斑节对虾Toll9受体基因免疫功能的研究与表达分析
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摘要
Toll受体蛋白(Toll receptors)是一类重要的模式识别受体,在无脊椎动物先天性免疫系统中发挥重要作用。文章对斑节对虾(Penaeus monodon)新型Toll9受体基因(Pm Toll9)进行了研究:以人源胚胎肾细胞(HEK293T)成功构建体外细胞免疫模型,通过免疫印迹方法证实Pm Toll9重组真核蛋白可在HEK293T中成功表达。双荧光素酶报告系统检测发现在200 ng转染浓度处Pm Toll9对NF-κB报告基因的激活效果显著。q RT-PCR数据证明PmToll9成功激活HEK293T细胞Toll-like receptor(TLR)信号通路,促进通路下游髓样分化因子(My D88)、肿瘤坏死因子(TNF-α)和白介素(IL-10)的上调表达,同时酶联免疫吸附测定结果证明Pm Toll9可促进TNF-α蛋白表达水平显著上调。斑节对虾体内细菌刺激实验结果显示无乳链球菌(Streptococcus agalactiae)可激活Pm Toll9在肝胰腺、肠、淋巴和鳃中的表达,哈维氏弧菌(Vibrio harveyi)可显著抑制Pm Toll9在肝胰腺中的表达。提示无乳链球菌可通过Pm Toll9激活Toll信号通路,引起机体免疫防御反应,而哈维氏弧菌对此过程具有一定的抑制作用。
Toll receptors are important pattern recognition receptors( PRRs) and play a vital role in the immune system of invertebrates. This paper investigates a new type of Toll receptor from Penaeus monodon( Pm Toll9). By constructing an in vitro cellular immune model in HEK293 T cells,we found that Pm Toll9 could express a recombinant eukaryotic protein in HEK293 T cells successfully with western blot technology. The dual-luciferase reporter system shows that Pm Toll9 could activate NF-κB reporter gene significantly when it was transfected with 200 ng DNA in HEK293 T cells. The q RT-PCR data reaveal that Pm Toll9 can activate TLR signaling pathway of HEK293 T cells and promote high expression level of the downstream factors( My D88,TNF-α and IL-10). Meanwhile,ELISA results confirm that Pm Toll9 could increase TNF-α protein level. The in vivo bacteria stimulation in P. mondon shows that Streptococcusagalactiae could activate the m RNA expression of Pm Toll9 in hepatopancreas,intestine,lymph and gill,while Vibrio harveyi could inhibit the m RNA expression of Pm Toll9 in hepatopancreas. It is revealed that Pm Toll9 could be activated and inhibited by S. agalactiae and V. harveyi via Toll signaling pathway to induce the immune defenses,respectively.
Toll receptors are important pattern recognition receptors( PRRs) and play a vital role in the immune system of invertebrates. This paper investigates a new type of Toll receptor from Penaeus monodon( Pm Toll9). By constructing an in vitro cellular immune model in HEK293 T cells,we found that Pm Toll9 could express a recombinant eukaryotic protein in HEK293 T cells successfully with western blot technology. The dual-luciferase reporter system shows that Pm Toll9 could activate NF-κB reporter gene significantly when it was transfected with 200 ng DNA in HEK293 T cells. The q RT-PCR data reaveal that Pm Toll9 can activate TLR signaling pathway of HEK293 T cells and promote high expression level of the downstream factors( My D88,TNF-α and IL-10). Meanwhile,ELISA results confirm that Pm Toll9 could increase TNF-α protein level. The in vivo bacteria stimulation in P. mondon shows that Streptococcusagalactiae could activate the m RNA expression of Pm Toll9 in hepatopancreas,intestine,lymph and gill,while Vibrio harveyi could inhibit the m RNA expression of Pm Toll9 in hepatopancreas. It is revealed that Pm Toll9 could be activated and inhibited by S. agalactiae and V. harveyi via Toll signaling pathway to induce the immune defenses,respectively.
引文
[1]苏建,戴习林,刘红,等.罗氏沼虾Toll样受体基因c DNA片段的克隆及序列分析[J].上海海洋大学学报,2014,23(3):338-344.
[2]KAWAI T,AKIRA S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors[J].Nat Immunol,2010,11(5):373-384.
[3]NUSSLEIN-VOLHARD C,LOHS-SCHARDIN M,SANDER K,et al.A dorso-ventral shift of embryonic primordia in a new maternaleffect mutant of Drosophila[J].Nature,1980,283(5746):474-476.
[4]ANDERSON K V,BOKLA L,NUSSLEIN-VOLHARD.Establishment of dorsal-ventral polarity in the Drosophila embryo:the induction of polarity by the Toll gene product[J].Cell,1985,42(3):791-798.
