猪δ冠状病毒核衣壳蛋白稳转细胞株的构建及其功能探究

doi:10.13560/j.cnki.biotech.bull.1985.2025-1397

生物技术通报 ›› 2026, Vol. 42 ›› Issue (10): 328-338.doi: 10.13560/j.cnki.biotech.bull.1985.2025-1397

• 研究报告 • 上一篇

猪δ冠状病毒核衣壳蛋白稳转细胞株的构建及其功能探究

杨泰然¹^,²^,³^,⁴, 郑鑫祺¹^,²^,³^,⁴, 胡超²^,³^,⁴^,⁵, 安婷婷²^,³^,⁴^,⁵, 文龙²^,³^,⁴^,⁵, 李有利²^,³^,⁴^,⁵, 陈峥宏²^,³^,⁵, 崔古贞²^,³^,⁵, 李博岩¹(), 田益明²^,³^,⁴^,⁵()

^1.贵州医科大学公共卫生与健康学院，贵阳 550000
^2.贵州医科大学基础医学院病原生物学系，贵阳 550000
^3.贵州省普通高等学校病原生物学特色重点实验室，贵阳 550000
^4.贵州医科大学?上海交通大学医学院病毒研究所，贵阳 550000
^5.贵州省微生物组与感染性疾病防控全省重点实验室，贵阳 550000

收稿日期:2025-12-22 出版日期:2026-10-26 发布日期:2026-10-15
通讯作者: 田益明tianym@gmc.edu.cn
李博岩boyan.li@gmc.edu.cn
基金资助:
国家自然科学基金项目(32260035);国家自然科学基金项目(32460042);贵州省科技计划项目（黔科合基础-ZK［2023］一般333），贵州省中央引导地方科技发展资金项目(黔科合中引地［2025］024)

Construction and Functional Characterization of Stable Cell Lines Expressing Nucleocapsid Protein of Porcine Deltacoronavirus

YANG Tai-ran¹^,²^,³^,⁴, ZHENG Xin-qi¹^,²^,³^,⁴, HU Chao²^,³^,⁴^,⁵, AN Ting-ting²^,³^,⁴^,⁵, WEN Long²^,³^,⁴^,⁵, LI You-li²^,³^,⁴^,⁵, CHEN Zheng-hong²^,³^,⁵, CUI Gu-zhen²^,³^,⁵, LI Bo-yan¹(), TIAN Yi-ming²^,³^,⁴^,⁵()

^1.School of Public Health, Guizhou Medical University, Guiyang 550000
^2.Department of Pathogen Biology, College of Basic Medicine, Guizhou Medical University, Guiyang 550000
^3.Guizhou Provincial Distinguished Key Laboratory of Pathogen Biology for Higher Education Institutions, Guiyang 550000
^4.Guizhou Medical University & Shanghai Jiao Tong University School of Medicine Joint Institute of Virology, Guiyang 550000
^5.Guizhou Provincial Key Laboratory of Microbiome and Infectious Disease Prevention and Control, Guiyang 550000

