重组表达载体plRES Rb94的构建及其对肺腺癌A549细胞株中SphK表达的影响

目的构建真核表达质粒pIRES Rb94,观察Rb94基因对鞘氨醇激酶（SphK）表达的影响。方法根据Rb94基因序列设计、合成引物，构建以内部核糖体进入位点（IRES）相连的Rb94基因的真核表达质粒pIRES Rb94，经脂质体转染A549细胞,G418筛选获得稳定转染的细胞株。采用Real time RT PCR和Western boltting法检测Rb94基因并观察其对人肺腺癌A549细胞株SphK表达的影响。结果成功构建重组表达质粒pIRES Rb94,转染A549细胞后获得稳定转染细胞株pIRES Rb94 A549，该细胞株中Rb94基因高效表达；SphK1表达下调而SphK2表达上调。结论真核表达质粒pIRES Rb94构建成功并可有效转染A549细胞，Rb94基因抑制SphK1的表达，增强SphK2的表达。

Construction of the recombination expression vector pIRES-Rb94 and its effect on expression of SphK in lung adenocarcinoma A549 cell line

Objective To construct a eukaryofic expression plasmid pIRES-Rb94 and investigate its effect on expression of SphK in lung adenocarcinoma A549 cell line. Methods A primer of Rb94 was designed and composed based on the Rb94 sequence. The Rb94 gene was cloned by PCR and was sequenced. The eukaryofic expression plasmid plRKS-Rb94 was correctly constructed and transfected into the A549 cells by lipofectamine 2O00,then the stably transfected pIRES-Rb94 cell line was selected through G418. Expression of the Rb94 ...