[5]ELDON E,KOOYER S,D'EVELYN D,et al.The Drosophila 18wheeler is required for morphogenesis and has striking similarities to Toll[J].Development,1994,120(4):885-899.
[6]YAGI Y,NISHIDA Y,IP Y T.Functional analysis of Toll-related genes in Drosophila[J].Dev Growth Differ,2010,52(9):771-783.
[7]OOI J Y,YAGI Y,HU X,et al.The Drosophila Toll-9 activates a constitutive antimicrobial defense[J].EMBO Rep,2002,3(1):82-87.
[8]BEUTLER B.Inferences,questions and possibilities in Toll-like receptor signaling[J].Nature,2004,430(6996):257-263.
[9]WEST A P,KOBLANSKY A A,GHOSH S.Recognition and signaling by toll-like receptors[J].Annu Rev Cell Biol,2006,22(1):409-437.
[10]李运东,周发林,黄建华,等.斑节对虾组织蛋白酶L基因的克隆及其表达分析[J].南方水产科学,2016,12(3):58-66.
[11]李晶晶,李云,刘红,等.感染溶藻弧菌及白斑综合症病毒后凡纳滨对虾不同组织的Toll样受体基因表达变化研究[J].海洋与湖沼,2016,47(2):476-483.
[12]张涛,黄建华,温为庚,等.WSSV+斑节对虾的血清免疫相关酶对人工感染WSSV粗提液的反应[J].南方水产科学,2013,9(1):35-42.
[13]YANG L S,YIN Z X,LIAO J X,et al.A Toll receptor in shrimp[J].Mol Immunol,2007,44(8):1999-2008.
[14]ARTS J A,CORNELISSEN F H,CIJSOUW T.Molecular cloning and expression of a Toll receptor in the giant tiger shrimp,Penaeus monodon[J].Fish Shellfish Immunol,2007,23(3):504-513.
[15]ASSAVALAPSAKUL W,PANYIM S.Molecular cloning and tissue distribution of the Toll receptor in the black tiger shrimp,Penaeus monodon[J].Genet Mol Res,2012,11(1):484-493.
[16]刘文静.斑节对虾TLR22及Relish基因的克隆与表达分析[D].上海:上海海洋大学,2012:13-44.
[17]YANG C,ZHANG J,LI F,et al.A Toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection[J].Fish Shellfish Immunol,2008,24(5):564-574.
[18]MEKATA T,KONO T,YOSHIDA T,et al.Identification of c DNA encoding Toll receptor,Mj Toll gene from kuruma shrimp,Marsupenaeus japonicas[J].Fish Shellfish Immunol,2008,24(1):122-133.
[19]SRISUK C,LONGYANT S,SENAPIN S,et al.Molecular cloning and characterization of a Toll receptor gene from Macrobrachium rosenbergii[J].Fish Shellfish Immunol,2014,36(2):552-562.
[20]WANG Z,CHEN Y H,DAI Y J,et al.A novel vertebrates Tolllike receptor counterpart regulating the antimicrobial peptides expression in the freshwater crayfish,Procambarus clarkia[J].Fish Shellfish Immunol,2015,43(1):219-229.
[21]WANG H C,TSENG C W,LIN H Y,et al.RNAi knock-down of the Litopenaeus vannamei Toll gene(Lv Toll)significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi[J].Dev Comp Immunol,2010,34(1):49-58.
[22]WANG P H,LIANG J P,GU Z H,et al.Molecular cloning,characterization and expression analysis of two novel Tolls(Lv Toll2and Lv Toll3)and three putative Spatzle-like Toll ligands(Lv Spz1-3)from Litopenaeus vannamei[J].Dev Comp Immunol,2012,36(2):359-371.
[23]YANG L S,LI X L,JIANG S G,et al.Characterization of Argonature2 gene from black tiger shrimp(Penaeus monodon)and its responses to immune challenges[J].Fish Shellfish Immunol,2014,36(1):261-269.
[24]史进选,傅明骏,赵超,等.斑节对虾GRP94基因的克隆及其在不同应激条件下的表达与分析[J].南方水产科学,2016,12(5):61-70.
[25]SUN R,WANG M Q,WANG L L,et al.The immune responses triggered by Cp G ODNs in shrimp Litopenaeus vannamei are associated with Lv Tolls[J].Dev Comp Immunol,2014,43(1):15-22.
[26]乔琨,陈贝,刘智禹,等.海洋贝类Toll样受体及信号通路的研究进展[J].渔业研究,2016,38(6):501-509.
[27]罗兵,李涛,徐元宏,等.Toll样受体的信号转导及抗感染免疫研究进展[J].免疫学杂志,2011,27(2):165-169.
[28]李世芳,刘泽众,常惠芸.细胞因子佐剂研究进展[J].中国畜牧兽医,2017,44(1):296-301.
