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Article(id=1259888462761702237, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1259888457367806489, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250874, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1763827200000, receivedDateStr=2025-11-23, revisedDate=null, revisedDateStr=null, acceptedDate=1768752000000, acceptedDateStr=2026-01-19, onlineDate=1778310417117, onlineDateStr=2026-05-09, pubDate=1777824000000, pubDateStr=2026-05-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1778310417117, onlineIssueDateStr=2026-05-09, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1778310417117, creator=13701087609, updateTime=1778310417117, updator=13701087609, issue=Issue{id=1259888457367806489, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='5', pageStart='2031', pageEnd='2556', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=0, articleOrder=1, issueType=-1, specialIssue=null, createTime=1778310415832, creator=13701087609, updateTime=1778320153326, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1259929299465921482, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1259888457367806489, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1259929299465921483, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1259888457367806489, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2384, endPage=2392, ext={EN=ArticleExt(id=1259888464842077028, articleId=1259888462761702237, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Construction of cell lines with ring finger protein 31 gene knockout and evaluation of its impact on the replication of foot-and-mouth disease virus, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the function of ring finger protein 31 (RNF31) in the replication of foot-and-mouth disease virus (FMDV) and to provide a theoretical basis for the research on the molecular mechanism by which the host protein RNF31 regulates FMDV replication. Methods CRISPR/Cas9 gene editing was employed to design two sgRNA sequences in the exon segment of RNF31, and recombinant plasmids were constructed by ligation with the pX459-puro vector. The recombinant plasmids pX459-RNF31-sgRNA were transfected into PK-15 cells, followed by screening under the action of puromycin to obtain the cell lines with RNF31 gene knockout. The effect of RNF31 gene knockout on FMDV replication was detected by Western blotting, RT-qPCR, and TCID50 methods. Results Compared with wild-type cells, the knockout of RNF31 significantly increased the protein level, mRNA level, and virus titer of FMDV. Conclusion We successfully construct the cell lines with RNF31 gene knockout and prove that RNF31 plays a key role in the replication of FMDV. This result provides data support for further research on the mechanism by which RNF31 inhibits FMDV replication.

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E-mail: PU Xiuying, ;
YANG Fan,
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目的 探究环指蛋白31 (ring finger protein 31, RNF31)在口蹄疫病毒(foot-and-mouth disease virus, FMDV)复制过程中的具体功能，旨在为宿主蛋白RNF31调控FMDV复制的分子机制提供理论支撑。 方法 运用CRISPR/Cas9基因编辑技术，在RNF31基因外显子区段设计2条sgRNA序列，并与pX459-puro载体连接构建重组质粒。将pX459-RNF31-sgRNA转染至PK-15细胞，经嘌呤霉素筛选，获得RNF31基因敲除的细胞系。通过Western blotting、RT-qPCR及TCID50方法检测敲除RNF31基因对FMDV复制的影响。 结果 与野生型细胞相比，RNF31基因敲除细胞系中FMDV的蛋白水平、mRNA水平及病毒滴度均显著升高。 结论 本研究成功构建了RNF31基因敲除细胞系，证实RNF31在FMDV复制过程中扮演关键角色，研究结果为进一步探究RNF31抑制FMDV复制的机制提供了数据支持。

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作者贡献声明

