论文部分内容阅读
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 eukaryotic expression plasmid pIRES-Rb94 and study the effect of Rb94 on the expression of sphingosine kinase (SphK). Methods According to the sequence of Rb94, we designed and synthesized the eukaryotic expression plasmid pIRES-Rb94 of Rb94 gene linked with internal ribosome entry site (IRES). The recombinant plasmid was transfected into A549 cells by lipofectamine. The stable transfected Cell lines. The Rb94 gene was detected by Real-time RT-PCR and Western blotting and its effect on SphK expression in human lung adenocarcinoma A549 cell line was observed. Results The recombinant plasmid pIRES-Rb94 was successfully constructed. The stable transfected cell line pIRES-Rb94-A549 was transfected into A549 cells. The expression of Rb94 gene was highly expressed in this cell line. SphK1 expression was up-regulated and SphK2 expression was up-regulated. Conclusion The eukaryotic expression plasmid pIRES-Rb94 was successfully constructed and transfected into A549 cells. The expression of SphK1 was inhibited by Rb94 gene and the expression of SphK2 was enhanced.