超声处理对羊瘙痒病脑组织中PrPSc聚集状态的影响

目的 研究超声处理对感染羊瘙痒症仓鼠脑组织中PrP^Sc聚集状态的影响，寻找产生PrP^Sc低聚体的条件。方法 裂解液制备脑组织提取物，用各种超声条件处理不同阶段的脑组织提取物；以蛋白酶消化后的Western blot方法和图象分析系统观测PrP^Sc蛋白的分布和聚集状态。结果 适当的超声处理(15s共30次)可增加脑组织匀浆上清中PrP^Sc含量1．29～1．58倍；同样条件下超声处理可明显增加羊瘙痒因子263K感染仓鼠脑组织匀浆上清中PrP蛋白总量，而对正常对照仓鼠脑组织匀浆上清中PrP蛋白总量影响不大；对经常规高速离心获得的PrP^Sc的超声处理显示约90％的PrP^Sc存在于离心上清液中。结论 对感染动物脑组织进行超声处理可增加PrP^Sc的提取量，利于实验室检测。适当的超声处理可破碎大的相对分子质量的PrP^Sc聚集物，产生小的相对分子质量的PrP^Sc产物。

Influence of ultrasonic processing on the aggregation of PrPSc in the brain extracts of the scrapie-infected hamsters

BACKGROUND: To evaluate the influence ultrasonic processing on the aggregation of PrP(Sc) in the brain extracts prepared from the scrapie-infected hamsters, and to seek for the way to prepare lower molecular PrP(Sc) polymer. METHODS: The extracts of infected brains were prepared with a lysis solution, and treated with ultrasonics at various conditions during the different phases. The distribution and aggregation state of PrP(Sc) were analyzed by proteinase K treated. Western blot, and afterwards, quantitatively calculated with a commercially supplied software Image Totaltech. RESULTS: The amount of PrP(Sc) in the supernatant of brain homogenates was 1.29-to 1.58-fold increased with appropriate sonication (15 s for 30 times). At the same conditions of ultrasound, the PrP amount in the supernatant prepared from the scrapie-infected hamster brain was significantly increased, whereas that prepared from healthy animal used as normal control showed little change. Comparative analyses of PrP(Sc) pellets prepared by high-speed centrifugation revealed that about 90% PrP(Sc) released into supernatant after ultrasound processing. CONCLUSION: Appropriate sonication of homogenate of scrapie-infected brain increases the extracted amount of PrP(Sc), being favorable to laboratory diagnosis. Larger molecular PrP(Sc) aggregates can be crashed by ultrasonic processing, engendering lower molecular PrP(Sc) polymers.