中华鳖IRF4基因克隆及免疫功能研究
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摘要
干扰素调节因子IRF4基因仅表达于免疫相关细胞,在先天性免疫和特异性免疫中发挥重要作用。本研究根据中华鳖高通量测序预测的IRF4基因序列(GenBank登录号:XM_014581444),设计特异性引物扩增片段验证其编码区;通过荧光定量检测IRF4基因在健康中华鳖中组织表达情况以及脂多糖、聚肌胞苷酸诱导后在外周血淋巴细胞中的表达情况;构建真核表达载体p3xflag-IRF4,检测过表达IRF4基因后细胞对中华鳖虹彩病毒的敏感性,以及对干扰素启动子、NF-κB启动子的活性。试验结果显示,IRF4基因编码区和GenBank中序列完全相同,其推测的氨基酸序列含有1个DNA结合结构域和1个IRF相关结构域;荧光定量结果分析显示,IRF4基因表达于所检测的组织和器官,其中在血液、肝、脾中表达较高,脂多糖和聚肌胞苷酸均能诱导外周血淋巴细胞中IRF4基因的表达;过表达IRF4基因后降低细胞对中华鳖虹彩病毒的敏感性,过表达IRF4基因能显著诱导干扰素启动子活性,抑制NF-κB启动子活性。上述结果为更深入研究中华鳖IRF4参与机体免疫应答提供基础。
A pair of specific primers was designed to qualify the open reading frame(ORF) of IRF4, limited in immune-related cells, deposited in Genbank(XM_014581444)by high-throughput sequencing to evaluate the immune function of IRF4 in soft-shelled turtle Pelodiscus sinensis. The tissue distribution and induction expression in peripheral blood lymphocytes(PBLs) of IRF4 after treatment with lipopolysaccharide(LPS) or polycytidine monophosphate(PolyI:C) were detected by real-time quantitative PCR to examine the sensitivity of STA cells to soft-shelled turtle iridovirus(STIV), and effects on IFN promoter and NF-κB promoter activity was examined in STA cells transfected with overexpression vector p3 xflagIRF4. The results showed that the amplified IRF4 ORF was identical to the sequence deposited in Genbank and the deduced amino acid sequence contains a DBD domain and a IAD domain. Rt-qPCR results indicated that IRF4 was mainly expressed in blood, liver and spleen and both LPS and PolyI:C induced IRF4 mRNA expression level in PBLs. Overexpression IRF4 led to decrease the sensitivity of STA cells to STIV, to activate the IFN promoter activity and inhibit the NF-κB promoter activity. These findings provided a fundation to investigate the mechanism of IRF4 involved in immune response in soft-shelled turtle.
A pair of specific primers was designed to qualify the open reading frame(ORF) of IRF4, limited in immune-related cells, deposited in Genbank(XM_014581444)by high-throughput sequencing to evaluate the immune function of IRF4 in soft-shelled turtle Pelodiscus sinensis. The tissue distribution and induction expression in peripheral blood lymphocytes(PBLs) of IRF4 after treatment with lipopolysaccharide(LPS) or polycytidine monophosphate(PolyI:C) were detected by real-time quantitative PCR to examine the sensitivity of STA cells to soft-shelled turtle iridovirus(STIV), and effects on IFN promoter and NF-κB promoter activity was examined in STA cells transfected with overexpression vector p3 xflagIRF4. The results showed that the amplified IRF4 ORF was identical to the sequence deposited in Genbank and the deduced amino acid sequence contains a DBD domain and a IAD domain. Rt-qPCR results indicated that IRF4 was mainly expressed in blood, liver and spleen and both LPS and PolyI:C induced IRF4 mRNA expression level in PBLs. Overexpression IRF4 led to decrease the sensitivity of STA cells to STIV, to activate the IFN promoter activity and inhibit the NF-κB promoter activity. These findings provided a fundation to investigate the mechanism of IRF4 involved in immune response in soft-shelled turtle.
引文
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[18] 郭琼林,贾伟章.中华鳖造血和免疫器官的个体发育[J].动物学报,2003,49(2),238-247.
[19] Xu Q Q,Yang D Q,Tuo R,et al.Gene cloning and induced expression pattern of IRF4 and IRF10 in the Asian swamp eel (Monopterus albus)[J].Zoological Research,2014,35(5):380-388.
