鸭维甲酸诱导基因Ⅰ克隆及其结构域功能分析

摘要

[目的]克隆鸭维甲酸诱导基因Ⅰ (retinoic acid inducible gene Ⅰ,RIG-Ⅰ),分析其不同结构域的功能.[方法]根据GenBank上公布的鸭RIG-Ⅰ序列设计引物,利用RT-PCR克隆鸭RIG-Ⅰ基因CDS (coding sequence)区,根据保守结构域预测结果,构建携带6*his组氨酸标签的不同结构域缺失突变体的真核表载体(RIG-Ⅰ-Full、RIG-Ⅰ-N和RIG-Ⅰ-C),转染鸡胚成纤维细胞DF1,经RT-PCR、间接免疫荧光方法鉴定重组质粒在细胞中转录与表达;同时,利用RT-qPCR检测RLR抗病毒信号通路中的IFN-β、Mx1和PKR等下游基因的表达变化.[结果]鸭RIG-Ⅰ基因CDS区全长2802 bp,共编码933个氨基酸；不同结构域缺失突变体的真核表载体转染DF1细胞后,重组蛋白均在DF1细胞中表达；RT-qPCR结果显示,N端能显著激活RLR通路上IFN-β、Mx1及PKR基因的表达上调.[结论]duRIG-Ⅰ及不同区段均能在DF1细胞中表达,其中N端在调节RLR抗病毒信号通路下游基因的表达过程中发挥了重要作用.%[Objective] Duck RIG-Ⅰ (duRIG-Ⅰ) gene was cloned and the functions of its different domains were analyzed preliminarily.[Method] The CDS of duRIG-Ⅰ gene was cloned on the basis of the sequence submitted to GenBank with RT-PCR and was analyzed by bioinformatics.The eukaryotic expression vectors of N-terminal,C-terminal and whole-length of duRIG-Ⅰ gene with 6*his tags were constructed to transfect DF1,and then the transcription and expression of the three recombinant plasmids in cells were detected via RT-PCR and indirect immunofluorescent assay,respectively.Meanwhile,the expressions of chicken IFN-β,Mxl and PKR mRNA were detected by real-time PCR.[Result] The whole-length of duRIG-Ⅰ CDS was 2802 bp encoding 933 amino acids.All the recombinant protein of duRIG-Ⅰ could express normally in DF1.The results of RT-qPCR indicated that CARDs significantly up-regulated the mRNA level of IFN-β,Mx1 and PKR genes.[Conclusion] The various domain fragments of duRIG-Ⅰ express normally in DF1.The N-terminal of duRIG-Ⅰ plays a vital role in regulating the expression of downstream genes of RLR antiviral signal pathway.