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目的 本试验采用CRISPR/Cas9基因编辑技术，成功构建干扰素诱导跨膜蛋白3(Interferon-induced transmembrane protein 3, IFITM3)基因敲除的LLC-PK1-IFITM3KO。以LLC-PK1-IFITM3KO细胞系为核心工具，系统探究IFITM3基因对猪δ冠状病毒(Porcine deltacoronavirus, PDCoV)感染宿主细胞及后续传播过程的调控作用，旨在为深入开展IFITM3基因的生物学功能研究，以及阐明其调控PDCoV感染与传播的分子机制提供坚实的理论依据与实验基础。方法 根据NCBI数据库中猪IFITM3基因序列，设计3条单链向导RNA(Single guide RNA,sgRNA)，构建重组慢病毒载体并包装成慢病毒，用以转导LLC-PK1细胞。经嘌呤霉素抗性筛选后，通过有限稀释法获得单克隆细胞系。采用DNA测序验证基因敲除情况，Western blotting检测IFITM3蛋白表达；利用RT-qPCR和Western blotting检测IFITM3基因敲除后PDCoV S基因拷贝数及N蛋白表达变化，分析病毒复制以及吸附和内化水平。结果 重组慢病毒载体测序验证构建成功，筛选获得IFITM3基因敲除的LLC-PK1单克隆细胞系(LLC-PK1-IFITM3KO)，测序显示细胞存在2个碱基缺失，Western blotting证实无IFITM3蛋白表达。以野生型LLC-PK1细胞为对照，针对LLC-PK1-IFITM3KO细胞系的PDCoV感染特性检测结果显示：在病毒复制阶段，PDCoV的S基因拷贝数显著升高(P<0.01)，同时其N蛋白表达水平亦显著上调，上述结果明确提示IFITM3基因敲除可增强PDCoV的复制能力；而在病毒吸附与入侵阶段，LLC-PK1-IFITM3KO细胞系中PDCoV的S基因拷贝数则呈显著下降趋势(P<0.01)，表明IFITM3基因缺失会显著削弱PDCoV对宿主细胞的吸附及入侵能力。结论 本研究利用CRISPR/Cas9技术成功构建了LLC-PK1-IFITM3KO细胞系，功能验证结果表明，IFITM3基因的敲除虽可显著抑制PDCoV对宿主细胞的吸附与入侵进程，却能明显促进该病毒在胞内的复制效率。LLC-PK1-IFITM3KO细胞系的成功建立，不仅为深入阐释IFITM3基因的生物学功能奠定了关键工具基础，更为后续解析IFITM3调控PDCoV感染的分子机制提供了核心细胞模型。
Abstract：Objective To generate an interferon-induced transmembrane protein 3(IFITM3) knockout porcine kidney cell line(LLC-PK1-IFITM3KO) using the CRISPR/Cas9 gene-editing system, and to systematically investigate the role of IFITM3 in regulating porcine deltacoronavirus(PDCoV) infection and subsequent viral dissemination. This study aims to provide a robust experimental platform for elucidating the biological functions of IFITM3 and the molecular mechanisms underlying its regulation of PDCoV infection.Methods Based on the porcine IFITM3 gene sequence retrieved from the NCBI database, three single guide RNA(sgRNA) were designed and cloned into a recombinant lentiviral CRISPR/Cas9 vector, which was subsequently packaged into lentiviral particles. LLC-PK1 cells were transduced with the recombinant lentivirus and subjected to puromycin selection, followed by isolation of monoclonal cell lines using the limiting dilution method. Gene knockout was confirmed by DNA sequencing, and IFITM3 protein expression was assessed by Western blotting. After IFITM3 knockout, PDCoV infection was evaluated by measuring viral S gene copy numbers using RT-qPCR and N protein expression by Western blotting, to assess viral replication as well as viral attachment and internalization.Results Sequencing analysis confirmed the successful construction of the recombinant lentiviral vector and the establishment of an IFITM3 knockout LLC-PK1 monoclonal cell line(LLC-PK1-IFITM3KO), which harbored a two-base pair deletion in the IFITM3 gene. Western blotting verified the complete absence of IFITM3 protein expression. Compared with wild-type LLC-PK1 cells, PDCoV infection assays revealed that: during the viral replication stage, PDCoV S gene copy numbers and N protein expression levels were significantly increased in LLC-PK1-IFITM3KO cells(P < 0.01), indicating enhanced viral replication. In contrast, during the viral attachment and internalization stages, PDCoV S gene copy numbers were significantly reduced in IFITM3 knockout cells(P < 0.01), suggesting that IFITM3 deficiency markedly impaired PDCoV attachment to and entry into host cells.Conclusion This study successfully established an IFITM3 knockout LLC-PK1 cell line using the CRISPR/Cas9 system. Functional analyses demonstrated that IFITM3 deficiency significantly suppresses PDCoV attachment and internalization, while paradoxically promoting viral replication within host cells. The LLC-PK1-IFITM3KO cell line represents a valuable cellular model for elucidating the biological functions of IFITM3 and for further dissecting the molecular mechanisms by which IFITM3 regulates PDCoV infection.
