人Rab12基因真核表达载体的构建及在HEK293细胞中的表达

Construction of Homo sapiens Rab12 Eukaryotic Expression Vector and Its Expression in HEK293 Cells

Rab蛋白参与细胞的囊泡运输过程。自噬体-溶酶体的融合需要活化的Rab12蛋白参与。本研究从HEK293细胞获取总RNA,采用RT-PCR方法扩增出Rab12基因的ORF全序列,将其克隆到含有增强型绿色荧光蛋白(EGFP)基因的真核表达载体pEGFP-N1上,成功构建重组质粒pEGFP-Rab12,然后采用lipofectamine 2000将重组质粒转染至HEK293细胞中并获得高表达。

Rab proteins are involved in vesicle trafficking processes. Autophagosome-lysosome fusion relies on the activity of Rab12 protein. In this study, total RNA was extracted from the HEK293 cells and the ORF sequences of Rab12 gene were amplified by RT-PCR. The PCR production was cloned into eukaryotic expression vector(p EGFP-N1) which expressed the enhanced green fluorescent protein to construct recombinant plasmid p EGFP-Rab12. Then, the recombinant plasmid was transfected into HEK293 cells with lipofectamine 2000. After transfection, the Rab12 was expressed successfully.