Cloning and expression of small G protein Rab7 in RAW264.7 cells

Objective To construct Rab7 eukaryotic expression plasmid and analyze its expression in murine macrophage cell line RAW264.Methods The expression of Rab7 in tissues and cells were detected by RT-PCR.Using the cDNA of RAW264.7 cells as template,the open reading frame of Rab7 was amplified by primers designed according to the full-length sequence of Rab7 published in Genbank.The target gene was connected with the eukaryotic expression vector pcDNA3.1 to get pcDNA-Rab7.The recombinant eukaryotic vector of Rab7-pcDNA were transiently transfected into Raw264.7 cells by liposome,and the over expression state of Rab7 were indentified by RT-PCR and Western blot.Results Sequence analysis showed that the Rab7-pcDNA plasmid was 100% homologous with the sequence of Rab7 in Genbank.The expression of Rab7 was increased significantly as verified by RT-PCR and Western blot.Conclusion The eukaryotic expression vector of Rab7-pcDNA is successfully constructed.This study may lay a foundation for further elucidating the function of Rab7 in macrophages.