鳜IRAK4基因的克隆、组织表达及病毒感染后表达分析
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摘要
为了研究鳜IRAK4生物学特性及其在抗病毒免疫应答中的作用,根据鳜转录组数据中筛选出的IRAK4 unigene序列设计引物,利用SMART-RACE技术克隆得到CDS全长为1389 bp的c DNA(命名为Sc IRAK),编码462个氨基酸,含有1个N端死亡结构域和1个保守的中央蛋白激酶结构域。采用荧光定量RT-PCR方法分析了Sc IRAK4在健康鳜各组织中的表达差异及病毒感染后在脾脏中的表达变化,结果显示,健康鳜中Sc IRAK4在肝脏中表达量最大,与其他组织差异显著,而在血液、脑和胃中表达量最低;传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)感染鳜后Sc IRAK4的表达量呈现下调趋势,24 h脾脏中的表达量达到最低,为对照组的45%;而鳜弹状病毒(siniperca chuatsi rhabdovirus,SCRV)感染鳜后Sc IRAK4的表达量呈现上调趋势,12 h脾脏中Sc IRAK4的表达量达到最高,为对照组的8.17倍,表明Sc IRAK4在抗ISKNV和SCRV的免疫应答中可能发挥不同的作用。本研究为进一步揭示Sc IRAK4的抗病毒免疫反应机制提供了依据。
As a pivotal signaling mediator of Toll-like receptor(TLR) and interleukin(IL)-1 receptor(IL-1R)signaling cascades, the IL-1R-associated kinase 4(IRAK4) is engaged in the activation of host immunity. To study its biological function in mandarin fish(Siniperca chuatsi), the expression profiling of the gene and its role in immune responses to the infection of infectious spleen and kidney necrosis virus(ISKNV) and S. chuatsi Rhabdovirus(SCRV) were investigated. Based on the unigene sequences of IRKA4 gene which was obtained from the transcriptomic results of S. chuatsi, specific primers were designed and the complete IRKA4 gene(named Sc IRAK) was cloned and sequenced by SMART-RACE. Bioinformatics analysis demonstrated that the CDS of Sc IRAK4 gene was 1389 bp, encoding a 462 amino acid with an N-terminal death domain(DD) and a central protein kinase domain(PKc). The transcription profiles of IRAK4 in the tissues of S. chuatsi were characterized by fluorescent quantitative RT-PCR. The results showed that the highest m RNA expression was found in the liver(P<0.05), followed by that in the muscle, blood, brain and stomach(P<0.05). The transcriptions of the IRAK4 in the spleen of mandarin fish infected with ISKNV or SCRV were furtherly analyzed. The m RNA of Sc IRAK4 was down-regulated in the mandarin fish infected with ISKNV and the lowest transcription was observed at 24 h post infection(P<0.01). By contrast, the m RNA of Sc IRAK4 was up-regulated in the mandarin fish infected with SCRV and the higest transcription was observed at 12 h post of the infection(P<0.01). These findings suggest that Sc IRAK4 plays a crucial and different role in the immune responses of Mandarin fish infected with different viruses.
