Characterization of unconventional agent (scrapie agent): analysis by dissociation and reconstitution of SAF fraction and trial study for detection of scrapie specific nucleic acid in the fraction.

为了表征羊瘙痒病病原体，对SAF进行了以下实验，SAF与羊瘙痒病感染性相关，由宿主蛋白PrP组成，并从脑中制备。1)用SAF抗血清对小鼠痒病潜伏期进行了观察。用抗血清处理后，潜伏期确实发生了变化。2)为了检验SAF与病原体的结合是否仅仅是病原体对SAF的吸附，在感染后的不同时间检测了羊瘙痒病感染性从感染早期就存在相当程度的羊瘙痒病感染性的脾和淋巴结中的PrP。PrP在临床前各器官中均能检测到，说明感染性与SAF密切相关，提示PrP检测可用于临床前痒病的诊断。3)由于SAF在正丁醇中有微溶，因此采用柱层析法对ASF的正丁醇溶液进行了分离。从分形 ...更多信息 在含有PrP的离子中，通过与水混合除去丁醇。通过电子显微镜在组分中观察到细原纤维，表明SAF部分重构。接种未分级丁醇样品的小鼠出现瘙痒症症状，但在一年内接种了馏分，并对其进行了观察。4)据认为，PrP自组装和形成SAF的纯化过程中，包括洗涤剂处理的脑膜。为了制备不暴露于洗涤剂的可溶性PrP级分，使用尿素溶解膜，并通过甘油梯度离心进行分级。甚至在由50%甘油组成的级分中也检测到PrP，并且在这样的级分中的PrP形成显示双折射的大聚集体。通过短暂暴露于洗涤剂，原纤维从聚集体中解离，表明PrP作为聚集体存在于羊瘙痒病脑中，并且通过洗涤剂处理从解离的聚集体中纯化SAF。5)为了检测SAF富集级分中的感染性核酸，通过苯酚提取从蛋白酶处理的SAF级分制备含有核酸的级分。通过磷酸钙沉淀法将该级分引入小鼠培养细胞中。2周后将细胞接种到小鼠体内。接种的小鼠在其寿命内没有出现瘙痒症症状。少

In order to characterize scrapie agent, following experiments were carried out on SAF which associate with scrapie infectivity, consist of a host protein, PrP, and have been prepared from brain. 1) Effect of antiserum to SAF was examined for incubation period of scrapie in mice. The incubation period did change by treatment with the antiserum. 2) To examine whether the association of SAF and the agent was mere adsorption of the agent to SAF, detection of PrP in slpeen and lymph node at various time after infection, in which considerable level of scrapie infectivity exists from early stage of infection, was carried out. PrP was detected in these organs from preclinical stages indicating that association of the infectivity and SAF was intimate.Moreover, the results suggest that detection PrP can be applied for scrapie diagnosis in preclinical stage. 3) Since we found that SAF dissolves slightly in butanol, butanol solution of ASF was fractionated by column chromatography. From the fracti … More ons containing PrP, butanol was removed by mixing with water. Fine fibrils were observed by an electron microscope in the fractions, indicating that SAF was partially reconstituted. Mice inoculated with unfractionated butanol sample showed scrapie signs but with the fractions within one year and they were under observation. 4) It has been thought that PrP self-assembles and forms SAF during purification procedure including detergent treatment of brain membrane. To prepare soluble PrP fraction without exposure to detergent, urea was used to dissolve the membrane, and fractionated by glycerol gradient centrifugation. PrP was detected even in fractions consisting with 50% glycerol, and PrP in such fractions formed large aggregates showing birefringence. Fibrils were dissociated from the aggregates by brief exposure to a detergent, indicating that PrP exists in scrapie brain as aggregates, and SAF is purified from the dissociated aggregates by detergent treatment. 5) To detect infectious nucleic acid in SAF enriched fraction, a fraction containing nucleic acids was prepared from protease treated SAF fraction by phenol extraction. The fraction was introduced in to mouse cultured cells by the calcium phosphate-precipitation method. The cells were inoculated in to mice after 2 weeks. No mice inoculated showed scrapie signs within their life span. Less