Establishment of maker for early diagnosis of systemic autoimmune diseases based on de-phosphorylation of autoimmogen U1-68k

基于自身免疫原U1-68k去磷酸化的系统自身免疫性疾病早期诊断标记物的建立

• 批准号: 24790564
• 负责人: NAGAI Kouhei
• 金额: $ 2.83万
• 依托单位: Kinki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2012
• 资助国家: 日本
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-24790564/
• 关键词: 自己免疫疾患; 翻訳後修飾; 診断マーカー; 自己抗体

In this study, we tried to establish an useful method for detecting a de-phosphorilated form of U1-68k autoimmunogen, which was found to be increased in peripheral blood mononuclear cells of patients of systemic lupus erythematosus or Mixed connective tissue diseases. As a substitution of a conventional 2D-western blot method, we examined three methods; (1) an immunological method, (2) a phos-tag electrophorsis, and (3)an isoelectric focusing electrophoresis-western blot. As a result, we found that the isoelectric focusing electrophoresis-western blot method is most suitable for the detection of the de-phosphorilated form of U1-68k. Moreover, we found that whole cell extract prepared by cell lysis buffer containing SDS can be used for detecting U1-68k. In conclusion, combination of isoelectric focusing electrophoresis-western blot and whole cell extract by SDS-containing cell lysis buffer can shorten analsys time from 5 days to 2.5 days.

在本研究中，我们试图建立一种有用的方法来检测系统性红斑狼疮或混合性结缔组织病患者外周血单个核细胞中发现的U1-68k自身免疫原去磷酸化形式。作为传统2D-Western印迹方法的替代，我们检查了三种方法：(1)免疫学方法，(2)PHOS-Tag电泳法，(3)等电聚焦电泳法-Western印迹法。结果发现，等电聚焦电泳-蛋白质印迹法最适合于U1-68k去磷酸化形式的检测。此外，我们还发现，用含有十二烷基硫酸钠的细胞裂解缓冲液制备的全细胞提取液可以用于检测U1-68k。综上所述，等电聚焦电泳-Western印迹结合含十二烷基硫酸钠的细胞裂解缓冲液提取全细胞可将分析时间从5天缩短至2.5天。

项目成果

Alterd posttranslational modification on U1 small nuclear ribonucleoprotein 68k in systemic autoimmune disease detected by 2D Western blot

2D Western blot 检测系统性自身免疫性疾病中 U1 小核核糖核蛋白 68k 的翻​​译后修饰发生改变

• 发表时间: 2012
• 期刊: Electrophoresis
• 影响因子: 2.9
• 作者: K Nagai;M Arito;Y Takakuwa;S Ooka;T Sato;MS Kurokawa;K Okamoto;T Uchida;N Suematsu;T Kato
• 通讯作者: T Kato

Altered posttranslational modification on U1 small nuclear ribonucleoprotein 68k in systemic autoimmune diseases detected by 2D Western blot.

通过 2D Western blot 检测系统性自身免疫性疾病中 U1 小核核糖核蛋白 68k 翻译后修饰的改变。

• DOI: 10.1002/elps.201200058
• 发表时间: 2012
• 期刊: Electrophoresis
• 影响因子: 2.9
• 作者: Kouhei Nagai; Mitsumi Arito;Yukiko Takakuwa;Seido Ooka;Toshiyuki Sato;Manae S Kurokawa;Kazuki Okamoto;Teisuke Uchida;Naoya Suematsu;Tomohiro Kato
• 通讯作者: Tomohiro Kato