臺灣博碩士論文加值系統

魚類先天性免疫與後天性免疫反應保護作用，是防禦外源病原感染的基礎，為了研究石斑魚免疫相關基因在發育早期的表現，我們首先以石斑魚腦細胞株 (GB)進行神經壞死病毒之繁殖與純化，所得純化之神經壞死病毒顆粒進行紐西蘭大白兔免疫接種，製備出具有中和神經壞死病毒活性且高效價之多株抗體。抗神經壞死病毒多株抗體以被動免疫的方式稀釋2,000倍，浸泡中和沾黏在石斑魚受精卵卵鞘外之可能的感染源後，以RT-PCR追蹤受精卵以及孵化後之魚苗，證實無神經壞死病毒之感染後，培育魚苗作為研究材料，分析不同發育時期魚苗免疫基因之表現，本實驗室以點帶石斑Mx與IgM重鏈基因，分別做為先天性與後天性免疫基因標幟，並轉錄成Digoxigenin-UTP標定之RNA探針，進行整體原位雜交法；發現Mx基因轉錄在受精卵受精後第21 (孵化前)至受精後第39小時 (孵化後第一天後期)有訊號表現，表現位置在胸腺與前腎，但在受精後第51小時 (孵化後第二天前期)訊號下降，而在RT-PCR的檢測中，則發現在受精後第5小時即可表現；IgM重鏈基因轉錄在受精卵受精後第21小時 (孵化前)至受精後第27小時 (孵化後第一天前期)，有訊號表現且位置在胸腺，但在受精後第39小時 (孵化後第一天後期)訊號下降，而RT-PCR檢測則在受精後第5小時偵測到表現。此結果表示石斑魚先天性與後天性免疫基因在早期受精卵孵化至幼苗時期在淋巴器官內已開始基因表現。

Fish innate and adaptive immunity is a protective mechanism to defense foreign pathogens infection. In order to study the grouper immune-related gene expression at early stage, we used GB (grouper brain) cells to propagate and purify the NNV (Nervous Necrosis Virus) viral particles. The immunization inoculation of NNV viral particles into New Zealand rabbits produced a high titer anti-NNV polyclonal antibody, with the ability of neutralizing virus. To investigate the feasibility of passive immunity, we dilute the antibody in 2,000 fold in sea water and soaked the grouper eggs for 1 hour to disinfect the possible pathogens like NNV which may adhere on the surface of egg sheath. RT-PCR was then performed to trace the eggs and hatched out larvae. The larvae which was confirmed without NNV infection were analyzed the immune gene transcripts by using Epinephelus cocoidies Mx and IgM-heavy chain gene as innate and adaptive immune marker gene,respectively. These 2 genes were than transcribed to Digoxigenin labeled RNA probes for whole-mount in situ hybridization. The transcription of Mx gene showed signal at 21 hpf to 39 hpf in thymus, head kidney, and decreased at 51 hpf . RT-PCR can also detect the signal at 5 hpf. IgM-Heavy chain gene was transcribed at 21 hpf to 27 hpf in thymus, and the signal decreased at 39 hpf. RT-PCR can also detect the signal at 5 hpf. These results indicate the grouper innate and adaptive immune genes are transcribed in lymphoid organ during embryo to early larva stage.

