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 o...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.展开更多
文摘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.
