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牙鲆趋化因子基因CXCL9的克隆、鉴定与表达分析
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作者:张永珍1 2 3  陈松林1 2  王磊1 2 
单位:1. 中国水产科学研究院黄海水产研究所, 农业部农村海洋渔业可持续发展重点实验室, 山东 青岛 266071;
2. 青岛海洋科学与技术试点国家实验室, 海洋渔业科学与食物产出过程功能实验室, 山东 青岛 266071;
3. 上海海洋大学水产与生命学院, 上海 201306
关键词:牙鲆 CXCL9 趋化因子 细菌感染 基因表达 
分类号:S917
出版年·卷·期(页码):2020·27·1(35-43)
摘要:
趋化因子是一类具有化学趋化功能的小分子分泌性蛋白,能够引导白细胞到损伤或者感染的部位,参与免疫调节和病理反应。为丰富鲆鲽鱼类趋化因子的相关基础研究,本研究克隆了牙鲆(Paralichthys olivaceus)趋化因子基因CXCL9,其全长为910 bp,开放阅读框为408 bp,编码135个氨基酸。该基因具有趋化因子超家族的典型特征,在35、37、60和77个氨基酸位置具有4个保守的半胱氨酸残基,形成两个二硫键。氨基酸序列分析表明,该基因与大菱鲆(Scophthalmus maximus)、半滑舌鳎(Cynoglossus semilaevis)的CXCL9基因具有较高的同源性,分别为78.5%和71.0%。实时荧光定量PCR结果显示,CXCL9在正常牙鲆肝脏和脾脏中的表达量非常高,在皮肤和鳃等组织中表达量次之,说明CXCL9特异性地在免疫相关组织中表达。经迟缓爱德华氏菌(Edwardsiella tarda)感染后,牙鲆肝脏中CXCL9 mRNA表达量在感染后6 h即出现高峰值,而头肾和脾脏中的高峰值出现较晚。经鳗弧菌(Vibrio anguillarum)感染后6 h,牙鲆肝脏中CXCL9 mRNA的表达量升高近8倍,头肾中的表达高峰值出现在感染后24 h,而脾脏中CXCL9 mRNA的表达量在感染后6 h迅速升高10倍,逐渐降低后48 h又出现高峰,为正常水平的20倍。上述研究结果说明CXCL9基因在病原菌感染过程中有快速响应,可能参与了免疫防御过程,将有可能发展为一种牙鲆细菌性疾病的生物标记。本研究为牙鲆趋化因子超家族免疫机制的深入研究奠定了基础。
Chemokines are small secreted cytokine peptides (8-14 kD), which were initially found to have the ability to recruit a wide range of immune cells to sites of infection and disease in mammals. In this study, we cloned the CXCL9 genes of Paralichthys olivaceus. The full-length CXCL9 cDNA is 910 bp, contains an open reading frame of 408 nucleotides encoding 135 amino acid residues. Four conserve cysteine residues at 35, 37, 60, and 77, and the first two cysteines are separated with leucine acid, which is exactly consistent with the features of CXC. Amino acid sequence analysis revealed that the CXCL9 protein of P. olivaceus shared a high homology with that of Scophthalmus maximus and Cynoglossus semilaevis at 78.5% and 71.0%, respectively. RT-PCR demonstrated that CXCL9 mRNA was expressed highest in the liver and spleen, and high in the skin and gills of healthy P. olivaceus. CXCL9 mRNA expression was upregulated in the liver after being infected with Edwardsiella tarda for 6 h, while in the head kidney and spleen, the peak value of CXCL9 expression was 24 h or 48 h after infection. After being infected with Vibrio anguillarum, the CXCL9 mRNA expression in the liver was upregulated at 6 h, and its expression in the head kidney was upregulated at 24 h. The expression model is different in the spleen, as V. anguillarum infection upregulated CXCL9 expression 10-fold after 6 h, then decreased gradually, before upregulating approximately 20-fold at 48 h. These results indicate that P. olivaceus CXCL9 genes play an important role in the immune response, and can be a biomarker of bacterial disease in P. olivaceus.
该文献标准引用格式:
ZHANG Yongzhen,, CHEN Songlin, WANG Lei,.Cloning, characterization, and expression analysis of a chemokine gene CXCL9 from the Japanese flounder (Paralichthys olivaceus)[J].Journal of Fishery Sciences of China,2020,27(1):35-43.[张永珍,, 陈松林, 王磊,.牙鲆趋化因子基因CXCL9的克隆、鉴定与表达分析[J].中国水产科学,2020,27(1):35-43.]
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