干扰素调节因子3基因敲除对伪狂犬病病毒增殖的影响
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摘要
试验旨在研究敲除干扰素调节因子3 (interferon regulatory factor 3,IRF3)对伪狂犬病病毒(pseudorabies virus,PRV)复制的影响。使用慢病毒介导的CRISPR/Cas9基因编辑技术建立了IRF3基因敲除PK15细胞系。通过构建重组质粒pIRF3-sgRNA,转染至HEK293T/17细胞,收获慢病毒并感染PK15细胞,经嘌呤霉素筛选获得多克隆细胞系,T7酶切鉴定后通过有限稀释法获得PK15-IRF3~(-/-)单克隆稳定细胞系。为验证敲除IRF3基因稳定细胞系是否构建成功,采用实时荧光定量PCR、Western blotting方法检测PRV相关基因及蛋白的表达,应用荧光显微镜和流式细胞术观察病毒复制情况并进行病毒滴度检测。结果显示,PK15-IRF3~(-/-)细胞系感染PRV-GFP后PK15-IRF3~(-/-)细胞荧光强度明显强于PK15细胞。感染PRV野毒(PRV-QXX)后PK15-IRF3~(-/-)病毒滴度明显强于PK15细胞,PRV的gE蛋白水平明显高于PK15细胞。在mRNA水平检测两种细胞中PRV TK基因的变化也得到相同的结果。进一步研究表明,在感染PRV-QXX后,PK15细胞中IFN-βmRNA会随着时间增加显著增高(P<0.05),但PK15-IRF3~(-/-)细胞中IFN-βmRNA则无明显变化。上述结果表明,敲除IRF3基因后显著促进PRV的增殖,IRF3在病毒的复制中发挥着重要作用,为伪狂犬病的防控提供了新的方法和策略。
The aim of the study was to investigate the effect of knockout of interferon regulatory factor 3(IRF3) on the replication of pseudorabies virus(PRV).The IRF3 knockout PK15 cell line was established using lentiviral-mediated CRISPR/Cas9 genome editing technology.The recombinant plasmid pIRF3-sgRNA was constructed and transfected into HEK293 T/17 cells.Lentivirus was obtained and infected with PK15 cells.The polyclonal cell line was obtained by puromycin screening.After T7 digestion,PK15-IRF3~(-/-) monoclonal stable cell line was obtained by limiting dilution method.To verify whether the stable cell line of IRF3 gene was successfully constructed,Real-time PCR and Western blotting were used to detect the expression of PRV-related genes and proteins.Fluorescence microscopy and flow cytometry were used to observe the virus replication.The results showed that the fluorescence intensity of PK15-IRF3~(-/-) cells was significantly stronger than that of PK15 cells after infection of PRV-GFP in PK15-IRF3~(-/-) cell line.The PK15-IRF3~(-/-) virus titer was significantly stronger than that of PK15 cells after infection with PRV-QXX,and the gE protein level of PRV was significantly higher than that of PK15 cells.The same results were obtained by detecting changes of PRV TK gene in the two cells at the mRNA level.Further studies showed that IFN-β mRNA in PK15 cells increased significantly with time after infection with PRV-QXX(P<0.05),but there was no significant change in IFN-β mRNA in PK15-IRF3~(-/-) cells.The above results indicated that knocking out IRF3 significantly promoted the proliferation of PRV,and IRF3 played an important role in virus replication,providing new methods and strategies for the prevention and control of pseudorabies.
The aim of the study was to investigate the effect of knockout of interferon regulatory factor 3(IRF3) on the replication of pseudorabies virus(PRV).The IRF3 knockout PK15 cell line was established using lentiviral-mediated CRISPR/Cas9 genome editing technology.The recombinant plasmid pIRF3-sgRNA was constructed and transfected into HEK293 T/17 cells.Lentivirus was obtained and infected with PK15 cells.The polyclonal cell line was obtained by puromycin screening.After T7 digestion,PK15-IRF3~(-/-) monoclonal stable cell line was obtained by limiting dilution method.To verify whether the stable cell line of IRF3 gene was successfully constructed,Real-time PCR and Western blotting were used to detect the expression of PRV-related genes and proteins.Fluorescence microscopy and flow cytometry were used to observe the virus replication.The results showed that the fluorescence intensity of PK15-IRF3~(-/-) cells was significantly stronger than that of PK15 cells after infection of PRV-GFP in PK15-IRF3~(-/-) cell line.The PK15-IRF3~(-/-) virus titer was significantly stronger than that of PK15 cells after infection with PRV-QXX,and the gE protein level of PRV was significantly higher than that of PK15 cells.The same results were obtained by detecting changes of PRV TK gene in the two cells at the mRNA level.Further studies showed that IFN-β mRNA in PK15 cells increased significantly with time after infection with PRV-QXX(P<0.05),but there was no significant change in IFN-β mRNA in PK15-IRF3~(-/-) cells.The above results indicated that knocking out IRF3 significantly promoted the proliferation of PRV,and IRF3 played an important role in virus replication,providing new methods and strategies for the prevention and control of pseudorabies.
引文
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[2] 青宁生.动物病毒学家——殷震[J].微生物学报,2018,58(5):955-956.QING N S.An animal virologist——Yin Z[J].Acta Microbiologica Sinica,2018,58(5):955-956.(in Chinese)
[3] GUZ Q,HOUC C,SUN H F,et al.Emergence of highly virulent pseudorabies virus in Southern China[J].Canadian Journal of Veterinary Research,2015,79(3):221-228.
