鳜T细胞表面受体CD4分子的克隆与表达分析

The cDNA sequence and expression analysis of CD4 in mandarin fish(Siniperca chuatsi)

克隆了鳜T细胞表面受体CD4分子的cDNA序列并对其在组织/器官的表达进行了分析。鳜CD4分子的cDNA序列全长为2 356 bp,编码469个氨基酸。该CD4分子由4个免疫球蛋白样结构域(V1-C1-V2-C2)构成的胞外区、一个跨膜区和一个包含保守的CXC基序的胞内区组成。系统进化分析表明,鳜CD4分子与同属于鲈形目的鲈CD4分子聚为一支,与鲈的相似性最高(57%),与鲤的相似性较低(34%)。序列比对分析显示,CD4分子胞外区存在多个保守性位点,如:C1结构域中的WTC基序、V2和C2结构域中的N-糖基化位点等。同鸟类和哺乳类不同的是,鳜CD4分子在V1结构域中缺少由半胱氨酸(Cys)残基对形成的二硫键,而在V2结构域中存在一个额外的二硫键,这可能会改变CD4分子的空间构象,进而影响到CD4与MHCⅡ的结合以及后续的抗原诱导的T细胞活化等。荧光定量PCR分析表明,CD4mRNA在鳜的胸腺中表达量最高,在脾脏中表达量最低。脂多糖腹腔注射8 h后,CD4 mRNA在鳜的胸腺、脾脏和头肾中都呈显著的上调表达(P<0.05)。

The cDNA sequence of CD4 homologue was cloned from the mandarin fish Siniperca chuatsi.It consists of 2 356 bp,encoding 469 amino acids,and this molecule has a typical four Ig-like domains（V1-C1-V2-C2）and shares some fundamental conserved structures,i.e.WTC motif in D2 domain,N-glycosylation site in D4 domian and the CXC motif in the cytoplasmic tail.Unlike avian and mammalian CD4 molecules,the mandarin fish CD4 sequence lacks the Cys pairs in the first Ig-like domain,but has a unique possible disulfide bond in the third domain.These differences suggest that mandarin fish CD4 molecule may have a different structure,which may in turn influence its binding with MHC Ⅱ molecule and probably subsequent T-cell activation.The CD4 was expressed mainly in thymus,and following stimulation with LPS,its significant increase（P0.05）at mRNA level was observed 8 hours post injection in thymus,spleen and head-kidney.The mandarin fish CD4 shared a relatively high level of similarity（57%）with that in sea bass Dicentrarchus labrax,and in the phylogenetic tree,the mandarin CD4 was clustered with that in sea bass.The cloning of CD4 in the mandarin fish may serve as a basis for further functional characterization of fish T lymphocytes.