与传染性支气管炎病毒核衣壳蛋白互作的宿主RNA结合蛋白的鉴定

doi:10.3969/j.issn.1004-1524.20250304

浙江农业学报 ›› 2026, Vol. 38 ›› Issue (4): 632-642.DOI: 10.3969/j.issn.1004-1524.20250304

与传染性支气管炎病毒核衣壳蛋白互作的宿主RNA结合蛋白的鉴定

代雪艳(), 肖鹏, 符雪珍, 胡洁, 常宇昕, 赵伟, 廖敏()

浙江大学动物科学学院, 农业农村部动物病毒学重点实验室, 浙江杭州 310058

收稿日期:2025-04-15 出版日期:2026-04-25 发布日期:2026-05-08
作者简介:*廖敏,E-mail: liaomin4545@zju.edu.cn
代雪艳,主要从事分子病毒学研究。E-mail: 3226410640@qq.com
通讯作者: 廖敏
基金资助:
浙江省基础公益计划项目(LGN22C18003);浙江省“三农九方”科技协作项目(2024SNJF046)

Identification of host RNA-binding proteins interacting with infectious bronchitis virus N protein

DAI Xueyan(), XIAO Peng, FU Xuezhen, HU Jie, CHANG Yuxin, ZHAO Wei, LIAO Min()

Key Laboratory of Animal Virology of Ministry of Agriculture and Rural Affairs, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

Received:2025-04-15 Published:2026-04-25 Online:2026-05-08
Contact: LIAO Min

摘要/Abstract

摘要：

筛选与传染性支气管炎病毒(infectious bronchitis virus,IBV)N蛋白相互作用的宿主RNA结合蛋白,为揭示病毒与宿主互作机制提供基础。本研究以制备的抗N蛋白单克隆抗体4H4为工具,采用免疫共沉淀(co-immunoprecipitation,Co-IP)联合质谱技术鉴定与N蛋白相互作用的宿主蛋白,对差异表达蛋白进行生物信息学分析,并利用Co-IP实验验证N蛋白与候选RNA结合蛋白的相互作用。结果显示,共鉴定出847个差异表达蛋白,主要参与转录、翻译与蛋白质修饰、蛋白质-RNA复合体组装、信号转导与能量代谢等过程。Co-IP实验结果表明,N蛋白与宿主RNA结合蛋白TARDBP、hnRNPK、FUBP3、hnRNPA1之间存在相互作用。本研究筛选到与IBV N蛋白相互作用的宿主蛋白,这些蛋白参与病毒复制周期的多个关键环节,N蛋白与RNA结合蛋白的相互作用为揭示宿主蛋白在病毒感染中的具体机制提供了新的研究方向。

关键词: 传染性支气管炎病毒, N蛋白, RNA结合蛋白, 病毒-宿主互作, 免疫共沉淀, 单克隆抗体

Abstract:

To screen host RNA-binding proteins interacting with the infectious bronchitis virus (IBV) N protein, providing a basis for elucidating the virus-host interaction mechanism, co-immunoprecipitation (Co-IP) combined with mass spectrometry was employed to identify host proteins interacting with the N protein with the monoclonal antibody 4H4 against IBV N protein. Differentially expressed proteins were subjected to bioinformatics analysis, and Co-IP experiments were conducted to validate the interactions between the N protein and candidate RNA-binding proteins. The results showed that a total of 847 differentially expressed proteins were identified, primarily involved in processes such as transcription, translation and protein modification, protein-RNA complex assembly, signal transduction, and energy metabolism. Co-IP experimental results indicated that interactions exist between the N protein and the host RNA-binding proteins TARDBP, hnRNPK, FUBP3, and hnRNPA1. This study identified host proteins interacting with the IBV N protein, which are involved in multiple key stages of the viral replication cycle. The interactions between the N protein and RNA-binding proteins provide new research directions for elucidating the specific mechanisms of host proteins in viral infection.

Key words: infectious bronchitis virus, N protein, RNA-binding protein, virus-host interaction, co-immunoprecipitation, monoclonal antibody

中图分类号:

S855.3

代雪艳, 肖鹏, 符雪珍, 胡洁, 常宇昕, 赵伟, 廖敏. 与传染性支气管炎病毒核衣壳蛋白互作的宿主RNA结合蛋白的鉴定[J]. 浙江农业学报, 2026, 38(4): 632-642.