謝誌 ----------------------------------------------------------------------------- Ⅰ
目錄 ----------------------------------------------------------------------------- Ⅲ
中文摘要 ----------------------------------------------------------------------- Ⅶ
英文摘要 ----------------------------------------------------------------------- Ⅸ
第一章 前言 ------------------------------------------------------------------ 1
1. 石斑魚之簡介 --------------------------------------------------------- 1
2. 神經壞死病毒之簡介與疾病症狀 --------------------------------- 2
3. 硬骨魚之免疫系統 --------------------------------------------------- 5
4. 抗黏液病毒之簡介 --------------------------------------------------- 9
5. 免疫球蛋白M之簡介 ---------------------------------------------- 11
6. 實驗目的 -------------------------------------------------------------- 12
第二章 材料與方法 -------------------------------------------------------- 14
1. 生物性材料 ----------------------------------------------------------- 14
2. 神經壞死病毒之純化、蛋白質電泳分析與多株抗體製備--- 14
1. 藥品試劑 -------------------------------------------------- 14
2. NNV之純化 ---------------------------------------------- 17
3. NNV蛋白質電泳分析 ---------------------------------- 20
4. NNV之病毒效價 ---------------------------------------- 23
5. 不同MOI條件下NNV CAV之蛋白質電泳分析 - 24
6. 不同MOI與天數下NNV RV之ELISA分析 ----- 24
7. 製備抗NNV多株抗體 --------------------------------- 25
8. 抗NNV多株抗體之ELISA分析 -------------------- 26
9. NNV與抗NNV多株抗體之中和試驗 -------------- 27
10. 抗NNV多株抗體之西方墨點法 --------------------- 28
11. NNV RV之ELISA分析 ------------------------------- 30
12. 石斑魚受精卵與魚苗之RNA抽取 ------------------ 30
13. NNV之RT-PCR分析 ---------------------------------- 31
3. 整體原位雜交法 ---------------------------------------------------- 33
1. 藥品試劑 -------------------------------------------------- 33
2. pBS-Mx2質體之構築 ---------------------------------- 38
3. pBS-Mx2-5’與pBS-Mx2-3’質體之構築 ------------ 39
4. DIG標定Mx2 RNA探針之製備 -------------------- 40
5. DIG標定Mx2-5’與Mx2-3’RNA探針之製備 ---- 41
6. DIG標定IgM Heavy chain RNA探針之製備 ---- 42
7. DIG標定 RNA探針之定量 -------------------------- 42
8. DIG標定 RNA探針之RNA電泳與轉印 --------- 43
9. 石斑魚受精卵粉之製備 -------------------------------- 45
10. 抗體以石斑魚受精卵粉進行前吸附作用 ----------- 46
11. 整體原位雜交法 ----------------------------------------- 46
12. RT-PCR偵測Mx基因之轉錄 ------------------------- 50
13. RT-PCR偵測IgM Heavy chain基因之轉錄 -------- 51
第三章 結果 ----------------------------------------------------------------- 52
抗神經壞死病毒多株抗體之開發 ---------------------------------- 52
1. NNV之純化 ---------------------------------------------- 52
2. 找尋純化NNV CAV最佳條件 ------------------------ 52
3. 製備抗NNV多株抗體與西方墨點法確認 ---------- 53
4. 中和試驗確認抗NNV多株抗體之效價 ------------- 54
5. ELISA 分析抗NNV多株抗體之效價 --------------- 54
6. ELISA 分析在不同MOI與天數下NNV RV
病毒量 ------------------------------------------------------ 55
7. 田間試驗中抗NNV多株抗體之應用 ---------------- 55
探討點帶石斑Mx與 IgM Heavy chain 基因之表現 ----------- 56
1. 構築pBS-Mx2質體 -------------------------------------- 56
2. 構築pBS-Mx2-5’、pBS-Mx2-3’質體 ---------------- 56
3. DIG標定之pBS-Mx2、pBS-Mx2-5’、pBS-Mx2-3’
與IgM-H RNA 探針製備與確認 --------------------- 57
4. 整體原位雜交法偵測石斑魚早期不同時期Mx
與IgM-Heavy chain 基因之RNA表現 ------------- 57
5. RT-PCR 偵測石斑魚早期不同時期Mx 與
IgM-Heavy chain 基因之RNA表現 ----------------- 59
第四章 討論 ------------------------------------------------------------------ 60
第五章 参考文獻 ------------------------------------------------------------ 65



圖 目次
圖一 NNV CAV的病毒帶 ---------------------------------------------- 74
圖二 SDS-PAGE分析純化後之NNV --------------------------------- 75
圖三 SDS-PAGE分析在不同MOI條件下純化出NNV CAV
之差異 --------------------------------------------------------------- 76
圖四 西方墨點法確認第一次免疫接種NNV後的免疫反應 ---- 77
圖五 西方墨點法確認第二次免疫接種NNV後的免疫反應 ---- 78
圖六 西方墨點法確認第三次免疫接種NNV後的免疫反應 ---- 79
圖七 西方墨點法確認第四次免疫接種NNV後的免疫反應 ---- 81
圖八 ELISA分析在不同MOI與天數下繁殖NNV RV ----------- 83
圖九 光學顯微鏡觀察NNV在不同MOI感染GB細胞的
CPE現象------------------------------------------------------------- 84
圖十 RT-PCR檢測受精卵和孵化後之魚苗感染NNV之情形 -- 85
圖十一 構築pBS-Mx2、pBS-Mx2-5’、pBS-Mx2-3’質體 ----------- 86
圖十二 RNA電泳確認DIG標定pBS-Mx2、pBS-Mx2-5’、
pBS-Mx2-3’與 IgM-H RNA 探針 ----------------------------- 88
圖十三 DIG標定之pBS-Mx2、pBS-Mx2-5’、pBS-Mx2-3’與
IgM-H RNA 探針 ------------------------------------------------- 89
圖十四 Dot blot 分析DIG標定pBS-Mx2、pBS-Mx2-5’、
pBS-Mx2-3’與IgM-H RNA 探針之濃度 --------------------- 91
圖十五 石斑魚受精卵至魚苗生長圖
圖十六 整體原位雜交法偵測石斑魚受精卵早期不同時期
Mx RNA之表現 --------------------------------------------------- 96
圖十七 整體原位雜交法偵測石斑魚受精卵早期不同時期
IgM Heavy chain RNA之表現 ---------------------------------- 98
圖十八 RT-PCR偵測石斑魚受精卵Mx RNA之表現 ---------------- 99
圖十九 RT-PCR偵測石斑魚受精卵IgM Heavy chain RNA之表現100

附圖目次
附圖一 pBluescript SK – 質體圖 ---------------------------------------- 101
附圖二 斑馬魚胸腺與腎臟位置圖 -------------------------------------- 102

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