Zhang Haiyan, Hu Guobin, Liu Qiuming, Zhang Shicui
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China.
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China; Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China.
Fish Shellfish Immunol. 2016 Dec;59:137-148. doi: 10.1016/j.fsi.2016.10.001. Epub 2016 Oct 3.
Toll-like receptor 2 (TLR2) in mammals is a member of the ancient Toll-like family of receptors that predominantly recognizes conserved components of Gram-positive bacteria. In the present study, a tlr2 gene and its 5'-flanking sequence were cloned from turbot, Scophthalmus maximus, its responsive expressions to various immunostimulants were subsequently studied in vivo. The turbot (sm)tlr2 gene spans over 9.0 kb with a structure of 12 exon-11 intron and encodes 816 amino acids. The deduced protein shows the highest sequence identity (76.1%) to Japanese flounder Tlr2 and possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 19 LRR motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other neoteleostei Tlr2as. A number of transcription factor binding sites known to be important for the basal transcriptional activity of TLR3 and response of TLR2 to lipopolysaccharide (LPS) signalling in mammals were predicted in the 5'-flanking sequence of smtlr2. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of smtlr2 mRNA in all twelve examined tissues with higher levels in the lymphomyeloid-rich tissues and liver. Further, smtlr2 expression was up-regulated following stimulation with LPS, peptidoglycan (PGN) or polyinosinic: polycytidylic acid [poly(I:C)] in the gills, head kidney, spleen and muscle. Finally, for all three immunostimulants, a two-wave induced smtlr2 expression was observed in the head kidney and spleen in a 7-day time course and the strongest inducibility in the head kidney. These findings suggest a possible role of Smtlr2 in the immune responses to the infections of a broad range of pathogens that include Gram-positive and Gram-negative bacteria and RNA virus.
哺乳动物中的Toll样受体2（TLR2）是古老的Toll样受体家族的成员，主要识别革兰氏阳性菌的保守成分。在本研究中，从大菱鲆（Scophthalmus maximus）中克隆了tlr2基因及其5'侧翼序列，随后在体内研究了其对各种免疫刺激剂的反应性表达。大菱鲆（sm）tlr2基因跨度超过9.0 kb，具有12个外显子-11个内含子的结构，编码816个氨基酸。推导的蛋白质与牙鲆Tlr2的序列同一性最高（76.1%），并具有信号肽序列、由19个富含亮氨酸重复序列（LRR）基序组成的LRR结构域、跨膜区和Toll/白细胞介素-1受体（TIR）结构域。系统发育分析将其与其他新硬骨鱼Tlr2归为一类。在smtlr2的5'侧翼序列中预测到许多已知对TLR3的基础转录活性和TLR2对哺乳动物脂多糖（LPS）信号反应很重要的转录因子结合位点。定量实时PCR（qPCR）分析表明，smtlr2 mRNA在所有十二个检测组织中均有组成性表达，在富含淋巴髓样组织和肝脏中的表达水平较高。此外，在用LPS、肽聚糖（PGN）或聚肌苷酸：聚胞苷酸[poly（I:C）]刺激鳃、头肾、脾脏和肌肉后，smtlr2表达上调。最后，对于所有三种免疫刺激剂，在7天的时间进程中，头肾和脾脏中观察到两波诱导的smtlr2表达，头肾中的诱导性最强。这些发现表明Smtlr2在对包括革兰氏阳性菌、革兰氏阴性菌和RNA病毒在内的多种病原体感染的免疫反应中可能发挥作用。