周婷婷：实验设计，数据分析，撰写文章；张伟：实验指导，提供撰写思路；刘华南：协助实验设计；陈治彤：协助实验操作，数据分析；董星艳：协助实验操作，样本处理；王松豪：提供稿件修改建议；曹伟军：提供资源；郑海学：监督管理；蒲秀瑛：论文讨论；杨帆：稿件指导和修改，获取基金。

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A: Analysis diagram of Sanger sequencing for gene deletion in PK-RNF31-KO-1 cells; B: Analysis diagram of Sanger sequencing for gene deletion in PK-RNF31-KO-2 cells; C: Western blotting identification of RNF31 gene knockout cells., figureFileSmall=dUK5sibcCWpcPNi08u44tw==, figureFileBig=vT1/1NAsdF5TwLJpvsKdXg==, tableContent=null), ArticleFig(id=1259928493639459224, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=CN, label=图2, caption=构建RNF31基因敲除细胞系及其鉴定, figureFileSmall=dUK5sibcCWpcPNi08u44tw==, figureFileBig=vT1/1NAsdF5TwLJpvsKdXg==, tableContent=null), ArticleFig(id=1259928494893556134, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=EN, label=Figure 3, caption=Detection of cell viability in the RNF31 gene knockout cell lines. ns indicates no statistical significance. ns: P>0.05., figureFileSmall=RZTf2ZJIzqTwWOwyZLdPJg==, figureFileBig=lcFcgEHkr8eg94Jc91vZcA==, tableContent=null), ArticleFig(id=1259928496550306219, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=CN, label=图3, caption=检测RNF31基因敲除细胞系的细胞活力, figureFileSmall=RZTf2ZJIzqTwWOwyZLdPJg==, figureFileBig=lcFcgEHkr8eg94Jc91vZcA==, tableContent=null), ArticleFig(id=1259928497041039795, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=EN, label=Figure 4, caption=Effect of RNF31 gene knockout cell lines on FMDV protein levels. A: Western blotting detection; B: Intensity analysis of Figure A. ***: P<0.001; ****: P<0.000 1., figureFileSmall=CQ98SCT8Pf5/GuoHZgzkXw==, figureFileBig=qExoCDxa48ZrwRkuTWuw7Q==, tableContent=null), ArticleFig(id=1259928498240610749, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=CN, label=图4, caption=RNF31基因敲除细胞系对FMDV蛋白水平的影响, figureFileSmall=CQ98SCT8Pf5/GuoHZgzkXw==, figureFileBig=qExoCDxa48ZrwRkuTWuw7Q==, tableContent=null), ArticleFig(id=1259928498718761415, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=EN, label=Figure 5, caption=Real-time relative quantitative detection of the effect of RNF31 gene knockout cell lines on FMDV mRNA levels. ***: P<0.001; ****: P<0.000 1., figureFileSmall=7p2E3B17LPZFOUGCbDMGeQ==, figureFileBig=xSDFB7IqJ0OfPMyd2OIC8Q==, tableContent=null), ArticleFig(id=1259928500836884940, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=CN, label=图5, caption=实时相对定量检测RNF31基因敲除细胞系对FMDV mRNA水平的影响, figureFileSmall=7p2E3B17LPZFOUGCbDMGeQ==, figureFileBig=xSDFB7IqJ0OfPMyd2OIC8Q==, tableContent=null), ArticleFig(id=1259928505362538977, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=EN, label=Figure 6, caption=Determination of the effect of RNF31 gene knockout cell lines on FMDV TCID50. *: P<0.05; **: P<0.01; ***: P<0.001., figureFileSmall=THceSQ76TKkjd2VwaTEOZg==, figureFileBig=UOJL4k+4s4a2E26WV0sapA==, tableContent=null), ArticleFig(id=1259928505861661159, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=CN, label=图6, caption=TCID50 测定RNF31基因敲除细胞系对FMDV的影响, figureFileSmall=THceSQ76TKkjd2VwaTEOZg==, figureFileBig=UOJL4k+4s4a2E26WV0sapA==, tableContent=null), ArticleFig(id=1259928506721493487, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=EN, label=Table 1, caption=

Primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Primer namesPrimer sequences (5′→3′)
RNF31-sgRNA1-FCACCGTGGTCCGCTGCAACGCTCAT
RNF31-sgRNA1-RAAACATGAGCGTTGCAGCGGACCAC
RNF31-sgRNA2-FCACCGACTTGACCCCGCGCCAGTAC
RNF31-sgRNA2-RAAACGTACTGGCGCGGGGTCAAGTC
RNF31-FTCTTCCCTAGTACTTCCTGTT
RNF31-RTCCTCTGTGTAGCCATATAATC
FMDV-FCACTGGTGACAGGCTAAGG
FMDV-RCCCTTCTCAGATTCCGAGT
GAPDH-FACATGGCCTCCAAGGAGTAAGA
