Quantitative evaluation of stress using metallothionein induced by stress

使用应激诱导的金属硫蛋白定量评估应激

• 批准号: 03557028
• 负责人: KOJIMA Yutaka
• 金额: $ 4.16万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03557028/
• 关键词: metallothionein; metal-binding protein; stress; ELISA; monoclonal antibody; evaluation of stress; human metallothionein; Radioimmunoassay; メタロチオネイン遺伝子; メタロチオネイン抗体; モノクロナル抗体; 大腸菌での発現; ヒト・メタロチオネインII

It has known that the synthesis of metallothioneins (MTs), metal-binding proteins, is induced by various physical and psychological stressors resulting in increases in MT contents in the liver, blood, and urine. The goal of this study is to establish quantitative evaluation of stress using metallothionein induced under stressful conditions, that is, it is to establish objective evaluation of human stress with high accuracy using MT as an indicator of stress. To establish the evaluation, firstly monoclonal anti MT antibody is to be obtained using human MT, which is used as the antigen, obtained abundantly by a genetic-engineering method. Secondly, MT contents in urine or blood is to be measured by an ELISA method using the anti MT antibody. The results obtained are as follows :1) we successfully obtained the results of MT gene expression by introduction of the human MT II gene into Escherichia coli.2) we obtained abundant refined MT by gel filtration chromatography and high-performance-liquid-chromatography on the crude MT obtained from mass culture of the Escherichia coli.3) we obtained monoclonal anti-MT antibody by cell fusion of mouse myeloma cell and spleen cell of a mouse injected with the human MT II.4) an ELISA method using the monoclonal anti-MT antibody is being developed.

已知金属硫蛋白（MTs）的合成是由各种生理和心理应激源诱导的，导致肝脏、血液和尿液中金属硫蛋白的含量增加。本研究的目的是利用应激条件下诱导的金属硫蛋白建立对应激的定量评价，即利用MT作为应激指标，建立对人体应激的高精度客观评价。为了建立评价，首先利用人MT作为抗原，通过基因工程方法获得丰富的MT单克隆抗MT抗体。其次，用抗MT抗体ELISA法测定尿或血中MT的含量。结果如下：1)将人MT II基因导入大肠杆菌，成功获得了MT基因的表达结果。2)通过凝胶过滤层析和高效液相层析对大肠杆菌大量培养的粗MT进行了丰富的精制MT。3)通过小鼠骨髓瘤细胞与小鼠脾细胞的细胞融合，注射人MT ii，获得了单克隆抗MT抗体。4)利用单克隆抗MT抗体的ELISA方法正在开发中。

项目成果

T.Nagashima: "A new method for quantification of metallothinein using immobilized antibody on blotting membrane"Trace Elements and Electrolytes. 15. 127-131 (1995)

T.Nagashima：“使用印迹膜上固定化抗体定量金属硫蛋白的新方法”微量元素和电解质。

T.Nagashima, M.Okabe, S.Saito, T.Saito, M.Kurasaki, T.Hirauchi, S.Kimura and T.Niioka: "A new method for quantification of meallothionein using immobilized antibody on blotting membrane"Trace Elements and Electrolytes. Vol.15. 127-131 (1998)

T.Nagashima、M.Okabe、S.Saito、T.Saito、M.Kurasaki、T.Hirauchi、S.Kimura 和 T.Niioka：“使用印迹膜上固定化抗体定量甲硫蛋白的新方法”微量元素和

Fumitomo Odawara, Masaaki Kurasaki, Mika Suzuki-Kurasaki, Shinji Oikawa, Tadasu Emoto, Futoshi Yamasaki, Ana Rosa Linde Arias and Yutaka Kojima: "Expression of Human Metallothionein-2 in Escherichia coli : Cadmium Tolerance of Transformed Cells"J. Biochem

Fumitomo Odawara、Masaaki Kurasaki、Mika Suzuki-Kurasaki、Shinji Oikawa、Tadasu Emoto、Futoshi Yamasaki、Ana Rosa Linde Arias 和 Yutaka Kojima：“人金属硫蛋白 2 在大肠杆菌中的表达：转化细胞的镉耐受性”J。

江本 匡,及川 伸二,小田原 史知,蔵崎 美佳,Ana Rosa LINDES ARIAS,山崎 太,小島 豊,蔵崎 正明,Elaine A,Mackay,J.H.R.Kogi: "大腸菌で発現させたセリン→ロイシン、イソロイシン置換人工メタロチオネイン" 日本衛生学雑誌. 47. 200- (1992)

Tadashi Emoto、Shinji Oikawa、Fumichi Odawara、Mika Kurasaki、Ana Rosa LINDES ARIAS、Futoshi Yamazaki、Yutaka Kojima、Masaaki Kurasaki、Elaine A、Mackay、J.H.R.Kogi：“人工丝氨酸 → 亮氨酸和异亮氨酸取代在大肠杆菌“金属硫蛋白”中表达卫生杂志。47。200-（1992）

江本匡 他: "大腸菌内で発現させたセリン→ロイシン置換人工メタロチオネインの性質" 日本衛生学雑誌. 49. 435 (1994)

Tadashi Emoto 等：“大肠杆菌中表达的丝氨酸->亮氨酸取代的人工金属硫蛋白的特性”日本卫生杂志 49. 435 (1994)。