Expression and antibody preparation of the TRAF-type zinc finger domain of myasthenia gravis related gene P9

Purpose: To express the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG) related gene P9 (simply named as P9-ZFD) and to prepare P9-ZFD polyclonal antibody. Methods: The cDNA fragment encoding P9-ZFD was amplified from the skeletal muscle of MG patient by RT-PCR and sequenced. The cloned P9-ZFD fragment was ligated into the expression vector pET-24a and P9-ZFD fusion protein was induced in E. coli BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antibody was prepared through immunizing Balb/c rats with purified P9-ZFD fusion protein. The specificity of antibody was determined by Western blot. Results: The cDNA sequence of amplified P9-ZFD fragment was the same as its sequence inscribed in GeneBank. The molecular weight of purified P9-ZFD fusion protein was about 30 000. Its purity was more than 95%. Western blot analysis revealed that P9-ZFD antibody produced specific immunologic reaction only with purified P9-ZFD fusion protein. Conclusions: The protein coded by P9-ZFD can be expressed in prokaryocyte. Polyclonal antibody of P9-ZFD which has peculiar immunoreactive can be prepared.