Received:2025-12-22 Published:2026-10-26 Online:2026-10-15

摘要/Abstract

摘要：

目的构建稳定表达猪δ冠状病毒（porcine deltacoronavirus, PDCoV）核衣壳蛋白（N）的单克隆细胞株，为解析其在病毒生命周期中调控宿主细胞微环境促进病毒增殖的致病机制提供细胞工具。方法基于系统发育分析选取3个不同谱系PDCoV代表株，构建N基因慢病毒真核表达质粒；利用慢病毒系统及极限稀释法，获得稳定表达N蛋白的LLC-PK单克隆细胞株；通过干扰素-β（IFN-β）启动子荧光素酶报告系统、实时荧光定量PCR、流式细胞术及病毒增殖实验，分析N蛋白稳定表达对I型干扰素（IFN-I）通路、宿主细胞周期及病毒增殖能力的影响。结果成功构建了30株稳定表达PDCoV N蛋白的单克隆LLC-PK稳转细胞系，其中包括北美谱系人源毒株HT/h0256来源的12株、中国谱系人源毒株HT/h0329来源的9株，以及东南亚谱系毒株THA/S5011来源的9株。功能探究结果显示，PDCoV N蛋白的稳定表达可显著抑制仙台病毒（SeV）诱导的IFN-β启动子活性及下游干扰素刺激基因（ISG56、ISG15和MX1）的激活，并且该蛋白能显著诱导LLC-PK细胞发生S期阻滞。此外，N蛋白的稳定表达还可显著促进病毒在宿主细胞中的增殖。结论成功构建了表达3个谱系PDCoV代表株N蛋白的LLC-PK单克隆稳转细胞株，其中N蛋白的稳定表达可显著抑制IFN-I通路、诱导宿主细胞S期阻滞，并促进病毒增殖，为深入解析N蛋白调控宿主细胞微环境的致病机制提供了重要细胞模型。

关键词: 猪δ冠状病毒, 核衣壳蛋白, 稳转细胞株, I型干扰素通路, S期阻滞, 病毒复制

Abstract:

Objective This study aims to establish monoclonal cell lines stably expressing the nucleocapsid (N) protein of porcine deltacoronavirus (PDCoV), which will provide a cellular tool to elucidate its pathogenic mechanism in modulating the host cellular microenvironment to enhance viral proliferation in the viral life cycle. Method Three representative PDCoV strains from different lineages were selected based on phylogenetic analysis, and eukaryotic lentiviral expression plasmids carrying the N gene were constructed. The lentiviral system combined with the limiting dilution method was then used to generate LLC-PK monoclonal cell lines stably expressing PDCoV N protein. Finally, the interferon-β (IFN-β) promoter luciferase reporter system, quantitative real-time PCR (RT-qPCR), flow cytometry, and viral proliferation assays were employed to analyze the effects of stable N protein expression on the type I interferon (IFN-I) pathway, host cell cycle, and viral proliferation ability. Result A total of 30 LLC-PK monoclonal cell lines stably expressing PDCoV N protein were successfully established, including 12 lines of the North American lineage (HT/h0256, a human-derived strain), 9 lines of the Chinese lineage (HT/h0329, a human-derived strain), and 9 lines of the Southeast Asian lineage (THA/S5011). Functional investigation revealed that the stable expression of PDCoV N protein significantly suppresses Sendai virus (SeV)-induced activation of the IFN-β promoter activity and the expression of downstream interferon-stimulated genes (ISG56, ISG15 and MX1), induces pronounced S-phase arrest in LLC-PK cells, and markedly enhances viral proliferation within host cells. Conclusion Stable LLC-PK monoclonal cell lines expressing the N protein of representative strains across three PDCoV lineages were successfully established. The sustained expression of N protein in these cell lines effectively antagonizes the IFN-I signaling pathway, induces host cell S-phase arrest, and enhances viral proliferation, thereby providing a critical cellular model for further dissection of the pathogenic mechanisms underlying N protein-mediated modulation of the host cellular microenvironment.

Key words: porcine deltacoronavirus, nucleocapsid protein, stable cell line, type I interferon pathway, S-phase arrest, virus replication

杨泰然, 郑鑫祺, 胡超, 安婷婷, 文龙, 李有利, 陈峥宏, 崔古贞, 李博岩, 田益明. 猪δ冠状病毒核衣壳蛋白稳转细胞株的构建及其功能探究[J]. 生物技术通报, 2026, 42(10): 328-338.

YANG Tai-ran, ZHENG Xin-qi, HU Chao, AN Ting-ting, WEN Long, LI You-li, CHEN Zheng-hong, CUI Gu-zhen, LI Bo-yan, TIAN Yi-ming. Construction and Functional Characterization of Stable Cell Lines Expressing Nucleocapsid Protein of Porcine Deltacoronavirus[J]. Biotechnology Bulletin, 2026, 42(10): 328-338.

