石斑魚為台灣高經濟養殖魚種；然而，神經壞死病毒的感染卻造
成魚苗近100％的死亡率，對養殖漁業造成嚴重的經濟損失。疫苗現
今為病毒性疾病最佳防治方式，利用本研究室發展之龍膽石斑神經壞
死病毒似病毒顆粒(VLP)，施打龍膽石斑魚苗，並以微量採血法與抗
原捕捉式點墨法搭配增強化學發光法，不僅可定性分析，也可以定量
抗體力價。當免疫一週後，龍膽石斑魚苗可偵測特異性抗體存在血清
中，且抗體力價隨時間增加而升高。龍膽石斑施打10 µg VLPs 後，
一個月抗體強度可達8.4x106 U，不過予追加注射卻無法再提升抗體
強度。施打50 與100 µg 劑量者，一個月抗體強度可達7.2x106 U 與
6.7x106 U，抗體強度可持續增強三週以上。在田間疫苗實驗結果顯
示，本研究製備的多價疫苗，同樣也能使龍膽石斑產生特異性抗體。
綜合以上結果顯示，以龍膽石斑神經壞死病毒之似病毒顆粒及死菌製
成多價疫苗，微量採血法可追蹤此疫苗所誘發魚體之特異性抗體的力
價。

The grouper is a high-value fish in Taiwan. However, nervous
necrosis virus, which contains two single strand RNAs and doesn’t have an
envelope, causes groupers die in early stages of development. It
economically impacts on the aquaculture of this marine fish. Vaccination is
one of the best methods to prevent viral diseases. Virus-like particles
(VLPs) were used for studying the ability and efficiency of producing
antibodies against dragon grouper nervous necrosis virus. Different
injection dosages and injection frequency of VLPs were performed on
Epinephelus lanceolatus. The anti-sera of vaccinated fish were acquired by
microvoluminal phlebotomization method and analyzed by antigen-capture
dot blotting with ECL detections, which are the best choices for qualitative
and quantitative assays. The signal of antibodies in the vaccinated fish was
detected every week after primary immunization. The antibody signal
reached 8.4x106 U in one month when the dragon groupers were injected
with 10 µg of VLPs, but giving additional injection of VLPs didn’t
increase production of antibodies after one month. Moreover, dragon
groupers that were injected with 50 µg and 100 µg of VLPs generated
antibody signals up to 7.2x106 U and 6.7x106 U, respectively. The antibody
signal can remain at a higher level in the presence of high dosage injection
for at least three weeks. Furthermore, a polyvalent vaccine that included
killed bacteria was applied in field to test dragon grouper immune response.
The results not only support the data that dragon grouper boots antibody
production within a short period after immunization, but also demostrates
that the production of antibody by dragon grouper against virus and
bacteria can be monitored by the microvoluminal phlebotomization
method.

致謝 Ⅰ
中文摘要 --------------------------------------------------------------------- Ⅱ
Abstract ------------------------------------------------------------------------ Ⅲ
目錄 --------------------------------------------------------------------------- Ⅳ
表目錄 ------------------------------------------------------------------------ VI
圖目錄-------------------------------------------------------------------------- Ⅶ
壹、前言
一、台灣養殖石斑現況 ------------------------------------------------------------- 1
二、魚類神經壞死病毒介紹------------------------------------------------------- 3
三、病毒性神經壞死症 (viral nervous necrosis)之臨床病徵 ---------------- 6
四、溫度對神經壞死病毒感染力之影響 ---------------------------------------- 6
五、Betanodavirus之檢測法--------------------------------------------------------- 8
六、魚類免疫系統-------------------------------------------------------------------- 10
七、魚類血球細胞------------------------------------------------------------------- 15
八、魚類細胞激素(cytokines)------------------------------------------------------ 17
九、魚類抗體------------------------------------------------------------------------- 18
十、疫苗------------------------------------------------------------------------------- 20
十一、似病毒顆粒(virus-like particles, VLPs)------------------------------------ 25
十二、研究目的 ---------------------------------------------------------------------- 26
貳、材料與方法
一、石斑魚之來源 ------------------------------------------------------------------- 28
二、石斑魚微量採血----------------------------------------------------------------- 28
三、病毒繼代培養與純化---------------------------------------------------------- 28
四、似病毒顆粒之製備與純化 --------------------------------------------------- 29
五、SDS-PAGE蛋白質電泳 ------------------------------------------------------- 30
六、西方點墨法(Western blotting)------------------------------------------------ 31
七、蛋白質定量(Lowry method) ------------------------------------------------- 32
八、抗VLP 之小鼠血清製備 ------------------------------------------------------ 33
九、石斑魚抗血清之微量檢測建立 --------------------------------------------- 33
(1)抗原捕捉式點墨法(Antigen-capture dot blotting)偵測抗體------------- 33
(2)間接螢光抗體染色法(Indirect fluorescent antibody test, IFAT)偵測抗
體----------------------------------------------------------------------------------
35
(3)抗原捕捉式冷光點墨法(Antigen-capture ECL Dot Blotting) ----------- 35
十、VLPs 誘發石斑魚之免疫效力 ------------------------------------------------ 36
十一、多價疫苗製備-----------------------------------------------------------------
(1)DGNNV 似病毒顆粒之製備--------------------------------------------------- 37
(2)Iridovirus 死毒疫苗製備-------------------------------------------------------- 37
(3)菌苗的製備----------------------------------------------------------------------- 37
參、結果
一、純化似病毒顆粒---------------------------------------------------------------- 39
二、似病毒顆粒誘發之抗體可用於偵測神經壞死病毒---------------------- 39
三、魚體抽血量與死亡率關係---------------------------------------------------- 40
四、微量偵測方法之選擇---------------------------------------------------------- 40
五、微量偵測定量方法------------------------------------------------------------- 41
六、VLPs 所誘發魚類抗體的有效性-------------------------------------------- 42
七、多價疫苗免疫龍膽石斑魚苗之有效性-------------------------------------
(1)抗VLPs 之有效性--------------------------------------------------------------- 43
(2)抗巴斯德菌(Pasteurella)之有效性------------------------------------------- 44
肆、討論 -----------------------------------------------------------------------
一、VLPs 誘發抗體之生成時間---------------------------------------------------- 45
二、劑量與次數影響龍膽石斑魚苗之免疫效力---------------------------- 47
三、石斑魚抗血清檢測法之優缺點------------------------------------------------ 49
四、多價疫苗探討-------------------------------------------------------------------- 50
伍、參考文獻 --------------------------------------------------------------- 52
陸、圖表 --------------------------------------------------------------------- 78
附錄A:蛋白質定量之標準檢測線------------------------------------ 100
附錄B:VLPs 結合的抗體數-------------------------------------------- 101
附錄C:細胞病變之程度判定------------------------------------------- 102
附錄D:田間實驗場-------------------------------------------------------- 103

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