大黄鱼(Larimichthys crocea)白细胞介素10(IL-10)基因克隆及溶藻弧菌(Vibrio alginolyticus)侵染后表达变化分析
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摘要
白细胞介素10(interleukin10,IL-10)是鱼类先天性免疫因子之一,在炎症反应和免疫调节中有着重要作用。本研究获得大黄鱼(Larimichthys crocea)IL-10基因cDNA序列,由899个核苷酸组成,含1个555bp的开放阅读框、编码184个氨基酸,预测编码蛋白分子量为21.24kDa、N端有22个氨基酸组成的信号肽序列。氨基酸序列多重比对表明大黄鱼IL-10具有IL-10家族特征性序列和构成2对二硫键的4个保守性半胱氨酸,与欧洲鲈(Dicentrarchuslabrax)IL-10序列相似性最高(78.5%);系统进化树分析显示大黄鱼IL-10和其他鱼类IL-10形成一个大簇,与欧洲鲈进化关系最近。荧光定量PCR(quantitative real-time PCR, qRT-PCR)检测结果显示健康大黄鱼IL-10基因在鳃和肠中高表达,肝和头肾中低表达;溶藻弧菌(Vibrio alginolyticus)侵染后大黄鱼肠、脑、脾、肝和头肾中IL-10基因表达量均显著上调(P<0.05),其中头肾和肝中上调幅度最大(分别为菌侵染前的10.06倍和8.79倍)。综上,大黄鱼IL-10基因表达与病原菌感染密切相关,为深入研究大黄鱼IL-10的生物学功能及免疫机制提供了基础资料。
In teleost, interleukin 10(IL-10) is an immune factors of the innate immunity system, and it plays an important role in inflammatory responses and immunoregulation. In this study, IL-10 cDNA was cloned and characterized from large yellow croaker Larimichthys crocea. The results showed that L. crocea IL-10 cDNA contained 899 nucleotides with an open reading frame of 555 nucleotides encoding a ploy peptide of 184 amino acids. The molecular mass of L. crocea IL-10 protein was 21.24 kDa, and the 22 amino acid of the N-terminal region formed signal peptide. The multiple sequence alignment of amino acid sequences indicated that L. crocea IL-10 possessed the IL-10 family signature motif, and it had four conserved cysteine residues to form two disulfide bridges. L. crocea IL-10 shared the highest amino acid similarity(78.5%) with IL-10 of European seabass Dicentrarchus labrax. Phylogenetic tree analysis also confirmed that L. crocea IL-10 fell into the fish IL-10 cluster and was most closely related to that of D. labrax. In health L. crocea, quantitative real-time PCR(qRT-PCR) analysis showed that IL-10 mRNA had high expression level in the gill and intestine, and the low expression level was shown in the liver and head kidney. Upon Vibrio alginolyticus infection, L. crocea IL-10 transcripts up-regulated significantly in the intestine, brain, spleen, liver, and head kidney compared to that before the infection(P<0.05). Among these tissues, the head kidney had the most increase amplitude of IL-10 expression(10.06 fold) after V. alginolyticus infection, and followed by the liver(8.79 fold). Therefore, IL-10 gene is closely related to the pathogenic bacteria infection of L. crocea, and these data could be helpful to understand the biological function and immunologic mechanism of L. crocea IL-10 gene.
In teleost, interleukin 10(IL-10) is an immune factors of the innate immunity system, and it plays an important role in inflammatory responses and immunoregulation. In this study, IL-10 cDNA was cloned and characterized from large yellow croaker Larimichthys crocea. The results showed that L. crocea IL-10 cDNA contained 899 nucleotides with an open reading frame of 555 nucleotides encoding a ploy peptide of 184 amino acids. The molecular mass of L. crocea IL-10 protein was 21.24 kDa, and the 22 amino acid of the N-terminal region formed signal peptide. The multiple sequence alignment of amino acid sequences indicated that L. crocea IL-10 possessed the IL-10 family signature motif, and it had four conserved cysteine residues to form two disulfide bridges. L. crocea IL-10 shared the highest amino acid similarity(78.5%) with IL-10 of European seabass Dicentrarchus labrax. Phylogenetic tree analysis also confirmed that L. crocea IL-10 fell into the fish IL-10 cluster and was most closely related to that of D. labrax. In health L. crocea, quantitative real-time PCR(qRT-PCR) analysis showed that IL-10 mRNA had high expression level in the gill and intestine, and the low expression level was shown in the liver and head kidney. Upon Vibrio alginolyticus infection, L. crocea IL-10 transcripts up-regulated significantly in the intestine, brain, spleen, liver, and head kidney compared to that before the infection(P<0.05). Among these tissues, the head kidney had the most increase amplitude of IL-10 expression(10.06 fold) after V. alginolyticus infection, and followed by the liver(8.79 fold). Therefore, IL-10 gene is closely related to the pathogenic bacteria infection of L. crocea, and these data could be helpful to understand the biological function and immunologic mechanism of L. crocea IL-10 gene.
