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T淋巴细胞分化抗原3(cluster of differentiation 3,CD3)分子表达于T细胞表面,并通过非共价键与T细胞受体(T cell receptor,TCR)连接,参与T细胞活化。本研究采用反转录PCR(RT-PCR)和c DNA末端快速扩增(rapid amplification of c DNA ends,RACE)技术从鲈鱼(Lateolabrax japonicus)肝脏中克隆了CD3γ/δ、CD3ε和CD3ζ基因全长。结果表明,CD3γ/δ全长c DNA为1 231 bp,包括99 bp的5’非翻译区(untranslated regions,UTR),583 bp的3’-UTR和549 bp的开放阅读框(open reading frame,ORF),共编码182个氨基酸;CD3ε全长c DNA为2 145 bp,包括186 bp的5’-UTR,1 434 bp的3’-UTR和525 bp的ORF,共编码174个氨基酸;CD3ζ全长1 281 bp,包括56 bp的5’-UTR,772 bp的3’-UTR和453 bp的ORF,共编码150个氨基酸。氨基酸序列分析发现,CD3γ/δ和CD3ε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1个免疫受体酪氨酸激活基序(Immunoreceptor tyrosine-based activation motif,ITAM)的胞浆区,而CD3ζ含有1个仅由5个氨基酸组成的胞外区、1个跨膜区和含3个ITAM序列的胞浆区。分析DNA和c DNA序列发现,CD3γ/δ的ORF区由6个外显子和5个内含子组成,CD3ε的ORF区由5个外显子和4个内含子组成。q RT-PCR结果表明,CD3γ/δ、CD3ε和CD3ζ在鳃、脾、头肾、肠中表达较高,在脂肪、肝、眼、脑等组织中表达较少。当腹腔注射哈维氏弧菌(Vibrio harveyi)3 h后,脾组织的3种CD3分子均显著上调(P<0.05),在肠和头肾也有显著上调(P<0.05),表明细菌感染会增加CD3的表达。研究结果为进一步研究鱼类CD3在病原菌感染免疫中的分子机制提供基础资料。
T lymphocyte differentiation antigen 3 (CD3) is expressed on the surface of T cells and is linked to T cell receptor (TCR) through non-covalent bond and participates in T cell activation. In this study, the CD3γ / δ, CD3ε and CD3ζ genes were cloned from the liver of Lateolabrax japonicus by RT-PCR and rapid amplification of c DNA ends (RACE) . The results showed that the CD3γ / δ full length cDNA was 1 231 bp, including 99 bp of 5 ’untranslated regions (UTRs), 583 bp of 3’-UTR and 549 bp of open reading frame ORF encoding a total of 182 amino acids. CD3ε full-length cDNA was 2 145 bp in length and contained 186 bp of 5’-UTR, 1 434 bp of 3’-UTR and 525 bp of ORF encoding a total of 174 amino acids. It is 1 281 bp long and consists of 56 bp 5’-UTR, 772 bp 3’-UTR and 453 bp ORF encoding a total of 150 amino acids. Amino acid sequence analysis revealed that CD3γ / δand CD3εhave similar structures and contain one extracellular region of immunoglobulin-like structure, one transmembrane region and one immunoreceptor tyrosine- based activation motif (ITAM), whereas CD3ζ contains an extracellular region consisting of only 5 amino acids, a transmembrane region and a cytoplasmic domain containing three ITAM sequences. Analysis of DNA and c DNA sequences found that the ORF region of CD3γ / δ consists of 6 exons and 5 introns, while the ORF region of CD3ε consists of 5 exons and 4 introns. q RT-PCR results showed that CD3γ / δ, CD3ε and CD3ζ were highly expressed in gill, spleen, head and kidney, intestine and less in adipose tissue, liver, eye and brain. After intraperitoneal injection of Vibrio harveyi for 3 h, all three CD3 molecules in spleen tissue were significantly up-regulated (P <0.05) and also significantly up-regulated in intestinal and head kidney (P <0.05), indicating that bacterial infection Increase CD3 expression. The results provide the basic information for further studying the molecular mechanism of fish CD3 infection in pathogenic bacteria.