鸭维甲酸诱导基因I克隆及其结构域功能分析

doi:10.3864/j.issn.0578-1752.2013.10.015

摘要/Abstract

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。

关键词: 鸭 , RIG-I基因 , 克隆 , RLR通路

Abstract: 【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.

Key words: duck , RIG-I gene , cloning , RLR signal pathway

陈阳, 黄正洋, 张扬, 李欣钰, 甄霆, 吴宁昭, 徐琪, 陈国宏. 鸭维甲酸诱导基因I克隆及其结构域功能分析[J]. 中国农业科学, 2013, 46(10): 2094-2102.

CHEN Yang, HUANG Zheng-Yang, ZHANG Yang, LI Xin-Yu, ZHEN Ting, WU Ning-Zhao, XU Qi, CHEN Guo-Hong. Molecular Cloning and Preliminary Functional Analysis of Domains of Duck Retinoic Acid Inducible Gene I[J]. Scientia Agricultura Sinica, 2013, 46(10): 2094-2102.

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