GAPDH-RGATCGAGTTGGGGCTGTGACT
), ArticleFig(id=1259928507870732793, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1259888462761702237, language=CN, label=表1, caption=

本研究所用引物

, figureFileSmall=null, figureFileBig=null, tableContent=
Primer namesPrimer sequences (5′→3′)
RNF31-sgRNA1-FCACCGTGGTCCGCTGCAACGCTCAT
RNF31-sgRNA1-RAAACATGAGCGTTGCAGCGGACCAC
RNF31-sgRNA2-FCACCGACTTGACCCCGCGCCAGTAC
RNF31-sgRNA2-RAAACGTACTGGCGCGGGGTCAAGTC
RNF31-FTCTTCCCTAGTACTTCCTGTT
RNF31-RTCCTCTGTGTAGCCATATAATC
FMDV-FCACTGGTGACAGGCTAAGG
FMDV-RCCCTTCTCAGATTCCGAGT
GAPDH-FACATGGCCTCCAAGGAGTAAGA
GAPDH-RGATCGAGTTGGGGCTGTGACT
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环指蛋白31基因敲除细胞系的构建及其对口蹄疫病毒复制的影响
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周婷婷 1, 2 , 张伟 2 , 刘华南 2 , 陈治彤 2 , 董星艳 2 , 王松豪 3 , 曹伟军 2 , 郑海学 1, 2 , 蒲秀瑛 1 , 杨帆 2
微生物学报 | 研究报告 2026,66(5): 2384-2392
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微生物学报 | 研究报告 2026, 66(5): 2384-2392
环指蛋白31基因敲除细胞系的构建及其对口蹄疫病毒复制的影响
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周婷婷1, 2, 张伟2, 刘华南2, 陈治彤2, 董星艳2, 王松豪3, 曹伟军2, 郑海学1, 2, 蒲秀瑛1 , 杨帆2
作者信息
  • 1.兰州理工大学 生命科学与工程学院，甘肃 兰州
  • 2.中国农业科学院兰州兽医研究所/兰州大学 动物医学与生物安全学院，动物疫病防控全国重点实验室, 甘肃 兰州
  • 3.郏县农业农村局，河南 平顶山
Construction of cell lines with ring finger protein 31 gene knockout and evaluation of its impact on the replication of foot-and-mouth disease virus
Tingting ZHOU1, 2, Wei ZHANG2, Huanan LIU2, Zhitong CHEN2, Xingyan DONG2, Songhao WANG3, Weijun CAO2, Haixue ZHENG1, 2, Xiuying PU1 , Fan YANG2
Affiliations
  • 1.School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu, China
  • 2.State Key Laboratory of Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China
  • 3.Jiaxian Bureau of Agriculture and Rural Affairs, Pingdingshan, Henan, China
出版时间: 2026-05-04 doi: 10.13343/j.cnki.wsxb.20250874
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摘要
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目的 探究环指蛋白31 (ring finger protein 31, RNF31)在口蹄疫病毒(foot-and-mouth disease virus, FMDV)复制过程中的具体功能，旨在为宿主蛋白RNF31调控FMDV复制的分子机制提供理论支撑。 方法 运用CRISPR/Cas9基因编辑技术，在RNF31基因外显子区段设计2条sgRNA序列，并与pX459-puro载体连接构建重组质粒。将pX459-RNF31-sgRNA转染至PK-15细胞，经嘌呤霉素筛选，获得RNF31基因敲除的细胞系。通过Western blotting、RT-qPCR及TCID50方法检测敲除RNF31基因对FMDV复制的影响。 结果 与野生型细胞相比，RNF31基因敲除细胞系中FMDV的蛋白水平、mRNA水平及病毒滴度均显著升高。 结论 本研究成功构建了RNF31基因敲除细胞系，证实RNF31在FMDV复制过程中扮演关键角色，研究结果为进一步探究RNF31抑制FMDV复制的机制提供了数据支持。

CRISPR/Cas9  /  环指蛋白31  /  敲除细胞系  /  口蹄疫病毒

Objective To investigate the function of ring finger protein 31 (RNF31) in the replication of foot-and-mouth disease virus (FMDV) and to provide a theoretical basis for the research on the molecular mechanism by which the host protein RNF31 regulates FMDV replication. Methods CRISPR/Cas9 gene editing was employed to design two sgRNA sequences in the exon segment of RNF31, and recombinant plasmids were constructed by ligation with the pX459-puro vector. The recombinant plasmids pX459-RNF31-sgRNA were transfected into PK-15 cells, followed by screening under the action of puromycin to obtain the cell lines with RNF31 gene knockout. The effect of RNF31 gene knockout on FMDV replication was detected by Western blotting, RT-qPCR, and TCID50 methods. Results Compared with wild-type cells, the knockout of RNF31 significantly increased the protein level, mRNA level, and virus titer of FMDV. Conclusion We successfully construct the cell lines with RNF31 gene knockout and prove that RNF31 plays a key role in the replication of FMDV. This result provides data support for further research on the mechanism by which RNF31 inhibits FMDV replication.

