Generation of monoclonal antibody recognized by the GXXXG motif (glycine zipper) of prion protein
Generation of monoclonal antibody recognized by the GXXXG motif (glycine zipper) of prion protein
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DOI：
10.1089/hyb.2006.25.271
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发表时间：
2006-10-01
期刊：
影响因子：
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通讯作者：
Kim, Yong-Sun
中科院分区：
文献类型：
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作者：
Choi, Jin-Kyu;Park, Seok-Ju;Kim, Yong-Sun
为研制能与正常蛋白(PrPC)和异常蛋白亚型(PrPSc)发生反应的单抗，从263k瘙痒病感染的仓鼠脑中分离PrPSc，免疫PrP基因敲除小鼠。我们建立了两株杂交瘤细胞株3F10和1C5(Ig_1)，用重组仓鼠Pron蛋白的谷胱甘肽S转移酶融合蛋白和合适的多肽筛选其表位图。3F10对仓鼠和小鼠PrP具有较高的亲和力，并能识别137-151残基。1C5识别与GXXXG基序相对应的119-130区域，这是一种在所有哺乳动物中保守的甘氨酸拉链区域。在免疫组织化学分析中，PrPSc阳性染色出现在瘙痒病感染脑组织的细胞外室，而在正常脑组织中未见表达。然而，在Western印迹中，这些抗体既识别正常的普恩蛋白，也识别异常的普恩蛋白。这些结果表明，所研制的鼠单抗是针对PrP蛋白的，在免疫组织化学中能比正常Pron蛋白更有效地识别异常Pron蛋白。因此，这些抗体可以作为分析PrPC和PrPSc之间生化、结构和功能性质的有用试剂。
To develop monoclonal antibodies (MAbs) to react with normal prion protein (PrPC) and abnormal isoform of prion protein (PrPSc), PrPSc was isolated from brains of 263K scrapie-infected hamsters and immunized to PrP knockout mice. We developed two hybridomas, 3F10 and 1C5 (IgG1), of which epitope mappings were screened by using glutathione S-transferase (GST) fusion proteins of recombinant hamster prion protein and suitable peptides. 3F10 showed a high affinity for hamster and mouse PrP and was demonstrated to recognize the residues 137-151. 1C5 recognizes the region 119-130 corresponding to the GXXXG motif, the glycine zipper region, conserved in all mammals. In the immunohistochemical analysis, the positive staining for PrPSc was observed in the extracellular compartment of scrapie-infected brains but not in the normal brains. However, in Western blot, these antibodies recognized both normal and abnormal prion proteins. These results suggested that the developed mouse MAbs are specific to prion protein and can recognize abnormal prion protein more effectively than normal prion protein in immunohistochemistry. Therefore, these antibodies could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties between PrPC and PrPSc.