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.

用免疫印迹和免疫组织化学方法研究了抗牛普鲁恩蛋白(PrP)和牛海绵状脑病的单抗。MAb小组很有用，因为他们在小鼠、绵羊和牛脑组织中识别正常(PrP^c)和异常(PrP^&lt；Tse&gt；)PrP亚型。其中，两株单抗识别构象或片段性PrP表位。有趣的是，通过用与牛PrP基因153-166密码子相对应的合成多肽免疫PrP基因敲除小鼠而制备的抗牛PrP单抗1D12显示了未感染PrP的神经细胞的新的反应模式。当检测mAb表位的酶联免疫吸附试验时，单抗1D12与对应于牛PrP的156-160和165个氨基酸的YEDRY和M反应。观察了不同固定方法对PrP缺陷型神经细胞(HpL3-4)内PrP^c分布的影响。共聚焦显微镜下观察福尔马林固定后1D12免疫荧光染色细胞表面，丙酮固定后细胞核内颗粒染色。核内未见表达PrP的HpL3-4或HpL3-4细胞反应。核内可与表达金黄地鼠或牛PrP的HpL3-4细胞反应。这是首次在未感染病毒的细胞系的细胞表面和细胞核中检测到PrPc。此外，由于核内PrP^c被1D12特异性识别，金黄地鼠或牛PrP的156-160和165位氨基酸可能在PrP^c核内定位中起重要作用。