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。

Study on the Protein Expressed by P9-ZFD Fragment in Myasthenia Gravis

Aim:To express and purify the protein coded by P9-ZFD fragment and to prepare P9-ZFD antibody for studying P9-ZFD protein expressed in skeletal muscle. Methods: Total RNA was extracted from the skeletal muscle of MG patient and the cDNA fragment encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD fragment was ligated into the expression vector pET-24a, P9-ZFD protein was induced via BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antibody was prepared and the protein component reacting with the P9-ZFD antibody in the skeletal muscles of MG and controls was determined by Western blot. Results:The protein component reacting with P9-ZFD antibody was about mass of 40 000, the expression levels of which were higher in the skeletal muscles of MG with thymic hyperplasia or thymoma than in those of controls ( P < 0.001). Conclusion:40 000 protein of skeletal muscle should be a product coded by P9-ZFD fragment, this protein is obviously upregulated in MG with thymic hyperplasia or thymoma, which may have improtant pathophysiological significance.