图/表 5

图1 3个谱系PDCoV毒株N蛋白功能域及氨基酸水平差异分析A：基于194条PDCoV全基因组序列的系统发育谱系分型（红色标记为所选取的3个谱系PDCoV代表株）；B：PDCoV N蛋白结构域示意图（绿色：N端结构域；青色：C端结构域；黄色：SR富集区）；C：3个谱系PDCoV N蛋白氨基酸序列差异比对

Fig. 1 Functional domain and amino acid levels analysis of N protein from three PDCoV lineagesA: Phylogenetic genotyping based on 194 complete-genome sequences of PDCoV (red indicates representative strains of PDCoV from three lineages). B: Schematic diagram of PDCoV N protein domain (Green: NTD. Cyan: CTD. Yellow: SR-rich motif). C: Amino acid sequence alignment of N proteins from three PDCoV lineages

图2 3个谱系PDCoV N蛋白稳转细胞株的构建与鉴定A：间接免疫荧光法检测3个谱系PDCoV N蛋白在多克隆细胞株中的表达与定位（比例尺：50 μm）；B：Western blot法检测3个谱系单克隆稳转细胞株中N蛋白的表达水平（M：LLC-PK对照细胞）；C：间接免疫荧光检测3个谱系单克隆稳转细胞株中N蛋白的表达（比例尺：50 μm）；D：CCK-8法检测3个谱系稳转细胞株的细胞增殖活性，纵坐标为细胞活力，横坐标为检测时间点；*P<0.05、**P<0.01、***P<0.001、****P<0.000 1；无星号表示组间差异无统计学意义。下同

Fig. 2 Construction and identification of stable cell lines expressing the N protein from three PDCoV lineagesA: Indirect immunofluorescence assay for detecting the expression and localization of PDCoV N protein from three lineages in polyclonal cell lines (scale bar: 50 μm). B: Western blot analysis of N protein expression levels in monoclonal stable cell lines of three PDCoV lineages (M: LLC-PK control cells). C: Indirect immunofluorescence assay for detecting the expression of N protein in monoclonal stable cell lines of three PDCoV lineages (scale bar: 50 μm). D: CCK-8 assay for evaluating the proliferative activity of stable cell lines of three PDCoV lineages; the ordinate represents cell viability, and the abscissa represents detection time points. *P<0.05, **P<0.01, ***P<0.001, ****P<0.000 1. No asterisk indicates no statistically significant difference between groups. The same below

图3 PDCoV N蛋白抑制IFN-β活化及下游刺激基因激活A‒C：双荧光素酶报告系统检测3个谱系PDCoV N蛋白对SeV诱导的IFN-β启动子活化的影响（MOCK：空白对照）；D‒G：RT-qPCR检测3个谱系稳转细胞株中SeV诱导的IFN-β1（D）、ISG56（E）、ISG15（F）及MX1（G）基因mRNA的相对表达水平；H：荧光显微镜观察PDCoV N蛋白对VSV-GFP复制的影响（绿色荧光强度代表VSV复制水平；比例尺：100 μm）

Fig. 3 PDCoV N protein suppresses IFN-β activation and downstream stimulated gene expressionA‒C: Analysis of the effect of N proteins from three PDCoV lineages on SeV-induced IFN-β promoter activity using a dual-luciferase reporter assay system (MOCK: Mock control). D‒G: Detection of the relative mRNA expression levels of SeV-induced IFN-β1 (D), ISG56 (E), ISG15 (F) and MX1 (G) genes in stable cell lines of different PDCoV lineages by RT-qPCR. H: Fluorescence microscopy observation of the effect of PDCoV N protein on VSV-GFP replication (green fluorescence intensity indicates VSV replication level. scale bar: 100 μm)