引文
丰培金,2006.鱼类的白细胞介素研究进展.安徽农业科学,34(17):4317-4318,4320
井申荣,邹全明,2006.白细胞介素10的研究进展.免疫学杂志,22(3):26-28,32
农业部渔业渔政管理局,2016.中国渔业统计年鉴.北京:中国农业出版社,28-32
肖凡书,昌鸣先,孙军等,2006.鲢(Hypophthalmichthys molitrix)IL-10基因的克隆及表达分析.自然科学进展,16(2):183-189
高珊,余涛,周景祥等,2014.鱼类白介素及其受体的研究.水产学杂志,27(03):62-64
曹飞飞,朱凝瑜,郑晓叶,2017.2014-2016年浙江省大黄鱼养殖病害测报及防治建议.浙江农业科学,58(6):1043-1047
Blanco P,Palucka A K,Pascual V et al,2008.Dendritic cells and cytokines in human inflammatory and autoimmune diseases.Cytokine&Growth Factor Reviews,19(1):41-52
Buonocore F,Randelli E,Bird S et al,2007.Interleukin-10expression by real-time PCR and homology modelling analysis in the European sea bass(Dicentrarchus labrax L.).Aquaculture,270(1-4):512-522
Ding Y,Ao J Q,Ai C X et al,2016.Molecular and functional identification of three interleukin-17A/F(IL-17A/F)homologues in large yellow croaker(Larimichthys crocea).Developmental&Comparative Immunology,55:221-232
Fiorentino D F,Zlotnik A,Mosmann T R et al,1991.IL-10inhibits cytokine production by activated macrophages.Journal of Immunology,147(11):3815-3822
Grayfer L,Hodgkinson J W,Hitchen S J et al,2011.Characterization and functional analysis of goldfish(Carassius auratus L.)interleukin-10.Molecular Immunology,48(4):563-571
Inoue Y,Kamota S,Ito K et al,2005.Molecular cloning and expression analysis of rainbow trout(Oncorhynchus mykiss)interleukin-10 cDNAs.Fish&Shellfish Immunology,18(4):335-344
Jiang X Y,Zhang C,Zhao Y J et al,2016.Immune effects of the vaccine of live attenuated Aeromonas hydrophila screened by rifampicin on common carp(Cyprinus carpio L).Vaccine,34(27):3087-3092
Kongchum P,Sandel E,Lutzky S et al,2011.Association between IL-10a single nucleotide polymorphisms and resistance to cyprinid herpesvirus-3 infection in common carp(Cyprinus carpio).Aquaculture,315(3-4):417-421
Li C,Yao C L,2013.Molecular and expression characterizations of interleukin-8 gene in large yellow croaker(Larimichthys crocea).Fish&Shellfish Immunology,34(3):799-809
Livak K J,Schmittgen T D,2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-(35)(35)CT method.Methods,25(4):402-408
Magnadóttir B,2006.Innate immunity of fish(overview).Fish&Shellfish Immunology,20(2):137-151
Moore K W,de Waal Malefyt R,Coffman R L et al,2001.Interleukin-10 and the Interleukin-10 receptor.Annual Review of Immunology,19:683-765
Mu Y N,Wang K R,Ao J Q et al,2015.Molecular characterization and biological effects of a CXCL8homologue in large yellow croaker(Larimichthys crocea).Fish&Shellfish Immunology,44(2):462-470
Peng Y H,Cai X H,Zhang G Y et al,2017.Molecular characterization and expression of interleukin-10 and interleukin-22 in golden pompano(Trachinotus ovatus)in response to Streptococcus agalactiae stimulus.Fish&Shellfish Immunology,65:244-255
Sabat R,Grutz G,Warszawska K et al,2010.Biology of interleukin-10.Cytokine&Growth Factor Reviews,21(5):331-344
Swain B,Samanta M,Basu M et al,2012.Molecular characterization,inductive expression and mechanism of interleukin-10 gene induction in the Indian major carp,catla(Catla catla).Aquaculture Research,43(6):897-907
Windsor W T,Syto R,Tsarbopoulos A et al,1993.Disulfide bond assignments and secondary structure analysis of human and murine interleukin 10.Biochemistry,32(34):8807-8815
Wu C W,Zhang D,Kan M Y et al,2014.The draft genome of the large yellow croaker reveals well-developed innate immunity.Nature Communication,5:5227
Wu J,Shi Y H,Zhang X H et al,2015.Molecular characterization of an IL-1?gene from the large yellow croaker(Larimichthys crocea)and its effect on fish defense against Vibrio alginolyticus infection.Zoological Research,36(1):133-141
Zhang D C,Shao Y Q,Huang Y Q et al,2005.Cloning,characterization and expression analysis of interleukin-10from the zebrafish(Danio rerion).Journal of Biochemistry and Molecular Biology,38(5):571-576
Zou J,Clark M S,Secombes C J,2003.Characterisation,expression and promoter analysis of an interleukin 10homologue in the puffer fish,Fugu rubripes.Immunogenetics,55(5):325-335
