Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung, Taiwan.
Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung, Taiwan; Department of Fisheries and Marine Science, University of Brawijaya, Malang, Indonesia.
Fish Shellfish Immunol. 2014 Feb;36(2):417-27. doi: 10.1016/j.fsi.2013.12.017. Epub 2013 Dec 27.
Cobia culture is hindered by bacterial infection (Photobacterium damselae subsp. piscicida) and in order to study the effect of P. damselae subsp. piscicida challenge and CpG ODN stimulation on cobia Toll like receptor 9 (RCTLR9), we used PCR to clone RCTLR9 gene and qRT-PCR to quantify gene expression. The results indicated that RCTLR9 cDNA contains 3141 bp. It encodes 1047 amino acids containing 16 typical structures of leucine-rich repeats (LRRs) including an LRRTYP, LRRCT and a motif involved in PAMP binding was identified at position 240-253 amino acid. Broad expression of RCTLR9 was found in larval, juvenile and adult stages irrespective of the tissues. In larval stage, RCTLR9 mRNA expression decreased at 5 d and then increased at 10 dph. At juvenile stage cobia, the expression was significantly high (p < 0.05) in spleen and intestine compared to gill, kidney, liver and skin. However, at adult stage, the significant high expression was found in gill and intestine. Cobia challenged with P. damselae subsp. piscicida showed significant increase in RCTLR9 expression at 24 h post challenge in intestine, spleen and liver, while in kidney the expression was peak at 12 h and later it decreased at 24 h. The highest expression was 40 fold increase in spleen and the lowest expression was ∼3.6 fold increase in liver. Cobia stimulated with CpG oligonucleotides showed that the induction of these genes was CpG ODN type and time dependent. In spleen and liver, CpG ODNs 1668 and 2006 injected group showed high expression of RCTLR9, IL-1β, chemokine CC compared to other groups. Meanwhile, CpG ODN 2006 has induced high expression of IgM. The CpG ODNs 2395 have induced significant high expression of Mx in spleen and liver. These results demonstrates the potential of using CpG ODN to enhance cobia resistance to P. damselae subsp. piscicida infection and use as an adjuvant in vaccine development.
石斑鱼养殖受到细菌性疾病（发光杆菌）的阻碍。为了研究发光杆菌对石斑鱼 Toll 样受体 9（RCTLR9）的影响以及 CpG ODN 的刺激作用，我们使用 PCR 克隆了 RCTLR9 基因，并通过 qRT-PCR 对基因表达进行定量分析。结果表明，RCTLR9 cDNA 包含 3141bp，编码 1047 个氨基酸，包含 16 个富含亮氨酸重复序列（LRR）的典型结构，其中 240-253 位氨基酸的基序与病原体相关分子模式（PAMP）的结合有关。RCTLR9 在幼虫、幼鱼和成鱼期的各个组织中均有广泛表达。在幼虫期，RCTLR9mRNA 表达在 5d 时下降，10dph 时增加。在幼鱼期，RCTLR9 在脾脏和肠道中的表达显著高于鳃、肾脏、肝脏和皮肤。然而，在成鱼期，RCTLR9 在鳃和肠道中的表达显著较高。石斑鱼感染发光杆菌后，在感染后 24 小时内，肠道、脾脏和肝脏中 RCTLR9 的表达显著增加，而肾脏中表达在 12 小时时达到峰值，随后在 24 小时时下降。脾脏中的表达最高增加了 40 倍，肝脏中的表达最低增加了约 3.6 倍。石斑鱼用 CpG 寡核苷酸刺激后，这些基因的诱导呈 CpG ODN 类型和时间依赖性。在脾脏和肝脏中，CpG ODN 1668 和 2006 注射组的 RCTLR9、IL-1β 和趋化因子 CC 的表达较高。同时，CpG ODN 2006 诱导了 IgM 的高表达。CpG ODN 2395 在脾脏和肝脏中诱导了 Mx 的显著高表达。这些结果表明，CpG ODN 具有增强石斑鱼对发光杆菌感染的抵抗力的潜力，并可作为疫苗开发的佐剂。
