斜带石斑鱼CD4基因克隆和组织表达分析

Cloning,Characterization,and Expression Analysis of CD4 Gene from Epinephelus coioides

CD4作为TCR的共受体可以提高TCR/抗原-MHC复合体的稳定性,辅助TCR识别抗原,并且参与T细胞活化。本研究从斜带石斑鱼Epinepheluscoioides头肾中克隆得到全长2240bp的CD4cDNA序列,该序列包含长1410bp的ORF,编码469个氨基酸,预测蛋白分子包含一段信号肽,4个Ig样区(D1～D4),一个疏水跨膜区和一段胞浆区,具有鱼类CD4典型的结构特征。同源比对显示,其推测的氨基酸序列与其他鱼类CD4氨基酸序列相似性为25%～51%,与人Homo sapiens和鼠Mus musculus的CD4氨基酸序列相似性为13%～15%。RT-PCR分析显示,斜带石斑鱼CD4在脾脏中表达量最高,其次是胸腺和头肾。受刺激隐核虫Cryptocaryon irritans感染后,斜带石斑鱼鳃和皮肤中CD4表达量并没有显著变化(P<0.05),头肾和脾脏CD4表达量在感染5d后显著升高(P<0.05),第7d再次回落。说明刺激隐核虫感染可引起头肾和脾脏CD4+T细胞的增殖,预示头肾和脾脏CD4+T细胞在宿主对刺激隐核虫感染的抗力机制中可能发挥着重要作用。

CD4,a co-receptor for the T-cell receptor（TCR）,is able to increase the stability of the TCR/peptide-MHC class Ⅱ complex and assist the T cell activation.In this study,the sequence of CD4 cDNA was cloned from the head kidney of Epinephelus coioides,which was 2204 bp,containing an open reading frame（ORF） of 1410 bp,encoding 469 amino acids.The predicted CD4 protein had a signal peptide of 20 amino acids,four extracellular immunoglobulin-like（Ig-like） domains（D1~D4）,a transmebrane domain and a cytoplasmic tail,and showed the fundamental structural features of teleost CD4s.The predicted CD4 showed 25%~51% identity of amino acid to teleost,while only 13%~15% identity to Homo sapiens and Mus musculus.RT-PCR analysis showed that the highest level of CD4 mRNA expression was found in spleen,followed by thymus and head kidney.After infection with Cryptocaryon irritans,no significant response of CD4 gene were observed in gill and skin（P0.05）,however,the head kidney and spleen CD4 mRNA expression were markedly up-regulated（P0.05） 5 days after infection and then down-regulated 7 days later,suggesting that head kidney and spleen CD4＋ T cells undergo clonal expansion after infection,and might play a dominant role in the antiparasite immunity.