红笛鲷(Lutjanus sanguineus)CD4和CD4-2基因克隆与诱导表达

MOLECULAR CLONING AND INDUCED EXPRESSION OF CD4 AND CD4-2 GENES FROM HUMPHEAD SNAPPER LUTJANUS SANGUINEUS

应用RT-PCR和RACE-PCR技术克隆了红笛鲷(Lutjanus sanguineus)CD4和CD4-2基因,并分析了它们在健康鱼不同组织的表达分布及免疫刺激物诱导后的表达变化。CD4基因c DNA序列全长2216bp,包含180bp的5′UTR(untranslated regions)、605bp的3′UTR和1431bp的开放阅读框(open reading frame,ORF),编码476个氨基酸;红笛鲷CD4-2基因c DNA序列全长为1520bp,包含62bp的5′UTR、525bp的3′UTR和933bp的ORF,编码310个氨基酸。CD4分子由信号肽、4个免疫球蛋白样结构域(D1—D4)构成的胞外区、跨膜区和胞浆区组成,CD4-2分子由信号肽、2个免疫球蛋白样结构域(D1—D2)构成的胞外区、跨膜区和胞浆区组成。荧光定量PCR(Real Time Quantitative PCR,q PCR)分析显示红笛鲷CD4和CD4-2基因在健康鱼的胸腺中表达量最高,其次是中肾、鳃、皮肤、脾、头肾和肠。红笛鲷头肾淋巴细胞体外经脂多糖(Lipopolysaccharide,LPS)和刀豆蛋白A(Concanavalin A,Con A)刺激12h后,CD4和CD4-2表达量显著上调(P<0.05)。哈维氏弧菌疫苗免疫24h后鳃、头肾、脾脏和肠的表达量显著上升(P<0.05)。研究结果为进一步研究鱼类CD4和CD4-2在抗菌免疫反应中的作用提供了基础资料。

The full-length c DNA sequences of CD4 and CD4-2 gene were obtained by rapid amplification of c DNA ends（RACE） from humphead snapper, Lutjanus sanguineus. The tissue distribution in healthy fish and expression profiles after induction with immunostimulants were analyzed by real time quantitative PCR（q PCR）. The total c DNA sequence of CD4 was 2216 bp, including 5？ untranslated regions（UTR） of 180 bp, 3？ UTR of 605 bp, an open reading frame（ORF） of 1431 bp encoding 476 amino acids. The total c DNA sequence of CD4-2 was 1520 bp, including 5＇ UTR of 62 bp, 3？ UTR of 525 bp, an ORF of 933 bp encoding 310 amino acids. The CD4 contained a signal peptide, four Ig-like extracellular domains, a transmembrane domain, and a cytoplasmic tail in structure. The CD4-2 molecule had a signal peptide, two Ig-like extracellular domains, a transmembrane domain, and a cytoplasmic tail in structure. The q PCR analysis showed that the highest levels of CD4 and CD4-2 m RNA expression were found in thymus, followed by kidney, skin, gill, spleen, and head kidney. Furthermore, the transcriptions of CD4 and CD4-2 in head kidney leucocytes were up-regulated in 12 h after lipopolysaccharide（LPS） and concanavalin A（Con A） stimulation. The expression levels in gill and intestine in 12 h were significantly increased after immunized with formalin-inactivated Vibrio harveyi, and expression levels in anterior kidney and spleen at 24 h were up-regulated after the immunization with formalin-inactivated V. harveyi. The results provide a theoretical basis for further studying the role of CD4 and CD4-2 in immune response of the fish.