DAI Xueyan, XIAO Peng, FU Xuezhen, HU Jie, CHANG Yuxin, ZHAO Wei, LIAO Min. Identification of host RNA-binding proteins interacting with infectious bronchitis virus N protein[J]. Acta Agriculturae Zhejiangensis, 2026, 38(4): 632-642.

图/表 10

表1 真核表达质粒的引物信息

Table 1 Primer information for eukaryotic expression plasmids

基因名称 Gene name	正向引物序列(5'→3') Forward primer sequence (5'→3')	反向引物序列(5'→3') Reverse primer sequence (5'→3')
TARDBP	GCCCGAATTCGGTCGAATGTCGGAGTACATCCGGGT	GGATCCCCGCGGCCGCGCTACATTCCCCAGCCTGACG
hnRNPK	GCCCGAATTCGGTCGACCATGGAGACTGAACAACAGGAGGA	GGATCCCCGCGGCCGCGTTAGAATCCTTCAACATCTGCATACT
hnRNPA1	GCCCGAATTCGGTCGACCATGGCCAAGTCGGAGTCTCCAAAGGA	GGATCCCCGCGGCCGCGTCAGAACCTCCTCCCTCCGCCGTA
FUBP3	GCCCGAATTCGGTCGAATGGCGGAGCTGGCGCA	GGATCCCCGCGGCCGCGCTACTGCTCCTGGCTGTGGGC

图1 IBV YC181031抗原浓缩样品的鉴定 A,IBV浓缩样品的RT-PCR鉴定结果;B,IBV的Western blot检测结果。M,蛋白质相对分子质量标准物;1,浓缩前样品;2,浓缩后样品;3,上清液样品。

Fig.1 Identification of concentrated IBV YC181031 antigen A, RT-PCR identification results of concentrated IBV samples; B, Western blot detection results of IBV. M, Protein marker; 1, Sample before concentration; 2, Sample after concentration; 3, Supernatant sample.

图2 N蛋白单克隆抗体的反应性 A,ELISA鉴定;B,Western blot鉴定;C,IFA鉴定。M,蛋白质相对分子质量标准物;1,YC181031病毒浓缩液;2,纯化的原核表达N蛋白。

Fig.2 Reactivity of the N protein monoclonal antibodies A, Identification by ELISA; B, Identification by Western blot; C, Identification by IFA. M, Protein marker; 1, Concentrated solution of YC181031 virus; 2, Purified prokaryotically expressed N protein.

图3 感染IBV后N蛋白表达量的变化

Fig.3 Changes of N protein expression levels after IBV infection

图4 感染细胞中IBV互作蛋白的鉴定 A,Western blot检测N蛋白与protein A/G磁珠的结合;B,银染鉴定差异条带(红色箭头指示差异蛋白);M,蛋白质相对分子质量标准物。

Fig.4 Identification of IBV-interacting proteins in infected cells A, Western blot detection of the binding between the N protein and protein A/G magnetic beads; B, Silver staining identification of differential bands (red arrows indicate differential proteins); M, Protein marker.

图5 与N蛋白互作的宿主蛋白的GO功能分析 BP,生物学过程;CC,细胞组分;MF,分子功能。

Fig.5 GO functional analysis of host cell proteins interacting with the N protein BP, Biological process; CC, Cellular component; MF, Molecular function.

图6 与N蛋白互作的宿主细胞蛋白的COG功能分类

Fig.6 COG functional classification of host cell proteins interacting with the N protein

图7 与N蛋白互作的宿主细胞蛋白的KEGG通路富集分析

Fig.7 KEGG metabolic pathway annotation of host cell proteins interacting with N protein

图8 与N蛋白互作的宿主细胞蛋白的互作图谱

Fig.8 Interaction map of host cell proteins interacting with N protein

图9 免疫共沉淀验证IBV N蛋白与宿主细胞RNA结合蛋白的相互作用 A,目的片段的PCR扩增;M,DNA marker;1,TARDBP;2,hnRNPK;3,FUBP3;4,hnRNPA1。B,免疫共沉淀鉴定蛋白互作。

Fig.9 Co-IP validation of the interaction between IBV N protein and host cell RBP A, PCR amplification of target fragments; M, DNA marker; 1, TARDBP; 2, hnRNPK; 3, FUBP3; 4. hnRNPA1. B. Co-immunoprecipitation for protein interaction identification.

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与传染性支气管炎病毒核衣壳蛋白互作的宿主RNA结合蛋白的鉴定

Identification of host RNA-binding proteins interacting with infectious bronchitis virus N protein

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