IFN-λR1与TRAF6的相互作用及对NF-κB与ISRE活性的影响  

Interaction between IFN- λR1 and TRAF6 Down- regulates the NF- κB and ISRE Signal Pathway

作　　者：杨霞[1] 张俊文[1] 黄秉仁[1] 陈虹[1] 

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院生物化学与分子生物学系,医学分子生物学国家重点实验室

出　　处：《医学研究杂志》2014年第10期53-58,共6页Journal of Medical Research

摘　　要：目的构建含有信号肽的全长IFN-λR1质粒,研究在配体IFN-λ1存在的情况下IFN-λR1与TRAF6的相互作用及对NF-κB与ISRE活性的影响。方法利用多次PCR将信号肽编码序列引入IFN-λR1序列中,构建表达载体,用免疫荧光及膜蛋白分离实验鉴定IFN-λR1的表达定位。用免疫共沉淀的方法研究IFN-λ1刺激下IFN-λR1与TRAF6间的相互作用。用双荧光素酶报告基因实验研究IFN-λR1与TRAF6相互作用后对下游NF-κB与ISRE活性的影响,采用实时定量PCR的方法检测此种作用对OAS1与Mx1基因表达的影响。结果成功构建了含信号肽的全长IFN-λR1表达载体,细胞内表达的IFN-λR1可正确定位于细胞膜上。细胞共转染IFN-λR1和TRAF6,在免疫沉淀物中可以检测到两个蛋白同时存在。细胞内共表达的IFN-λR1能够抑制TRAF6诱导的NF-λB的激活,而共表达的TRAF6可以抑制IFN-λR1下游ISRE的活性,并下调OAS1与Mx1基因的表达。结论证实了在IFN-λ1存在下IFN-λR1与TRAF6的相互作用及二者相互作用对各自细胞信号转导通路的影响,为TRAF6作为IFN-λ受体信号通路中的相互作用蛋白提供了新的实验依据。Objective To construct an expression vector for the full length IFN-λR1 containing a signal peptide in order to investigate the interaction between IFN-λR1 and TRAF6 under the stimulation of IFN-λ1,and to observe how the interaction between IFN-λR1 and TRAF6 influence the activities of NF-κB and ISRE.Methods DNA cloning technique was employed to construct expression vector for the full length IFN-λR1 containing a signal peptide.Immunofluorescence assay and membrane protein separation were used to confirm the expression of IFN-λR1.Co-transfection and immunoprecipitation were used to study the interaction between IFN-λR1 and TRAF6 spurred by IFN-λ1.Dual Luciferase Reporter Gene Assay was also used to research the NF-κB and ISRE activities.OAS1 and Mx1 mRNA levels were detected by quantitative real time PCR.Results The full-length expression vector of IFN-λR1 with signal peptide was successfully constructed which can be expressed on the cell membrane correctly.The interaction between IFN-λR1 and TRAF6 at presence of IFN-λ1 was confirmed by co-transfection and immunoprecipitation.IFN-λR1 can repress the activity of NF-κB induced by TRAF6 overexpression in HEK293T and Hela cells.Whereas,TRAF6 can inhibite IFN-λR1 downstream signal activity of IFN-λ1 stimulation and down-regulate the expression of OAS1 and Mx1.Conclusion These results provide the further evidences for the IFN-λR1 interacting with TRAF6,and how this interaction effects on these two signal transduction pathways.They also provide a new experimental proof of TRAF6 as a binding protein in the signal pathway of IFN-λR1.

关 键 词：IFN-λR1 TRAF6 NF-ΚB ISRE 相互作用 

分 类 号：R3[医药卫生—基础医学]