日本鳗鲡TLR3基因的克隆及其免疫功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2017-0656

摘要/Abstract

摘要： Toll样受体3（TLR3）是用于识别双链RNA的一种重要模式识别受体。利用RT-PCR和RACE技术克隆了日本鳗鲡TLR3（AjTLR3）基因cDNA全长序列,并采用实时荧光定量PCR（qRT-PCR）检测分析该基因在日本鳗鲡各组织器官以及体外肝脏细胞的表达水平变化,以期对日本鳗鲡TLR3 基因的序列特征及其功能进行研究。结果表明：AjTLR3基因全长3 383 bp,开放阅读框为2 766 bp,编码921个氨基酸。该蛋白具有16个LRR的胞外结构域、跨膜结构域以及TIR结构域,其中在TIR结构域含有一个高度保守的氨基酸残基Tyr⁷⁷⁸。AjTLR3基因在日本鳗鲡各组织器官中均有表达,其中肝脏表达量最高;经poly I∶C刺激后在血、肠、肝脏、脾脏、皮肤、心脏及肌肉组织中均显著提高;而LPS刺激后,仅在肝脏和肠中有显著提高。日本鳗鲡肝脏细胞体外实验结果显示：poly I∶C处理后12 h和24 h表达水平显著增高,CpG-DNA和肽聚糖处理后24 h表达量均有显著增加;而细菌浓度达到10⁷CFU/mL和10⁸ CFU/mL后,AjTLR3基因表达水平分别在24 h、12 h显著增高并达到峰值。以上研究表明,AjTLR3在日本鳗鲡抵御病毒及细菌的免疫应答过程中具有重要作用。

关键词: 日本鳗鲡, TLR3, 基因克隆, 荧光定量, 肝脏细胞

Abstract: Toll-like receptor 3（TLR3）is one of the most significant members in TLRs family which is renowned as pattern recognition receptor（PRR）to recognize double-stranded RNA（dsRNA）. To identify the structure and function of TLR3 gene in Japanese eel（Anguilla japonica）,a full-length cDNA sequence of AjTLR3 was cloned by RT-PCR and RACE. The expressions of AjTLR3 in various tissues of Japanese eel as well as in the Japanese eel liver cells were analyzed and examined via quantitative real-time polymerase chain reaction（qRT-PCR）. The full-length cDNA sequence of AjTLR3 was composed of 3 383 bp with an open reading frame（ORF）of 2 766 bp encoding a polypeptide of 921 amino acid residues. Analysis of the deduced amino acid sequence indicated that AjTLR3 protein had three main structural domains,extracellular domain which contained 16 leucine-rich repeats（LRRs）motifs,a transmembrane region and a Toll/interleukin-1 receptor（TIR）domain where had a highly conserved amino acid residue Tyr⁷⁷⁸. qRT-PCR analysis revealed a broad expression for AjTLR3 in a wide range of tissues,with the predominant expression in liver. The AjTLR3 expressions in blood,intestine,liver,spleen,skin,heart,and muscle were significantly induced after injection with the viral mimic poly I∶C,while only in the liver and intestine significantly improved with the stimulation LPS. In vitro,the AjTLR3 transcripts of Japanese eel liver cells significantly up-regulated with poly I∶C treatment at 12 h and 24 h,whereas there were significantly enhanced after 24 h by PGN and CpG-DNA stimulation. The expression level of AjTLR3 gene significantly increased and reached the peak at 24 h and 12 h after the bacterial concentration was 10⁷CFU/mL and 10⁸CFU/mL,respectively. These results collectively suggest that AjTLR3 play an important role in Japanese eel’s immune responses to viral and bacterial infection.

Key words: Anguilla japonica, toll-like receptor 3（TLR3）, gene cloning, qRT-PCR, liver cells

余丽丽, 林鹏, 郭松林, 王艺磊, 张子平, 王婷婷, 冯建军. 日本鳗鲡TLR3基因的克隆及其免疫功能分析[J]. 生物技术通报, 2018, 34(1): 160-171.

YU Li-li, LIN Peng, GUO Song-lin, WANG Yi-lei, ZHANG Zi-ping, WANG Ting-ting, FENG Jian-jun. Molecular Cloning and Immune Function Analysis of TLR3 Gene in Anguilla japonica[J]. Biotechnology Bulletin, 2018, 34(1): 160-171.

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