文章摘要
侯月娥,冯 娟,郭志勋,郭吉余,徐力文,苏友禄.军曹鱼Ig μ重链的全长克隆及在各组织中的定量分析[J].水产学报,2012,36(6):838~848
军曹鱼Ig μ重链的全长克隆及在各组织中的定量分析
Full-length cDNA cloning of IgM heavy chain of cobia Rachycentron canadium and quantitative analysis in different tissues
投稿时间:2011-11-04  修订日期:2011-12-21
DOI:10.3724/SP.J.1231
中文关键词: 军曹鱼  免疫球蛋白  IgM  cDNA全长  荧光定量PCR
英文关键词: Rachycentron canadium  immunoglobulin  IgM  cDNA full-length  real time RT-PCR
基金项目:中央级公益性科研院所基本科研业务费专项资金项目(2007ZD10)
作者单位
侯月娥 中国水产科学研究院南海水产研究所 
冯 娟 中国水产科学研究院南海水产研究所 
郭志勋 中国水产科学研究院南海水产研究所 
郭吉余 广东海大集团股份有限公司畜牧水产研究中心 
徐力文 中国水产科学研究院南海水产研究所 
苏友禄 中国水产科学研究院南海水产研究所 
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中文摘要:
      采用同源克隆和RACE技术, 克隆了军曹鱼Ig μ重链的cDNA全序列, 并通过荧光定量技术分析其在组织中的表达。军曹鱼Ig μ的cDNA全长1 933 bp, 3¢的非编码区域(UTR)为164 bp, 5¢UTR为26 bp, 开放阅读框为1 743 bp, 编码580个氨基酸, 分子量为64.831 ku, 理论等电点6.6。推测的军曹鱼Ig μ全长氨基酸序列与已报道的鱼类的相似性最高为60%,最低为30%, 同两栖类的相似度在30%~33%, 同其他高等的动物相似度不足30%。较保守的CH4区和鱼类相似性在64%~71%, 同其他生物的相似度仅为31%~33%, 具有较强的物种特异性。军曹鱼Ig μ中存在半胱氨酸和色氨酸的保守位点以及FR2骨架区的GKGLEW和在FR3的YYCAR保守序列。 Ig μ链基因在健康鱼体中除了肾以外在其它各组织中均有表达, 在肠中的表达量最高。经鲨鱼弧菌刺激48、96、192 h后, 在采样的各个组织中均有表达, 心脏、鳃、肠道在刺激后48 h表达量明显增加, 脾脏、胃和脑0 h时的表达量较高, 肝脏、头肾以及肾脏192 h后表达较显著。从时间上看, 黏膜免疫屏障最先抵御外来抗原, 其后,依次是肝脏、肾、头肾产生免疫应答, 说明系统免疫在长时间的抵御外来抗原时发挥着重要作用。
英文摘要:
      Full-length cDNA of IgM haevy chain of cobia (Rachycentron canadium Linnaeus) was amplified by the techniques of Rapid Amplification of cDNA ends (RACE) and PCR, and expression of target gene was analyzed by the method of real time RT-PCR in different tissues. The full-length cDNA of μ of cobia is 1 933 bp, with a 5¢terminus untranslated region (UTR) of 26 bp and a 3¢terminus UTR of 164 bp, carried an open reading frame (ORF) of 1 743 bp, encoding 580 amino acids with estimated molecular weight of 64.83 ku and the theoretical isoelectric point of 6.6. The similarity of the deduced Igμ amino acid sequences of cobia was less than 30%, when compared with those of other animals. Moreover, similarities of the conservative CH4 region of cobia,to other fish were 64%-71%. But, to other living things, similarity was only 31%-33%, having a strong specificity. Clustal X analysis indicated that there existed conserved amino acid sites (cysteines, tryptophans) and two E-box motifs GKGLEW at framework region two and YYCAR at framework region three, respectively in the IgM heavy chain of cobia. Ig μ gene was expressed in all the tissues but kidney in healthy cobia and the expression with the highest quantity was present in intestine by real time RT-PCR. The target gene was respectively expressed in all tissues of cobia after stimulation by intraperitoneal injection with Vibrio carchariae for 48 h, 96 h and 192 h. The expression of Igμ gene was significantly increased in heart, gill and intestine after 48 h. Increased expression of gene in spleen, stomach, brain was after 0 h and in liver, head kidney and kidney was after 192 h. Conclusion could be drawn that mucosal immune defended against foreign pathogens as the first barrier, followed by the liver, spleen, kidney, head kidney immune response, which indicated that immune system plays an important role in defense against foreign pathogens for a long time.
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