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.

为了确定瘙痒病病原体的特性，对从脑部制备的、与瘙痒病感染性有关的、由宿主蛋白PrP组成的SAF进行了以下实验。1)观察SAF抗血清对小鼠皮肤瘙痒病潜伏期的影响。经抗血清处理后，潜伏期确实发生了变化。2)检测感染后不同时间皮肤和淋巴组织中PrP的含量，以确定SAF与病原体之间的相互作用是否仅仅是病原体对SAF的吸附。从临床前期的这些脏器中检测到PrP，表明传染性与SAF有密切的联系，并提示PrP的检测可用于临床前期的瘙痒病诊断。3)由于我们发现SAF在丁醇中略有溶解，因此用柱层析法对ASF的丁醇溶液进行了分离。来自Fratii…用水混合除去更多含有PrP、丁醇的ONS。电子显微镜观察到组分中有细小的纤维，表明SAF部分重组。接种普通丁醇样本的小鼠出现了瘙痒症状，但在一年内出现了分级，正在观察中。4)认为PrP在纯化过程中自组装形成SAF，包括脑膜的去污剂处理。为了制备不需洗涤剂洗涤的可溶性PrP组分，用尿素溶解膜，用甘油梯度离心法进行分级。即使在含有50%甘油的组分中也能检测到PrP，并且这种组分中的PrP形成了大的聚集体，呈现双折射。短时间接触洗涤剂，纤维从聚集体中解离，表明PrP以聚集体的形式存在于瘙痒病脑中，SAF通过洗涤剂处理从解离的聚集体中得到纯化。5)为了检测SAF富集组分中的感染性核酸，用苯酚提取法从SAF富集组分中提取了含有核酸的组分。用磷酸钙沉淀法将其导入小鼠培养细胞中。2周后将细胞接种于小鼠体内。接种的小鼠在其寿命内都没有表现出瘙痒的迹象。较少

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