[20] Liu D,Chen J,Zhang H,et al.Interferon regulatory factor 4b (IRF4b) in Japanese flounder,Paralichthys olivaceus:sequencing,ubiquitous tissue distribution and inducible expression by poly(I:C) and DNA virus[J].Developmental and Comparative Immunology,2016(62):127-133.
[21] Tang J T,Jiang L H,Liu W,et al.Expression and functional characterization of interferon regulatory factors 4,8,and 9 in large yellow croaker (Larimichthys crocea)[J].Developmental & Comparative Immunology,2018(78):35-41.
[22] 罗凯,田光明,郭利伟,等.草鱼干扰素调节因子IRF4亚家族的表达研究[J].生物技术,2017,27(1):22-28.
[23] 张珺,杨菁.干扰素调节因子4与T细胞分化及免疫性疾病的研究进展[J].医学综述,2016,22(1):9-12.
[24] Meraro D,Gleit-Kielmanowicz M,Hauser H,et al.IFN-stimulated gene 15 is synergistically activated through interactions between the myelocyte/lymphocyte-specific transcription factors,PU.1,IFN regulatory factor-8/IFN consensus sequence binding protein,and IFN regulatory factor-4:characterization of a new subtype of IFN-stimulated response element[J].The Journal of Immunology,2002,168(12):6224.
[25] Forero A,Moore P S,Sarkar S N.Role of IRF4 in IFN-stimulated gene induction and maintenance of Kaposi sarcoma-associated herpesvirus latency in primary effusion lymphoma cells[J].Journal of Immunology,2013,191(3):1476-1485.
[26] 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.
[27] Brass A L,Kehrli E,Eisenbeis C F,et al.Pip,a lymphoid-restricted IRF,contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1[J].Genes & Development,1996,10(18):2335-2347.
[28] 陈在贤,郑坚川,江育林.从患“红脖子病”甲鱼体分离到虹彩病毒[J].中国兽医学报,1998,18(2):32-36.
[29] 尹梦雅.中华鳖暴发性死亡症流行性调查及病原菌的致病性、耐药性研究[D].武汉:华中农业大学,2018.
[30] 薛俊敬,张飞,阳钢,等.中华鳖“红脖子病”组织病理观察、病原鉴定及药敏试验[J].南昌大学学报:理科版,2017,41(5):504-510.
[31] 赵运旺,朱嘉宁.NF-κB信号通路研究进展[J].甘肃科技,2016,32(21):117-123.
[32] Li S,Guo X,Lu L F,et al.Regulation pattern of fish irf4 (the gene encoding IFN regulatory factor 4) by STAT6,c-Rel and IRF4[J].Developmental & Comparative Immunology,2015,51(1):65-73.
[2] Sen G C.Viruses and interferons[J].Annual Review of Microbiology,2001(55):255-281.
[3] 戴伟,许梓荣.鱼类干扰素系统的研究进展[J].水产科学,2008,27(5):266-270.
[4] Huang B,Qi Z T,Xu Z,et al.Global characterization of interferon regulatory factor (IRF) genes in vertebrates:glimpse of the diversification in evolution[J].BMC Immunology,2010(11):22.
[5] Eisenbeis C F,Singh H,Storb U.Pip,a novel IRF family member,is a lymphoid-specific,PU.1-dependent transcriptional activator[J].Genes & Development,1995,9(11):1377-1387.
[6] Politis A D,Sivo J,Driggers P H,et al.Modulation of interferon consensus sequence binding protein mRNA in murine peritoneal macrophages.Induction by IFN-gamma and down-regulation by IFN-alpha,dexamethasone,and protein kinase inhibitors[J].Journal of Immunology,1992,148(3):801-807.
[7] Shaffer A L,Emre N C,Romesser P B,et al.IRF4:Immunity.Malignancy! Therapy[J].Clinical Cancer Research,2009,15(9):2954-2961.
[8] Xu W D,Pan H F,Ye D Q,et al.Targeting IRF4 in autoimmune diseases[J].Autoimmunity Reviews,2012,11(12):918-924.
[9] Bathige S D,Whang I,Umasuthan N,et al.Interferon regulatory factors 4 and 8 in rock bream,Oplegnathus fasciatus:structural and expressional evidence for their antimicrobial role in teleosts[J].Fish & Shellfish Immunology,2012,33(4):857-871.
[10] Ai K T,Luo K,Li Y S,et al.Expression pattern analysis of IRF4 and its related genes revealed the functional differentiation of IRF4 paralogues in teleost[J].Fish & Shellfish Immunology,2017(60):59-64.
