一种朊蛋白错误折叠循环扩增方法的建立  

Establishment of a protein misfolding cyclic amplification for PrP^sc

作　　者：韩俊[1] 韩露[1] 石琦[1] 石菘[1] 王新[1] 张宝云[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：《中华实验和临床病毒学杂志》2007年第3期202-204,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金委资助项目（30500018、30571672）;欧盟资助项目（QLRT200001441）;国家科技攻关计划资助项目（2003BA712A04.02）

摘　　要：目的建立一种类似于PCR的蛋白质扩增方法-蛋白错误折叠循环扩增技术（PMCA），用于朊病毒病脑组织中PrP^sc的检测。方法将不同浓度的羊瘙痒因子263K毒株原液与正常仓鼠脑组织匀浆混合，经反复孵育／超声，共10～15个循环。Western Blot检测扩增产物中蛋白酶K抗性PrP^sc信号。结果在本研究试验体系下，263K毒株可以利用仓鼠脑组织为基质在体外迅速复制。所建立的PrP^sc-PMCA技术可检测到10^-5稀释的毒株原液中的PrP^sc。与常规的脑组织免疫印记方法相比，敏感度提高了10^5～10^6倍。研究还显示PrP^sc。还可利用小脑和脑于为基质进行体外扩增复制。结论成功建立了PrP^sc-PMCA技术，为朊病毒病的早期诊断和朊病毒生物学特性的研究提供了一种新的手段。Objective To establish a methodology of protein misfolding cyclic amplification （PMCA） and utilize in the detection of PrP^sc in brain tissues from prion diseases. Methods Different amounts of Scrapie 263K agent bulk were mixed with brain homogenates of health hamsters and treated with repeated incubation/sonication for 10 to 15 cycles. The proteinase K-resistant PrP^sc was evaluated with Western Blot. Results In this experimental situation, 263K agent replicated rapidly in vitro, utilizing hamsters＇ brains as the medium. With the established PrP^sc-PMCA technique, PrP^sc signals in the preparations containing less than 10^-5 diluted 263K bulk could be detected. Compared with conveniently used immuno-blot assay, the sensitivity of PrP^sc-PMCA for PrP^sc was 10^5 to 10^6-fold increased. It has been also shown that homogenates of cerebuller and brain stem could be used as the medium for PrP^sc replication. Conclusion A rapidly replicating method for PrP^sc, PrP^sc-PMCA, was successfully established, providing a new approach for early diagnosis of prion diseases and research on the biological features of prion.

关 键 词：朊病毒 蛋白错误折叠循环扩增技术 

分 类 号：R3[医药卫生—基础医学]