Analysis of a novel RNA epitope recognized by sera in patients with rheumatic diseases using randomized RNA epitope library

使用随机 RNA 表位库分析风湿性疾病患者血清识别的新 RNA 表位

• 批准号: 10670427
• 负责人: TAKASAKI Yoshinari
• 金额: $ 1.22万
• 依托单位: Juntendo 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 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670427/
• 关键词: RNA; autoantibody; randomized RNA library; epitope; rheumatic disease; 自己免疫

We defined a novel RNA epitope recognized by serum from a patient with Sjogren's syndrome (SjS) from a randomized RNA epitope library, and investigated the epitope reactivity of the anti-RNA antibodies in patients with various connective tissue diseases. Serum from SjS patient TS was used to select ligands from the library of RNA oligomers with a central region of 25 degenerate nucleotides. Bound RNA was recovered by reverse transcription, PCR amplification and subcloning. Sera from 32 of 61 patients with SS (52.4%) precipitated with one of the selected RNA (TS1・RNA ; CGAAAGUCCGAUCGGCGUAAUGCA), whereas sera from 8 of 41 patients with systemic lupus erythematosus (19.5%) and 3 of 25 patients with rheumatoid arthritis (12.0%) precipitated. Frequency of anti-SS-A antibodies was significantly higher in anti-TS1・RNA positive patients but there was no cross-reactivity between anti-SS-A and anti-TS1・RNA.The epitopes on this RNA were further analyzed by immunoprecipitation using mutants of TS1・RNA.The reaction of patient sera with the RNAs declined when any part of the TS1・RNA was mutated. This finding indicates that the selected TS1・RNA is a novel sequence-specific RNA epitope that binds specifically sera with MCTD and SjS.Then we attempted to study the clinical significance of antibodies to TS1・RNA.Whereas there was no significant correlation between anti-TS1・RNA and clinical features in SjS patients, anti-TS1・RNA antibodies significantly correlated with the laboratory findings such as anti-dsDNA, Sm and U1 RNP, and low serum level of complement in MCTD patients. In addition, the activities of renal involvement was significantly correlated with the titer of anti-TS1・RNA.These results suggested that anti-TS1・RNA antibody is a novel antibody against the sequence specific RNA in SiS and MCTD patients and may play certain roles associated with autoantibody-production and pathogenesis in MCTD.

我们从随机RNA表位文库中定义了一种新的可被干燥综合征患者血清识别的RNA表位，并研究了抗RNA抗体在各种结缔组织疾病患者中的表位反应性。用SjS患者的血清从RNA低聚体文库中选择中心区域为25个简并核苷酸的配基。通过逆转录、聚合酶链式反应和亚克隆获得结合的RNA。61例SS患者中有32例(52.4%)与所选择的RNA(TS1·RNA；CGAAGUCCGAUCGGCGUAAUGCA)发生沉淀，而41例系统性红斑狼疮患者中有8例(19.5%)和25例类风湿关节炎患者中有3例(12.0%)出现沉淀。抗TS1·RNA阳性患者抗SS-A抗体的出现频率明显高于抗TS1·RNA阳性患者，但抗SS-A与抗TS1·RNA之间无交叉反应。用TS1·RNA突变体进行免疫沉淀进一步分析该RNA上的表位。因此，我们试图研究抗TS1·RNA抗体的临床意义。抗TS1·RNA抗体与SjS患者的临床特征无明显相关性，而抗TS1·RNA抗体与MCTD患者的实验室指标如抗dsDNA、Sm、U1 RNP及低补体水平显著相关。提示抗TS1·RNA抗体是SIS和MCTD患者中一种新的针对序列特异性RNA的抗体，可能在MCTD自身抗体的产生和发病机制中起一定作用。

项目成果

Takasaki Y,Kogure T,Takeuchi K,Kaneda K,Yano T,Hirokawa K,Hirose S,Shirai T,Hashimoto H,: "Reactivity of anti-proliferating cell nuclear antigen(PCNA)murine monoclonal antibodies to the PCNA multiprotein complexes involved in cell proliferation"J Immunol.

Takasaki Y、Kogure T、Takeuchi K、Kaneda K、Yano T、Hirokawa K、Hirose S、Shirai T、Hashimoto H：“抗增殖细胞核抗原 (PCNA) 鼠单克隆抗体对参与 PCNA 多蛋白复合物的反应性

Abe K,Takasaki Y,Ushiyama C,Asakawa J,Fukazawa T,Seki M,Hirashima M,Ogaki M,: "Expression of CD8O and CD86 on peripheral blood T lymphocytes in patients with systemic lupus erythematosus."J Clin Immunol. 9. 58-66 (1999)

Abe K，Takasaki Y，Ushiyama C，Asakawa J，Fukazawa T，Seki M，Hirashima M，Ogaki M，：“系统性红斑狼疮患者外周血 T 淋巴细胞上 CD8O 和 CD86 的表达。”J Clin Nutrition。

Isoyama T,Kamoshita N,Yasui K,Iwai A,Shiroki K,Toyada H,Yamada A,Takasaki Y,Nomoto A: "Lower concentration of La protein required for internal ribosome entry on hepatitis C virus RNA than on poliovirus RNA"J Gen Virol. 80. 2319-2327 (1999)

Isoyama T、Kamoshita N、Yasui K、Iwai A、Shiroki K、Toyada H、Yamada A、Takasaki Y、Nomoto A：“丙型肝炎病毒 RNA 内部核糖体进入所需的 La 蛋白浓度低于脊髓灰质炎病毒 RNA”J Gen

Shiroki K,Isoyama T,Kuge S,Ishii T,Ohmi S,Hata S,Suzuki K,Takasaki Y,Nomoto A: "Intracellular redistribution of truncutated La protein produced by Poiovirus 3Cpro-mediated cleavage."J Virol. 73. 2193-2200 (1999)

Shiroki K、Isoyama T、Kuge S、Ishii T、Ohmi S、Hata S、Suzuki K、Takasaki Y、Nomoto A：“Poiovirus 3Cpro 介导的切割产生的截短 La 蛋白的细胞内重新分布。”J Virol。

高崎芳成: "混合性結合組織病、内科学エッセンス"鈴木一、橋本博史編、朝倉書店. 3

高崎吉成：《混合性结缔组织病，内科精华》，铃木肇、桥本博主编，朝仓书店3。