图4 PDCoV N蛋白诱导LLC-PK细胞S期阻滞A：流式细胞术检测血清饥饿同步化处理后，不同时间点（0、12和24 h）野生型LLC-PK与PDCoV N蛋白稳转细胞株的细胞周期代表性图谱（蓝色：G0/G1期；棕色：S期；绿色：G2/M期）；B：不同时间点G0/G1期、S期及G2/M期细胞占比的统计结果

Fig. 4 PDCoV N protein induces S-phase arrest in LLC-PK cellsA: Representative cell cycle profiles of wild-type LLC-PK cells and PDCoV N protein stably transfected cell lines detected by flow cytometry at different time points (0, 12 and 24 h) after serum starvation synchronization (Blue: G0/G1 phase. Brown: S phase. Green: G2/M phase). B: Statistical results of the proportions of cells in G0/G1, S and G2/M phases at different time points

图5 PDCoV N蛋白促进PDCoV的复制及增殖A：qPCR绝对定量法检测3个谱系N蛋白稳转细胞株上清中PDCoV的一步生长曲线；B：基于S蛋白鼠单克隆抗体的间接免疫荧光法检测PDCoV的复制增殖情况（比例尺：50 μm）

Fig. 5 PDCoV N protein enhances PDCoV replication and proliferationA: Replication kinetics of PDCoV in supernatants of N protein stable cell lines from three lineages using absolute quantitative qPCR. B: Immunofluorescence detection of PDCoV proliferation in stable cell lines expressing the N protein using an anti-spike monoclonal antibody (scale bar: 50 μm)