[1] Yang H, Rao Z. Structural biology of SARS-CoV-2and implications for therapeutic development[J]. Nat Rev Microbiol, 2021, 19(11):685-700. DOI:10. 1038/s41579-021-00630-8.
[2] Yan Q, Liu X, Sun Y, et al. Swine enteric coronavirus:diverse pathogen-host interactions[J]. Int J Mol Sci, 2022, 23(7):3953. DOI:10. 3390/ijms23073953.
[3] Lin CN, Chan KR, Ooi EE, et al. Animal coronavirus diseases:parallels with COVID-19 in humans[J].Viruses, 2021, 13(8):1507. DOI:10. 3390/v13081507.
[4]方谱县，方六荣，董楠，等.猪δ冠状病毒的研究进展[J].病毒学报，2016, 32(2):243-248. DOI:10. 13242/j. cnki. bingduxuebao. 002914.
[5]喻康，戚洋睿，粟硕等. 2010—2024年中国猪δ冠状病毒S蛋白密码子使用偏好及选择分析[J].病毒学报，2025, 41(3):835-844. DOI:10. 13242/j. cnki.bingduxuebao. 250082.
[6] Han F, Shan F, Hou J, et al. Establishment and application of PDCoV antigen-specific DAS-ELISA detection method[J]. BMC Vet Res, 2024, 20(1):342. DOI:10. 1186/s12917-024-04201-w.
[7] Wang N, Wang Z, Ma M, et al. Expression of codonoptimized PDCoV-RBD protein in baculovirus expression system and immunogenicity evaluation in mice[J]. Int J Biol Macromol, 2023, 252:126113. DOI:10. 1016/j. ijbiomac. 2023. 126113.
[8] Yánez DC, Ross S, Crompton T. The IFITM protein family in adaptive immunity[J]. Immunology, 2020,159(4):365-372. DOI:10. 1111/imm. 13163.
[9] Du S, Wang Y, Wang J, et al. IFITM3 inhibits severe fever with thrombocytopenia syndrome virus entry and interacts with viral Gc protein[J]. J Med Virol, 2024,96(3):e29491. DOI:10. 1002/jmv. 29491.
[10]Ma J, Tu Z, Du S, et al. IFITM3 restricts RABV infection through inhibiting viral entry and mTORC1-dependent autophagy[J]. Vet Microbiol, 2023, 284:109823. DOI:10. 1016/j. vetmic. 2023. 109823.
[11]Liu Y, Ma J, Gao P, et al. IFITM3 reduces infectious bursal disease virus proliferation by regulating interferon expression[J]. Microb Pathog, 2024, 194:106802.DOI:10. 1016/j. micpath. 2024. 106802.
[12]McMichael TM, Zhang Y, Kenney AD, et al. IFITM3restricts human metapneumovirus infection[J]. J Infect Dis, 2018, 218(10):1582-1591. DOI:10. 1093/infdis/jiy361.
[13]Wang Z, Tuo X, Zhang J, et al. Antiviral role of IFITM3 in prototype foamy virus infection[J]. Virol J,2022, 19(1):195. DOI:10. 1186/s12985-022-01931-x.
[14]Kenney AD, Zani A, Kawahara J, et al. Interferoninduced transmembrane protein 3(IFITM3)limits lethality of SARS-CoV-2 in mice[J]. EMBO Rep,2023, 24(4):e56660. DOI:10. 15252/embr. 202256660.