井申荣,邹全明,2006.白细胞介素10的研究进展.免疫学杂志,22(3):26-28,32
农业部渔业渔政管理局,2016.中国渔业统计年鉴.北京:中国农业出版社,28-32
肖凡书,昌鸣先,孙军等,2006.鲢(Hypophthalmichthys molitrix)IL-10基因的克隆及表达分析.自然科学进展,16(2):183-189
高珊,余涛,周景祥等,2014.鱼类白介素及其受体的研究.水产学杂志,27(03):62-64
曹飞飞,朱凝瑜,郑晓叶,2017.2014-2016年浙江省大黄鱼养殖病害测报及防治建议.浙江农业科学,58(6):1043-1047
Blanco P,Palucka A K,Pascual V et al,2008.Dendritic cells and cytokines in human inflammatory and autoimmune diseases.Cytokine&Growth Factor Reviews,19(1):41-52
Buonocore F,Randelli E,Bird S et al,2007.Interleukin-10expression by real-time PCR and homology modelling analysis in the European sea bass(Dicentrarchus labrax L.).Aquaculture,270(1-4):512-522
Ding Y,Ao J Q,Ai C X et al,2016.Molecular and functional identification of three interleukin-17A/F(IL-17A/F)homologues in large yellow croaker(Larimichthys crocea).Developmental&Comparative Immunology,55:221-232
Fiorentino D F,Zlotnik A,Mosmann T R et al,1991.IL-10inhibits cytokine production by activated macrophages.Journal of Immunology,147(11):3815-3822
Grayfer L,Hodgkinson J W,Hitchen S J et al,2011.Characterization and functional analysis of goldfish(Carassius auratus L.)interleukin-10.Molecular Immunology,48(4):563-571
Inoue Y,Kamota S,Ito K et al,2005.Molecular cloning and expression analysis of rainbow trout(Oncorhynchus mykiss)interleukin-10 cDNAs.Fish&Shellfish Immunology,18(4):335-344
Jiang X Y,Zhang C,Zhao Y J et al,2016.Immune effects of the vaccine of live attenuated Aeromonas hydrophila screened by rifampicin on common carp(Cyprinus carpio L).Vaccine,34(27):3087-3092
Kongchum P,Sandel E,Lutzky S et al,2011.Association between IL-10a single nucleotide polymorphisms and resistance to cyprinid herpesvirus-3 infection in common carp(Cyprinus carpio).Aquaculture,315(3-4):417-421
Li C,Yao C L,2013.Molecular and expression characterizations of interleukin-8 gene in large yellow croaker(Larimichthys crocea).Fish&Shellfish Immunology,34(3):799-809
Livak K J,Schmittgen T D,2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-(35)(35)CT method.Methods,25(4):402-408
Magnadóttir B,2006.Innate immunity of fish(overview).Fish&Shellfish Immunology,20(2):137-151
Moore K W,de Waal Malefyt R,Coffman R L et al,2001.Interleukin-10 and the Interleukin-10 receptor.Annual Review of Immunology,19:683-765
Mu Y N,Wang K R,Ao J Q et al,2015.Molecular characterization and biological effects of a CXCL8homologue in large yellow croaker(Larimichthys crocea).Fish&Shellfish Immunology,44(2):462-470
Peng Y H,Cai X H,Zhang G Y et al,2017.Molecular characterization and expression of interleukin-10 and interleukin-22 in golden pompano(Trachinotus ovatus)in response to Streptococcus agalactiae stimulus.Fish&Shellfish Immunology,65:244-255
Sabat R,Grutz G,Warszawska K et al,2010.Biology of interleukin-10.Cytokine&Growth Factor Reviews,21(5):331-344
Swain B,Samanta M,Basu M et al,2012.Molecular characterization,inductive expression and mechanism of interleukin-10 gene induction in the Indian major carp,catla(Catla catla).Aquaculture Research,43(6):897-907
Windsor W T,Syto R,Tsarbopoulos A et al,1993.Disulfide bond assignments and secondary structure analysis of human and murine interleukin 10.Biochemistry,32(34):8807-8815
Wu C W,Zhang D,Kan M Y et al,2014.The draft genome of the large yellow croaker reveals well-developed innate immunity.Nature Communication,5:5227
Wu J,Shi Y H,Zhang X H et al,2015.Molecular characterization of an IL-1?gene from the large yellow croaker(Larimichthys crocea)and its effect on fish defense against Vibrio alginolyticus infection.Zoological Research,36(1):133-141
Zhang D C,Shao Y Q,Huang Y Q et al,2005.Cloning,characterization and expression analysis of interleukin-10from the zebrafish(Danio rerion).Journal of Biochemistry and Molecular Biology,38(5):571-576
Zou J,Clark M S,Secombes C J,2003.Characterisation,expression and promoter analysis of an interleukin 10homologue in the puffer fish,Fugu rubripes.Immunogenetics,55(5):325-335