Molecular Cloning and Immune Function Analysis of TLR3 Gene in Anguilla japonica

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

Abstract

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

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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