[11] Zhang J,Li Y X,Hu Y H.Molecular characterization and expression analysis of eleven interferon regulatory factors in half-smooth tongue sole,Cynoglossus semilaevis[J].Fish & Shellfish Immunology,2015,44(1):272-282.
[12] Laghari Z A,Li L,Chen S N,et al.Composition and transcription of all interferon regulatory factors (IRFs),IRF1-11 in a perciform fish,the mandarin fish,Siniperca chuatsi[J].Developmental & Comparative Immunology,2018(81):127-140.
[13] Fu J P,Luo Z,Yan Y,et al.Establishment of a cell line from Chinese soft-shelled turtle Pelodiscus sinensis with the practicability of transfection and viral replication[J].Fish Pathology,2013,48(4):126-134.
[14] 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.
[15] Reed L J,Muench H.A simple method of estimating fifty percent endpoints[J].American Journal of Epidemiology,1938,27(3):493-497.
[16] Grumont R J,Gerondakis S.Rel induces interferon regulatory factor 4 (IRF-4) expression in lymphocytes:modulation of interferon-regulated gene expression by rel/nuclear factor kappaB[J].Journal of Experimental Medicine,2000,191(8):1281-1292.
[17] Gupta S,Jiang M,Anthony A,et al.Lineage-specific modulation of interleukin 4 signaling by interferon regulatory factor 4[J].Journal of Experimental Medicine,1999,190(12):1837-1848.
[18] 郭琼林,贾伟章.中华鳖造血和免疫器官的个体发育[J].动物学报,2003,49(2),238-247.
[19] Xu Q Q,Yang D Q,Tuo R,et al.Gene cloning and induced expression pattern of IRF4 and IRF10 in the Asian swamp eel (Monopterus albus)[J].Zoological Research,2014,35(5):380-388.
[20] Liu D,Chen J,Zhang H,et al.Interferon regulatory factor 4b (IRF4b) in Japanese flounder,Paralichthys olivaceus:sequencing,ubiquitous tissue distribution and inducible expression by poly(I:C) and DNA virus[J].Developmental and Comparative Immunology,2016(62):127-133.
[21] Tang J T,Jiang L H,Liu W,et al.Expression and functional characterization of interferon regulatory factors 4,8,and 9 in large yellow croaker (Larimichthys crocea)[J].Developmental & Comparative Immunology,2018(78):35-41.
[22] 罗凯,田光明,郭利伟,等.草鱼干扰素调节因子IRF4亚家族的表达研究[J].生物技术,2017,27(1):22-28.
[23] 张珺,杨菁.干扰素调节因子4与T细胞分化及免疫性疾病的研究进展[J].医学综述,2016,22(1):9-12.
[24] Meraro D,Gleit-Kielmanowicz M,Hauser H,et al.IFN-stimulated gene 15 is synergistically activated through interactions between the myelocyte/lymphocyte-specific transcription factors,PU.1,IFN regulatory factor-8/IFN consensus sequence binding protein,and IFN regulatory factor-4:characterization of a new subtype of IFN-stimulated response element[J].The Journal of Immunology,2002,168(12):6224.
[25] Forero A,Moore P S,Sarkar S N.Role of IRF4 in IFN-stimulated gene induction and maintenance of Kaposi sarcoma-associated herpesvirus latency in primary effusion lymphoma cells[J].Journal of Immunology,2013,191(3):1476-1485.
[26] 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.
[27] Brass A L,Kehrli E,Eisenbeis C F,et al.Pip,a lymphoid-restricted IRF,contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1[J].Genes & Development,1996,10(18):2335-2347.
[28] 陈在贤,郑坚川,江育林.从患“红脖子病”甲鱼体分离到虹彩病毒[J].中国兽医学报,1998,18(2):32-36.
[29] 尹梦雅.中华鳖暴发性死亡症流行性调查及病原菌的致病性、耐药性研究[D].武汉:华中农业大学,2018.
[30] 薛俊敬,张飞,阳钢,等.中华鳖“红脖子病”组织病理观察、病原鉴定及药敏试验[J].南昌大学学报:理科版,2017,41(5):504-510.
[31] 赵运旺,朱嘉宁.NF-κB信号通路研究进展[J].甘肃科技,2016,32(21):117-123.
[32] Li S,Guo X,Lu L F,et al.Regulation pattern of fish irf4 (the gene encoding IFN regulatory factor 4) by STAT6,c-Rel and IRF4[J].Developmental & Comparative Immunology,2015,51(1):65-73.