重症肌无力P9-ZFD蛋白的骨骼肌亚细胞定位

目的 :表达、纯化重症肌无力相关P9基因TRAF型锌指结构 (P9TRAF typezincfingerdomain ,简称P9 ZFD)编码的蛋白质 ,制备P9 ZFD多克隆抗体 ,研究P9 ZFD蛋白的骨骼肌亚细胞定位。 方法 :应用逆转录 聚合酶链反应 (RT PCR )克隆编码P9 ZFD的cDNA序列 ,构建pET2 4a P9 ZFD表达载体 ,在E .coliBL2 1(DE3)中胞内诱导表达P9 ZFD蛋白 ,组氨酸亲和色谱法纯化P9 ZFD蛋白并免疫Balb/c小鼠制备其多克隆抗体。ELISA和Westernblot鉴定抗体效价及其免疫特异性。免疫组织化学及免疫电镜观察P9 ZFD蛋白在骨骼肌中的表达部位及其亚细胞分布。结果 :表达、纯化的P9 ZFD蛋白相对分子质量约 30kD ,纯度达 95 %以上。制备的P9 ZFD抗体具有特异的免疫反应性。免疫组织化学和免疫电镜结果显示 ,P9 ZFD蛋白的免疫染色部位集中沿MG骨骼肌细胞膜分布。结论 :重症肌无力相关的P9 ZFD蛋白在MG骨骼肌细胞膜处表达 ,是一种骨骼肌细胞膜蛋白组分。

EXPRESSION AND SUBCELLULAR LOCALIZATION OF P9-ZFD PROTEIN IN PATIENTS WITH MYASTHENIA GRAVIS

To express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-re-lated gene P9 ( P9-ZFD ) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG. Methods The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET-24a, and the P9-ZFD recombinant protein was induced via E.coli. BL21 (DE3) and purified by histidine affinity chromato-graphy. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expres-sion and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied. Results The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control. Conclusion P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.

重症肌无力P9-ZFD基因片段表达蛋白的研究

目的:探讨P9-ZFD基因片段在人骨骼肌中表达的蛋白质。方法:以MG骨骼肌RNA为模板,扩增编码P9-ZFD片段的cDNA,构建pFT24a-P9-ZFD表达载体,经BL21(DE3)诱导表达P9-ZFD蛋白和组氨酸亲和层析法进行纯化,并制备P9-ZFD抗体。Western blot鉴定MG和对照组骨骼肌中与P9-ZFD抗体产生特异性免疫反应的蛋白组分。结果:骨骼肌中与P9-ZFD抗体产生特异性免疫反应的蛋白质相对分子质量约40000,在伴胸腺增生或胸腺瘤MG骨骼肌中的表达水平明显高于对照组(P<0.001)。结论:骨骼肌中40 000的蛋白是P9-ZFD基因片段的编码产物,该蛋白在伴胸腺增生或胸腺瘤MG骨骼肌中的表达水平明显上调,可能具有重要的病理生理意义。