一种基于链霉素沉淀的PrP^(Sc)检测方法的建立  

Establishment of an Assay for PrP^(Sc) Detection Based on Streptomycin Precipitation

作　　者：董辰方[1] 黄银霞[1] 安润[1] 陈建明[1] 王小凡[1] 单冰[1] 雷艳君[1] 韩露[1] 张宝云[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

出　　处：《病毒学报》2008年第3期185-189,共5页Chinese Journal of Virology

基　　金：国家自然科学基金委重点项目(No30500018);国家自然科学基金委项目(No30571672);欧盟项目(NoQLRT200001441);国家科技攻关计划项目(No2003BA712A04-02)资助

摘　　要：建立一种新的基于链霉素沉淀的PrPSc的Western blot检测方法,用终浓度为60mmol/L的链霉素处理蛋白酶K消化的PrPSc贮存液,通过离心沉淀PrPSc,用Western blot对PrPSc的链霉素富集效果进行检测。结果显示,链霉素能够与PrPSc结合形成高分子量复合物,但不影响糖基化形式。此外,基于链霉素沉淀的Western blot,无论是在低浓度或是大容积的条件下,均可显著地提高对PrPSc检测的敏感性。基于链霉素沉淀的Western blot试验是一种敏感、特异、快速及灵活的检测方法,有潜力用于脑组织、外周组织及体液中低水平的PrPSc的检测。To establish a new Western blotting assay for PrP^Sc detection, we optimized the Western blotting assay with a precipitation procedure of streptomycin sulfate. After digestion with PK, 10% scrapie infected hamster brain homogenates were incubated with 60 mmol/L streptomycin and the precipitated PrP^Sc was recovered by centrifugation. The enrichment of PrP^Sc by streptomycin sulfate precipitation was evaluated using Western blotting assay. The results showed streptomycin could bind to PK-treated PrP^Sc , forming high molecular masses, but not influence the glycosylated patterns on SDS-PAGE. Western blot assay revealed that the detective sensitivity of the streptomycin-precipitation PrP^Sc was remarkably improved. As a sensitive, specific, rapid and flexible protocol for PrP^Sc, the protocol in this study has the potential utility, alone or combined with other techniques, for the detection of low level PrP^Sc in the specimens from central nerve system, or from peripheral organs or body fluids.

关 键 词：朊病毒 链霉素沉淀 WESTERN BLOT 分子诊断技术 

分 类 号：R373.9[医药卫生—病原生物学] Q78[医药卫生—基础医学]