CRISPR/Cas9  /  ring finger protein 31  /  knockout cell lines  /  foot-and-mouth disease virus
周婷婷, 张伟, 刘华南, 陈治彤, 董星艳, 王松豪, 曹伟军, 郑海学, 蒲秀瑛, 杨帆. 环指蛋白31基因敲除细胞系的构建及其对口蹄疫病毒复制的影响. 微生物学报, 2026 , 66 (5) : 2384 -2392 . DOI: 10.13343/j.cnki.wsxb.20250874
Tingting ZHOU, Wei ZHANG, Huanan LIU, Zhitong CHEN, Xingyan DONG, Songhao WANG, Weijun CAO, Haixue ZHENG, Xiuying PU, Fan YANG. Construction of cell lines with ring finger protein 31 gene knockout and evaluation of its impact on the replication of foot-and-mouth disease virus[J]. Acta Microbiologica Sinica, 2026 , 66 (5) : 2384 -2392 . DOI: 10.13343/j.cnki.wsxb.20250874
正文
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口蹄疫(foot-and-mouth disease, FMD)是由口蹄疫病毒(foot-and-mouth disease virus, FMDV)感染引发的高度接触性动物疫病，严重影响猪、牛、羊等偶蹄类动物，在全球范围内给畜牧业造成了巨大经济损失[1]。FMDV属于小核糖核酸病毒科口蹄疫病毒属，目前FMDV的传播途径主要有2种，即接触传播和气溶胶传播[2]。FMDV为了在宿主细胞中高效复制，不断进化出新的拮抗和逃避天然免疫的策略，因此深入研究病毒-宿主蛋白相互作用是开发新型抗病毒药物的关键。
E3泛素连接酶家族分为homologous to the E6-AP C-terminus (HECT)、really interesting new gene (RING)和ring between ring (RBR) 3种类型，RBR家族包含多种蛋白，常见的有parkin RBR E3 ubiquitin protein ligase (PARKIN)、环指蛋白19B (ring finger protein 19B, RNF19B)、环指蛋白144A (ring finger protein 144A, RNF144A)、环指蛋白144B (ring finger protein 144B, RNF144B)、环指蛋白216 (ring finger protein 216, RNF216)，环指蛋白31 (ring finger protein 31, RNF31)是RBR型E3泛素连接酶，由1 072个氨基酸构成。RNF31的结构组成包括1个假定的泛素结合结构域(putative ubiquitin binding domain, PUB)、1个含泛素相关结构域(ubiquitin-associate domain, UBA)、3个锌指结构域、1个RING与RING间结构域(RBR)和1个线性泛素链决定域(linear ubiquitin chain-determining domain, LDD)[3-5]。RNF31是一种多功能蛋白，定位于细胞质和细胞核，已证实与多种炎症性疾病和癌症相关。RNF31的各结构域可分别与不同蛋白质相互作用，展现出多元化的生理功能。RNF31 N端的PUB结构域介导蛋白质间相互作用，如RNF31与淋巴细胞Yes相关蛋白(Yes-associated protein, YAP)结合，能够促进YAP蛋白的泛素化和降解[6]。UBA结构域可识别并结合游离泛素或泛素化底物，参与泛素信号的传递[7]。C末端的RBR结构域和LDD结构域是泛素连接酶催化底物线性泛素化的重要结构域，若该结构域缺失，则会丧失泛素化功能[8-9]。例如，RNF31缺失RBR结构域可减少雌激素受体α (estrogen receptor α, Erα)的表达量，影响乳腺癌细胞的增殖[10]。目前，关于RNF31在调控病毒复制方面的研究报道较少，需进一步探索RNF31在病毒复制中的潜在作用。
本研究利用CRISPR/Cas9基因编辑技术构建RNF31基因敲除的细胞系，在该细胞系中进行FMDV感染试验，通过Western blotting、RT-qPCR及TCID50方法研究RNF31对FMDV复制的影响，探究RNF31在FMDV复制过程中的关键作用，以期为进一步揭示RNF31与FMDV相互作用的分子机制提供理论依据，也为抗病毒药物研究提供新的靶点。
1 材料与方法
