Identification of antigen for cause of ITP by the new molecular biological method (SEREX)

通过新分子生物学方法（SEREX）鉴定 ITP 病因抗原

• 批准号: 10670943
• 负责人: WADA Hideo
• 金额: $ 0.64万
• 依托单位: Mie University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670943/
• 关键词: ITP; SEREX; cDNA; specific antibody; sequence; UT-7; Molecular biology; recombinant protein; 細み替え蛋白; dipA; IgG抗体; 遺伝子解析; 遺伝子クローニング

To identify the cause of specific antigen in idiopathic thrombocytopenic purpura (ITP), we directly analyzed the gene of specific antigen in ITP by high sensitivity of genetic cloning method ; serological identification of antigens by recombinant expression cloning (SEREX). We extracted the RNA from UT-7/thrombopoietin and made the cDNA. After expression of protein from the cDNA in E-Coli, 38 clones which were reacted to the serum of patients with ITP were obtained. Out of them, 9 clones were unknown and 19 were known by the analysis of sequence of gene. We focused human hepatitis delta antigen interacting protein A (dip A), because it strongly reacted to the ITP serum. By RT-PCR method, we detected the expression of dip A in all cell line. We examined the anti-dip A antibody in patients with ITP by western blot method. Sixty percent of ITP patients were positive for dip A antibody, but only 20% of healthy volunteers were positive. The possibility of point mutation in dip-A and role of anti-dip-A antibody in the onset of ITP should be studied furthermore.

为了查明特发性血小板减少性紫癜（ITP）特异性抗原的病因，我们采用高灵敏度的基因克隆方法直接分析了ITP特异性抗原的基因;通过重组表达克隆（SEREX）进行抗原血清学鉴定。我们从UT-7/血小板生成素中提取RNA并制备cDNA。将该cDNA在大肠杆菌中表达后，获得38个与ITP患者血清反应的克隆。其中9个克隆为未知克隆，19个克隆为已知克隆。我们重点研究人丁型肝炎抗原相互作用蛋白A（dip A），因为它对ITP血清有强烈反应。用RT-PCR方法检测dip A在所有细胞系中的表达。用免疫印迹法检测ITP患者血清中抗dip A抗体。60%的ITP患者dip A抗体阳性，而健康志愿者仅20%阳性。dip-A基因点突变的可能性及抗dip-A抗体在ITP发病中的作用有待进一步研究。

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