Molecular cloning, characterization and expression of goose Toll-like receptor 5
Molecular cloning, characterization and expression of goose Toll-like receptor 5
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鹅Toll样受体5的分子克隆、表征和表达
DOI：
10.1016/j.molimm.2012.05.005
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发表时间：
2012-10-01
影响因子：
3.6
通讯作者：
Jiao, Xinan
中科院分区：
文献类型：
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作者：
Fang, Qiang;Pan, Zhiming;Jiao, Xinan
Toll样受体（TLR）是模式识别受体（PRR），其通过识别病原体相关分子模式（PAMP）对响应入侵病原体的先天免疫系统的激活至关重要。TLR 5负责脊椎动物中细菌鞭毛蛋白的识别。本研究采用cDNA末端快速扩增法（RACE）克隆了鹅TLR 5基因。鹅TLR 5 cDNA的开放阅读框（ORF）长2583 bp，编码860个氨基酸。成功地从基因组DNA中扩增了TLR 5基因的整个编码区，并包含单个外显子。鹅TLR 5的氨基酸序列由信号肽序列、11个LRR结构域、LRR-CT结构域、跨膜结构域和细胞内Toll-白细胞介素-1受体（TIR）结构域组成。鹅TLR 5的氨基酸序列与人（Homosapiens）、小鼠（Mus musculus）和鸡（Gallus gallus）的同源性分别为50.5%、49.8%和82.7%。鹅TLR 5基因在脾脏、肝脏和脑中高度表达;在PBMC、肾脏、肺、心脏、骨髓、小肠和大肠中中度表达;在盲肠中最低表达。用含有鹅TLR 5和NF-κ B-荧光素酶的质粒转染的HEK 293细胞对来自鼠伤寒沙门氏菌的鞭毛蛋白显著应答，表明其是功能性TLR 5同源物。在感染S.结果表明，感染后1d，PBMCs中TLR 5 mRNA水平显著高于对照组（P. i.）在感染后1d或3d，脾脏中的含量略有升高。IL-6在PBMC中的表达低于对照水平，但在脾脏中上调。与IL-6相比，IL-8在感染后1 d或3 d在PBMC和脾脏中的表达水平均明显降低。SE攻击还导致PBMC和脾脏中IL-18和IFN-γ的mRNA表达增加。这些结果表明，鹅TLR 5的表达在不同组织中的差异调节，并可能参与对细菌病原体的免疫反应。(C)2012爱思唯尔有限公司保留所有权利。
Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that are vital to activation of the innate immune system in response to invading pathogens through their recognition of pathogen-associated molecular patterns (PAMPs). TLR5 is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the goose TLR5 gene using rapid amplification of cDNA ends (RACE). The open reading frame (ORF) of goose TLR5 cDNA is 2583 bp in length and encodes an 860 amino acid protein. The entire coding region of the TLR5 gene was successfully amplified from genomic DNA and contained a single exon. The putative amino acid sequence of goose TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR-CT) domain, a transmembrane domain and an intracellular Toll-interleukin-1 receptor (TIR) domain. The amino acid sequence of goose TLR5 shared 50.5% identity with human (Homo sapiens), 49.8% with mouse (Mus musculus) and 82.7% with chicken (Gallus gallus). The goose TLR5 gene was highly expressed in the spleen, liver and brain: moderately expressed in PBMCs, kidney, lung, heart, bone marrow, small intestine and large intestine; and minimally expressed in the cecum. HEK293 cells transfected with goose TLR5 and NF-kappa B-luciferase containing plasmids significantly responded to flagellin from Salmonella typhimurium indicating that it is a functional TLR5 homologue. In response to infection with S. enterica serovar Enteritidis (SE), the level of TLR5 mRNA significantly increased over the control in PBMCs at 1 d post infection (p.i.) and was slightly elevated in the spleen at 1 d or 3d p.i. IL-6 was expressed below control levels in PBMCs but was upregulated in the spleen. In contrast to IL-6, an evident decrease in the expression level of IL-8 was observed in both PBMCs and spleens at 1 d or 3d p.i. SE challenge also resulted in an increase in the mRNA expression of IL-18 and IFN-gamma in PBMCs and the spleen. These results imply that the expression of goose TLR5 is differentially regulated in various tissues and may participate in the immune response against bacterial pathogens. (C) 2012 Elsevier Ltd. All rights reserved.