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1.1 载体、细胞及病毒
PK-15细胞、BHK-21细胞、pX459-puro质粒、FMDV (O/BY/CHA/2010株)均保存于中国农业科学院兰州兽医研究所国家口蹄疫参考实验室。
1.2 主要试剂和仪器
大肠杆菌DH5α感受态细胞、T4 DNA连接酶，宝生物工程(大连)有限公司；Bbs I，New England Biolabs公司；RNA isolater Total RNA Extraction Reagent、M-MLV (H-) Reverse Transcriptase，南京诺唯赞生物科技股份有限公司；Realtime PCR Super mix plus，北京聚合美生物科技有限公司；Beta Actin Monoclonal antibody，武汉三鹰生物技术有限公司；Anti-RNF31/HOIP antibody，Abcam Plc公司；兔抗O型FMDV多克隆抗体由本课题组Zhang等[11]提供。
电泳仪、PCR仪、实时荧光定量PCR仪，Bio-Rad公司；多功能酶标仪，Agilent Technologies公司。
1.3 sgRNA引物的设计与合成
在NCBI数据库查询猪源RNF31基因序列，按照CRISPR/Cas9基因编辑设计原则，对基因序列的21个外显子区段进行标注。选择含有特定靶基因位点的第1、2区段，设计特异性sgRNA，相应引物序列见表1，设计的2对引物由北京擎科生物科技有限公司西安分公司合成。
1.4 构建重组质粒pX459-sgRNA
1.4.1 单酶切pX459-puro质粒
使用限制性核酸内切酶Bbs I对载体进行单酶切，使其形成线性DNA分子。相应酶切体系和程序参照Bbs I说明书，酶切产物加入Gel Loading Dye (6×) 0.006 mL，混合均匀后进行琼脂糖凝胶电泳，随后进行胶回收。
1.4.2 重组质粒pX459-sgRNA构建
将2对sgRNA的上、下游引物均稀释至10 μmol/L，相应扩增体系和程序参照LA 10×PCR buffer说明书，形成2个双链DNA片段。将这2个DNA片段分别与单酶切后的pX459-puro载体通过T4 DNA连接酶进行连接，相应连接体系和程序参照T4 DNA连接酶说明书。将2个连接产物分别转化至大肠杆菌DH5α感受态细胞，涂布于含有氨苄抗性的LB培养板，37 ℃培养12 h。培养板中形成可见菌落后，选取单克隆菌落置于含有氨苄抗性的LB培养液中，37 ℃、220 r/min培养14 h。通过菌液PCR鉴定筛选阳性克隆，并提取质粒进行测序，将构建成功的2个质粒分别命名为pX459-RNF31-sgRNA-1和pX459-RNF31-sgRNA-2。
1.5 细胞转染与筛选
1.5.1 细胞转染
复苏PK-15细胞，进行1-2次传代，将生长旺盛的细胞接种于6孔板中。待细胞密度达到70%-80%时，进行转染。在1.5 mL离心管中加入jetPRIME® buffer 0.1 mL，其中一支离心管中加入质粒pX459-RNF31-sgRNA-1 2 μg，另一支离心管中加入质粒pX459-RNF31-sgRNA-2 2 μg，涡旋振荡混匀。提前将jetPRIME® reagent涡旋振荡混匀，取0.004 mL加入至质粒混合液中，涡旋振荡混匀，静置孵育10 min形成复合物。更换6孔板中的细胞培养基，将复合物贴近液面缓慢均匀加入，置于37 ℃、5% CO2培养箱中培养24 h。
1.5.2 细胞筛选
取出已完成质粒转染的6孔板，弃去细胞上清液，用1 mL PBS清洗细胞1遍，加入0.5 mL胰酶，室温静置30 s后弃去，置于37 ℃温箱中消化至细胞变圆。加入1 mL培养基将细胞均匀分散，再补加2 mL培养基使细胞混合均匀，接种至6孔板中，分别加入终浓度为1.5、2、3 μg/mL的嘌呤霉素，继续培养3 d，弃去上清液，更换2 mL新的培养基促进细胞生长。1 d后用胰酶消化细胞并计数，按10个/mL的密度接种于96孔板中，7 d后观察细胞状态。通过有限稀释法挑选正常形态的单克隆细胞，逐级扩大培养。
1.6 细胞鉴定
1.6.1 PCR扩增鉴定
将待测细胞于4 ℃、12 000 r/min离心3 min，弃去上清液，提取基因组DNA。鉴定RNF31基因的引物见表1，相应PCR扩增体系和程序参照2×Phanta Max Master Mix (Dye Plus)说明书，扩增产物经琼脂糖凝胶电泳后，送北京擎科生物科技有限公司西安分公司进行测序分析。将RNF31基因出现缺失、插入导致移码的细胞株命名为PK-RNF31-KO，将RNF31基因碱基序列未发生改变的细胞株命名为PK-RNF31-WT。
1.6.2 Western blotting鉴定
