Analysis of the expression pattern of Rab GTPase 9 and construction of the eukaryotic expression vector

This study is to learn the expression patterns of Rab GTPase 9 and construction of its eukaryotic expression vector. The expression profile of Rab9 in different murine tissues was examined by RT-PCR and Real-time PCR. RAW264.7 macrophages were stimulated with LPS (100ng/ml)or CpG(0.3μM) for different times, and relative mRNA expression of Rab9 was measured by RT-PCR and Quantitative- PCR. Using the cDNA of RAW264.7 cells as a template, the full-length of Rab9 was amplified and introduced into the restriction site of eukaryotic expression vector pc-DNA. The positive clone of recombinant Rab9 vector was then verified by double restriction enzyme digestion and sequence analysis. Rab9 eukaryotic expression vector were transiently transfected into Raw264.7cells by jetPEI, and the overexpression state was examined. RT-PCR and Real-time PCR results showed that Rab9 was expressed in the liver, lung, kidney, spleen, testicle, ileum and colon of mouse, and the highest expression of Rab9 was found in ileum and colon. After CpG stimulation, Rab9 mRNA expression in RAW 264.7 cells began to decrease at 8-12h, and then resumed slightly the expression at 24h but still lower compared with the original expression. Quantitative-PCR results verified the induced expression pattern of Rab9. Sequence analysis on the recombinant Rab9 vector has showed that the sequence of full-length Rab9 was 100% homologous with the sequence in Genbank. Compared with the control group, Rab9 expression was significantly increased in cells transfected with Rab9 eukaryotic expression vector. Widely expressed Rab9 mRNA is negatively regulated by CpG in the mouse macrophage cells. Rab9 eukaryotic expression vector was successfully constructed and expressed in RAW264.7 cells.