鲈鱼CD3基因cDNA克隆及诱导表达

发布时间：2018-06-24 10:35

  本文选题：鲈鱼 + CDγ/δ　； 参考：《农业生物技术学报》2016年03期

【摘要】：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(P0.05),在肠和头肾也有显著上调(P0.05),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。
[Abstract]:T-lymphocyte differentiation antigen 3 (cluster of differentiation 3 + CD3) is expressed on the surface of T cells and is associated with T cell receptor (T cell receptor) via non-covalent bonds. In this study, the full length of CD3 纬 / 未 T CD3 蔚 and CD3 味 genes were cloned from the liver of Lateolabrax japonicus by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of (rapid amplification of c DNA endsrace. The results show that the full-length cDNA of CD3 纬 / 未 is 1 231 BP, including a untranslated region of 99 BP (583 BP) and an open reading frame (open reading frame ORF) of 583 BP. The full-length c DNA encoding 182 amino acid sequence CD3 蔚 is 2 145 BP, including 186 BP of 5 '-UTR1 434 BP of 3FT-UTR and 525 BP of BP. ORF, encoding 174 amino acids CD3 味 with a total length of 1 281 BP, including 56 BP 5- UTR772 BP 3- UTR772 BP 3-UTR and 453-BP ORF, encoding a total of 150 amino acids. Amino acid sequence analysis showed that the molecular structures of CD3 纬 / 未 and CD3 蔚 were similar, with one extracellular region with immunoglobulin-like structure, one transmembrane region and one cytoplasmic domain containing immune receptor tyrosine-based activation motifitm. CD3 味 contained an extracellular domain consisting of only 5 amino acids, a transmembrane region and a cytoplasmic domain containing three ITAM sequences. The ORF region of CD3 纬 / 未 was composed of 6 exons and 5 introns. Q RT-PCR showed that CD3 纬 / 未 CD3 蔚 and CD3 味 were expressed in Gill, spleen, head kidney, intestine, fat, liver and liver. Eye, brain and other tissues are less expressed. After intraperitoneal injection of Vibrio harveyi for 3 h, three kinds of CD3 molecules in spleen tissue were significantly up-regulated (P0.05), as well as in intestine and cephalic kidney (P0.05), indicating that bacterial infection could increase the expression of CD3. The results provide basic data for further study of the molecular mechanism of fish CD3 in pathogen infection immunity.
【作者单位】： 宁波大学海洋学院;
【基金】：浙江省科技厅创新团队项目(No.2012R10029-08) 宁波大学“水产”浙江省重中之重开放基金资助(xkzsc1513)
【分类号】：S917.4
，

本文编号：2061237