Expression and subcellular localization of P9-ZFD protein in patients with myasthenia gravis.
- Author:
Ming-shan REN
1
;
Chuan-zhen LU
;
Jian QIAO
;
Hui-min REN
;
Ren XU
;
Ren-bao GAN
Author Information
- Publication Type:Journal Article
- MeSH: Adult; Cell Membrane; metabolism; Escherichia coli; metabolism; Female; Humans; Muscle Proteins; biosynthesis; genetics; Muscle, Skeletal; metabolism; pathology; Myasthenia Gravis; metabolism; Peptide Fragments; biosynthesis; genetics; Recombinant Proteins; biosynthesis; genetics; Transfection; Zinc Fingers
- From: Chinese Medical Sciences Journal 2004;19(3):221-224
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-related 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.
METHODSThe cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET24a, and the P9-ZFD recombinant protein was induced via E. coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied.
RESULTSThe 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.
CONCLUSIONP9-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.
