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尼罗罗非鱼ICAM-1基因克隆及其mRNA表达分析

Cloning and mRNA Expression Analysis of ICAM-1 Gene in Nile Tilapia(Oreochromis niloticus)

  • 摘要: 细胞间黏附分子-1 (intercellular adhesion molecule-1, ICAM-1)是介导细胞识别、粘附的重要粘附分子,参与细胞的信号转导与活化、炎症反应和免疫应答等一系列重要生理病理过程。本研究克隆鉴定了尼罗罗非鱼(Oreochromis niloticus) ICAM-1 cDNA全长(命名为On-ICAM-1),并对该基因进行生物信息学分析,运用荧光定量PCR方法对无乳链球菌(Streptococcus agalactiae)刺激后On-ICAM-1在各组织中的m RNA表达模式进行了研究。结果显示,该基因ORF为1 059 bp,编码352个氨基酸,分子质量为38.8 kD,等电点为5.55。On-ICAM-1序列与美妊丽鱼(Astatotilapia calliptera)、布氏朴丽鱼(Haplochromis burtoni)和三湖慈鲷(Pundamilia nyererei)的基因序列相似度较高,分别为89.80%、88.10%和71.02%。该基因编码蛋白氨基酸序列的的比对结果表明,ICAM-1在慈鲷科其他鱼类中高度保守。On-ICAM-1基因在健康尼罗罗非鱼的10种组织中均有表达,在胸腺组织中的表达量最高,其次是鳃、肠道和皮肤,在头肾组织的表达量最低。经无乳链球菌刺激后,On-ICAM-1基因在鳃、肠道、脾、脑、胸腺中的表达显著下调,且呈时序性表达。鳃、脾均在12 h表达量最低,肠道、脑、胸腺也在12 h时表达量下调,且都在24 h开始上调。该研究表明On-ICAM-1表达可以被无乳链球菌诱导并参与尼罗罗非鱼的免疫应答过程。

     

    Abstract: intercellular adhesion molecule-1(ICAM-1) is an important adhesion molecule mediating cell recognition and adhesion, and it is involved in a series of important physiological and pathological processes including cell signal transduction and activation, inflammatory response and immune response. The full length of ICAM-1 cDNA of Nile tilapia(Oreochromis niloticus)(named On-ICAM-1) was cloned and identified in this study. The bioinformatics analysis of this gene was carried out, and the expression pattern of On-ICAM-1 m RNA in various tissues after the stimulation of Streptococcus agalactiae was studied by using fluorescence quantitative PCR method. The results showed that the ORF was 1 059 bp, encoded 352 amino acids, with a molecular weight of 38.8 kD and a theoretical pI of 5.55. The sequence of On-ICAM-1 was similar to that of Astatotilapia calliptera, Haplochromis burtoni and Pundamilia nyererei in 89.80%, 88.10% and 71.02%. Amino acid sequence of the protein of the gene alignment showed that ICAM-1 was highly conserved in other cichlid fishes. Moreover, the mRNA expression levels in different tissues were analyzed by real time quantitative PCR, it was found that the expression of On-ICAM-1 could be detected in all the 10 examined tissues and the highest expression level was in the thymus, with lower level detected in gill, intestine, skin, and the lowest level was detected in head kidney. Under Streptococcus agalactiae stimulation, the expression of On-ICAM-1 gene was significantly down-regulated in gill, intestine, spleen, brain and thymus, and the expression was temporal. The expression levels in gill and spleen were the lowest at 12 h, and decreased expressions in intestine, brain and thymus at 12 h, all of which were upregulated at 24 h. This study showed that On-ICAM-1 expression could be induced by Streptococcus agalactiae and was involved in the immune response of Nile tilapia.

     

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