将PK-RNF31-WT和PK-RNF31-KO细胞接种至12孔板，待细胞密度生长至80%时，每孔加入2×SDS-PAGE loading buffer 0.2 mL，裂解并收集细胞样品。于100 ℃加热10 min，涡旋振荡混匀，室温12 000 r/min离心1 min，取上清进行SDS-PAGE，转印至NC膜上。以Anti-RNF31/HOIP antibody、Beta Actin Monoclonal antibody为一抗，Goat Anti-Rabbit IgG H&L (HRP)、Goat Anti-Mouse IgG H&L (HRP)为二抗，进行Western blotting分析，检测RNF31蛋白的表达情况。
1.7 CCK-8检测细胞活力
将PK-RNF31-WT、PK-RNF31-KO-1和PK-RNF31-KO-2细胞以4×104个/mL的密度接种至96孔板，每孔0.1 mL。置于培养箱中培养8 h，观察细胞生长状态，弃去细胞上清液，加入含有CCK-8溶液的培养基(每孔0.01 mL)。置于培养箱中培养4 h，用酶标仪测量450 nm处的吸光度。计算2组细胞存活率，分析敲除RNF31基因对PK-15细胞生长活力的影响。
1.8 Western blotting检测病毒蛋白
将PK-RNF31-WT、PK-RNF31-KO-1和PK-RNF31-KO-2细胞接种至12孔板，待细胞密度生长至80%时，感染等量FMDV。在0、6、12 h后弃去细胞上清液，每孔加入2×SDS-PAGE loading buffer 0.2 mL，裂解并收集细胞样品。于100 ℃加热10 min，涡旋振荡混匀，室温12 000 r/min离心1 min，取上清进行SDS-PAGE，转印至NC膜上。以Anti-RNF31/HOIP antibody、兔抗O型FMDV多克隆抗体、Beta Actin Monoclonal antibody为一抗，Goat Anti-Rabbit IgG H&L (HRP)、Goat Anti-Mouse IgG H&L (HRP)为二抗，进行Western blotting分析，检测RNF31蛋白和病毒蛋白的表达情况。
1.9 RT-qPCR检测病毒mRNA
将PK-RNF31-WT、PK-RNF31-KO-1和PK-RNF31-KO-2细胞接种至12孔板，待细胞密度生长至80%时，感染等量FMDV。在0、6、12 h后弃去细胞上清液，每孔加入RNA isolater Total RNA Extraction Reagent 1 mL，室温裂解5-10 min，收集细胞样品提取RNA。相应RNA反转录体系和程序参照M-MLV (H-) Reverse Transcriptase说明书，获得cDNA。相应实时荧光定量PCR体系参照Realtime PCR Super mix plus说明书，GAPDH、FMDV上、下游引物见表1。检测FMDV的相对mRNA水平，每组设置3个重复，以2-ΔΔCt法分析试验数据。
1.10 TCID50 检测病毒滴度
将PK-RNF31-WT、PK-RNF31-KO-1和PK-RNF31-KO-2细胞接种至12孔板，待细胞密度生长至80%时，感染等量FMDV。在0、6、12 h后收集细胞上清液，将上清液反复冻融3次，4 ℃、12 000 r/min离心3 min，取上清测定病毒滴度。将BHK-21细胞接种至96孔板，每孔0.1 mL。用DMEM培养基将离心后的病毒液按10倍倍比稀释，加入到BHK-21细胞中，每孔0.1 mL，每个稀释度设置8个重复。在48-72 h期间观察细胞病变情况，根据Reed-Muench法计算TCID50。
1.11 统计学分析
使用GraphPad Prism 10.3.1软件进行统计学分析，并绘制相关图表。ns表示P>0.05，*表示P<0.05，**表示P<0.01，***表示P<0.001，****表示P<0.000 1。
2 结果与分析
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2.1 重组质粒pX459-RNF31-sgRNA的鉴定
利用CRISPR/Cas9基因编辑技术在猪RNF31基因序列的第1个外显子区段的5 529 bp位置、第2个外显子区段的5 956 bp位置设计sgRNA序列(图1)。通过序列测定，确认sgRNA成功构建至pX459-puro载体中，将重组质粒分别命名为pX459-RNF31-sgRNA-1、pX459-RNF31-sgRNA-2。
2.2 敲除RNF31基因的PK-15细胞系的构建与鉴定
DNA测序结果如图2A、2B所示，第一条sgRNA打靶成功，PK-RNF31-KO-1在第1个外显子区段插入1个碱基对，PK-RNF31-KO-2在第1个外显子区段敲除1个碱基对。同时，Western blotting检测发现，2株对应细胞中均未能检测到RNF31蛋白表达(图2C)，表明成功构建了敲除RNF31基因的猪PK-15细胞系。
2.3 敲除RNF31基因对细胞活力的影响
如图3所示，PK-RNF31-KO-1和PK-RNF31-KO-2细胞活力与野生型对照细胞活力相比无明显差异，这为后续研究RNF31在FMDV复制中的功能提供了良好的工具细胞。