[29]俞敏,田兆方.急性肺损伤时损伤标志物的研究进展[J].中国当代儿科杂志,2014,16(1):94-98.
[30]XIAO W H,HODGE D R,WANG L H,et al.NF-kappa B activates IL-6 expression through cooperation with c-Jun and IL6-AP1site,but is independent of its IL6-NFkappa B regulatory site in autocrine human multiple myeloma cells[J].Cancer Biol Ther,2004,3(10):1007-1017.
[31]SONG L S,ZHAO J M,ZHANG L,et al.A primitive Toll-like receptor signaling pathway in mollusk Zhikong scallop,Chlamys farreri[J].Dev Comp Immunol,2011,35(4):511-520.
[32]DEEPIKA A,SREEDHATAN K,PARIA A,et al.Toll-pathway in tiger shrimp(Penaeus monodon)responds to white spot syndrome virus infection:evidence through molecular characterisation and expression profiles of My D88,TRAF6 and TLR genes[J].Fish Shellfish Immunol,2014,41(2):441-454.
[33]SHI G F,ZHAO C,FU M J,et al.The immune response of the C-Jun in the black tiger shrimp(Penaeus monodon)after bacterial infection[J].Fish Shellfish Immunol,2017,61:181-186.
[34]JANG I H,CHOSA N,KIM S H,et al.A Sptzle-processing enzyme required for toll signaling activation in Drosophila innate immunity[J].Dev Cell,2006,10(1):45-55.
[35]LAN J F,ZHAO L J,WEI S,et al.Pc Toll2 positively regulates the expression of antimicrobial peptides by promoting Pc ATF4translocation into the nucleus[J].Fish Shellfish Immunol,2016,58:59-66.
[36]韩明.果蝇先天性免疫Toll通路与IMD信号通路相关基因进化机制研究[D].南京:南京师范大学,2014:4-6.
[37]刘甜,罗开臖.果蝇Toll和IMD信号通路中的功能结构域[J].环境昆虫学报,2011,33(3):388-395.
[2]KAWAI T,AKIRA S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors[J].Nat Immunol,2010,11(5):373-384.
[3]NUSSLEIN-VOLHARD C,LOHS-SCHARDIN M,SANDER K,et al.A dorso-ventral shift of embryonic primordia in a new maternaleffect mutant of Drosophila[J].Nature,1980,283(5746):474-476.
[4]ANDERSON K V,BOKLA L,NUSSLEIN-VOLHARD.Establishment of dorsal-ventral polarity in the Drosophila embryo:the induction of polarity by the Toll gene product[J].Cell,1985,42(3):791-798.
[5]ELDON E,KOOYER S,D'EVELYN D,et al.The Drosophila 18wheeler is required for morphogenesis and has striking similarities to Toll[J].Development,1994,120(4):885-899.
[6]YAGI Y,NISHIDA Y,IP Y T.Functional analysis of Toll-related genes in Drosophila[J].Dev Growth Differ,2010,52(9):771-783.
[7]OOI J Y,YAGI Y,HU X,et al.The Drosophila Toll-9 activates a constitutive antimicrobial defense[J].EMBO Rep,2002,3(1):82-87.
[8]BEUTLER B.Inferences,questions and possibilities in Toll-like receptor signaling[J].Nature,2004,430(6996):257-263.
[9]WEST A P,KOBLANSKY A A,GHOSH S.Recognition and signaling by toll-like receptors[J].Annu Rev Cell Biol,2006,22(1):409-437.
[10]李运东,周发林,黄建华,等.斑节对虾组织蛋白酶L基因的克隆及其表达分析[J].南方水产科学,2016,12(3):58-66.
[11]李晶晶,李云,刘红,等.感染溶藻弧菌及白斑综合症病毒后凡纳滨对虾不同组织的Toll样受体基因表达变化研究[J].海洋与湖沼,2016,47(2):476-483.
[12]张涛,黄建华,温为庚,等.WSSV+斑节对虾的血清免疫相关酶对人工感染WSSV粗提液的反应[J].南方水产科学,2013,9(1):35-42.
[13]YANG L S,YIN Z X,LIAO J X,et al.A Toll receptor in shrimp[J].Mol Immunol,2007,44(8):1999-2008.
[14]ARTS J A,CORNELISSEN F H,CIJSOUW T.Molecular cloning and expression of a Toll receptor in the giant tiger shrimp,Penaeus monodon[J].Fish Shellfish Immunol,2007,23(3):504-513.
[15]ASSAVALAPSAKUL W,PANYIM S.Molecular cloning and tissue distribution of the Toll receptor in the black tiger shrimp,Penaeus monodon[J].Genet Mol Res,2012,11(1):484-493.
