【摘要】 以人的胚胎cDNA为模板 ,用PCR方法筛选获得rab13基因 ,并克隆到真核表达载体pcDNA3和原核表达载体pGEX、pET 15b和 pMAL c2中 .把rab13基因转染入牛肾上腺毛细血管内皮细胞 (BCE细胞 )中 ,免疫荧光染色显示Rab13定位于BCE细胞胞质内膜性细胞器上 .在大肠杆菌DH5a或BL2 1:DE3中表达的原核质粒表达系统均显示rab13基因的高表达 .这些结果为rab13基因对膜通道调节功能的进一步研究打下了基础更多还原

【Abstract】 Small Rab GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways. Rab proteins may function as “molecular switches” to regulate the formation of protein complexes necessary for the targeting, docking and/or fusion of accuracy of vesicle targeting events. Rab13 protein may be essential for protein transport in polarized cells as well as in epithelial cells.In the present paper, human rab13 was obtained by PCR method from a human embryo cDNA library. It was further subcloned into the mammalian expression vector pcDNA3.0 and E. coli expression vector pGEX, pET 15b and pMAL c2. The sequence of HA(a small peptide of nine amino acid)was subcloned into pcDNA3.0/rab13 and a kind of pcDNA3.0/HA rab13 plasmid was obtained. The bovine adrenal capillary endothelial cell (BCE cell) was transfected with pcDNA3.0/HA rab13 plasmid by calcium phosphate method. Immunofluorescence localization of Rab13 in BCE cells showed that Rab13 was localized in the intracellular organelles and Rab13 was found to associate with vesicles dispersed throughout the cytoplasm. Rab 13 was lighly expressed in E.coli DH 5a or Bl21:DE3 which were transformed with recombinant plasmid of pGEX/rab13, pMAL c2/rab13, pET 15b/rab13. Molecular weight of Rab13/GST fusion protein is about 50?kD.Molecular weight of Rab13/MBP fusion protein is about 62.6?kD . Molecular weight of rab13 with six histidine residues at the N terminal is about 25?kD, and Rab13 will be purified with affinity chromatography. The results of present study will benefit the further study of the function of rab13 gene in the membrane traffic.更多还原