关键词: GRIM19, 克隆, 真核表达载体, 转染, 凋亡

Abstract: Objective: To construct pcDNA3.1-GRIM-19 eukaryotic expression vector and to investigate its function in mouse prostate cancer rm-1 cells. METHODS RT-PCR was performed on the total RNA extracted from mouse spleen tissue to obtain the cDNA of GRIM-19,which was inserted into pMD-18T vector .DNA sequencing was tested before the amplified products were cloned into pcDNA3.1. The recombinant vector was transfected into rm-1 cells and its expression examined by RT-PCR. The apoptotic effects of cells were observed by laser scanning confocal microscope (LSCM) and DNA ladder assay. Then, using the RT-PCR detected caspase -3 activity in rm-1 cells after transfection . RESULTS The amplified products were confirmed as the cDNA of rm-1 by DNA sequencing, being capable of expression in rm-1 cells. Typical apoptosis was observed by LSCM and DNA ladder in recombinant plasmid group after 48h of transfection in rm-1 cells. Overexpression of GRIM-19 could increase caspase -3 expression. CONCLUSION The eukaryotic expression vector for GRIM-19 was successfully constructed, which can be expressed in rm-1 cells and induce the cells apoptosis and its apoptosis mechanism maybe related with caspase -3 activity.

Key words: GRIM19 gene, Cloning, Eukaryotic expression vector, Transfection, Apoptosis