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

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

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。

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 eukaryofie expression plasmid plRKS-Rb94 was correctly constructed and transfected into the A549 cells by lipofectamine 2000, then the stably transfeeted pIRES-Rb94 cell line was selected through CA18. Expression of the Rb94 gene was detected by real time RT-PCR and Western blotting. Results The recombination ex- pression vector containing the Rb94 gene was successfully constructed and a new cell hne with stable and high expression of Rb94 was established. Real time RT-PCR and Western Blotting analysis demonstrated that the Rb94 gene could be effectively expressed in the A549 cells. Moreover, expression of SphK1 was down-regulated and of SphK2 was up-regulated. Conclusion The plRES-Rb94 expression plasmid was successfully constructed and could be effectively transfected into A549 cells. After transfec- tion, Rb94 can efficiendy inhibit expression of SphK1, and enhances expression of SphK2 in the A549 cell line.