Dong Chen-Fang, Huang Yin-Xia, An Run, Chen Jian-Ming, Wang Xiao-Fan, Shan Bing, Lei Yan-Jun, Han Lu, Zhang Bao-Yun, Han Jun, Dong Xiao-Ping
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Bing Du Xue Bao. 2008 May;24(3):185-9.
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.
为建立一种用于检测PrP(Sc)的新型蛋白质印迹分析方法，我们用硫酸链霉素沉淀法对蛋白质印迹分析进行了优化。用蛋白酶K消化后，将10%的羊瘙痒病感染仓鼠脑匀浆与60 mmol/L链霉素孵育，通过离心回收沉淀的PrP(Sc)。使用蛋白质印迹分析评估硫酸链霉素沉淀对PrP(Sc)的富集效果。结果表明，链霉素可与经蛋白酶K处理的PrP(Sc)结合，形成高分子质量物质，但不影响SDS-PAGE上的糖基化模式。蛋白质印迹分析显示，链霉素沉淀PrP(Sc)的检测灵敏度显著提高。作为一种用于PrP(Sc)的灵敏、特异、快速且灵活的方法，本研究中的方法单独使用或与其他技术联合使用，有潜力用于检测来自中枢神经系统、外周器官或体液的标本中的低水平PrP(Sc)。