参考文献 37

[1]	Cui J, Li F, Shi ZL. Origin and evolution of pathogenic coronaviruses [J]. Nat Rev Microbiol, 2019, 17(3): 181-192.
[2]	Ye ZW, Yuan SF, Yuen KS, et al. Zoonotic origins of human coronaviruses [J]. Int J Biol Sci, 2020, 16(10): 1686-1697.
[3]	Ruiz-Aravena M, McKee C, Gamble A, et al. Ecology, evolution and spillover of coronaviruses from bats [J]. Nat Rev Microbiol, 2022, 20(5): 299-314.
[4]	Miranda C, Silva V, Igrejas G, et al. Genomic evolution of the human and animal coronavirus diseases [J]. Mol Biol Rep, 2021, 48(9): 6645-6653.
[5]	Zhang JQ. Porcine deltacoronavirus: Overview of infection dynamics, diagnostic methods, prevalence and genetic evolution [J]. Virus Res, 2016, 226: 71-84.
[6]	Wang QH, Vlasova AN, Kenney SP, et al. Emerging and re-emerging coronaviruses in pigs [J]. Curr Opin Virol, 2019, 34: 39-49.
[7]	Shan XT, Li R, Ma X, et al. Epidemiology, pathogenesis, immune evasion mechanism and vaccine development of porcine Deltacoronavirus [J]. Funct Integr Genomics, 2024, 24(3): 79.
[8]	He WT, Ji X, He W, et al. Genomic epidemiology, evolution, and transmission dynamics of porcine deltacoronavirus [J]. Mol Biol Evol, 2020, 37(9): 2641-2654.
[9]	Tang P, Cui EH, Song YH, et al. Porcine deltacoronavirus and its prevalence in China: a review of epidemiology, evolution, and vaccine development [J]. Arch Virol, 2021, 166(11): 2975-2988.
[10]	Bahoussi AN, Wang PH, Shah PT, et al. Evolutionary plasticity of zoonotic porcine Deltacoronavirus (PDCoV): genetic characteristics and geographic distribution [J]. BMC Vet Res, 2022, 18: 444.
[11]	Jung K, Hu H, Saif LJ. Porcine deltacoronavirus infection: Etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis [J]. Virus Res, 2016, 226: 50-59.
[12]	Vlasova AN, Kenney SP, Jung K, et al. Deltacoronavirus evolution and transmission: current scenario and evolutionary perspectives [J]. Front Vet Sci, 2021, 7: 626785.
[13]	Tian YY, Sun JQ, Hou XH, et al. Cross-species recognition of two porcine coronaviruses to their cellular receptor aminopeptidase N of dogs and seven other species [J]. PLoS Pathog, 2025, 21(1): e1012836.
[14]	Lednicky JA, Tagliamonte MS, White SK, et al. Independent infections of porcine deltacoronavirus among Haitian children [J]. Nature, 2021, 600(7887): 133-137.
[15]	Su S, Wong G, Shi WF, et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses [J]. Trends Microbiol, 2016, 24(6): 490-502.
[16]	Ye QZ, West AMV, Silletti S, et al. Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein [J]. Protein Sci, 2020, 29(9): 1890-1901.
[17]	Chen H, Cui Y, Han XL, et al. Liquid-liquid phase separation by SARS-CoV-2 nucleocapsid protein and RNA [J]. Cell Res, 2020, 30(12): 1143-1145.
[18]	Cong YY, Ulasli M, Schepers H, et al. Nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronaviral life cycle [J]. J Virol, 2020, 94(4): e01925-e01919.
[19]	Mu JF, Fang YH, Yang Q, et al. SARS-CoV-2 N protein antagonizes type I interferon signaling by suppressing phosphorylation and nuclear translocation of STAT1 and STAT2 [J]. Cell Discov, 2020, 6: 65.
[20]	Pan P, Shen MM, Yu ZY, et al. SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation [J]. Nat Commun, 2021, 12: 4664.
[21]	Hu H, Jung K, Vlasova AN, et al. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States [J]. J Clin Microbiol, 2015, 53(5): 1537-1548.
[22]	Ji LK, Li SS, Zhu WX, et al. Porcine deltacoronavirus nucleocapsid protein suppressed IFN-β production by interfering porcine RIG-I dsRNA-binding and K63-linked polyubiquitination [J]. Front Immunol, 2019, 10: 1024.
[23]	Ji LK, Wang N, Ma JJ, et al. Porcine deltacoronavirus nucleocapsid protein species-specifically suppressed IRF7-induced type I interferon production via ubiquitin-proteasomal degradation pathway [J]. Vet Microbiol, 2020, 250: 108853.
[24]	Hu YT, Hao CL, Wang DH, et al. Porcine deltacoronavirus nucleocapsid protein antagonizes JAK-STAT signaling pathway by targeting STAT1 through KPNA2 degradation [J]. J Virol, 2024, 98(7): e00334-e00324.
[25]	Ji LK, Zhou LY, Wang Y, et al. FBXW8 suppresses PDCoV proliferation via the NPD52-dependent autophagic degradation of a viral nucleocapsid protein [J]. Front Immunol, 2024, 15: 1457255.
[26]	Yang XY, Kong N, Qin WZ, et al. PGAM5 degrades PDCoV N protein and activates type I interferon to antagonize viral replication [J]. J Virol, 2023, 97(11): e01470-e01423.
[27]	Wu XH, Lan SJ, Wang Y, et al. LDHB suppresses the PDCoV proliferation by targeting viral nucleocapsid protein for autophagic degradation [J]. Microbiol Spectr, 2025, 13(5): e02787-e02724.
[28]	Huson DH, Bryant D. Application of phylogenetic networks in evolutionary studies [J]. Mol Biol Evol, 2006, 23(2): 254-267.
[29]	Abramson J, Adler J, Dunger J, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3 [J]. Nature, 2024, 630(8016): 493-500.
[30]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method [J]. Methods, 2001, 25(4): 402-408.
[31]	El-Maradny YA, Badawy MA, Mohamed KI, et al. Unraveling the role of the nucleocapsid protein in SARS-CoV-2 pathogenesis: From viral life cycle to vaccine development [J]. Int J Biol Macromol, 2024, 279: 135201.
[32]	Zhao YH, Sui LY, Wu P, et al. A dual-role of SARS-CoV-2 nucleocapsid protein in regulating innate immune response [J]. Sig Transduct Target Ther, 2021, 6: 331.
[33]	Liu JM, Liu MY, Yu XY, et al. SARS-CoV-2 nucleocapsid protein delays cell cycle in S-phase [J]. Int J Med Microbiol, 2025, 320: 151671.
[34]	Costa-Mattioli M, Walter P. The integrated stress response: From mechanism to disease [J]. Science, 2020, 368(6489): eaat5314.
[35]	Luftig MA. Viruses and the DNA damage response: activation and antagonism [J]. Annu Rev Virol, 2014, 1: 605-625.
[36]	Bagga S, Bouchard MJ. Cell cycle regulation during viral infection [M]//Noguchi E, Gadaleta MC. Cell Cycle Control: Mechanisms and Protocols. New York: Springer, 2014: 165-227.
[37]	Bezerra PR, Almeida FCL. Structural basis for the participation of the SARS-CoV-2 nucleocapsid protein in the template switch mechanism and genomic RNA reorganization [J]. J Biol Chem, 2024, 300(11): 107834.

