为了揭示硬骨鱼C型凝集素受体 (C-type lectin receptor，CTLR)的生物学功能，实验以从大黄鱼转录组数据库中筛选出的一个CTLR基因—C型凝集素结构域家族4成员E基因 (Clec4e)为研究对象，研究其分子特征、表达分布和凝集特性。结果显示，LcClec4e cDNA全长1 546 bp，开放阅读框 (ORF)771 bp，编码254个氨基酸。LcClec4e的N端有一个跨膜区，无信号肽，C端含有一个糖识别结构域 (CRD)，其中含有糖结合位点EPN和WFD以及6个可形成二硫键的保守半胱氨酸。系统发育分析表明，LcClec4e与多种鲈形目鱼类Clec4e具有较近的亲缘关系。荧光定量PCR结果显示，LcClec4e在所检测的10种组织中呈组成型分布，且在肝脏中表达量最高；LcClec4e在来源于大黄鱼头肾组织的原代巨噬细胞、淋巴细胞和粒细胞中均有表达，且在巨噬细胞中表达量最高；经灭活溶藻弧菌刺激后，LcClec4e在3种免疫细胞中的表达均极显著上调。原核表达的重组LcClec4e胞外段 (recombinant LcClec4e-extracellular domain，rLcClec4e-ex)具有Ca²⁺依赖性的凝集活性，可凝集小鼠、家兔的红细胞，以及嗜水气单胞菌、变形假单胞菌、溶藻弧菌和坎氏弧菌等4种水产常见的革兰氏阴性菌。D-葡萄糖、D-果糖、D-甘露糖、D-麦芽糖、α-乳糖和脂多糖均可抑制rLcClec4e-ex对大黄鱼重要病原菌变形假单胞菌的凝集作用，说明LcClec4e可能与变形假单胞菌表面的糖类物质结合。上述结果提示，LcClec4e可能作为一种模式识别受体，通过结合病原菌表面的糖类病原体相关分子模式来识别病原，参与大黄鱼抗细菌感染的免疫防御。

C-type lectin receptor (CTLR) is a kind of pattern recognition receptors that can specifically bind to carbohydrates pathogen-associated molecular patterns (PAMPs) and plays an important role in innate immunity. In order to elucidate the biological function of CTLR in teleost fish, a CTLR gene-Clec4e (C-type lectin domain family 4 member E gene, Clec4e), which was screened from the transcriptome database of large yellow croaker (Larimichthys crocea), was taken as the object of the present study, and its molecular features, expression distribution and agglutination characteristics were studied. The results showed that the full-length cDNA of LcClec4e was 1 546 bp, with an open reading frame (ORF) of 771 bp, encoding 254 amino acids. The N-terminus of LcClec4e had a transmembrane region without a signal peptide, and the C-terminus had a carbohydrate recognition domain (CRD), which contained carbohydrate-binding sites EPN and WFD and 6 conserved cysteines what could form disulfide bonds. Phylogenetic analysis showed that LcClec4e was closely related to CTLRs of a variety of fishes of the order Perciformes. The results of real-time quantitative PCR showed that LcClec4e was constitutively distributed in the 10 tissues tested, and the expression level was the highest in the liver; LcClec4e was expressed in primary macrophages, lymphocytes and granulocytes from the head kidney tissues of L. crocea, and the expression level was the highest in macrophages; the expression levels of LcClec4e in these 3 kinds of immune cells were significantly upregulated after stimulation by inactivated Vibrio alginolyticus. The extracellular segment of LcClec4e (rLcClec4e-extracellular domain, rLcClec4e-ex) expressed in Escherichia coli BL21 had Ca²⁺-dependent agglutination activity and could agglutinate mouse, rabbit erythrocytes and 4 Gram-negative bacteria common in aquaculture, such as Aeromonas hydrophila, Pseudomonas plecoglossicida, V. alginolyticus, V. campbellii. D-glucose, D-fructose, D-mannose, D-maltose, α-lactose, and lipopolysaccharide all had inhibitory effects on the agglutination of rLcClec4e-ex toward P. plecoglossicida, suggesting that LcClec4e could bind to carbohydrates of P. plecoglossicida. The results above indicated that LcClec4e might be an PPR which could recognize bacteria by binding to carbohydrates PAMPs, thus involved in the immune defense of L. crocea against bacterial infection.

S 942

国家自然科学基金(31802333)