[4] WU R,BAI C Y,SUN J Z,et al.Emergence of virulent pseudorabies virus infection in Northern China[J].Journal of Veterinary Science,2013,14(3):363-365.
[5] ZHAO G N,JIANG D S,LI H.Interferon regulatory factors:At the crossroads of immunity,metabolism,and disease[J].Biochimica et Biophysica Acta,2015,1852(2):365-378.
[6] JUANG Y T,LOWTHER W,KELLUM M,et al.Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3[J].Proceedings of the National Academy of Sciences,1998,95(17):9837-9842.
[7] BELLINGHAM J,GREGORY-EVANS K,GREGORY-EVANS C Y,et al.Mapping of human interferon regulatory factor 3 (IRF3) to chromosome 19q13.3-13.4 by an intragenic polymorphic marker[J].Annals of Human Genetics,1998,62(Pt 3):231-234.
[8] TANIGUCHI T,OGASAWARA K,TAKAOKA A,et al.IRF family of transcription factors as regulators of host defense[J].Annual Review of Immunology,2001,19(1):623-655.
[9] OZATO K,TAILOR P,KUBOTA T.The interferon regulatory factor family in host defense:Mechanism of action[J].Journal of Biological Chemistry,2007,282(28):20065-20069.
[10] LIN R,HEYLBROECK C,PITHA P M,et al.Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation,transactivation potential,and proteasome-mediated degradation[J].Molecular and Cellular Biology,1998,18(5):2986-2996.
[11] 李帅,赵一霏,沈国顺.一例猪伪狂犬病的诊断与防控建议[J].黑龙江畜牧兽医,2018,22:122-124.LI S,ZHAO Y Z,SHEN G S.A diagnosis and prevention and control of a case of pseudorabies in pigs[J].Heilongjiang Animal Science and Veterinary Medicine,2018,22:122-124.(in Chinese)
[12] SAITO T,GALE M JR.Principles of intracellular viral recognition[J].Current Opinion Immunology,2007,19(1):17-23.
[13] WANG H,YANG H,SHIVALILA C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell,2013,153(4):910-918.
[14] KORKMAZ G,LOPES R,UGALDE A P,et al.Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9[J].Nature Biotechnology,2016,34(2):192-198.
[15] MAZ A,PERETZ M,AHARONY A,et al.Defining essential genes for human pluripotent stem cells by CRISPR-Cas9 screening in haploid cells[J].Nature Cell Biology,2018,20(5):610-619.
[16] KLANN T S,BLACK J B,CHELLAPPAN M,et al.CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome[J].Nature Biotechnology,2017,35(6):561-568.
[17] NELSON C A,FREMONT D H,VIRGIN H W,et al.Discovery of a proteinaceous cellular receptor for a norovirus[J].Science,2016,353(6302):933-936.
[18] HAGA K,FUJIMOTO A,TAKAI-TODAKA R,et al.Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(41):E6248-E6255.
[19] VIRREIRA WINTER S,ZYCHLINSKY A,BARDOEL B W.Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity[J].Scientific Reports,2016,6:242-244.
[20] ZHANG R,MINER J J,GORMAN M J,et al.A CRISPR screen defines a signal peptide processing pathway required by flaviviruses[J].Nature,2016,535(7610):164-168.
[21] MA H,DANG Y,WU Y,et al.A CRISPR-based screen identifies genes essential for West-Nile-virus-induced cell death[J].Cell Reports,2015,12(4):673-683.
[22] MARCEAU C D,PUSCHNIK A S,MAJZOUB K,et al.Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens[J].Nature,2016,535(7610):159-163.
[23] SIDIK S M,HUET D,GANESAN S M A,et al.A genome-wide CRISPR screen in toxoplasma identifies essential apicomplexan genes[J].Cell,2016,166(6):1423-1435.
[24] MODELL J W,JIANG W,MARRAFFINI L A.CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity[J].Nature,2017,544(7648):101-104.
[25] MOCH C,FROMANT M,BLANQUET S,et al.DNA binding specificities of Escherichia coli Cas1-Cas2 integrase drive its recruitment at the CRISPR locus[J].Nucleic Acids Research,2017,45(5):2714-2723.
[26] WRIGHT A V,LIU J J,KNOTT G J,et al.Structures of the CRISPR genome integration complex[J].Science,2017,357(6356):1113-1118.
[27] SHALEM O,SANJANA N E,HARTENIAN E,et al.Genome-scale CRISPR-Cas9 knockout screening in human cells[J].Science,2014,343(6166):84-87.
[28] SHALEM O,SANJANA N E,ZHANG F.High-throughput functional genomics using CRISPR-Cas9[J].Nature Reviews Genetics,2015,16(5):299-311.
[29] MILLER L C,ZANELLA E L,WATERS W R,et al.Cytokine protein expression levels in tracheobronchial lymph node homogenates of pigs infected with pseudorabies virus[J].Clinical Vaccine Zmmanol,2010,17(5):728-734.
[30] VAN DER SANDEN S M,WU W,DYBDAHL-SISSOKO N,et al.Engineering enhanced vaccine cell lines to eradicate vaccine-preventable diseases:The polio end game[J].Journal of Virology,2015,90(4):1694-1704.
[31] BIKARD D,EULER C W,JIANG W,et al.Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials[J].Nature Biotechnology,2014,32(11):1146-1150.
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