病毒学报》2008年第4期282-286,共5页Chinese Journal of Virology

基　　金：国家科技攻关项目(2006BAD06A13-2);国家973项目(2007CB310505);国家自然科学基金委项目(No.30500018、30571672)资助

摘　　要：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。In order to establish an amplification system in vitro with which the PrP^Sc is able to convert PrP^C 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 prp^Sc. 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 PrP^Sc from hamsters＂ brain tissues of scrapie agent 263K could replicate efficiently and infinitely with serial PMCA compared with finite propagation of PrP^Sc with conventional PMCA system. The study of PrP^Sc 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 PrP^Sc.

关 键 词：PRION病 PrP^Sc Prp^C 蛋白质错误折叠循环扩增 

分 类 号：R373.9[医药卫生—病原生物学]