As a pivotal signaling mediator of Toll-like receptor(TLR) and interleukin(IL)-1 receptor(IL-1R)signaling cascades, the IL-1R-associated kinase 4(IRAK4) is engaged in the activation of host immunity. To study its biological function in mandarin fish(Siniperca chuatsi), the expression profiling of the gene and its role in immune responses to the infection of infectious spleen and kidney necrosis virus(ISKNV) and S. chuatsi Rhabdovirus(SCRV) were investigated. Based on the unigene sequences of IRKA4 gene which was obtained from the transcriptomic results of S. chuatsi, specific primers were designed and the complete IRKA4 gene(named Sc IRAK) was cloned and sequenced by SMART-RACE. Bioinformatics analysis demonstrated that the CDS of Sc IRAK4 gene was 1389 bp, encoding a 462 amino acid with an N-terminal death domain(DD) and a central protein kinase domain(PKc). The transcription profiles of IRAK4 in the tissues of S. chuatsi were characterized by fluorescent quantitative RT-PCR. The results showed that the highest m RNA expression was found in the liver(P<0.05), followed by that in the muscle, blood, brain and stomach(P<0.05). The transcriptions of the IRAK4 in the spleen of mandarin fish infected with ISKNV or SCRV were furtherly analyzed. The m RNA of Sc IRAK4 was down-regulated in the mandarin fish infected with ISKNV and the lowest transcription was observed at 24 h post infection(P<0.01). By contrast, the m RNA of Sc IRAK4 was up-regulated in the mandarin fish infected with SCRV and the higest transcription was observed at 12 h post of the infection(P<0.01). These findings suggest that Sc IRAK4 plays a crucial and different role in the immune responses of Mandarin fish infected with different viruses.
引文
[1]Akira S,Uematsu S,Takeuchi O.Pathogen recognition and innate immunity[J].Cell,2006,124(4):783-801.
[2]Krishnan J,Selvarajoo K,Tsuchiya M,et al.Toll-like receptor signal transduction[J].Experimental and Molecular Medicine,2007,39(4):421-438.
[3]Janssens S,Beyaert R.Functional diversity and regulation of different interleukin-1 receptor-associated kinase(IRAK)family members[J].Molecular Cell,2003,11(2):293-302.
[4]Cao Z,Henzel W J,Gao X.IRAK:a kinase associated with the interleukin-1 receptor[J].Science,1996,271(5252):1128-1131.
[5]Wang H,El maadidi S,FIscher J,et al.A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance[J].Hepatology,2015,62(5):1375-1387.
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[7]Wesche H,Gao X,Li X,et al.IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase(IRAK)family[J].Journal of Biological Chemistry,1999,274(27):19403-19410.
[8]Flannery S,Bowie A G.The interleukin-1 receptorassociated kinases:critical regulators of innate immune signalling[J].Biochemical Pharmacology,2010,80(12):1981-1991.
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[11]Kim T W,Staschk E K,Bulek K,et al.A critical role for IRAK4 kinase activity in Toll-like receptor-mediated innate immunity[J].Journal of Experimental Medicine,2007,204(5):1025-1036.
[12]Qin J,Jiang Z,Qian Y,et al.IRAK4 kinase activity is redundant for interleukin-1(IL-1)receptor-associated kinase phosphorylation and IL-1 responsiveness[J].Journal of Biological Chemistry,2004,279(25):26748-26753.
[13]Qin J,Yao J,Cui G,et al.TLR8-mediated NF-kappa Band JNK activation are TAK1-independent and MEKK3-dependent[J].Journal of Biological Chemistry,2006,281(30):21013-21021.
[14]Song K W,Talamas F X,Suttmann R T,et al.The kinase activities of interleukin-1 receptor associated kinase(IRAK)-1 and 4 are redundant in the control of inflammatory cytokine expression in human cells[J].Molecular Immunology,2009,46(7):1458-1466.
[15]Phelan P E,Mellon M T,Kim C H.Functional characterization of full-length TLR3,IRAK-4,and TRAF6 in zebrafish(Danio rerio)[J].Molecular Immunology,2005,42(9):1057-1071.
[16]Yu Y,Zhong Q,Li C,et al.Identification and characterization of IL-1 receptor-associated kinase-4(IRAK-4)in half-smooth tongue sole Cynoglossus semilaevis[J].Fish&Shellfish Immunology,2012,32(4):609-615.
[17]Liu Y,Yu S,Chai Y,et al.Lipopolysaccharide-induced gene expression of interleukin-1 receptor-associated kinase 4 and interleukin-1βin roughskin sculpin(Trachidermus fasciatus)[J].Fish&Shellfish Immunology,2012,33(4):690-698.