[15]胡艺欣，吴兴一，李泽辉，等.利用CRISPR/Cas9系统构建ITGαV及ITGβ3敲除细胞系[J].病毒学报，2023, 39(2):491-498. DOI:10. 13242/j. cnki.bingduxuebao. 004292.
[16]支大龙，牛昱宇，季维智.人工核酸酶介导的基因组编辑技术[J].中国生物化学与分子生物学报，2015, 31(11):1132-1137. DOI:10. 13865/j. cnki.cjbmb. 2015. 11. 02.
[17]宋梓虢.基于Un1Cas12f1的高效微型基因编辑工具开发及在绵羊细胞中的应用[D].杨凌：西北农林科技大学，2025.
[18]王森.邓肯巴贝斯虫基于同源重组的基因编辑方法的建立与应用[D].武汉：华中农业大学，2024.
[19]Xue C, Greene EC. DNA repair pathway choices in CRISPR-Cas9-mediated genome editing[J]. Trends Genet, 2021, 37(7):639-656. DOI:10. 1016/j.tig. 2021. 02. 008.
[20]刘佳琪，李梦瑶，郝晨琳，等.利用CRISPR/Cas9技术构建RIPK1基因敲除的Neuro-2a细胞系及其功能研究[J].病毒学报，2025, 41(4):1121-1130. DOI:10. 13242/j. cnki. bingduxuebao. 240259.
[21]Zin Mar Oo. CRISPR/Cas9介导的小鼠Dip2c基因敲除及脑转录组分析[D].长春：东北师范大学，2020.
[22]Hryhorowicz M, Lipiński D, Zeyland J, et al.CRISPR/Cas9 immune system as a tool for genome engineering[J]. Arch Immunol Ther Exp, 2017, 65(3):233-240. DOI:10. 1007/s00005-016-0427-5.
[23]Hossain MA. CRISPR-Cas9:a fascinating journey from bacterial immune system to human gene editing[J].Prog Mol Biol Transl Sci, 2021, 178:63-83. DOI:10. 1016/bs. pmbts. 2021. 01. 001.
[24]Gómez-Herranz M, Taylor J, Sloan RD. IFITM proteins:Understanding their diverse roles in viral infection, cancer, and immunity[J]. J Biol Chem,2023, 299(1):102741. DOI:10. 1016/j.jbc. 2022. 102741.
[25]Jiménez-Munguía I, Beaven AH, Blank PS, et al.Interferon-induced transmembrane protein 3(IFITM3)and its antiviral activity[J]. Curr Opin Struct Biol,2022, 77:102467. DOI:10. 1016/j. sbi. 2022. 102467.
[26]王凯瑜，雷雪裴，黄艺滢，等. Ifitm3敲除抑制小鼠神经干细胞增殖和分化[J].中国实验动物学报，2024,32(6):691-701. DOI:10. 3969/j. issn. 1005-4847. 2024. 06. 002.
[27]Lv L, Luo H, Yi J, et al. IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus[J]. J Virol, 2025, 99(6):e02028-e02024. DOI:10. 1128/jvi. 02028-24.
[28]Prelli Bozzo C, Nchioua R, Volcic M, et al. IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro[J]. Nat Commun, 2021, 12(1):4584. DOI:10. 1038/s41467-021-24817-y.
[29]Zhao X, Guo F, Liu F, et al. Interferon induction of IFITM proteins promotes infection by human coronavirus OC43[J]. Proc Natl Acad Sci U S A,2014, 111(18):6756-6761. DOI:10. 1073/pnas. 1320856111.
基本信息:
中图分类号:S852.651
引用信息:
[1]邢云飞,李士文,李泽辉,等.利用CRISPR/Cas9系统构建IFITM3敲除细胞系[J].病毒学报().
基金信息:
国家自然科学基金（项目号：32402922）,题目：PDCoV N蛋白通过调节Keap1-NRF2信号通路诱导细胞铁死亡的分子机制研究; 中原科技创新领军人才（项目号：254200510033）,题目：仔猪肠道粘膜免疫系统发育对PDCoV感染的影响
2026-02-09
2026-02-09
2026-02-09