红笛鲷（Lutjanus sanguineus）CD8基因的克隆与诱导表达

doi:10.13560/j.cnki.biotech.bull.1985.2017-0095

摘要/Abstract

摘要： 探讨红笛鲷（Lutjanus sanguineus）CD8基因的基本特性。应用同源克隆和cDNA末端快速扩增（Rapid amplification of cDNA ends，RACE）技术克隆了红笛鲷CD8α和CD8β基因，并分析了其在不同组织的表达分布及免疫刺激物诱导后的表达模式。结果显示，CD8α cDNA序列全长1 576 bp，编码225个氨基酸。红笛鲷CD8β基因cDNA序列全长为1 486 bp，编码210个氨基酸。氨基酸序列分析结果显示，CD8α和CD8β均由信号肽、免疫球蛋白超家族（Immunoglobulin superfamily，IgSF）可变区、铰链区、跨膜区和胞浆区组成。荧光定量PCR（Real time quantitative PCR，qRT-PCR）分析显示红笛鲷CD8α和CD8β基因在胸腺表达量最高，其次为中肾、鳃、肠、皮肤、脾和头肾。红笛鲷头肾淋巴细胞体外经LPS、ConA和PolyI﹕C刺激8 h后CD8α和CD8β表达量显著上调（P<0.01）。哈维氏弧菌疫苗刺激24 h后鳃、头肾、脾脏、和肠的表达量上升。

关键词: 红笛鲷, CD8, 基因克隆, 诱导表达

Abstract: This work aims to characterize CD8 gene of humphead snapper，Lutjanus sanguineus. The full length cDNA sequences of CD8α and CD8β genes were cloned by homologous cloning and rapid amplification of cDNA ends（RACE）from humphead snapper，and their tissue distribution and expression pattern after induction with immunostimulants were also analyzed by real time quantitative PCR（qRT-PCR）. The CD8α’s cDNA consisted of 1 576 bp encoding 225 amino acids，and the CD8β’s cDNA consisted of 1 486 bp encoding 210 amino acids. The predicted CD8α and CD8β proteins were similar in structure，both contained a signal peptide，immunoglobulin superfamily variable domain，hinge region，transmembrane domain and cytoplasmic tail. The qRT-PCR analysis showed that the highest level of CD8α and CD8β mRNA expression was found in thymus，followed by kidney，gill，intesine，skin，spleen，and head kidney. Furthermore，the mRNA levels of CD8α and CD8β in head kidney leucocytes was significantly up-regulated after 8 h treated with the stimulants lipopolysaccharide（LPS），concanavalin A（ConA），and polyinosinic-polycytidylic acid（PolyI∶C），respectively（P < 0.01）. And their expression levels in gill，anterior kidney，spleen，and intesine also increased at 24 h after stimulated with formalin-inactivated Vibrio harveyi.

Key words: Lutjanus sanguineus, CD8, gene cloning, induced expression

黄郁葱,梁秀全,蔡双虎,鲁义善,简纪常,吴灶和. 红笛鲷（Lutjanus sanguineus）CD8基因的克隆与诱导表达[J]. 生物技术通报, 2017, 33(7): 169-178.

HUANG Yu-cong, LIANG Xiu-quan ,CAI Shuang-hu,LU Yi-shan,JIAN Ji-chang, WU Zao-he. Molecular Cloning and Induced Expression Analysis of CD8 Gene from Humphead Snapper[J]. Biotechnology Bulletin, 2017, 33(7): 169-178.

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