基于茎环法的RT-qPCR检测哺乳动物抗病毒siRNA的表达
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摘要
抗病毒RNA干扰(RNA interference,RNAi)机制在哺乳动物中保守存在,宿主通过病毒源siRNA(virus-derived small interfering RNA,vsiRNA)引导Ago2蛋白剪切病毒RNA,从而发挥抗病毒作用.为了建立快速检测宿主抗流感病毒(PR8ΔNS1)和野田村病毒(NoVΔB2)特异性siRNA分子的方法,根据病毒特异性siRNAs序列设计茎环引物和扩增引物,筛选出具有高特异性和灵敏度的茎环引物和PCR扩增引物(16768,3280和24～45),成功建立了茎环法RT-qPCR检测哺乳动物病毒源siRNA表达水平的系统.该方法快速简便,准确性高,兼具较高的特异性与灵敏度,可作为小RNA测序替代方案检测siRNA或在测序前对宿主产生的病毒源siRNA进行有效监测与评价.
Antiviral RNA interference(RNAi)is conserved in mammals.Diverse hosts produce virus-derived small interfering RNAs(vsiRNAs)to guide Ago2 to cleave virus RNA genome,directing antiviral immunity by RNAi.To establish a real-time RT-PCR assay for detection of specific siRNAs derived from influenza A virus(PR8ΔNS1)and nodamura virus(NoVΔB2),a real-time RT-PCR method was developed with a stem-loop primer and a pair of PCR primers targeting the viral siRNAs sequence in PR8ΔNS1 and NoVΔB2.Three groups stem-loop primer and PCR primers(16768,3280 and 24—45)was screened out as indicators after evaluating ΔCt and vsiRNA expression level between mock and treatment groups,a system for detection mammalian antiviral vsiRNAs by stem-loop primer was built.The method was capable of fast-quantitative detection viral siRNAs,which showed high specificity and sensitivity simultaneously.Hence,the system may be used as the alternative solution of small sequence or monitor host antiviral siRNAs before sequencing.
Antiviral RNA interference(RNAi)is conserved in mammals.Diverse hosts produce virus-derived small interfering RNAs(vsiRNAs)to guide Ago2 to cleave virus RNA genome,directing antiviral immunity by RNAi.To establish a real-time RT-PCR assay for detection of specific siRNAs derived from influenza A virus(PR8ΔNS1)and nodamura virus(NoVΔB2),a real-time RT-PCR method was developed with a stem-loop primer and a pair of PCR primers targeting the viral siRNAs sequence in PR8ΔNS1 and NoVΔB2.Three groups stem-loop primer and PCR primers(16768,3280 and 24—45)was screened out as indicators after evaluating ΔCt and vsiRNA expression level between mock and treatment groups,a system for detection mammalian antiviral vsiRNAs by stem-loop primer was built.The method was capable of fast-quantitative detection viral siRNAs,which showed high specificity and sensitivity simultaneously.Hence,the system may be used as the alternative solution of small sequence or monitor host antiviral siRNAs before sequencing.
引文
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[28] CHENG A,LI M,LIANG Y,et al.Stem-loop RT-PCR quantification of siRNAs in vitro and in vivo[J].Oligonucleotides,2009,19(2):203-208.
[29] LU R,MADURO M,LI F,et al.Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans[J].Nature,2005,436(7053):1040-1043.
[30] PELTIER H J,LATHAM G J.Normalization of microRNA expression levels in quantitative RT-PCR assays:Identification of suitable reference RNA targets in normal and cancerous human solid tissues[J].RNA,2008,14(5):844-852.
[31] GUO Z,LI Y,DING S W.Small RNA-based antimicrobial immunity[J].Nat Rev Immunol,2018,19(1):31-44.
[32] LINDENBACH B D,SGRO J Y,AHLQUIST P.Long-distance base pairing in flock house virus RNA1regulates subgenomic RNA3synthesis and RNA2replication[J].Journal of Virology,2002,76(8):3905-3919.
[33] HEID C A,STEVENS J,LIVAK K J,et al.Real time quantitative PCR[J].Genome Res,1996,6(10):986-994.
[34] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method[J].Methods,2001,25(4):402-408.
[35]徐静,谢翊,管大伟,等,含内参的登革病毒与基孔肯雅病毒3重荧光定量PCR检测方法的建立与评估[J].中国病原生物学杂志,2016,11(4):301-311.
[2] EAMENS A,WANG M B,SMITH N A,et al.RNA silencing in plants:Yesterday,today,and tomorrow[J].Plant Physiol,2008,147(2):456-468.
[3] GORDON K H and WATERHOUSE P M.RNAi for insect-proof plants[J].Nat Biotechnol,2007,25(11):1231-1232.
[4] ROSA C,KUO Y W,WURIYANGHAN H,et al.RNA Interference mechanisms and applications in plant pathology[J].Annu Rev Phytopathol,2018,25(56):581-610
[5] ALIYARI R,WU Q,LI H-W,et al.Mechanism of induction and suppression of antiviral immunity directed by virus-derived small RNAs in Drosophila[J].Cell Host&Microbe,2008,4(4):387-397.
