F-Box蛋白家族新成员FBXO30基因的融合表达

采用非定向克隆技术将FBXO3 0 (F boxonlyprotein 3 0 )基因编码区cDNA序列克隆到哺乳类融合表达载体pEGFP C2中 ,通过脂质体转染 ,将pEGFP C2 /FBX0 3 0导入NIH 3T3细胞株 ,并在荧光显微镜下观察融合蛋白的表达。采用定向克隆技术将FBXO3 0基因编码区cDNA序列克隆到表达载体pGEX 4T 2中 ,转化大肠杆菌。结果显示FBXO3 0蛋白主要存在于胞浆中。十二烷基硫酸钠 聚丙烯酰胺凝胶电泳和免疫印迹均检测到一条约 6 5 5kD左右的新增蛋白泳带。此外 ,还用谷胱甘肽Sepharose 4B亲和层析柱获得了纯化的该融合表达产物。FBXO3 0蛋白为胞浆蛋白 ,以可溶性的形式存在 ,它能在原核表达体系中稳定表达 ,这对制备其特异性抗体和从蛋白水平研究其功能均具有十分重要的意义

Study on the fusion expression of FBXO30: a novel member of F-Box protein family

Objective The cDNA sequence of the coding region of FBXO30 (F box only protein 30), which is a novel member of F box protein family, was cloned into the mammalian expression vector pEGFP C2 by non directional cloning method and introduced into the NIH 3T3 cells by liposome transfection. Observation under the fluorescent microscopy after transfection showed that EGFP (enhanced green fluorescent protein)/FBXO30 was expressed and existed mainly in the cytoplasm. At the same time, the cDNA sequence of the coding region of FBXO30 was cloned into prokaryotic expression vector pGEX 4T 2 by directional cloning strategy. The GST (glutathione S transferase)/FBXO30 fusion protein was expressed under the induction of IPTG (isopropylthio β D galactoside) in E. coli. A new band (approximately 65.5 kD) was detected by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS PAGE) and immunoblotting. Purification of the GST fused FBXO30 was carried out by affinity chromatography with glutathione sepharose 4B. The fusion expression of FBXO30 indicates that: FBXO30 protein is a cytoplasmic soluble protein, and the stable fusion expression of FBXO30 in the prokaryotic expression system can provide a base for the preparation of the specific antibody of FBXO30 and further functional study of FBXO30.