CAJ | 학술논문

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。
본연구극륭료중화별(Pelodiscus sinensis)T세포표면표기분자 CD3γ/δ、CD3ε화 CD3ζ적기인，분석료기재조직/기관표체적분포，병진일보게시료기수기단포균(Aeromanas hydrophila)감염후저사기인적표체변화。중화별CD3γ/δ화ε분자결구상사，균함유1개면역구단백양결구적포외구、1개과막구화함유1개ITAM결구역적포장구。여지불동적시， CD3ζ함유1개부유10개안기산조성적포외구、1개과막구화함유3개ITAM결구역적포장구。통과생물신식학분석현시， CD3γ/δ유6개외현자화5개내함자조성， CD3ε유7개외현자화6개내함자조성，차CD3γ/δ화CD3ε위우염색체Scaffold JH208224중반향배렬차상거9.9 kb。CD3ζ위우염색체Scaffold JH209116.1상，유8개외현자화7개내함자조성。형광정량PCR분석현시， CD3γ/δ、ε화ζ재비、간、장、혈액중표체량교고，재흉선、신、심장、기육화폐중표체량저。복강주사기수기단포균12 h후， CD3γ/δ、ε화ζ재흉선중도정현저적상조표체，분별위대조조적69.3배、85.7배화163.4배，표명세균감염가이유도CD3분자적표체。
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,ζ(η)] 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, CD3εand CD3ζin 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, CD3εand CD3ζencoded 170 aa, 183 aa and 168 aa, respectively. The CD3γ/δand CD3εare 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 (N81TS) were also found in CD3γ/δ amino acid sequence, and a proline-rich motif (R155PPPVP) and ER rention motif (Y175AGLDSR) were found in CD3εamino acid sequence with the absence of the potential N-glycosylation site. The deduced CD3ζamino 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/δgene, containing 6 exons and 5 introns, and CD3εgene, containing 7 exons and 6 introns located and transcribed oppositely in the Chromosome Scaffold JH208224 and CD3ζgene, 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, CD3εand CD3ζwere 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), CD3ε(85.7 fold) and CD3ζ(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 important basis for T lymphocyte function analysis in Chinese soft-shelled turtle.