[6] NAKAYASHIKI H.RNA silencing in fungi:Mechanisms and applications.[J].FEBS Lett,2005,579(26):5950-5957.
[7] ZAMORE P D.Plant RNAi:How a viral silencing suppressor inactivates siRNA[J].Curr Biol,2004,14(5):198-200.
[8] LI Y,LU J,HAN Y,et al.RNA interference functions as an antiviral immunity mechanism in mammals[J].Science,2013,342(6155):231-234.
[9] MAILLARD P V,CIAUDO C,MARCHAIS A,et al.Antiviral RNA interference in mammalian cells[J].Science,2013,342(6155):235-238.
[10] DING S W,HAN Q,WANG J,et al.Antiviral RNA interference in mammals[J].Curr Opin Immunol,2018,54:109-114.
[11] XU Y P,QIU Y,ZHANG B,et al.Zika virus infection induces RNAi-mediated antiviral immunity in human neural progenitors and brain organoids[J].Cell Res,2019,29(4):265-273.
[12] MAILLARD P V,VAN DER VEEN A G,DEDDOUCHE-GRASS S,et al.Inactivation of the typeⅠinterferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells[J].EMBO J,2016,35(23):2505-2518.
[13] CULLEN B R,CHERRY S,TENOEVER B R.Is RNA interference a physiologically relevant innate antiviral immune response in mammals?[J].Cell Host Microbe,2013,14(4):374-378.
[14] ALI P S,JOHN J,SELVARAJ M,et al.Nodamura virus B2amino terminal domain sensitivity to small interfering RNA[J].Microbiol Immunol,2015,59(5):299-304.
[15] FAN X,DONG S,LI Y,et al.RIG-I-dependent antiviral immunity is effective against an RNA virus encoding apotent suppressor of RNAi[J].Biochem Biophys Res Commun,2015,460(4):1035-1040.
[16] LI Y,BASAVAPPA M,LU J,et al.Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells[J].Nat Microbiol,2016,2:16250.
[17] BUCHER E,HEMMES H,DE HAAN P,et al.The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants[J].J Gen Virol,2004,85(4):983-991.
[18] GARCIA-SASTRE A,EGOROV A,MATASSOV D,et al.Influenza A virus lacking the NS1gene replicates in interferon-deficient systems[J].Virology,1998,252(2):324-330.
[19]刘帅,李江南,袁婷,等,茎环法RT-qPCR定量检测细胞抗猪瘟病毒siRNA的表达[J].生物工程学报,2012,28(1):26-36.
[20] NAPOLI C,LEMIEUX C,JORGENSEN R.Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans[J].Plant Cell,1990,2(4):279-289.
[21] FIRE A,XU S,MONTGOMERY M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811.
[22] NAKAMINAMI H,NOGUCHI N,IKEDA M,et al.Molecular epidemiology and antimicrobial susceptibilities of 273exfoliative toxin-encoding-gene-positive Staphylococcus aureus isolates from patients with impetigo in Japan[J].J Med Microbiol,2008,57(10):1251-1258.
[23] ALBARIO C G,ECKERLE L D,BALL L A.The cis-acting replication signal at the 3′end of Flock House virus RNA2is RNA3-dependent[J].Virology,2003,311(1):181-191.
[24] CASTORENA K M,WEEKS S A,STAPLEFORD K A,et al.A functional heat shock protein 90chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells[J].J Virol,2007,81(16):8412-8420.
[25] MILLER D J,AHLQUIST P.Flock House virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion[J].Journal of Virology,2002,76(19):9856-9867.
[26] PALL G S,CODONY-SERVAT C,BYRNE J,et al.Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA,miRNA and piRNA by northern blot[J].Nucleic Acids Res,2007,35(8):e60.
[27] SHI R,CHIANG V L.Facile means for quantifying microRNA expression by real-time PCR[J].Biotechniques,2005,39(4):519-525.
[28] CHENG A,LI M,LIANG Y,et al.Stem-loop RT-PCR quantification of siRNAs in vitro and in vivo[J].Oligonucleotides,2009,19(2):203-208.
[29] LU R,MADURO M,LI F,et al.Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans[J].Nature,2005,436(7053):1040-1043.
[30] PELTIER H J,LATHAM G J.Normalization of microRNA expression levels in quantitative RT-PCR assays:Identification of suitable reference RNA targets in normal and cancerous human solid tissues[J].RNA,2008,14(5):844-852.
[31] GUO Z,LI Y,DING S W.Small RNA-based antimicrobial immunity[J].Nat Rev Immunol,2018,19(1):31-44.
[32] LINDENBACH B D,SGRO J Y,AHLQUIST P.Long-distance base pairing in flock house virus RNA1regulates subgenomic RNA3synthesis and RNA2replication[J].Journal of Virology,2002,76(8):3905-3919.
[33] HEID C A,STEVENS J,LIVAK K J,et al.Real time quantitative PCR[J].Genome Res,1996,6(10):986-994.
[34] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method[J].Methods,2001,25(4):402-408.
[35]徐静,谢翊,管大伟,等,含内参的登革病毒与基孔肯雅病毒3重荧光定量PCR检测方法的建立与评估[J].中国病原生物学杂志,2016,11(4):301-311.