Construction of eukaryotic expression plasmid carrying interleukin-18 receptor alpha,beta and establishment of cell line stably expressing interleukin-18 receptor beta / 中国组织工程研究

ABSTRACT

AIM:To construct human interleukin-18 receptor ?(IL-18R?) and interleukin-18 receptor ?(IL-18R?) eukaryotic expression vectors named pcDNA3.1-zeocin/IL-18R? and pcDNA3.0/IL-18R? respectively,and then stably transfect pcDNA3.0/IL-18R? into 293 cells in order to establish the cell line stably expressing IL-18R? 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-18R? or IL-18R? was amplified and cloned into pcDNA3.1-zeocin or pcDNA3.0 vector,respectively. The recombinant of pcDNA3.0/IL-18R? was transfectd into 293 cells. After screening culture by G418,stable transfected cell line was established and the expression of IL-18R? was identified by Western-blot assay. The stable cell line with the combinant eukaryotic expression vector pcDNA3.1-zeocin /IL-18R? to establish the IL-18 signaling pathway,which was detected by NF-?B-dependent Luciferase. RESULTS:The eukaryotic expression vectors of IL-18R? and IL-18R? were constructed successfully. The gene of IL-18R? 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-?B-activated cell model in vitro which responds to IL-18 can provide solid foundation for further experimental studies on IL-18 signaling transduction pathway.