西伯利亚鲟MHCⅡβ基因克隆、组织分布及细菌感染后的表达变化
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摘要
为研究鲟鱼主要组织相容性复合体(Major histocompatibility complex,MHC)基因的分子特征和表达,利用RACE技术克隆获得了西伯利亚鲟Acipernser baerii MHCⅡβ cDNA序列1443 bp,包括开放阅读框(OFR)801 bp,编码184个氨基酸。Blast分析显示,由MHCⅡβcDNA基因推导的氨基酸序列同其他物种的一致性为46%～83%,通过Smart站点分析,该序列具有SMART MHC_Ⅱ_beta结构域、SMART IGc1结构域和跨膜螺旋结构域;实时定量分析表明,正常情况下MHCⅡβ mRNA在西伯利亚鲟10种组织中均有表达,其中脾、头肾和血液中表达量较高,皮肤和肉中表达量极低(P<0.05);感染维氏气单胞菌Aeromonas veronii 40 h后,脾脏中MHCⅡβ mRNA的表达量显著低于未感染的对照组(P<0.05),直至93 h时恢复至对照组水平且有显著性变化(P<0.05)。研究表明,MHCⅡβ基因参与了西伯利亚鲟的免疫反应。
In this study,major histocompatibility(MHC)Ⅱβ cDNA was cloned from Siberian sturgeon Acipenser baerii by RACE. Sequence comparison indicated that the deduced amino acid sequence with 1443 bp including 801 bp open reading frame deducing 184 amino acids had 46%-83% identity with those of other species. The smart sequence analysis revealed that it had signal peptide, SMART MHC_Ⅱ_beta domain, SMART ⅠGc1 domain and transmembrane helix region. Real-time quantitative RT-PCR indicated that the MHCⅡβ was ubiquitously expressed in ten normal tissues including skin, meat, heart, gill, liver, spleen, intestine, kidney, brain and blood, higher in spleen,kindy and blood, and lower in liver, heart, intestine and brain,and nearly no in muscle and skin. There was significantly lower MHC Ⅱβ expression in spleen of the Siberian sturgeon challenged with bacterium Aeromonas veronii for 40 h than that in the control fish(P<0.05), recover to normal level in 96 h, indicating that MHCⅡβ is involved in the immunity of Sibreian sturgeon.
In this study,major histocompatibility(MHC)Ⅱβ cDNA was cloned from Siberian sturgeon Acipenser baerii by RACE. Sequence comparison indicated that the deduced amino acid sequence with 1443 bp including 801 bp open reading frame deducing 184 amino acids had 46%-83% identity with those of other species. The smart sequence analysis revealed that it had signal peptide, SMART MHC_Ⅱ_beta domain, SMART ⅠGc1 domain and transmembrane helix region. Real-time quantitative RT-PCR indicated that the MHCⅡβ was ubiquitously expressed in ten normal tissues including skin, meat, heart, gill, liver, spleen, intestine, kidney, brain and blood, higher in spleen,kindy and blood, and lower in liver, heart, intestine and brain,and nearly no in muscle and skin. There was significantly lower MHC Ⅱβ expression in spleen of the Siberian sturgeon challenged with bacterium Aeromonas veronii for 40 h than that in the control fish(P<0.05), recover to normal level in 96 h, indicating that MHCⅡβ is involved in the immunity of Sibreian sturgeon.
引文
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[22] Zhou F,Dong Z,Fu Y,et al.Molecular cloning,genomic structure,polymorphism and expression analysis of major histocompatibility complex class Ⅱ B gene of Nile tilapia(Oreochromis niloticus)[J].Aquaculture,2013,372-375(1):149-157.
[23] 田照辉,徐绍刚,胡红霞,等.西伯利亚鲟免疫球蛋白M重链区mRNA的组织分布和细菌感染后的表达[J].大连海洋大学学报,2014,29(3):217-220.
[24] 刘德财,王雪,兰莹,等.林茜草根黄酮对S-180小鼠肿瘤TG-3、MHC-Ⅰ和MHC-Ⅱ蛋白表达的影响[J].黑龙江医药科学,2017(6):22-24.
[25] Bannai H P,Nonaka M.Comprehensive analysis of medaka major histocompatibility complex(MHC) class Ⅱ genes:implications for evolution in teleosts[J].Immunogenetics,2013,65(12):883-895.
[26] Venkatesh B,Ning Y,Brenner S.Late changes in spliceosomal introns define clades in vertebrate evolution[J].Proc Natl Acad Sci U S A,1999,96(18):10267-10271.
[2] 何球藻,吴厚生,曹雪涛.细胞与分子免疫学[M].上海:上海科学技术文献出版社,1997:77-79.
[3] Neilson E G,Couser W G,主编.韩瑞发,姚智,王林主,译.免疫肾脏病学[M].沈阳:辽宁科学技术出版社,2006:177.
