評估pCpG ODN 1668 對海鱺免疫之影響

Evaluation of pCpG ODN 1668 effect on immunal response on Cobia

方季

CpG ODN 1668 ； 不活化細菌 ； 佐劑 ； 海鱺 ； CpG ODN 1668 ； inactivated vaccine ； adjuvant ； cobia

屏東科技大學熱帶農業暨國際合作系學位論文

2016年

碩士

鄭達智

英文

人工合成無甲基化的細菌DNA序列cytosine phosphorothioate guanine oligodeoxynucleotides (CpG ODNs)具有免疫刺激與佐劑效用，且CpG ODNs會誘發脊椎動物的先天及後天性免疫反應。本研究評估含有CpG ODN 1668序列的質體 (pCpG 1668) 與發光桿菌疫苗混和注射海鱺後的免疫反應。不同劑量(50μ g或100μ g)的質體(pSB1C3)或pCpG 1668與福馬林去活化的致病性發光桿菌(FKB)混和，注射到體重30克海鱺幼魚的腹腔後採樣分析免疫參數。在第1、3、6、14、20 及 28天注射後 (dpi) 分析血清中lysozyme、peroxidase與myeloperoxidase的活性。肝、脾與腸則在第1、5與10 dpi採樣分析Toll-like receptor 9 (TLR9)與Interleukin 1 beta (IL-1β )的基因表現量。同時評估肝與脾組織是否有明顯的變化。結果顯示: lysozyme的活性在第6 dpi時pCpG 1668 (50μ g)+FKB處理組顯著增加，然而peroxidase與myeloperoxidase的活性則皆無顯著變化。在脾的TLR9基因表現量上，第1 dpi時以pCpG 1668 (50) + FKB的處理組有顯著增加，在肝則是第1 dpi與5 dpi時，以pCpG1668 (100) + FKB的處理組有顯著增加，在腸則是在第1 dpi時，pCpG 1668 (100) + FKB處理組有顯著增加。而在各組織IL-1β基因的表現量，則在第1 dpi與10 dpi時，pCpG 1668 (100)+FKB處理組皆有顯著增加。各處理組的肝及脾的組織並沒有顯著變化。結論是pCpG 1668+FKB會增加TLR9與IL-1β的表現量，同時增加血清中lysozyme的活性，且肝及脾組織無顯著的變化。

Synthetic unmethylated cytosine phosphorothioate guanine oligodeoxynucleotides (CpG ODNs) which mimics bacterial DNA is one of the options that can be used as an immunostimulant and an adjuvant in inactivated vaccines. They activate both the innate and adaptive immune responses in vertebrate organisms. The B class of CpG ODN 1668 was used in the study to evaluate its effects on the immune response in juvenile cobia fish when used as a plasmid on vaccine. In the study juvenile cobia approximately 25 cm weighing 30 grams were injected with formalin killed bacteria (FKB), pSB1C3 50 µg, pSB1C3 100 µg, pSB1C3 50 µg + FKB, pSB1C3 100 µg + FKB, pCpG 1668 50 µg + FKB and pCpG 1668 100 µg + FKB. Blood serum from cobia was extracted for the analysis of lysozyme, peroxidase and myeloperoxidase activities at 1, 3, 6, 14, 20 and 28 days .The liver, spleen and intestines were sampled for the evaluation of Toll like receptor 9 (TLR 9) and Interleukin 1 Beta (IL- 1β) gene expression at 1, 5 and 10 days. Histopathology analysis of tissues was assessed to check any histology changes after vaccination. The lysozyme activity was significantly increased at 6 dpi after vaccinating with pCpG 1668 (50) + FKB. Myeloperoxidase and peroxidase activities were not increased after vaccinating with pCpG 1668 + FKB at (50) and (100). The expression of TLR9 was significantly increased at 1 dpi in spleen after vaccinating with pCpG 1668 (50) + FKB. In the liver, a significant expression of TLR 9 was observed at 1 dpi and 5 dpi vaccinated with pCpG 1668 (100) + FKB and in intestines a significant expression was at 1 dpi vaccinated with pCpG 1668 (100) + FKB. The expression of IL-1β was significantly increased at 1dpi and 10 dpi in all tissues after vaccinating with pCpG 1668 (100) + FKB. There was no or mild histological change observed in pCpG 1668 (100) + FKB and pCpG 1668 (50) + FKB treatments in the liver and spleen. In conclusion pCpG 1668 can increase the expression of TLR 9 and IL-1β and also increase the level of humoral response and has no or mild histological change in cobia.

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