pcDNA3.1/eIF5A真核表达载体的构建及其在真核细胞CCRF-CEM中的表达

The construction and expression of eukaryotic expression vector pcDNA3.1/eIF5A

目的:摸索真核细胞翻译启动因子5A(eIF5A)编码基因的合成、pcDNA3.1/eIF5A真核表达载体的构建及其在真核细胞CCRF-CEM中的表达。方法:用PCR合成eIF5A基因,将基因分别接在克隆载体pMD 18-T的EcoRⅤ多克隆位点上和真核表达载体pcDNA3.1的HindⅢ和EcoRⅠ的多克隆位点之间,构建eIF5A基因的克隆载体和真核表达载体,分别在大肠杆菌E.coliDH5α中转化并提取质粒,将真核表达载体pcDNA3.1/eIF5A的质粒提取后,用脂质体包合并转染真核细胞CCRF-CEM,用流式细胞仪对eIF5A的表达水平进行检测。结果:eIF5A在CCRF-CEM细胞中的表达水平为107.03,eIF5A在转染细胞CCRF-CEM/trans中的表达水平为114.27,表达升高。结论:本实验构建了pcDNA3.1/eIF5A真核表达载体,发现其在CCRF-CEM细胞中高表达。本实验结果将有助于探讨肿瘤治疗过程中的新靶标。

Objective： To investigate synthesis of the gene sequence encoding eukaryotic initiation factor 5A（eIF5A） , construction of the eukaryotic cloning vectors pcDNA3.1/eIF5A and its expression in eukaryocyte CCRF-CEM cells. Methods： The eIF5A sequence was synthesized by PCR. The construction of the cloning vector and eukaryotic expression vector containing eIF5A was performed by an- nealing of eIF5A fragments to the EcoR V polycloning sequence region of the clone vector PMD18-T followed by subcloning into Hind Ⅲ and EcoR Ⅰ polycloning sequence regions of the eukaryotic vector pcDNA3.1. The recombinant eukaryotic vector pcDNA3. 1/eIF5A was transformed into competent E. coli DH5α. The plasmids were extracted from the pcDNA3.1/eIF5A and encapsulated by liposomes. Subsequently, the plasmid liposomes were transfected into the eukaryotic CCRF-CEM cells. The expression level of eIF5A was measured by flow cytometry. Results： The expression level of eIF5A in CCRF-CEM and CCRF-CEM/trans was 107.03 and 114.27, respectively. Conclusion： The recombinant expression vector pcDNA3.1/eIF5A had a high-throughput expression in CCRF-CEM cells, which might have the potential for the further layout of targeted therapy against cancers.