PcDNA3．1（-）-edPSMA载体的构建及其稳转鉴定

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

目的构建人前列腺特异性膜抗原胞外区（edPSMA）真核表达质粒，并建立稳定表达edPSMA的小鼠前列腺癌（RM-1-edPSMA）的细胞株。方法构建PcDNA3．1（-）．edPSMA真核表达载体并测序，脂质体转染法分别将PcDNA3．1（-）-edPSMA质粒和PcDNA3．1（-）空载质粒转染至RM-1细胞，G418筛选后获得了稳定生长的阳性克隆细胞株，逆转录-聚合酶链反应（RT—PCR）、Westernblot检测edPSMA蛋白的表达。结果质粒经酶切测序，结果与GeneBank序列进行Blast比对分析，同源性为99．7％，RT—PCR和Westernblot均证明转染了PcDNA3．1（-）-edPS—MA质粒的RM-1细胞表达edPSMA。结论成功构建了PcDNA3．1（-）-edPSMA质粒，并建立了稳定表达edPSMA的细胞株。

Objective To construct the eukaryotic expression plasmid about extraeellular 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 Lipofectamine^TM 2000. These transfected cells 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 amplified 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. Conclusion The PcDNA 3.1 （ - ）-edPSMA plasmid and the RM-1 cells stablely expressing edPSMA,were successfully constructed, which provides the basis for the research and application of the new vaccine of dendritic cells.