尼罗罗非鱼IgT重链的蛋白表达及表达分析
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摘要
IgT(Immunoglobulin T)作为新型免疫球蛋白在粘膜免疫中具有重要作用.文中根据尼罗罗非鱼IgT基因序列构建原核表达载体获得IgT重组蛋白,相对分子量约75 k Da,并制备出效价较高的小鼠抗罗非鱼IgT的多克隆抗体.荧光定量PCR检测证明:健康尼罗罗非鱼中IgT在多种组织中都有表达,其中脾脏、皮肤、肠和鳃中表达量相对较高.无乳链球菌感染后,皮肤和头肾中IgT mRNA的相对表达量分别在12 h和4 d显著上调.免疫组化结果显示:IgT蛋白在头肾和皮肤中均有少量分布,在无乳链球菌刺激后,在皮肤中出现大量分布.以上结果表明:IgT在尼罗罗非鱼皮肤粘膜免疫中具有重要作用.
As a new type of immunoglobulin,Ig T(Immunoglobulin T) plays an important role in mucosal immunity. The prokaryotic expression vector of Nile tilapia Ig T was constructed according to its gene sequence to obtain the Ig T recombinant protein,weighing 75×103 Da approximately,and the mouse polyclonal antibody against Ig T with high titer was prepared. Expression analysis showed that Ig T was expressed in various tissues,and the relatively high expression was in spleen,skin,intestine and gills. The relative expression of Ig T mRNA in the head kidney and skin was significantly up-regulated in 12 h and 4 d after the infection of S.agalactiae. The results of immunohistochemistry revealed that Ig T protein had a small amount of distribution in the head kidney and skin; however,there was an obvious up-regulation in the skin after the stimulation of S. agalactiae. Taken together,the results of this study indicate that Ig T plays a main role in mucosal immunity of Nile tilapia.
As a new type of immunoglobulin,Ig T(Immunoglobulin T) plays an important role in mucosal immunity. The prokaryotic expression vector of Nile tilapia Ig T was constructed according to its gene sequence to obtain the Ig T recombinant protein,weighing 75×103 Da approximately,and the mouse polyclonal antibody against Ig T with high titer was prepared. Expression analysis showed that Ig T was expressed in various tissues,and the relatively high expression was in spleen,skin,intestine and gills. The relative expression of Ig T mRNA in the head kidney and skin was significantly up-regulated in 12 h and 4 d after the infection of S.agalactiae. The results of immunohistochemistry revealed that Ig T protein had a small amount of distribution in the head kidney and skin; however,there was an obvious up-regulation in the skin after the stimulation of S. agalactiae. Taken together,the results of this study indicate that Ig T plays a main role in mucosal immunity of Nile tilapia.
引文
[1]WARR G W.The immunoglobulin genes of fish[J].Developmental&Comparative Immunology,1995,19(1):1-12.
[2]罗晓春,谢明权,黄玮,等.鱼类粘膜免疫研究进展[J].水产学报,2005,29(3):411-416.LUO X C,XIE M Q,HUANG W,et al.Review of fish mucosal immunity research[J].Journal of Fisheries of China,2005,29(3):411-416.
[3]DANILOVA N,BUSSMANN J K,STEINER L.The immunoglobulin heavy-chain locus in zebrafish:identification and expression of a previously unknown isotype,immunoglobulin Z[J].Nature Immunology,2005,6(3):295-302.
[4]HANSEN J D,LANDIS E D,PHILLIPS R B.Discovery of a unique Ig heavy-chain isotype(Ig T)in rainbow trout:implications for a distinctive B cell developmental pathway in teleost fish[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(19):6919-6924.
[5]SAVAN R,AMAN A,SATO K,et al.Discovery of a new class of immunoglobulin heavy chain from fugu[J].European Journal of Immunology,2005,35(11):3320-3331.
[6]YASUIKE M,de BOER J,von SCHALBURG K R,et al.Evolution of duplicated Ig H loci in Atlantic salmon,Salmo salar[J].BMC Genomics,2010,11(1):486.
[7]RYO S,WIJDEVEN R H M,TYAGI A,et al.Common carp have two subclasses of bonyfish specific antibody Ig Z showing differential expression in response to infection[J].Developmental&Comparative Immunology,2010,34(11):1183-1190.
[8]ZHANG Y A,SALINAS I,SUNYER J O.Recent findings on the structure and function of teleost Ig T[J].Fish&Shellfish Immunology,2011,31(5):627-634.
[9]HU Y L,XIANG L X,SHAO J Z.Identification and characterization of a novel immunoglobulin Z isotype in zebrafish:implications for a distinct B cell receptor in lower vertebrates[J].Molecular Immunology,2010,47(4):738-746.
[10]ZHANG Y A,SALINAS I,LI J,et al.Ig T,a primitive immunoglobulin class specialized in mucosal immunity[J].Nature Immunology,2010,11(9):827-835.
[11]XU Z,PARRA D,GMEZ D,et al.Teleost skin,an ancient mucosal surface that elicits gut-like immune responses[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(32):13097-13102.
[12]XU Z,TAKIZAWA F,PARRA D,et al.Mucosal immunoglobulins at respiratory surfaces mark an ancient association that predates the emergence of tetrapods[J].Nature Communications,2016,7:10728.
[13]WANG B,JIAN J,LU Y,et al.Complete genome sequence of Streptococcus agalactiae ZQ0910,a pathogen causing meningoencephalitis in the GIFT strain of nile tilapia(Oreochromis niloticus)[J].Journal of Bacteriology,2012,194(18):5132-5133.
[14]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[15]TIANJ Y,SUN B J,LUO Y P,et al.Distribution of Ig M,Ig D and Ig Z in mandarin fish,Siniperca chuatsi lymphoid tissues and their transcriptional changes after Flavobacterium columnare stimulation[J].Aquaculture,2009,288(1):14-21.
