摘要
目的 研究超声处理对感染羊瘙痒症仓鼠脑组织中PrPSc聚集状态的影响 ,寻找产生PrPSc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Westernblot方法和图象分析系统观测PrPSc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PrPSc含量 1 2 9～ 1 5 8倍 ;同样条件下超声处理可明显增加羊瘙痒因子 2 6 3K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PrPSc的超声处理显示约 90 %的PrPSc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PrPSc 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PrPSc聚集物 ,产生小的相对分子质量的PrPSc产物。
Objective 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′s 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.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2004年第2期118-121,共4页
Chinese Journal of Experimental and Clinical Virology
基金
国家自然科学基金项目 (3 0 0 70 0 3 8)
国家自然科学基金重点项目 (3 0 13 0 0 70 )
国家 863计划项目 (2 0 0 1AA2 15 3 91)
欧盟项目资助 (QLRT2 0 0 0 0 14 41)
