Construction of PcDNA 3.1 (-) -edPSMA vector and identification of its expression in RM.1 cell line after stable transfection

Abstract

Objective To construct the eukaryotic expression plasmid about extracellular domain of PSMA (edPSMA) and establish an edPSMA-expressing RM-1-edPSMA cells to provide materials for the research of the new vaccine of dendritic cells and the exploration of the immune mechanism. Methods A eukaryotic expression vector pcDNA 3.1(- )- edPSMA was constructed and sequenced. The pcDNA 3.1(-)-edPSMA and pcDNA3.1(-)plasmids were transfected into RM-1 cell lines using Lipofectami- neTM 2000. These transfected ceils were cultured by G418 (200 mg/L). The expression of edPSMA in the survival cells was detected by RT-PCR and Western blot. Results Plasmid was digested, and the ampli-fied fragments had 99.7% homology with the human PSMA published in the Gene bank. The results of RT-PCR and Western blot demonstrated that the RM-1 cells expressing edPSMA had been obtained. Conclu- sion The PcDNA 3.1(- )-edPSMA plasmid and the RM-1 cells stablely expressing edPSMA,were suc-cessfully constructed, which provides the basis for the research and application of the new vaccine of den-dritic cells. Key words: PSMA; Vector; Transfection; Gene expression