2.4 敲除RNF31基因对FMDV蛋白水平的影响
如图4所示，随着感染时间的延长，PK-RNF31-KO-1和PK-RNF31-KO-2细胞中FMDV的蛋白丰度显著高于野生型对照细胞，表明敲除RNF31基因促进FMDV的蛋白水平。
2.5 敲除RNF31基因对FMDV mRNA水平的影响
如图5所示，随着感染时间的延长，6 h和12 h时PK-RNF31-KO-1和PK-RNF31-KO-2细胞中FMDV的相对mRNA水平显著高于野生型对照细胞，表明敲除RNF31基因可促进FMDV的mRNA水平。
2.6 敲除RNF31基因对FMDV病毒滴度的影响
如图6所示，随着感染时间的延长，6 h和12 h时PK-RNF31-KO-1和PK-RNF31-KO-2细胞中FMDV的病毒滴度显著高于野生型对照细胞，表明敲除RNF31基因可促进FMDV的病毒滴度。
3 讨论与结论
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CRISPR/Cas作为近年来基因编辑领域的核心技术之一，与锌指核酸酶技术(zinc finger nuclease, ZFN)、转录激活因子样效应核酸酶技术(transcription activator-like effector nuclease, TALEN)相比，可对目的基因进行插入、删除和替换操作，从而改变生物体的基因组序列和遗传信息；该技术具有成本低、简单高效的优势特性，广泛应用于癌症、病毒、遗传疾病和农作物改良育种等研究领域[12]。在病毒学领域应用较多，如研究病毒-宿主的相互作用、不同病毒基因的功能、重组疫苗的研发以及构建敲除细胞系等，为实验提供了重要的工具和方法[13]。CRISPR全基因组筛选可快速找到与病毒相关的基因，如参与病毒进入、复制、释放等过程的宿主蛋白。Li等[14]通过CRISPR/Cas全基因组筛选与甲型流感病毒(influenza A virus, IAV)感染相关的宿主蛋白，在121个蛋白中鉴定出宿主蛋白WD repeat-containing protein 7 (WDR7)、卷曲螺旋结构域蛋白115 (coiled-coil domain-containing protein 115, CCDC115)和跨膜蛋白199 (transmembrane protein 199, TMEM199)与病毒感染有关。Peng等[15]根据猪全基因组设计CRISPR/Cas9敲除库，通过筛选并鉴定ERBB2转录因子1 (transducer of ErbB2.1, TOB1)敲除可抑制FMDV感染。Khasa等[16]利用人胶质母细胞瘤细胞系SNB-19进行CRISPR/Cas9全基因组筛选，确定受体酪氨酸激酶AXL (receptor tyrosine kinase AXL, AXL)是寨卡病毒(Zika virus, ZIKV)感染的关键宿主因子，敲除AXL可抑制ZIKV的感染。
研究发现E3泛素连接酶在抗病毒方面发挥重要作用，多种E3泛素连接酶被鉴定为机体本身的抗病毒效应物，可通过泛素-蛋白酶体系统(ubiquitin-proteasome system, UPS)调控病毒蛋白，从而影响病毒复制。RNF31作为线性泛素链组装复合体(linear ubiquitin chain assembly complex, LUBAC)不可或缺的重要组成部分，也是目前发现并证实能够通过特定分子机制影响底物蛋白线性泛素化修饰过程的E3泛素连接酶[17]。例如，丙型肝炎病毒(hepatitis C virus, HCV)感染后，非结构蛋白3 (non-structural protein 3, NS3)与LUBAC相互作用，使核因子κB (nuclear factor-kappaB, NF-κB)必需调节蛋白(NF-κB essential modulator, NEMO)无法进行线性泛素化，抑制NF-κB的激活，从而使HCV逃避天然免疫[18]。人冠状病毒(human coronavirus, HCoV)的非结构蛋白14 (non-structural protein 14, NSP14)与RNF31结合，利用线性泛素化途径激活NF-κB，泛素化的NSP14招募NEMO从而引发促炎反应[19]。这些结果初步揭示了RNF31在病毒感染后的炎症反应中发挥作用，但RNF31参与病毒复制的功能和分子机制仍不清楚。
为了探究RNF31在病毒复制中的功能，本研究利用CRISPR/Cas9基因编辑技术构建了RNF31基因敲除的PK-15细胞系模型，以探究RNF31对FMDV复制的影响。对PK-RNF31-KO细胞与野生型细胞感染FMDV的样本进行分析后发现，PK-RNF31-KO细胞感染FMDV后FMDV的蛋白水平、mRNA水平和病毒滴度均显著升高，这证实了RNF31具有抑制FMDV复制的功能。推测RNF31可能调控FMDV生命环节中的关键步骤，如吸附、内化、病毒颗粒脱壳和基因组释放等；它可能利用E3泛素连接酶靶向FMDV结构蛋白或非结构蛋白泛素化降解，从而阻断病毒粒子的组装和释放等；还可能催化线性泛素链修饰NEMO等信号分子，激活NF-κB与I型干扰素(type I interferon, IFN-I)信号通路，诱导IFN-I与促炎因子的产生，进而促进抗病毒天然免疫等。目前关于RNF31调控FMDV的分子机制尚不清楚，可利用该细胞系模型揭示RNF31抑制FMDV复制的作用机制。
