中华鳖CD3γ/δ、CD3ε和CD3ζ分子的克隆与表达分析

Cloning and expression analysis of CD3γ/δ, CD3ε and CD3ζ chains in Chinese soft-shelled turtle, Pelodiscus sinensis

本研究克隆了中华鳖(Pelodiscus sinensis)T细胞表面标记分子CD3γ/δ、CD3ε和CD3ζ的基因,分析了其在组织/器官表达的分布,并进一步揭示了嗜水气单胞菌(Aeromanas hydrophila)感染后这些基因的表达变化。中华鳖CD3γ/δ和ε分子结构相似,均含有1个免疫球蛋白样结构的胞外区、1个跨膜区和含有1个ITAM结构域的胞浆区。与之不同的是,CD3ζ含有1个仅由10个氨基酸组成的胞外区、1个跨膜区和含有3个ITAM结构域的胞浆区。通过生物信息学分析显示,CD3γ/δ由6个外显子和5个内含子组成,CD3ε由7个外显子和6个内含子组成,且CD3γ/δ和CD3ε位于染色体Scaffold JH208224中反向排列且相距9.9 kb。CD3ζ位于染色体Scaffold JH209116.1上,由8个外显子和7个内含子组成。荧光定量PCR分析显示,CD3γ/δ、ε和ζ在脾、肝、肠、血液中表达量较高,在胸腺、肾、心脏、肌肉和肺中表达量低。腹腔注射嗜水气单胞菌12 h后,CD3γ/δ、ε和ζ在胸腺中都呈显著的上调表达,分别为对照组的69.3倍、85.7倍和163.4倍,表明细菌感染可以诱导CD3分子的表达。

The Chinese soft-shelled turtle, Pelodiscus sinensis has a wide geographical distribution ranging from south-eastern Siberia and Korea through most parts of China to Vietnam. Besides its nutritive and medical value, the turtle also became a model animal for investigating the development and evolution of their unique body and evolution of other biological as well as immune functions. CD3 molecules which are composed of four chains [γ,δ,ε,ξ] act as markers of T lymphocytes and play important roles in T cell signaling, TCR-CD3 complex assembly and T cell mature. To understand its molecular structures, function and evolution in reptile, we cloned the cDNA sequences of CD3γ/δ, CD3ε and CD3ξ in the Chinese soft-shelled turtle using the SMART RACE technology and analyzed its expression pat- tern in normal and bacterial infected individuals using real time quantitative PCR （rt-qPCR）. The deduced CD3γ/δ, CD3ε and CD3ξ encoded 170 aa, 183 aa and 168 aa, respectively. The CD3γ/δ and CD3ε are structurally related with an extracellular immunoglobulin-like domain, a transmembrane region and an ITAM motif containing cytoplasmic tail. The Di-leucine based internalization motif （D138RQNLI） and a potential N-glycosylation site （NSlTS） were also found in CD3γ/δ amino acid sequence, and a proline-rich motif （RlSSpPPVP） and ER rention motif （Y175AGLDSR） were found in CD3e amino acid sequence with the absence of the potential N-glycosylation site. The deduced CD3ξ amino acid sequence has a different structure with a 10 aa extracellular part, a transmembrane region and three ITAM motifs containing cytoplasmic tail. The phylogenetic tree showed that each chain was clustered together and Chinese soft-shelled turtle had a closer relationship with birds and amphibians. Bioinformatics analysis in genomic sequences showed that CD3γ/δ gene, containing 6 exons and 5 introns, and CD3e gene, containing 7 exons and 6 introns located and transcribed oppositely in the Chromosome Scaffold JH208224 and CD3ξ gene, containing 8 exons and 7 introns with a big intron 1 （66.3 kB） located in Chromosome Scaffold JH209116.1. These conserved motifs in amino acid se- quence and genomic arrangement suggested they were conserved in evolution. Real time quantitative PCR results showed that CD3γ/δ, CD3ε and CD3ξ were highly expressed in spleen, liver, intestine and blood, with lower expression level in thymus, kidney, heart, muscle and lung. However, obviously increased expression of CD3γ/δ （69.3 fold）, CD3e （85.7 fold） and CD3ξ （163.4 fold） were observed in thymus at 12 h after the challenge with Aeromonas hydrophila, in- dicating an immune response involving CD3 T cells after bacterial infection. This obtained information will provide an imnortant basis for T lvmohocvte function analysis in Chinese soft-shelled turtle.