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2 802 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,Mx1 and PKR mRNA were detected by real-time PCR.【Result】The whole-length of duRIG-I CDS was 2 802 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.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第10期2094-2102,共9页
Scientia Agricultura Sinica
基金
现代农业产业技术体系建设专项(CARS-43-3)
江苏高校优势学科建设工程资助项目(苏政办发[2011]137号)
