Study for atypical BSE in Italy

意大利非典型疯牛病研究

• 批准号: 17405042
• 负责人: ONODERA Takashi
• 金额: $ 7.36万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17405042/
• 关键词: Italy; atypical; BSE; Western Blot; ELISA; monoclonal antibody; immunohistochemistry; bcl3 facilyty; ウエスタン・ブロッティング

A monoclonal antibody (mAb) panel to bovine prion protein (PrP) was studied by immunoblotting and immunohistochemistry for scrapie, and bovine spongiform encephalopathy. The mAb panel is useful because they recognized both normal (PrP^c) and abnormal (PrP^<TSE>) isoforms of PrP in murine, ovine and bovine brain tissues. Among them, two mAbs recognized a conformational or fragmented PrP epitope. Interestingly, an anti-bovine PrP mAb, 1D12, prepared by immunizing PrP gene-knockout mice with a synthetic polypeptides corresponding to codons 153-166 of the bovine PrP gene showed the novel patterns of reactivity for prion-uninfected neuronal cells. When an enzyme-linked immunosorbent assay-mapping of the mAb epitopes was examined, monoclonal 1D12 reacted with YEDRY and M corresponding to amino acids 156-160 and 165 of bovine PrP. Several patterns of bovine PrP^c distribution in PrP-deficient neuronal cells (HpL3-4) transfected with bovine PrP were observed after different fixation methods. Staining of cell surface was observed after formalin fixation by immunofluorescent assay of 1D12 with confocal microscopy, whereas granules in nucleus were stained after acetone fixation. No reactivity in the nucleus was observed to HpL3-4 or HpL3-4 cells expressing mouse PrP. Reactivity in the nucleus was observed to HpL3-4 cells expressing hamster or bovine PrP. It is the first report to detect the PrPc at both cell surface and nuclei of prion-uninfected cell line. Furthermore, as nuclear PrP^c was specifically recognized by 1D12, amino acids 156-160 and 165 of hamster or bovine PrP may play important role in localization of PrP^c into nucleus.

通过免疫印迹和免疫组织化学研究了牛朊病毒蛋白 (PrP) 的单克隆抗体 (mAb) 组合对痒病和牛海绵状脑病的影响。 mAb 组合非常有用，因为它们可以识别小鼠、绵羊和牛脑组织中 PrP 的正常 (PrP^c) 和异常 (PrP^<TSE>) 同工型。其中，两种 mAb 识别构象或片段化 PrP 表位。有趣的是，用与牛 PrP 基因密码子 153-166 相对应的合成多肽对 PrP 基因敲除小鼠进行免疫接种而制备的抗牛 PrP mAb 1D12，显示出未感染朊病毒的神经元细胞的新反应模式。当检查 mAb 表位的酶联免疫吸附测定图谱时，单克隆 1D12 与对应于牛 PrP 氨基酸 156-160 和 165 的 YEDRY 和 M 发生反应。在不同的固定方法后，观察到转染牛 PrP 的 PrP 缺陷神经元细胞 (HpL3-4) 中牛 PrP^c 的几种分布模式。福尔马林固定后，通过共聚焦显微镜对 1D12 进行免疫荧光测定，观察​​细胞表面染色，而丙酮固定后，细胞核内的颗粒染色。未观察到细胞核对表达小鼠 PrP 的 HpL3-4 或 HpL3-4 细胞有反应性。观察到表达仓鼠或牛 PrP 的 HpL3-4 细胞的细胞核反应性。这是首次在未感染朊病毒的细胞系的细胞表面和细胞核中检测到PrPc。此外，由于核PrP^c被1D12特异性识别，仓鼠或牛PrP的氨基酸156-160和165可能在PrP^c定位到细胞核中发挥重要作用。