- 學位論文
含多套CpG-ODNs的質體對海鱺(Rachycentron canadum) 的免疫調節效應
Immunomodulatory Effect of Plasmid Containing Multi-Copy CpG-ODNs on Cobia (Rachycentron canadum)
摘要
人工合成未甲基化的cytosine phosphorothioate guanine oligodeoxynucleotides (CpG-ODNs)具有誘發脊椎動物先天及後天免疫反應的效果。CpG-ODNs 以Toll like receptor (TLR9)為受體，引起與Myeloid differentiation factor 88 (MyD88)及Tumor necrosis factor receptor-associated factor 6 (TRAF6)相關的訊息傳導路徑。海鱺的TLR9 (RCTLR9)基因已在本研究被選殖出來，但是含有多套CpG的質體對RCTLR9基因表現的影響則尚未被研究。因此本研究先選殖MyD88與TRAF6的部分基因序列，並評估含有多套CpG-ODN 1668的質體(pCpG 1688)對海鱺免疫作用。結果顯示MyD88與TRAF6基因序列與其他魚類極為相似。無論在幼苗、幼魚或成魚的階段，MyD88與TRAF6基因在各檢測的組織皆有表現。當以pCpG 1668+FKB+Alum注射至魚體，在注射第5與第10天候，脾及肝的TLR9，MyD88與TRAF6的基因表現達到最高，尤其是在注射劑量100 µg的魚。同時在注射後的第10天，海鱺的體液免疫參數亦顯著增加。本研究的結論是含有多套CpG 1668 ODNs的質體可促進TLR9、MyD88與TRAF6等基因的表現，並且亦會刺激海鱺體液免疫反應。
並列摘要
Synthetic unmethylated cytosine phosphorothioate guanine oligodeoxynucleotides (CpG-ODNs) can activate both innate and adaptive immune responses in vertebrate organisms. CpG ODNs are recognized by the host through Toll like receptor 9 (TLR9) signaling pathway involving myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). In our previous study, we cloned cobia Toll like receptor 9 but there were no supporting evidences demonstrating the effect of CpG-ODNs on TLR9 expression. Therefore, this study was carried out to clone MyD88 and TRAF6 and to determine the adjuvanticity of plasmid containing multi-copy pCpG-ODNs 1668 (pCpG 1668) on cobia by evaluating immune gene expression and humoral response. As the result, both of two partial gene sequences of MyD88 and TRAF6 shared a remarkable high degree of homology with fish species counterparts. Broad expression of MyD88 and TRAF6 was found in larval, and in all tissues of juvenile and adult stages. For gene expression, the high TLR9, MyD88 and TRAF6 expressions were observed at 5 and 10 dpi in fish treated with pCpG 1668 + FKB + alum in both spleen and liver, especially in group treated with 100 µg pCpG 1668, compared with those of controls. For MyD88 and TRAF6, the high gene expressions were recorded at both 5 and 10 dpi. With a similar result, pCpG 1668 + FKB + alum enhanced a significant humoral response in cobia at 10dpi. In conclusion, multi-copy CpG-ODN 1668 can up-regulate MyD88, TRAF6 genes and stimulates humoral response in cobia.