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牙鲆NOD2基因的表达分析及在抗迟缓爱德华氏菌感染过程中的功能
曹丹丹, 刘金相, 王志刚, 张全启, 齐 洁, 王旭波, 于海洋
中国海洋大学海洋生命学院 海洋生物遗传学与育种教育部重点实验室 青岛 266003
摘要:
本研究利用实验室已建牙鲆(Paralichthys olivaceus)转录组数据库预测得到牙鲆NOD2基因(PoNOD2),并利用PCR技术进行序列验证。同时,设计迟缓爱德华氏菌注射感染牙鲆成鱼和体外免疫刺激牙鲆鳃细胞系实验,探究PoNOD2基因在抗菌免疫反应中的作用。PoNOD2基因的开放阅读框长度为2964 bp,编码988个氨基酸。PoNOD2蛋白有3种保守结构域,包括C-末端LRR,中心NACHT和N-末端CARD结构域。实时荧光定量PCR结果显示,在所检测牙鲆组织中,迟缓爱德华氏菌的侵染能显著上调PoNOD2的表达。体外免疫刺激牙鲆鳃细胞系实验显示,在PGN、Poly Ⅰ:C和迟缓爱德华氏菌刺激下,PoNOD2表达上调。亚细胞定位显示,PoNOD2蛋白定位于牙鲆鳃细胞的细胞质中。在迟缓爱德华氏菌侵染牙鲆鳃细胞过程中,PoNOD2基因的过表达能够抑制细菌生长,并引起IL-1β、IL-6和IL-8等炎性细胞因子的表达上调。结果表明,PoNOD2在抑制迟缓爱德华氏菌生长以及调节牙鲆对病原菌的免疫应答中发挥重要作用。
关键词:  牙鲆  模式识别受体  NOD2  迟缓爱德华氏菌  牙鲆鳃细胞系
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Expression Analysis and Functional Characterization of NOD2 in the Resistance of Japanese Flounder (Paralichthys olivaceus) to Edwardsiella tarda Infection
CAO Dandan, LIU Jinxiang, WANG Zhigang, ZHANG Quanqi, QI Jie, WANG Xubo, YU Haiyang
Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences,Ocean University of China, Qingdao 266003
Abstract:
The innate immune system acts as the first defense against invading pathogens through three classes of pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs), including the Toll-like receptors (TLRs), RIG-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). The nucleotide-binding oligomerization domain-like receptors are a family of innate immune receptors that link cytosolic sensing of microbes and danger stimuli to activate immune responses. As a member of the NOD-like receptor family, NOD2 can recognize bacterial peptidoglycan and activate immune responses via nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). In the present study, the Japanese flounder (Paralichthys olivaceus) NOD2 (PoNOD2) was identified by searching the transcriptome sequence of Japanese flounder based on the available NOD2 cDNA sequences of other fishes. The complete open reading frame (ORF) of PoNOD2 is 2964 bp and encodes a polypeptide of 988 amino acids. The gene PoNOD2 is composed of three main domains: an N-terminal domain containing two adjacent CARDs, a central NACHT domain, and a multiple C-terminal LRRs. The phylogenetic tree analysis suggests that the gene PoNOD2 is closely related to that of the Fugu rubripes (Takifugu rubripes). Tissue expression analysis by qRT-PCR showed that the gene PoNOD2 is constitutively expressed in all the examined tissues, predominantly in the spleen and liver. Real-time PCR showed that the infection of Edwardsiella tarda can significantly upregulate the expression of PoNOD2 in the tissue of Japanese flounder. In vitro immune stimulation experiments showed that PoNOD2 expression could be upregulated by peptidoglycan, polyinosinic: polycytidylic acid, and E. tarda infection. Subcellular localization of the PoNOD2 protein is in the cytoplasm of the gill cells of flounder. When the flounder gill (FG) cells were infected by E. tarda, the overexpression of PoNOD2 can inhibit the bacterial replication and upregulate the transcription of pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-8. These results suggest that PoNOD2 plays a key role in the resistance of P. olivaceus to E. tarda infection and in the modulation of immune responses.
Key words:  Japanese flounder (Paralichthys olivaceus)  PRR  NOD2  Edwardsiella tarda  FG cells