Study for atypical BSE in Italy

应用免疫印迹和免疫组化技术对牛朊蛋白（PrP）单克隆抗体（mAb）进行了羊瘙痒病和牛海绵状脑病的研究。mAb组是有用的，因为它们识别鼠、羊和牛脑组织中PrP的正常（PrP^c）和异常（PrP^<TSE>）亚型。其中，两个单克隆抗体识别构象或片段化的PrP表位。有趣的是，通过用对应于牛PrP基因密码子153-166的合成多肽免疫PrP基因敲除小鼠制备的抗牛PrP mAb 1D 12显示出对朊病毒未感染的神经元细胞的新型反应模式。当检查mAb表位的酶联免疫吸附测定图谱时，单克隆抗体1D 12与YEDRY和M反应，对应于牛PrP的氨基酸156-160和165。用不同的固定方法，观察了牛PrP^c在PrP缺陷神经细胞（HpL 3 -4）中的分布。用共聚焦显微镜对1D 12进行免疫荧光分析，福尔马林固定后观察到细胞表面染色，而丙酮固定后细胞核中的颗粒被染色。在细胞核中未观察到对表达小鼠PrP的HpL 3 -4或HpL 3 -4细胞的反应性。在细胞核中观察到对表达仓鼠或牛PrP的HpL 3 -4细胞的反应性。这是第一次在未感染朊病毒的细胞系中同时检测到PrPc在细胞表面和细胞核中的存在。此外，由于PrP^c能被1D 12特异性识别，因此，156-160和165位氨基酸可能对PrP^c的核定位起重要作用。

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.