[18]Brietzke A,Goldammer T,Rebl H,et al.Characterization of the interleukin 1 receptor-associated kinase 4(IRAK4)-encoding gene in salmonid fish:the functional copy is rearranged in Oncorhynchus mykiss and that factor can impair TLR signaling in mammalian cells[J].Fish&Shellfish Immunology,2014,36(1):206-214.
[19]Umasuthan N,Bathige S D,Whang I,et al.Insights into molecular profiles and genomic evolution of an IRAK4homolog from rock bream(Oplegnathus fasciatus):immunogen-and pathogen-induced transcriptional expression[J].Fish&Shellfish Immunology,2015,43(2):436-448.
[20]黄郁葱,鲁义善,简纪常,等.红笛鲷IRAK-4基因c DNA全长的克隆及组织表达分析[J].广东海洋大学学报,2015,35(1):18-27.Huang Y C,Lu Y S,Jian J C,et al.Full-length c DNAcloning and tissue expression analysis of IRAK-4 gene from humphead snapper(Lutjanus sanguineus)[J].Journal of Zhanjiang Ocean University,2015,35(1):18-27(in Chinese).
[21]Hu X Q,Fu X Z,Li N Q,et al.Transcriptomic analysis of mandarin fish brain cells infected with infectious spleen and kidney necrosis virus with an emphasis on retinoic acid-inducible gene 1-like receptors and apoptosis pathways[J].Fish&Shellfish Immunology,2015,45(2):619-629.
[22]吴明皇,常藕琴,石存斌,等.鳜鱼TCRα基因实时荧光定量RT-PCR检测方法的建立[J].广东农业科学,2011,38(6):1-5.Wu M H,Chang O Q,Shi C B,et al.Establishment of a real-time fluorescent quantitative RT-PCR assay for detection of Siniperca chuatsi’s TCRαgene[J].Guangdong Agricultural Sciences,2011,38(6):1-5(in Chinese).
[23]时云朵,任燕,张德锋,等.山莨菪碱提高嗜水气单胞菌灭活疫苗浸泡免疫鲫的效果[J].水产学报,2015,39(5):720-727.Shi Y D,Ren Y,Zhang D F,et al.Anisodamine increases efficacy of Aeromonas hydrophila inactivated vaccine in gibel carp Carassius auratus gibelio by immersion immunization[J].Journal of Fishery of China,2015,39(5):720-727(in Chinese).
[24]Fu X Z,Li N Q,Lin Q,et al.Protective immunity against infectious spleen and kidney necrosis virus induced by immunization with DNA plasmid containing mcp gene in Chinese perch Siniperca chuatsi[J].Fish&Shellfish Immunology,2014,40(1):259-266.
[25]Ye H,Arron J R,Lamothe B,et al.Distinct molecular mechanism for initiating TRAF6 signalling[J].Nature,2002,418(6896):443-447.
[26]Ohnishi H,Tochio H,Kato Z,et al.Structural basis for the multiple interactions of the My D88 TIR domain in TLR4 signaling[J].Proceedings of the Ntional Aademy of Siences of The United States of America,2009,106(25):10260-10265.
[27]Bakkemo K R,Mikkelsen H,Bordevik M,et al.Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod(Gadus morhua)macrophages[J].Fish&Shellfish Immunology,2011,31(6):993-1004.
[28]Yamashita M,Yamashita Y,Ando T,et al.Identification and determination of selenoneine,2-Selenyl-N-alpha,N-alpha,N-alpha-Trimethyl-L-Histidine,as the major organic selenium in blood cells in a fish-eating population on remote Japanese Islands[J].Biological Trace Element Research,2013,156(1-3):36-44.
[29]MEnke a L,Spitsbergen J M,Wolterbeek A P,et al.Normal anatomy and histology of the adult zebrafish[J].Toxicologic Pathology,2011,39(5):759-775.
