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Identification, expression profiling of a grass carp TLR8 and its inhibition leading to the resistance to reovirus in CIK cells

文献类型： 外文期刊

作者： Chen, Xiaohui 1 ; Wang, Qing 2 ; Yang, Chunrong 1 ; Rao, Youliang 1 ; Li, Qingmei 1 ; Wan, Quanyuan 1 ; Peng, Limin 1 ; W 1 ;

作者机构： 1.Northwest A&F Univ, Coll Anim Sci & Technol, Shaanxi Key Lab Mol Biol Agr, Yangling 712100, Peoples R China

2.Chinese Acad Fishery Sci, Pearl River Fishery Res Inst, Minist Agr, Key Lab Fishery Drug Dev, Guangzhou 510380, Guangdong, Peoples R China

3.Chinese Acad Fishery Sci, Pearl River Fishery Res Inst, Minist Agr, Key Lab Fishery Drug Dev, Guangzhou 510380, Guangdong

关键词： Antiviral activity;CIK cells;Gene cloning;Grass carp (Ctenopharyngodon idella);Grass carp reovirus;TLR8

期刊名称：DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY （ 影响因子：3.636； 五年影响因子：3.654 ）

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收录情况： SCI

摘要： TLR8 (toll-like receptor 8), a homolog of TLR3, TLR7 and TLR9 as prototypical intracellular members of TLR family, is generally associated with sensing single stranded RNA and plays a pivotal role in antiviral immune response. In this study, a TLR8 gene from grass carp Ctenopharyngodon idella (designated as CiTLR8) was obtained and characterized. The full-length cDNA of CiTLR8 was of 3766. bp. The open reading frame was of 3072. bp and encoded a polypeptide of 1023 amino acids, including seventeen LRR (leucine-rich repeat) motifs, one transmembrane domain and one TIR (toll/interleukin-1 receptor) domain. A single intron with the size of 839. bp was found on the neck of start codon (ATG). CiTLR8 mRNA was ubiquitously expressed in the 15 tested tissues and the expression level in gas bladder, spleen, brain, hindgut and trunk kidney tissues was high. Besides, the CiTLR8 expression in spleen and head kidney was significantly up-regulated and reached peak at 24. h post-injection of grass carp reovirus (GCRV). CiTLR8 transcription reached peak at 8. h and then declined below the normal level post-GCRV infection in the C. idella kidney (CIK) cell line; and it was rapidly and significantly down-regulated by the stimulation of the synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid sodium salt (poly I:C) in CIK cells in a dose and time-dependent manner. The inhibitor expression vectors were constructed and transfected into CIK cell line to obtain stably expressing shRNA targeting TLR8. In CiTLR8-knockdown cells, CiTLR7 transcript weakly increased, CiIFN-I mRNA was significantly down-regulated, and the expression of CiMyD88, CiIRF7 and CiMx1 scarcely changed. Post poly I:C stimulation, CiTLR8, CiTLR7 and CiMyD88 transcripts significantly increased, CiIRF7 was down-regulated after an initial phase of increase, and CiIFN-I and CiMx1 transcripts were up-regulated. After GCRV infection, the transcripts of CiTLR8, CiTLR7, CiMyD88 and CiIRF7 were up-regulated, but CiIFN-I and CiMx1 transcripts were inhibited. Nevertheless, cells transfected with pshTLR8 vectors had strong resistance against GCRV injection, suggesting CiTLR8 might play a negative role in antiviral immune response. These results collectively suggested that CiTLR8 was a novel member of TLR gene family, engaging in antiviral innate immune defense in C. idella, which laid a foundation for the further mechanism research of TLR8 in fishes.

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