摘要
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。
Objective: To express and purify the protein encoding the TRAF-type zinc finger domain of myastheniagravis ( MG ) related gene P9 ( P9-ZFD ) and to prepare P9-ZFD polyclonal antibody for studying subcellular localization of P9-ZFD protein in skeletal muscle. Methods: The cDNA fragment encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA fragment was ligated into the expression vector pET-24a, and the P9-ZFD recombinant protein was induced via E.coli BL21(DE3)and purified by histidine affinity chromatography. P9-ZFD antibody was prepared through immunizing Balb/c rats with purified P9-ZFD protein. Its titer and specificity were determined with ELISA and Western blot. Subcellular distribution of skeletal muscle P9-ZFD protein was studied with immunohistochemitry and immunoelectron microscopy. Results:The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95 percent. The antibody was specific for P9-ZFD protein. Immunostaining of P9-ZFD protein distributed along cellular membrane of skeletal muscle of MG. Conclusion: P9-ZFD protein was expressed in cellular membrane of skeletal muscle. It should be a membrane component.
基金
国家自然科学基金资助项目 (3 0 0 70 716)