[16]刘文静.斑节对虾TLR22及Relish基因的克隆与表达分析[D].上海:上海海洋大学,2012:13-44.
[17]YANG C,ZHANG J,LI F,et al.A Toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection[J].Fish Shellfish Immunol,2008,24(5):564-574.
[18]MEKATA T,KONO T,YOSHIDA T,et al.Identification of c DNA encoding Toll receptor,Mj Toll gene from kuruma shrimp,Marsupenaeus japonicas[J].Fish Shellfish Immunol,2008,24(1):122-133.
[19]SRISUK C,LONGYANT S,SENAPIN S,et al.Molecular cloning and characterization of a Toll receptor gene from Macrobrachium rosenbergii[J].Fish Shellfish Immunol,2014,36(2):552-562.
[20]WANG Z,CHEN Y H,DAI Y J,et al.A novel vertebrates Tolllike receptor counterpart regulating the antimicrobial peptides expression in the freshwater crayfish,Procambarus clarkia[J].Fish Shellfish Immunol,2015,43(1):219-229.
[21]WANG H C,TSENG C W,LIN H Y,et al.RNAi knock-down of the Litopenaeus vannamei Toll gene(Lv Toll)significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi[J].Dev Comp Immunol,2010,34(1):49-58.
[22]WANG P H,LIANG J P,GU Z H,et al.Molecular cloning,characterization and expression analysis of two novel Tolls(Lv Toll2and Lv Toll3)and three putative Spatzle-like Toll ligands(Lv Spz1-3)from Litopenaeus vannamei[J].Dev Comp Immunol,2012,36(2):359-371.
[23]YANG L S,LI X L,JIANG S G,et al.Characterization of Argonature2 gene from black tiger shrimp(Penaeus monodon)and its responses to immune challenges[J].Fish Shellfish Immunol,2014,36(1):261-269.
[24]史进选,傅明骏,赵超,等.斑节对虾GRP94基因的克隆及其在不同应激条件下的表达与分析[J].南方水产科学,2016,12(5):61-70.
[25]SUN R,WANG M Q,WANG L L,et al.The immune responses triggered by Cp G ODNs in shrimp Litopenaeus vannamei are associated with Lv Tolls[J].Dev Comp Immunol,2014,43(1):15-22.
[26]乔琨,陈贝,刘智禹,等.海洋贝类Toll样受体及信号通路的研究进展[J].渔业研究,2016,38(6):501-509.
[27]罗兵,李涛,徐元宏,等.Toll样受体的信号转导及抗感染免疫研究进展[J].免疫学杂志,2011,27(2):165-169.
[28]李世芳,刘泽众,常惠芸.细胞因子佐剂研究进展[J].中国畜牧兽医,2017,44(1):296-301.
[29]俞敏,田兆方.急性肺损伤时损伤标志物的研究进展[J].中国当代儿科杂志,2014,16(1):94-98.
[30]XIAO W H,HODGE D R,WANG L H,et al.NF-kappa B activates IL-6 expression through cooperation with c-Jun and IL6-AP1site,but is independent of its IL6-NFkappa B regulatory site in autocrine human multiple myeloma cells[J].Cancer Biol Ther,2004,3(10):1007-1017.
[31]SONG L S,ZHAO J M,ZHANG L,et al.A primitive Toll-like receptor signaling pathway in mollusk Zhikong scallop,Chlamys farreri[J].Dev Comp Immunol,2011,35(4):511-520.
[32]DEEPIKA A,SREEDHATAN K,PARIA A,et al.Toll-pathway in tiger shrimp(Penaeus monodon)responds to white spot syndrome virus infection:evidence through molecular characterisation and expression profiles of My D88,TRAF6 and TLR genes[J].Fish Shellfish Immunol,2014,41(2):441-454.
[33]SHI G F,ZHAO C,FU M J,et al.The immune response of the C-Jun in the black tiger shrimp(Penaeus monodon)after bacterial infection[J].Fish Shellfish Immunol,2017,61:181-186.
[34]JANG I H,CHOSA N,KIM S H,et al.A Sptzle-processing enzyme required for toll signaling activation in Drosophila innate immunity[J].Dev Cell,2006,10(1):45-55.
[35]LAN J F,ZHAO L J,WEI S,et al.Pc Toll2 positively regulates the expression of antimicrobial peptides by promoting Pc ATF4translocation into the nucleus[J].Fish Shellfish Immunol,2016,58:59-66.
[36]韩明.果蝇先天性免疫Toll通路与IMD信号通路相关基因进化机制研究[D].南京:南京师范大学,2014:4-6.
[37]刘甜,罗开臖.果蝇Toll和IMD信号通路中的功能结构域[J].环境昆虫学报,2011,33(3):388-395.