线纹海马Hippocampus erectus的tlr21基因结构及其对CpG-ODN的应答特征

doi:10.11978/2018034

摘要/Abstract

摘要：

作为先天性免疫反应中重要的模式识别受体之一, TLR(toll-like receptor)基因家族介导的先天性免疫是脊椎动物抵御病原微生物的重要防线。研究分析了线纹海马Hippocampus erectus tlr21基因的序列特征, 并基于抗原注射实验探讨了海马tlr21的免疫功能。线纹海马tlr21基因的开放阅读框长度为2946bp, 编码981个氨基酸, 预测其编码蛋白相对分子量为246.98kDa, 理论等电点为4.78。tlr21编码蛋白主要包含1个信号肽和3个功能结构域: 胞外区具有14个富含亮氨酸重复序列的结构域(LRR), 跨膜区具有富含半胱氨酸的结构域, 胞内区则具有TIR结构域。同源性比较和系统进化分析表明, 线纹海马tlr21基因与虎尾海马Hippocampus comes tlr21基因的同源性最高, 其次与斜带石斑鱼Epinephelus coioides、牙鲆Paralichthys olivaceus、大菱鲆Scophthalmus maximus和红鳍东方鲀Takifugu rubripes的tlr21基因聚为一枝。tlr21基因在线纹海马的脑、鳃、肝、肠、肾、性腺、肌肉和育儿袋各组织均有分布, 肾脏中表达量最高。抗原注射实验结果发现, 线纹海马Tlr21对不同类型CpG寡脱氧核苷酸(CpG-ODNs)的识别能力存在差异, 其中, CpG-2007和CpG-HC4040对海马肾脏tlr21的mRNA表达量具有显著的促进作用(P<0.05)。研究表明, 线纹海马Tlr21能够通过识别含CpG序列的DNA发挥免疫识别作用, 研究结果将有助于系统认识海马免疫系统中TLR家族基因的功能特征, 为建立海马病害防治策略提供理论依据。

关键词: tlr21, 基因结构, CpG-ODN, 免疫, 线纹海马, 组织表达差异

Abstract:

Toll-like receptors (TLRs) are the major pattern recognition receptors (PRR) of the innate immune system in vertebrates, which can initiate host immune responses by recognizing potential pathogens. The present study focused on the role of Tlr21 involved in immune responses in lined seahorse (Hippocampus erectus). The full-length sequence of the lined seahorse tlr21 gene was cloned. The open reading frame (ORF) of seahorse tlr21 gene includes 2964 base pairs, which is predicted to encode 981 amino acids. The encoding protein had a predicted molecular mass of 246.98 kDa and an estimated isoelectric point of 4.78. Homology comparison and phylogenetic analysis showed that lined seahorse tlr21 shared high sequence identity with its homologs from other species, especially those species in Syngnathidae. Quantitative real-time PCR analysis showed that tlr21 mRNA was widely expressed in various tissues and expressed highest in the kidney. We conducted intraperitoneal CpG-ODNs injections to the lined seahorses. Both CpG-2007 and CpG-HC4040 increased expression levels of tlr21 mRNA in seahorse kidney significantly. In conclusion, these findings indicated that tlr21 initiated the innate immunity in lined seahorse by recognizing CpG DNA.

Key words: tlr21, gene structure, CpG-ODN, immunity, Lined seahorse, tissue expression difference

张媛, 秦耿, 张博, 林强. 线纹海马Hippocampus erectus的tlr21基因结构及其对CpG-ODN的应答特征[J]. 热带海洋学报, 2019, 38(1): 67-75.

Yuan ZHANG, Geng QIN, Bo ZHANG, Qiang LIN. The gene structure of tlr21 in lined seahorse, Hippocampus erectus, and its responses to CpG-ODN treatment[J]. Journal of Tropical Oceanography, 2019, 38(1): 67-75.

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名称	序列 (5′-3′)	用途
HE tlr21-1F	GCAACCAGCGAAGGGAGA	克隆全长
HE tlr21-1R	GGACACGGAACGGACGAT
HE tlr21-2F	ATCGTCCGTTCCGTGTCC
HE tlr21-2R	CGAGCGCAAGTTGAGGTAG
HE tlr21-3F	CGTCCGTTCCGTGTCCAA
HE tlr21-3R	CAGCGTCACGACGTTTGG
HE tlr21-4F	CCGTTACAACCGCATCCT
HE tlr21-4R	GTCCGTCAGCTCGTCCAA
HE tlr21-5F	CGTCCCTTTATCTCACCAAC
HE tlr21-5R	CAGCATGACCCAGTCTTCG
HE tlr21-6F	CGTCCCTTTATCTCACCAAC
HE tlr21-6R	GTGATGCAGGCACAGTTTC
HE tlr21-7F	GCGTTTCGCTCCTGGTTC
HE tlr21-7R	CCGCCCGAGTTTCTTCAT
HE tlr21-8F	AAATCAAGTACGGCTACTACGC
HE tlr21-8R	TGAGTGTCAGAATCTCCCAAA
tlr13bF	CTTCGCCTGCTTCGGTTAGACAA	荧光定量PCR
tlr13bR	GCTCAAGTTGGACACGGAACGG	荧光定量PCR
β-actinF	TGGGCGTACAACAGGTATCG	内参基因
β-actinR	AGGAGTAGCCACGTTCAGTG	内参基因

CpG-ODN	序列 (5′-3′)
CpG-2000	TCCATGACGTTCCTGCAGTTCCTGACTGG
CpG-2006	TCGTCGTTTTGTCGTTTTGTCGTT
CpG-2007	TCGTCGTTGTCGTTTTGTCGTT
CpG-HC4040	TGACTGTGAACGTTCGAGATGA