Immunochemical analysis of DMD gene product dystrophin

本研究旨在建立抗肌营养不良蛋白不同结构域的抗体，并分析其生理和病理意义。1)合成了215 ~ 264 （N端结构域）氨基酸序列的3个多肽。10125-10138和10209-10229（富含半胱氨酸和C端结构域）。我们成功地产生了一株A1C杂交瘤，分泌针对N端结构域的单克隆抗体IgG2a。然而，我们不能制造针对其他域的抗体。2)我们分析了肌营养不良蛋白在肌纤维中的精确定位。我们可以在神经肌肉和肌肌腱连接处显示密集的积累。10;405-409.1989)以及肌膜。3)起初，我们认为肌营养不良蛋白在DMD中存在缺陷，在BMD中要么数量减少，要么以异常大小表达。然而，我们证明了在DMD中存在表达全尺寸肌营养不良蛋白的反向纤维（Proc Japan academy .ser. b 64; 205-208.1988）。我们成功地在23例骨髓瘤患者中展示了每个骨髓瘤基因等位基因的不同剪接。121年;183 - 189, 1994)。4) DRP在妊娠期下调，而肌营养不良蛋白在妊娠期上调。在各种先天性肌病中，尽管细胞核迁移、线粒体成熟和肌球蛋白分化受到不同程度的干扰，但肌营养不良蛋白的表达良好。在先天性肌强直性营养不良中，肌营养不良蛋白的出现大大延迟。结果证实肌纤维发育不成熟。5)从家兔体内分离到肌营养不良蛋白。重膜和肌原纤维部分的triton X提取物在羟基磷灰石、WGA和DEAE柱上依次处理，得到纯度为90%的肌营养不良蛋白。旋转阴影演示了哑铃型杆。尺寸为-10nm厚，3nm宽。结果与提出的反平行同型二聚体模型（proc . japa . acad . ser）吻合较好。B, 66;96 - 99, 1990)。

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).