[30]Uribe C,Folch H,Enriquez R,et al.Innate and adaptive immunity in teleost fish:a review[J].Veterinarni Medicina,2011,56(10):486-503.
[31]Ellis A E.Innate host defense mechanisms of fish against viruses and bacteria[J].Development and Comparative Immunology,2001,25(8-9):827-839.
[32]M?ller A M,Korytá?T,K?llner B,et al.The teleostean liver as an immunological organ:intrahepatic immune cells(IHICs)in healthy and benzo[a]pyrene challenged rainbow trout(Oncorhynchus mykiss)[J].Development and Comparative Immunology,2014,46(2):518-529.
[33]Raida M K,Buchmann K.Innate immune response in rainbow trout(Oncorhynchus mykiss)against primary and secondary infections with Yersinia ruckeri O1[J].Developmental&Comparative Immunology,2009,33(1):35-45.
[34]Roca F J,Sepulcre M P,López-Castejón G,et al.The colony-stimulating factor-1 receptor is a specific marker of macrophages from the bony fish gilthead seabream[J].Molecular Immunology,2006,43(9):1418-1423.
[35]Tsujita T,Tsukada H,Nakao M,et al.Sensing bacterial flagellin by membrane and soluble orthologs of Toll-like receptor 5 in rainbow trout(Onchorhynchus mikiss)[J].Journal of Biological Chemistry,2004,279(47):48588-48597.
[36]Chen W J,Guo C J,Zhou Z C,et al.Molecular cloning of IKKβfrom the mandarin fish(Siniperca chuatsi)and its up-regulation in cells by ISKNV infection[J].Veterinary Immunology and Immunopathology,2011,139(1):61-66.
[37]Suzuki N,Suzuki S,Duncan G S,et al.Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4[J].Nature,2002,416(6882):750-756.
[38]Krug A,French A R,Barchet W,et al.TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NKcell function[J].Immunity,2004,21(1):107-119.
[39]Yang K,Puel A,Zhang S,et al.Human TLR-7-,-8-,and-9-mediated induction of IFN-alpha/beta and-lambda Is IRAK-4 dependent and redundant for protective immunity to viruses[J].Immunity,2005,23(5):465-478.
[2]Krishnan J,Selvarajoo K,Tsuchiya M,et al.Toll-like receptor signal transduction[J].Experimental and Molecular Medicine,2007,39(4):421-438.
[3]Janssens S,Beyaert R.Functional diversity and regulation of different interleukin-1 receptor-associated kinase(IRAK)family members[J].Molecular Cell,2003,11(2):293-302.
[4]Cao Z,Henzel W J,Gao X.IRAK:a kinase associated with the interleukin-1 receptor[J].Science,1996,271(5252):1128-1131.
[5]Wang H,El maadidi S,FIscher J,et al.A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance[J].Hepatology,2015,62(5):1375-1387.
[6]Li Y W,Mo X B,Zhou L,et al.Identification of IRAK-4 in grouper(Epinephelus coioides)that impairs My D88-dependent NF-κB activation[J].Developmental and Comparative Immunology,2014,45(1):190-197.
[7]Wesche H,Gao X,Li X,et al.IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase(IRAK)family[J].Journal of Biological Chemistry,1999,274(27):19403-19410.
[8]Flannery S,Bowie A G.The interleukin-1 receptorassociated kinases:critical regulators of innate immune signalling[J].Biochemical Pharmacology,2010,80(12):1981-1991.
[9]Lin S C,Lo Y C,Wu H.Helical assembly in the My D88-IRAK4-IRAK2 complex in TLR/IL-1Rsignalling[J].Nature,2010,465(7300):885-890.
[10]Li S,Strelow A,Fontana e J,et al.IRAK-4:a novel member of the IRAK family with the properties of an IRAK-kinase[J].Proceedings of the Ntional Aademy of Siences of The United States of America,2002,99(8):5567-5572.
