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Immunochemical analysis of DMD gene product dystrophin

DMD基因产物肌营养不良蛋白的免疫化学分析

基本信息

批准号：
01480238
负责人：
SHIMIZU Teruo
金额：
$ 3.97万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480238/
关键词：
Dystrophin immunohistochemistry Rotary shadowing revertant fibers in DMD abnormal splicing in BMD immature fibers in cogenital myotonic dystrophy DRP 単クロ-ン抗体免疫組織 rotary shadowing DMD

项目摘要

The reseach was aimed to establish antibodies to varied domains of dystrophin and to analyze the physiological and pathogenetic significance of dystrophin.1) We synthesized three peptides of the amino acid sequences 215-264 (N terminal domain).10125-10138 and 10209-10229 (cyteine rich and C terminal domains). We succeeded to produce a hybridoma A1C secreting a monoclonal antibody IgG2a against the N terminal domain. However, We could not make antibodies to other domains.2) We analyzed the precise localization of dystrophin in myofibers. We could show a dense accumulation onto neuromuscular and myotendon junctions (Biomed.Res.10 ; 405-409.1989) as well as on sarcolemma. 3) At first, dystrophin was believed to be defective in DMD and either to be decreased in the amount or to be expressed in the abnormal size in BMD.However, we dimonstrated the pesence of revertant fibers in DMD which expressed the full size of dystrophin (Proc Japan Acad.ser.B 64 ; 205-208.1988). We succeeded in presenting various splicings of each BMD gene allele in 23 BMD patients (J.Neurol.Sci.121 ; 183-189,1994). 4) During myogenesis DRP was downregulated whereas dystrophin was upregulated during gestation. In various congenital myopathies dystrophin was well expressed although nucleus migration, mitochondrion maturity and myosin differentiation were variously disturbed. In congenital myotonic dystrophy, appearance of dystrophin was enormously delayd. The results confirmed the immaturity of the myofibers. 5) We isolated dystrophin from rabbit. The triton X extract of the heavy-membrane and myofibril fraction was sequentially processed in hydroxyapatite, WGA and DEAE column and we got 90% purity of dystrophin. The rotary shadowing demonstrated a dumbbell type rod. The size was -10nm thick and 3 nm wide. The result was in good accordance with the proposed model of antiparallel homodimer (Proc.Jap.Acad.Ser.B,66 ; 96-99,1990).

本研究的目的是建立抗肌营养不良蛋白（dystrophin）不同结构域的抗体，分析dystrophin的生理和致病意义。1）合成了三个氨基酸序列的肽段，分别为215-264（N端结构域）、10125 -10138和10209-10229（C端富含半胱氨酸结构域）。我们成功地产生了杂交瘤A1 C分泌的单克隆抗体IgG 2a的N端结构域。2）分析了抗肌萎缩蛋白在肌纤维中的精确定位。我们可以显示在神经肌肉和肌腱连接处（Biomed.Res.10 ; 405-409.1989）以及肌膜上的密集积聚。3)起初，认为DMD中的肌营养不良蛋白是缺陷的，在BMD中要么数量减少，要么以异常大小表达，然而，我们证明了DMD中表达肌营养不良蛋白完整大小的回复突变纤维的存在（Proc Japan Acad.ser.B 64 ; 205-208.1988）。我们成功地在23名BMD患者中呈现了每个BMD基因等位基因的各种剪接（J.Neurol.Sci.121; 183- 189，1994）。4)在肌形成期间，DRP下调，而肌营养不良蛋白在妊娠期间上调。在各种先天性肌病肌营养不良蛋白表达良好，虽然细胞核迁移，成熟和肌球蛋白分化受到不同程度的干扰。在先天性强直性肌营养不良中，肌营养不良蛋白的出现大大延迟。结果证实了肌纤维的不成熟。5)我们从家兔中分离出抗肌萎缩蛋白。经羟基磷灰石柱、WGA柱和DEAE柱层析，得到纯度为90%的肌营养不良蛋白。旋转阴影显示哑铃型杆。尺寸为约10 nm厚和3 nm宽。结果与日本科学院学报B辑66; 96- 99，1990提出的反平行同源二聚体模型一致。