Induced expresssion pattern of Rab9 in CpG — Activated macrophages and construciton of Rab9 eukaryotic expression vector

This study is to learn the expression patterns of small Rab GTPase Rab9 in macrophages induced by CpG and construction of Rab9 eukaryotic expression vector. Rab9 expression in different murine tissues was detected by RT-PCR and Real-time PCR. RAW264.7 cells were stimulated with 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-DNA3.1(-)/Flag(-)B. The recombinant Rab9 vector was then verified by double restriction enzyme digestion and sequence analysis. RT-PCR and Real-time PCR results have revealed 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. In the mouse macrophage cells, Rab9 mRNA expression is negatively regulated by CpG. Rab9 eukaryotic expression vector was successfully constructed.