Preparation of monoclonal antibodies for monitoring radiation effects

用于监测辐射效应的单克隆抗体的制备

• 批准号: 07557237
• 负责人: SUZUKI Norio
• 金额: $ 1.47万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557237/
• 关键词: MOLT-4; X-rays; Radiation-induced proteins; p41; Anti-p41 antibody; Cell death; Predictive assay; X線誘導蛋白p41; p41抗体; Set遺伝子; MOLT4白血病細胞; 放射線誘導蛋白; 抗ペプチド抗体; SET

The aims of the project were (1) to characterize the appearance of a protein after X-irradiation in radiosensitive human T cell leukemia MOLT-4 cells, and (2) to prepare monoclonal antibodies against the protein.By two-dimensional electrophoresis and silver staining, a new spot was detected after irradiation. The location was 41 kDa in the mass and 4.0 of pI.The protein (p41) was identified from its partial sequences as a set gene product, whose gene translocation was previously found in a case of acute undifferentiated leukemia. Polyclonal rabbit antibodies against p41 were prepared ; the antibodies detected p41 and a 42kDa protein (p42). p42 was present also in non-irradiated control cells. Photoimage analyzes of the silver stained gels and Western blots indicated that appearance of p41 was time dependent, starting 4 hrs after irradiation and reaching a plateau by 10 hrs, and dose dependent between 1 and 10 Gy.By the following evidences, p41 was suggested to be derived from p42 through post-translational modification ; (1) p41 appeared in the presence of cycloheximide, a protein synthesis inhibitor, (2) radioactivity was not found in p41 after post-irradiation labeling with ^<35>S-amino acids, and (3) in cells which had been pre-labeled with ^<35>S-amino acids, the radioactivity was found in p41 and p42 after irradiation. Suppression of p41 appearance after irradiation by vanadate, an inhibitor of protein phosphatase, suggested that phosphorylation/dephosphorylation would be involved in the post-translational modification.The anti-p41 polyclonal antibodies were prepared and have been successfully utilized, as described above, for the analyzes of p41/p42. The preparation of monoclonal antibodies against p41 and p42 has been under way.

本课题的目的是（1）对放射敏感的人T细胞白血病MOLT-4细胞中X射线照射后出现的蛋白质进行表征，（2）制备针对该蛋白质的单克隆抗体。p41蛋白的分子量为41 kDa，pI为4.0，经部分序列分析，确定为一组基因产物，其基因易位曾在1例急性未分化白血病中发现。制备抗p41的多克隆兔抗体;该抗体检测p41和42 kDa蛋白（p42）。p42也存在于未照射的对照细胞中。银染凝胶图像分析和Western印迹分析表明，p41的出现具有时间依赖性，从照射后4小时开始，10小时达到稳定，在1 ~ 10戈伊剂量范围内，p41的出现具有剂量依赖性。（1）p41在蛋白质合成抑制剂放线菌酮存在下出现，（2）放射后用^S-氨基酸标记后，在p41中未发现放射性<35>，（3）在用^S-氨基酸预先标记的细胞中<35>，放射后在p41和p42中发现放射性。用钒酸盐（一种蛋白磷酸酶抑制剂）抑制p41的表达，提示p41的磷酸化/去磷酸化参与了翻译后修饰，制备了抗p41多克隆抗体，并成功地用于p41/p42的分析。目前正在制备抗p41和p42的单克隆抗体。

项目成果

Morimatsu,A.et al.: "Identification and characterization of a protein appeared after X-irradiation in human T cell leukemia." J.Radiat.Res.37. 1-11 (1996)

Morimatsu, A. 等人：“人类 T 细胞白血病中 X 射线照射后出现的蛋白质的鉴定和表征。”

Takeuchi,K.: "cDNA cloning of mouse VLA-3 αsubunit" J.Cellular Biochemistry. 57. 371-377 (1995)

Takeuchi, K.：“小鼠 VLA-3 α 亚基的 cDNA 克隆”J.Cellular Biochemistry 57. 371-377 (1995)。

Matsumoto,Y.et al.: "A possible mechanism for hyperthermic radiosensitization mediated through hyperthermic lability of Ku subunits in DNA-dependent protein kinase." Biochemical and Biophysical Research Communications. (in press). (1997)

Matsumoto,Y.et al.：“通过 DNA 依赖性蛋白激酶中 Ku 亚基的高温不稳定性介导的高温放射增敏的可能机制。”

Morimatsu, A., Suzuki, N., Hirano, K., Matsumoto, Y., and Sakai, K.: "Identification and characterization of a protein appeared after X-irradiation in human T cell leukemia." J.Radiat.Res.37. 1-11 (1996)

Morimatsu, A.、Suzuki, N.、Hirano, K.、Matsumoto, Y. 和 Sakai, K.：“人类 T 细胞白血病中 X 射线照射后出现的蛋白质的鉴定和表征。”

Matsumoto, Y., Suzuki, N., Sakai, K., Hirano, K., Morimatsu, A., and Murofushi, H.: "A possible mechanism for hyperthermic radiosensitization mediated through hyperthermic lability of Ku subunits in DNA-dependent protein kinase." Biochemical and Biophysic

Matsumoto, Y.、Suzuki, N.、Sakai, K.、Hirano, K.、Morimatsu, A. 和 Murofushi, H.：“通过 DNA 依赖性蛋白中 Ku 亚基的高温不稳定性介导的高温放射增敏的可能机制