鸭干扰素因子7基因克隆及组织表达特异性分析

doi:10.16431/j.cnki.1671-7236.2023.07.002

摘要/Abstract

摘要： 【目的】构建樱桃谷鸭干扰素调节因子7（interferon regulatory factor 7，IRF7）基因CDS区序列的真核表达载体，分析IRF7蛋白结构和生物特性，检测不同组织内IRF7基因的表达水平，为进一步对鸭天然免疫系统开展研究奠定基础。【方法】利用RT-PCR扩增樱桃谷鸭IRF7基因的CDS区，构建真核表达质粒，利用Western blotting检测蛋白表达。使用荧光显微镜观察Poly （I：C）对IRF7蛋白核移位的影响。对IRF7基因序列进行核苷酸相似性比对，构建系统进化树，并通过生物信息学方法在线分析预测IRF7蛋白的分子和结构特征。实时荧光定量PCR检测不同组织中IRF7基因的表达特异性。【结果】成功克隆樱桃谷鸭IRF7基因CDS区，长1 536 bp，共编码512个氨基酸，通过对重组质粒的真核表达，提取细胞总蛋白，Western blotting检测蛋白表达，结果显示重组蛋白大小约为110 ku。Poly （I：C）处理鸭胚成纤维细胞促使pCMV-C-EGFP-IRF7的核内定位增加。核苷酸相似性结果显示，樱桃谷鸭与绿头鸭和鸡的IRF7基因核苷酸序列相似性分别为99.1%和80.8%，与非洲爪蛙的相似性仅为41.8%。系统进化树结果显示，樱桃谷鸭与绿头鸭和鸡亲缘关系最近，与非洲爪蛙亲缘关系最远。IRF7蛋白表现为结构不稳定的疏水性蛋白，主要由α-螺旋、β-折叠和无规则卷曲构成。IRF7基因在各个组织内均有表达，在胆囊中表达量最高，在肺脏中表达量最低。【结论】樱桃谷鸭IRF7基因的CDS区序列大小为1 536 bp，编码512个氨基酸，IRF7蛋白为结构不稳定的疏水性蛋白，Poly （I：C）刺激可引起IRF7发生核移位，该基因在不同组织表达差异较大。

关键词: 干扰素调节因子7(IRF7); 蛋白结构; 亚细胞定位

Abstract: 【Objective】 The purpose of this study was to obtain the eukaryotic expression vector of CDS of interferon regulatory factor 7 (IRF7) gene in Cherry Valley ducks, to study the structure and biological characteristics of IRF7 protein, and to understand the expression level of IRF7 gene in different tissues, so as to lay a foundation for further study of duck innate immune system.【Method】 The CDS of IRF7 gene in Cherry Valley ducks was amplified by RT-PCR, and the eukaryotic expression plasmid was constructed.The protein expression was detected by Western blotting.The effect of Poly(I:C) on the nuclear translocation of IRF7 protein was observed by fluorescence microscope.The nucleotide similarity alignment was performed on the IRF7 gene sequence, and the phylogenetic tree was constructed, and the molecular and structural characteristics of IRF7 protein were analyzed and predicted online by bioinformatics method.Real-time quantitative PCR was used to detect the expression specificity of IRF7 gene in different tissues.【Result】 The CDS of IRF7 gene in Cherry Valley ducks was 1 536 bp and encoded 512 amino acids.Through the eukaryotic expression of the recombinant plasmid, the total protein was extracted and the protein expression was detected by Western blotting.The size of the recombinant protein was about 110 ku.The treatment of duck embryo fibroblasts with Poly (I:C) could cause in the localization of pCMV-C-EGFP-IRF7 in the nucleus.The results of nucleotide similarity showed that the nucleotide sequence similarity of the IRF7 gene between Cherry Valley ducks, Anas platyrhynchos and Gallus gallus was 99.1% and 80.8%, respectively, while the similarity with Xenopus laevis was only 41.8%.The phylogenetic tree results showed that Cherry Valley ducks had the closest genetic relationship with Anas platyrhynchos and Gallus gallus, and the most distant genetic relationship with Xenopus laevis.IRF7 protein was a structurally unstable hydrophobic protein, mainly composed of alpha helix, beta turn and random coil.IRF7 gene was expressed in various tissues, with the highest expression in gallbladder and the lowest in lung.【Conclusion】 The size of the CDS region of IRF7 gene in Cherry Valley ducks was 1 536 bp, encoding 512 amino acids.The IRF7 protein was a structurally unstable hydrophobic protein, and Poly (I:C) stimulation could cause nuclear translocation of IRF7.The expression of this gene varies greatly in different tissues.

Key words: interferon regulatory factor 7(IRF7); protein structure; subcellular localization

中图分类号:

S834

邓志超, 章程, 罗沛, 詹晓芝, 江丹莉, 阳佑天, 刘文俊. 鸭干扰素因子7基因克隆及组织表达特异性分析[J]. 中国畜牧兽医, 2023, 50(7): 2606-2616.

DENG Zhichao, ZHANG Cheng, LUO Pei, ZHAN Xiaozhi, JIANG Danli, YANG Youtian, LIU Wenjun. Cloning and Tissue Expression Specificity Analysis of Interferon Regulatory Factor 7 Gene in Ducks[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2606-2616.

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