[11]Kim T W,Staschk E K,Bulek K,et al.A critical role for IRAK4 kinase activity in Toll-like receptor-mediated innate immunity[J].Journal of Experimental Medicine,2007,204(5):1025-1036.
[12]Qin J,Jiang Z,Qian Y,et al.IRAK4 kinase activity is redundant for interleukin-1(IL-1)receptor-associated kinase phosphorylation and IL-1 responsiveness[J].Journal of Biological Chemistry,2004,279(25):26748-26753.
[13]Qin J,Yao J,Cui G,et al.TLR8-mediated NF-kappa Band JNK activation are TAK1-independent and MEKK3-dependent[J].Journal of Biological Chemistry,2006,281(30):21013-21021.
[14]Song K W,Talamas F X,Suttmann R T,et al.The kinase activities of interleukin-1 receptor associated kinase(IRAK)-1 and 4 are redundant in the control of inflammatory cytokine expression in human cells[J].Molecular Immunology,2009,46(7):1458-1466.
[15]Phelan P E,Mellon M T,Kim C H.Functional characterization of full-length TLR3,IRAK-4,and TRAF6 in zebrafish(Danio rerio)[J].Molecular Immunology,2005,42(9):1057-1071.
[16]Yu Y,Zhong Q,Li C,et al.Identification and characterization of IL-1 receptor-associated kinase-4(IRAK-4)in half-smooth tongue sole Cynoglossus semilaevis[J].Fish&Shellfish Immunology,2012,32(4):609-615.
[17]Liu Y,Yu S,Chai Y,et al.Lipopolysaccharide-induced gene expression of interleukin-1 receptor-associated kinase 4 and interleukin-1βin roughskin sculpin(Trachidermus fasciatus)[J].Fish&Shellfish Immunology,2012,33(4):690-698.
[18]Brietzke A,Goldammer T,Rebl H,et al.Characterization of the interleukin 1 receptor-associated kinase 4(IRAK4)-encoding gene in salmonid fish:the functional copy is rearranged in Oncorhynchus mykiss and that factor can impair TLR signaling in mammalian cells[J].Fish&Shellfish Immunology,2014,36(1):206-214.
[19]Umasuthan N,Bathige S D,Whang I,et al.Insights into molecular profiles and genomic evolution of an IRAK4homolog from rock bream(Oplegnathus fasciatus):immunogen-and pathogen-induced transcriptional expression[J].Fish&Shellfish Immunology,2015,43(2):436-448.
[20]黄郁葱,鲁义善,简纪常,等.红笛鲷IRAK-4基因c DNA全长的克隆及组织表达分析[J].广东海洋大学学报,2015,35(1):18-27.Huang Y C,Lu Y S,Jian J C,et al.Full-length c DNAcloning and tissue expression analysis of IRAK-4 gene from humphead snapper(Lutjanus sanguineus)[J].Journal of Zhanjiang Ocean University,2015,35(1):18-27(in Chinese).
[21]Hu X Q,Fu X Z,Li N Q,et al.Transcriptomic analysis of mandarin fish brain cells infected with infectious spleen and kidney necrosis virus with an emphasis on retinoic acid-inducible gene 1-like receptors and apoptosis pathways[J].Fish&Shellfish Immunology,2015,45(2):619-629.
[22]吴明皇,常藕琴,石存斌,等.鳜鱼TCRα基因实时荧光定量RT-PCR检测方法的建立[J].广东农业科学,2011,38(6):1-5.Wu M H,Chang O Q,Shi C B,et al.Establishment of a real-time fluorescent quantitative RT-PCR assay for detection of Siniperca chuatsi’s TCRαgene[J].Guangdong Agricultural Sciences,2011,38(6):1-5(in Chinese).
