香鱼凝血因子X基因表达与鳗利斯顿氏菌感染的相关性

Alteration on the expression of ayu coagulation factor X gene upon Listonella anguillarum infection

凝血途径的关键因子--凝血因子X(coagulation factor X,FX),是一种与免疫调控密切相关的维生素K依赖型丝氨酸蛋白酶。该研究克隆了香鱼(Plecoglossus altivelis)FX基因全长cDNA序列,它由1817个核苷酸组成,包含一个大的开放阅读框,编码一个由453个氨基酸组成的相对分子质量为5.07×104的蛋白。香鱼FX与已知的哺乳动物FX的结构相似,N端24个残基为信号肽序列。序列比较表明,香鱼FX与斑马鱼FX的氨基酸同一性最高,为53%。在健康香鱼中,FX基因mRNA主要在肝组织中表达,脑和鳃中也有少量表达。实时荧光定量PCR分析揭示,鳗利斯顿氏菌感染后香鱼肝组织中FX基因mRNA表达显著上调,16h时达到5.43倍。通过原核表达系统表达了香鱼FX丝氨酸蛋白酶结构域,并制备了抗血清。Western blotting分析显示,鳗利斯顿氏菌感染后香鱼血清中FX含量显著增加,并随着时间延长上调倍数不断增大,在36h时达到3.68倍。据此,FX基因mRNA及蛋白的表达与鳗利斯顿氏菌感染香鱼的过程紧密相关,揭示它可能在鱼类抗细菌感染的免疫反应中起重要作用。

Coagulation factor X（FX） plays an important role in the immune response of mammals.In this study,the full length cDNA sequence of the ayu FX gene,1817 bp in length excluding 3＇-polyA tail,was determined for the first time.The sequence contained an open reading frame,which encoded a protein of 453 amino acids with a molecular weight of 5.07 × 104.The predicted protein had motifs typical of animal FX,and its N-terminal 24 residues were the signal peptides.Sequence comparison showed that ayu FX shared 53% amino acid sequence identity with zebrafish FX.In healthy ayu,FX mRNA was expressed mainly in the liver and weakly in the brain and gill.After Listonella anguillarum infection,liver FX transcriptions significantly increased,and peaked at 16 h post infection.The serine protease motif of ayu FX was expressed in Escherichia coli and was subsequently used for antiserum preparation.Western blotting analysis revealed that serum FX significantly increased in bacterially infected ayu fish.In conclusion,the ayu FX gene expression was significant in the progress of bacterial infection,which suggests FX＇s role in fish immune response.