[16]王片片,刘春,李凯彬,等.剑尾鱼Ig Z基因的克隆及疫苗免疫对其在组织中表达的影响[J].水产学报,2015,39(3):318-326.WANG P P,LIU C,LI K B,et al.Identification and expression analysis of Ig Z gene in swordtail fish(Xiphophorus hellri)responses to vaccine challenges[J].Fisheries of China,2015,39(3):318-326.
[17]KATO G,TAKANO T,SAKAI T,et al.Cloning and expression analyses of a unique Ig T in ayu Plecoglossus altivelis[J].Fisheries Science,2015,81(1):29-36.
[18]SALINAS I,ZHANG Y A,SUNYER J O.Mucosal immunoglobulins and B cells of teleost fish[J].Developmental&Comparative Immunology,2011,35(12):1346-1365.
[19]严伟,肖凡书,聂品.草鱼Ig M、Ig D和Ig Z的抗体制备与组织表达分析[J].水生生物学报,2012,36(1):35-40.YAN W,XIAO F S,NIE P.Preparation of polyclonal antibody antibody against Ig M,Ig Z and Ig D in grass crap(Ctenopharyngodon idellus)and expression analysis in tissues[J].Acta Hydrobiologica Sinica,2012,36(1):35-40.
[2]罗晓春,谢明权,黄玮,等.鱼类粘膜免疫研究进展[J].水产学报,2005,29(3):411-416.LUO X C,XIE M Q,HUANG W,et al.Review of fish mucosal immunity research[J].Journal of Fisheries of China,2005,29(3):411-416.
[3]DANILOVA N,BUSSMANN J K,STEINER L.The immunoglobulin heavy-chain locus in zebrafish:identification and expression of a previously unknown isotype,immunoglobulin Z[J].Nature Immunology,2005,6(3):295-302.
[4]HANSEN J D,LANDIS E D,PHILLIPS R B.Discovery of a unique Ig heavy-chain isotype(Ig T)in rainbow trout:implications for a distinctive B cell developmental pathway in teleost fish[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(19):6919-6924.
[5]SAVAN R,AMAN A,SATO K,et al.Discovery of a new class of immunoglobulin heavy chain from fugu[J].European Journal of Immunology,2005,35(11):3320-3331.
[6]YASUIKE M,de BOER J,von SCHALBURG K R,et al.Evolution of duplicated Ig H loci in Atlantic salmon,Salmo salar[J].BMC Genomics,2010,11(1):486.
[7]RYO S,WIJDEVEN R H M,TYAGI A,et al.Common carp have two subclasses of bonyfish specific antibody Ig Z showing differential expression in response to infection[J].Developmental&Comparative Immunology,2010,34(11):1183-1190.
[8]ZHANG Y A,SALINAS I,SUNYER J O.Recent findings on the structure and function of teleost Ig T[J].Fish&Shellfish Immunology,2011,31(5):627-634.
[9]HU Y L,XIANG L X,SHAO J Z.Identification and characterization of a novel immunoglobulin Z isotype in zebrafish:implications for a distinct B cell receptor in lower vertebrates[J].Molecular Immunology,2010,47(4):738-746.
[10]ZHANG Y A,SALINAS I,LI J,et al.Ig T,a primitive immunoglobulin class specialized in mucosal immunity[J].Nature Immunology,2010,11(9):827-835.
[11]XU Z,PARRA D,GMEZ D,et al.Teleost skin,an ancient mucosal surface that elicits gut-like immune responses[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(32):13097-13102.
[12]XU Z,TAKIZAWA F,PARRA D,et al.Mucosal immunoglobulins at respiratory surfaces mark an ancient association that predates the emergence of tetrapods[J].Nature Communications,2016,7:10728.
[13]WANG B,JIAN J,LU Y,et al.Complete genome sequence of Streptococcus agalactiae ZQ0910,a pathogen causing meningoencephalitis in the GIFT strain of nile tilapia(Oreochromis niloticus)[J].Journal of Bacteriology,2012,194(18):5132-5133.
[14]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[15]TIANJ Y,SUN B J,LUO Y P,et al.Distribution of Ig M,Ig D and Ig Z in mandarin fish,Siniperca chuatsi lymphoid tissues and their transcriptional changes after Flavobacterium columnare stimulation[J].Aquaculture,2009,288(1):14-21.
[16]王片片,刘春,李凯彬,等.剑尾鱼Ig Z基因的克隆及疫苗免疫对其在组织中表达的影响[J].水产学报,2015,39(3):318-326.WANG P P,LIU C,LI K B,et al.Identification and expression analysis of Ig Z gene in swordtail fish(Xiphophorus hellri)responses to vaccine challenges[J].Fisheries of China,2015,39(3):318-326.
[17]KATO G,TAKANO T,SAKAI T,et al.Cloning and expression analyses of a unique Ig T in ayu Plecoglossus altivelis[J].Fisheries Science,2015,81(1):29-36.
[18]SALINAS I,ZHANG Y A,SUNYER J O.Mucosal immunoglobulins and B cells of teleost fish[J].Developmental&Comparative Immunology,2011,35(12):1346-1365.
[19]严伟,肖凡书,聂品.草鱼Ig M、Ig D和Ig Z的抗体制备与组织表达分析[J].水生生物学报,2012,36(1):35-40.YAN W,XIAO F S,NIE P.Preparation of polyclonal antibody antibody against Ig M,Ig Z and Ig D in grass crap(Ctenopharyngodon idellus)and expression analysis in tissues[J].Acta Hydrobiologica Sinica,2012,36(1):35-40.