猪δ冠状病毒核衣壳蛋白稳转细胞株的构建及其功能探究

Construction and Functional Characterization of Stable Cell Lines Expressing Nucleocapsid Protein of Porcine Deltacoronavirus

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 37

相关文章 11

编辑推荐

Metrics

本文评价

[1]	任柱平, 杨泰然, 雷元三, 金留飞, 崔古贞, 田益明, 陈峥宏. 具备高效CRISPR协同激活活性的HIEC6-dCas9-SAM稳转细胞株构建[J]. 生物技术通报, 2025, 41(5): 52-61.
[2]	金浙东, 李惠宜, 包汶鑫, 崔彩霞, 袁运生. 表达猴痘病毒表面蛋白 A35R和E8L稳转细胞株的构建与应用[J]. 生物技术通报, 2024, 40(10): 315-322.
[3]	张西西, 张怡青, 李玉林, 韩笑, 王国强, 王晓军, 王旭东, 王云龙. 新型冠状病毒（SARS-CoV-2）N蛋白C端重组蛋白的原核表达、纯化及应用[J]. 生物技术通报, 2021, 37(5): 92-97.
[4]	陈汭, 付嘉钰, 刘浩宇, 李成, 赵玉佳, 胡靖飞, 瞿欢, 曹三杰, 文心田, 文翼平, 赵勤, 伍锐, 黄小波. 猪δ冠状病毒（PDCoV）N蛋白的原核表达及多克隆抗体制备[J]. 生物技术通报, 2020, 36(8): 104-110.
[5]	张荷;代淑燕;毛君婷;王开功;周碧君;文明;. DEV-NP基因酵母双杂交诱饵载体的构建及自激活检测[J]. , 2011, 0(03): 196-199.
[6]	秦春圃;. PRRSV血液抗体间接ELISA检测方法的建立[J]. , 2008, 0(01): 132-132.
[7]	朱中泽;张志芳;何家禄. 杆状病毒复制相关基因突变株研究进展[J]. , 2002, 0(02): 21-24.
[8]	田桂英. 植物抗病毒的基因工程[J]. , 1995, 0(05): 3-6.
[9]	Karen Beth G. 利用具病原诱导抗性的转基因植物防治植物病毒病[J]. , 1995, 0(03): 1-4.
[10]	郭殿瑞;. 研究者们在探求“反义”(Anti-Sense)技术在医学和农业上的应用[J]. , 1989, 0(07): 7-7.
[11]	李思经;. 将病毒随体RNA插入植物可减轻病毒病[J]. , 1988, 0(07): 10-11.