抗凝血酶(antithrombin,AT)主要抑制凝血酶(thrombin)及其它凝血因子的活性,并具有重要的抗炎活性。本实验从香鱼(Plecoglossus altivelis)肝组织cDNA文库中成功获得了香鱼AT基因的cDNA全序列(EMBL登录号:FN429980)。测序结果表明,香鱼AT基因cDNA全长1487个核苷酸,包含1个大的开放阅读框(ORF),推测编码1个由453个氨基酸组成的蛋白,N端23个氨基酸为信号肽序列。成熟AT具有与哺乳动物AT相似的特征结构,包括:羧基末端附近的丝氨酸蛋白酶活性中心、6个保守的Cys残基形成的3个二硫键结构和4个N-糖基化位点。氨基酸序列分析表明,香鱼AT与大西洋鲑(Salmo salar)AT氨基酸序列同源性最高,为81%。系统进化树分析表明,物种AT的进化关系与目前接受的物种分类关系基本一致,香鱼AT位于鱼类AT簇中,与大西洋鲑、红鳍东方豚(Takifugu rubripes)和斑马鱼(Danio rerio)AT进化相关性最高。AT mRNA在健康香鱼的肝组织中表达量最大,在脾、肾和脑中表达量较低。实时荧光定量PCR结果表明,在鳗利斯顿氏菌(Listonella anguillarum)侵染4和8h的香鱼肝组织中AT mRNA表达量显著下调(P〈0.05),但随着病程发展,12~36h,AT mRNA表达水平显著上调(P〈0.05),揭示AT是一个病程相关蛋白,并参与香鱼细菌感染导致的急性期反应。
Antithrombin(AT) is the major inhibitor of thrombin and other serine proteinases comprising the coagulation cascade enzymes. Furthermore,it has anti-inflammatory properties. The nucleotide sequence of full-length cDNA clone of ayu(Plecoglossus altivelis) AT gene (EMBL accession:No. FN429980) was obtained from the constructed liver cDNA of ayu. The whole cDNA length of ayu AT gene was 1 487 bp,consisting of a 1 362 bp open reading frame(ORF). The predicted mature ayu AT consisted of 453 amino acids preceded by a signal peptide of 23 residues. The structure of mature AT in ayu was similar with those in the mammalian AT,including serine protease active site near the carboxyl-terminal,three disulfide bonds formed by six conserved Cys residues and the four N-glycosylation sites. Sequence comparison showed that ayu AT had the highest amino acid indentity to Atlantic salmon(Salmo salar),which was 81%. Phylogenetic analysis showed that the evolutionary relationship of AT among different species was identical with the classification relationship of different species accepted currently and ayu AT grouped constantly with those previously reported from Atlantic salmon,tiger pufferfish(Takifugu rubripes),and zebrafish(Danio rerio). In healthy ayu,AT mRNA was mainly expressed in the liver,and weakly in the spleen,the kidney and the brain. Following Listonella anguillarum infection,liver AT mRNA expression change was determined by quantitative Real-time PCR(qRT-PCR) method. AT transcripts were significantly down-regulated in ayu liver at 4 and 8 h post injection(hpi) (P0.05). However,AT transcripts were significantly up-regulated at 12~36 h when bacteria developing(P0.05),suggesting that AT may be involved in the immune response of Ayu.