人Rab26基因的克隆和表达

Cloning and Expression of Human Rab26 Gene

以完整EST为参考序列设计引物,以人的胎脑cDNA为模板,用PCR方法筛选获得Rab26基因全长序列,并亚克隆到载体pGEM-T,真核表达载体pEGFP-N1和原核表达载体pET-32a(+)中.RT-PCR显示该基因在不同组织的肿瘤细胞株中表达量有明显的差异.把Rab26基因转染入HeLa细胞中,通过与绿色荧光蛋白(GFP)基因融合表达,显示Rab26定位于胞内膜性细胞器上.在大肠杆菌表达系统获得Rab26基因的高表达.这些结果为进一步研究Rab26基因在细胞内吞和运输功能等方面打下了基础.

According to the complete EST reference sequences,the clone primers were designed to obtain human full-length rab26 gene by PCR amplification method from a human embryo brain cDNA library. It was further subcloned into the cloning vector pGEM-T, the eukaryotic expression vector pEGFP-N1 and E. coli expression vector pET-32a（＋） respectively. RT-PCR displayed that the expression of rab26 gene was distinct difference in various human tumor cells. The human HeLa cells were transfected with pEGFPN1-Rab26 plasmid by liposome method. Fluorescence localization of Rab26 in HeLa cells showed that Rab26 was localized in the intracellular organelles and Rab26 was found to associate with vesicles dispersed throughout the cytoplasm. Rab26 was highly expressed in E. coli BL21 ： DE3 with recombinant plasmid of pET32a-Rab26. Western blotting with anti-HIS antibodies assay confirmed that fusion protein was expected. The results of present study will benefit the further study of the function of Rab26 gene in the endocytic pathway and membrane traffic.