[23]时云朵,任燕,张德锋,等.山莨菪碱提高嗜水气单胞菌灭活疫苗浸泡免疫鲫的效果[J].水产学报,2015,39(5):720-727.Shi Y D,Ren Y,Zhang D F,et al.Anisodamine increases efficacy of Aeromonas hydrophila inactivated vaccine in gibel carp Carassius auratus gibelio by immersion immunization[J].Journal of Fishery of China,2015,39(5):720-727(in Chinese).
[24]Fu X Z,Li N Q,Lin Q,et al.Protective immunity against infectious spleen and kidney necrosis virus induced by immunization with DNA plasmid containing mcp gene in Chinese perch Siniperca chuatsi[J].Fish&Shellfish Immunology,2014,40(1):259-266.
[25]Ye H,Arron J R,Lamothe B,et al.Distinct molecular mechanism for initiating TRAF6 signalling[J].Nature,2002,418(6896):443-447.
[26]Ohnishi H,Tochio H,Kato Z,et al.Structural basis for the multiple interactions of the My D88 TIR domain in TLR4 signaling[J].Proceedings of the Ntional Aademy of Siences of The United States of America,2009,106(25):10260-10265.
[27]Bakkemo K R,Mikkelsen H,Bordevik M,et al.Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod(Gadus morhua)macrophages[J].Fish&Shellfish Immunology,2011,31(6):993-1004.
[28]Yamashita M,Yamashita Y,Ando T,et al.Identification and determination of selenoneine,2-Selenyl-N-alpha,N-alpha,N-alpha-Trimethyl-L-Histidine,as the major organic selenium in blood cells in a fish-eating population on remote Japanese Islands[J].Biological Trace Element Research,2013,156(1-3):36-44.
[29]MEnke a L,Spitsbergen J M,Wolterbeek A P,et al.Normal anatomy and histology of the adult zebrafish[J].Toxicologic Pathology,2011,39(5):759-775.
[30]Uribe C,Folch H,Enriquez R,et al.Innate and adaptive immunity in teleost fish:a review[J].Veterinarni Medicina,2011,56(10):486-503.
[31]Ellis A E.Innate host defense mechanisms of fish against viruses and bacteria[J].Development and Comparative Immunology,2001,25(8-9):827-839.
[32]M?ller A M,Korytá?T,K?llner B,et al.The teleostean liver as an immunological organ:intrahepatic immune cells(IHICs)in healthy and benzo[a]pyrene challenged rainbow trout(Oncorhynchus mykiss)[J].Development and Comparative Immunology,2014,46(2):518-529.
[33]Raida M K,Buchmann K.Innate immune response in rainbow trout(Oncorhynchus mykiss)against primary and secondary infections with Yersinia ruckeri O1[J].Developmental&Comparative Immunology,2009,33(1):35-45.
[34]Roca F J,Sepulcre M P,López-Castejón G,et al.The colony-stimulating factor-1 receptor is a specific marker of macrophages from the bony fish gilthead seabream[J].Molecular Immunology,2006,43(9):1418-1423.
[35]Tsujita T,Tsukada H,Nakao M,et al.Sensing bacterial flagellin by membrane and soluble orthologs of Toll-like receptor 5 in rainbow trout(Onchorhynchus mikiss)[J].Journal of Biological Chemistry,2004,279(47):48588-48597.
[36]Chen W J,Guo C J,Zhou Z C,et al.Molecular cloning of IKKβfrom the mandarin fish(Siniperca chuatsi)and its up-regulation in cells by ISKNV infection[J].Veterinary Immunology and Immunopathology,2011,139(1):61-66.
[37]Suzuki N,Suzuki S,Duncan G S,et al.Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4[J].Nature,2002,416(6882):750-756.
[38]Krug A,French A R,Barchet W,et al.TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NKcell function[J].Immunity,2004,21(1):107-119.
[39]Yang K,Puel A,Zhang S,et al.Human TLR-7-,-8-,and-9-mediated induction of IFN-alpha/beta and-lambda Is IRAK-4 dependent and redundant for protective immunity to viruses[J].Immunity,2005,23(5):465-478.