[4] Hashimoto K,Nakanishi T,Kurosawa Y.Isolation of carp genes encoding major histocompatibility complex antigens[J].Proceedings of the National Academy of Sciences of the United States of America,1990,87(17):6863-6867.
[5] Sültmann H,Mayer W E,Figueroa F,et al.Organization of Mhc,Class Ⅱ B genes in the zebrafish(Brachydanio rerio )[J].Genomics,1994,23(1):1-14.
[6] Xu T J,Chen S L,Ji X S,et al.Molecular cloning,genomic structure,polymorphism and expression analysis of major histocompatibility complex class ⅡA and ⅡB genes of half-smooth tongue sole(Cynoglossus semilaevis)[J].Fish & Shellfish Immunology,2009,27(2):192-201.
[7] Pang J C,Gao F Y,Lu M X,et al.Major histocompatibility complex class ⅡA and ⅡB genes of Nile tilapia Oreochromis niloticus:genomic structure,molecular polymorphism and expression patterns[J].Fish & Shellfish Immunology,2013,34(2):486.
[8] Chen S L,Zhang Y X,Xu M Y,et al.Molecular polymorphism and expression analysis of MHC class Ⅱ B gene from red sea bream(Chrysophrys major)[J].Developmental & Comparative Immunology,2006,30(4):407-418.
[9] 张玉喜,陈松林.牙鲆MHCⅡB基因多态性及其与鱼体抗病力关系的分析[J ].水产学报,2006,30(5):633-639.
[10] 赵凤岐,曹谨玲,刘青.西伯利亚鲟败血症病理学观察与病原学研究[J].水生生物学报,2009,33(2):316-323.
[11] 马志宏,杨慧,李铁梁,等.西伯利亚鲟(Acipenser baerii)致病性维氏气单胞菌的分离鉴定[J].微生物学报,2009,49(10):1289-1294.
[12] 储卫华,于勇.鲟鱼嗜水气单胞菌的分离与鉴定[J].淡水渔业,2003,33(2):16-17.
[13] 曹海鹏,杨先乐,王玉洁,等.鲟源致病性豚鼠气单胞菌的分离及其生长特性[J].动物学杂志,2007,42(6):1-6.
[14] Petersen T N,Brunak S,von Heijne G,et al.Signal P 4.0:discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8:785-786.
[15] 王重庆.分子免疫学基础[M].北京:北京大学出版社,1997,155-159.
[16] 董忠典,周芬娜,王慧.MHC基因的遗传变异及其与鱼类抗病性研究进展[J].水生态学杂志,2011,32(2):6-10.
[17] 宋德利.日本七鳃鳗中MHCⅡB基因的克隆、表达与生物信息学分析[D].大连:辽宁师范大学,2011:40-45.
[18] 田照辉,徐绍刚,王巍,等.西伯利亚鲟热休克蛋白HSP70 cDNA的克隆、序列分析和组织分布[J].大连海洋大学学报,2012,27(2):150-157.
[19] F?nge R.Lymphoid organs in sturgeons(Acipenseridae)[J].Veterinary Immunology & Immunopathology,1986,12(1-4):153-161.
[20] Wolk K,Do¨cke W D,Von B V,et al.Impaired antigen presentation by human monocytes during endotoxin tolerance[J].Blood,2000,96(1):218-223.
[21] Kunz S,Crompton N E A,et al.Multiple mechanisms of reduced major histocompatibility complex class Ⅱ expression in endotoxin tolerance [J].Journal of Biological Chemistry,2003,278(20):18030-18036.
[22] Zhou F,Dong Z,Fu Y,et al.Molecular cloning,genomic structure,polymorphism and expression analysis of major histocompatibility complex class Ⅱ B gene of Nile tilapia(Oreochromis niloticus)[J].Aquaculture,2013,372-375(1):149-157.
[23] 田照辉,徐绍刚,胡红霞,等.西伯利亚鲟免疫球蛋白M重链区mRNA的组织分布和细菌感染后的表达[J].大连海洋大学学报,2014,29(3):217-220.
[24] 刘德财,王雪,兰莹,等.林茜草根黄酮对S-180小鼠肿瘤TG-3、MHC-Ⅰ和MHC-Ⅱ蛋白表达的影响[J].黑龙江医药科学,2017(6):22-24.
[25] Bannai H P,Nonaka M.Comprehensive analysis of medaka major histocompatibility complex(MHC) class Ⅱ genes:implications for evolution in teleosts[J].Immunogenetics,2013,65(12):883-895.
[26] Venkatesh B,Ning Y,Brenner S.Late changes in spliceosomal introns define clades in vertebrate evolution[J].Proc Natl Acad Sci U S A,1999,96(18):10267-10271.