综上所述，本研究利用CRISPR/Cas9基因编辑技术在FMDV易感细胞PK-15中构建了敲除RNF31基因的细胞系，发现敲除RNF31可显著促进FMDV的蛋白水平、mRNA水平和病毒滴度升高。本研究为深入阐明RNF31调控FMDV复制的机制提供了依据和细胞模型，也为FMD的防控提供新的理论基础和潜在干预策略。
基金
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  • 国家自然科学基金(32330107)
  • 甘肃省联合科研基金(25JRRA1088)
  • 甘肃省联合科研基金(25JRRA1087)
  • 甘肃省研产融合科技攻关赋能计划(25FNNA002)
  • 甘肃省科技重大专项计划(22ZD6NA001)
  • 国家生猪产业技术体系项目(CARS-35)
  • 国家生猪技术创新中心项目(NCTIP-XD/C03)
文献
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参考文献 引证文献
排序方式：
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郑海学, 刘湘涛. 口蹄疫病毒遗传进化研究进展[J]. 生命科学，2016，28(3): 311-324.
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2026年第66卷第5期
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doi: 10.13343/j.cnki.wsxb.20250874
  • 接收时间：2025-11-23
  • 首发时间：2026-05-09
  • 出版时间：2026-05-04
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出版历史
  • 收稿日期：2025-11-23
  • 录用日期：2026-01-19
基金
The National Natural Sciences Foundation of China(32330107)
国家自然科学基金(32330107)
The Gansu Provincial Joint Research Fund(25JRRA1088)
甘肃省联合科研基金(25JRRA1088)
The Gansu Provincial Joint Research Fund(25JRRA1087)
甘肃省联合科研基金(25JRRA1087)
The Gansu Province Research and Development-Industry Integration and Technology Empowerment Program(25FNNA002)
甘肃省研产融合科技攻关赋能计划(25FNNA002)
The Gansu Provincial Major Science and Technology Special Program(22ZD6NA001)
甘肃省科技重大专项计划(22ZD6NA001)
The Earmarked Fund for CARS-35, and the Project of National Center of Technology Innovation for Pigs(CARS-35)
国家生猪产业技术体系项目(CARS-35)
The Earmarked Fund for CARS-35, and the Project of National Center of Technology Innovation for Pigs(NCTIP-XD/C03)
国家生猪技术创新中心项目(NCTIP-XD/C03)
作者信息
    1.兰州理工大学 生命科学与工程学院，甘肃 兰州
    2.中国农业科学院兰州兽医研究所/兰州大学 动物医学与生物安全学院，动物疫病防控全国重点实验室, 甘肃 兰州
    3.郏县农业农村局，河南 平顶山
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https://castjournals.cast.org.cn/joweb/wswxb/CN/10.13343/j.cnki.wsxb.20250874
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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