文章摘要
周芬娜,董忠典,李同明,傅 咏,王 慧.尼罗罗非鱼MHC ⅡA基因的克隆、表达及多态性分析[J].水产学报,2012,36(8):1167~1178
尼罗罗非鱼MHC ⅡA基因的克隆、表达及多态性分析
Molecular cloning, expression and polymorphism analysis of major histocompatibility complex (MHC) class II A of Nile tilapia (Oreochromis niloticus)
投稿时间:2011-12-25  修订日期:2012-03-08
DOI:DOI:10.3724/SP.J.1231.2012.27903
中文关键词: 尼罗罗非鱼  表达  多态性  基因  克隆  MHC
英文关键词: Oreochromis niloticus  expression  polymorphism  gene  cloning  major histocompatibility complex (MHC)
基金项目:中国博士后创新基金项目(20100481295); 山东省自然科学基金(ZR2009DQ021, Y2008D32); 山东省博士后创新基金项目(200703044); 国家重大专项科技计划项目(2001BA804A29)
作者单位
周芬娜 山东农业大学动物科技学院 
董忠典 山东农业大学动物科技学院 
李同明 山东农业大学动物科技学院 
傅 咏 山东农业大学动物科技学院 
王 慧 山东农业大学动物科技学院 
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中文摘要:
      为进一步了解鱼类MHC ⅡA基因的特点及其在免疫反应中的功能,采用同源克隆、RACE-PCR、巢式PCR等技术, 从健康的尼罗罗非鱼体获得1 205 bp的MHC ⅡA基因cDNA全序列(Orni-DBA-0101, Genebank登录号:JF719813)及1 388 bp的基因组序列。序列分析发现, 尼罗罗非鱼MHC ⅡA基因含4个外显子和3个内含子, 开放阅读框长720 bp, 编码239个氨基酸。从4尾尼罗罗非鱼中共得到8条不同的cDNA序列, 分别编码不同的氨基酸序列。氨基酸序列比对后发现, 序列间存在丰富的多态性, 且主要集中在α-1区, 多态性位点数远远高于半滑舌鳎MHC ⅡA基因。生物信息学分析表明, 尼罗罗非鱼MHC ⅡA编码的蛋白质分子包含1个信号肽、2个胞外结构域、1个跨膜区和1个胞质区, 存在4个保守的半胱氨酸残基以及丰富的磷酸化位点, 与其他物种的相似性为23%~65%。RT-PCR结果表明, MHC ⅡA基因在脾、肾、肠、鳃、性腺、肝、心脏表达量很高, 在鳔和肌肉中表达量最低。人工感染嗜水气单胞菌后, 肝、脾、肾、鳃、肠5个组织中MHC ⅡA基因的mRNA水平均发生了不同程度的变化, 提示MHC ⅡA分子作为一种重要的免疫因子, 在清除病原的免疫反应中起着重要作用。
英文摘要:
      The major histocompatibility complex (MHC) class II A gene plays an important role in the immune response of teleost. In this study, the full length of MHC IIA cDNA and genomic sequences were obtained from healthy Oreochromis niloticus by homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length of cDNA sequence (Orni-DBA*0101, GenBank No: JF719813) comprises 1 205 bp with a 720 bp open reading frame (ORF) encoding a predicted protein with 239 amino acid residues. The genomic sequence was further identified to be 1 388 bp in length, which contains four exons and three introns. Eight class IIA alleles were identified from four healthy O. niloticus individuals. The variability that existed in the α-1 region was higher than that of half-smooth tongue sole. All the characteristic domains present in MHC IIA of other species could be found in O. niloticus MHC IIA sequence, including a leader peptide, two extracellular domains, a transmembrane region, and a cytoplasmic domain. In addition, there are four conserved cysteine residues and abundant phosphorylation sites. The deduced amino acid sequence shares 23%-65% identity with those of other vertebrates. Semi-quantitative reverse transcriptase PCR (RT-PCR) demonstrated that O. niloticus MHC IIA mRNA was ubiquitously expressed in all tested tissues, which is highly expressed in spleen, kidney, gills, liver, heart, gonad and gut, however, lowly expressed in muscle and bladder. Challenge of O. niloticus with pathogenic bacteria, Aeromonas hydrophila, results in a significant change of the expression of MHC IIA in the liver, spleen, kidney, gills and intestine. The result implied that MHC IIA might play an important role in the immune system.
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