【摘要】 目的:构建人白细胞介素18受体IL-18Rα,β真核表达载体pcDNA3.1-zeocin/IL-18RαpcDNA3.0/IL-18Rβ,并将pcDNA3.0/IL-18Rβ稳定转染293细胞,构建IL-18Rβ稳定表达细胞株,用于建立IL-18信号转导的体外细胞模型。方法:实验于2006-06/2007-05在北京大学医学部免疫系实验室完成。扩增并克隆人IL-18Rα和IL-18RβcDNA,构建真核表达载体pcDNA3.1-zeocin/IL-18Rα,pcDNA3.0/IL-18Rβ。将pcDNA3.0/IL-18Rβ转染293细胞,经G418筛选建立起稳定表达IL-18Rβ的细胞株,Western-blot方法检测稳定细胞株IL-18Rβ的表达,挑选表达较高的细胞转染IL-18Rα,通过NF-κB依赖的Luciferase检测IL-18信号转导通路的建立。结果:成功构建了IL-18Rα和IL-18Rβ的真核表达载体,IL-18Rβ的基因稳定转染到了293细胞中并获得了表达,通过瞬时转染IL-18Rα真核表达载体,在293细胞中建立了IL-18信号转导的体外细胞模型。结论:成功建立起了IL-18诱导NF-κB活化的体外细胞模型,为进一步研究IL-18信号转导途径奠定了基础。更多还原

【Abstract】 AIM:To construct human interleukin-18 receptor α(IL-18Rα) and interleukin-18 receptor β(IL-18Rβ) eukaryotic expression vectors named pcDNA3.1-zeocin/IL-18Rα and pcDNA3.0/IL-18Rβ respectively,and then stably transfect pcDNA3.0/IL-18Rβ into 293 cells in order to establish the cell line stably expressing IL-18Rβ and cell model which responds IL-18 signal transduction in vitro. METHODS:The experiment was performed at the laboratory of Department of Immunology,Peking University Health Science Center from June 2006 to May 2007. DNA encoding IL-18Rα or IL-18Rβ was amplified and cloned into pcDNA3.1-zeocin or pcDNA3.0 vector,respectively. The recombinant of pcDNA3.0/IL-18Rβ was transfectd into 293 cells. After screening culture by G418,stable transfected cell line was established and the expression of IL-18Rβ was identified by Western-blot assay. The stable cell line with the combinant eukaryotic expression vector pcDNA3.1-zeocin /IL-18Rα to establish the IL-18 signaling pathway,which was detected by NF-κB-dependent Luciferase. RESULTS:The eukaryotic expression vectors of IL-18Rα and IL-18Rβ were constructed successfully. The gene of IL-18Rβ stably expressed in 293 cells and cell model of IL-18 signaling transduction in vitro could be established in these stable 293 cell lines. CONCLUSION:The establishment of NF-κB-activated cell model in vitro which responds to IL-18 can provide solid foundation for further experimental studies on IL-18 signaling transduction pathway.更多还原