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研究生:林思維
研究生(外文):Lin,Si-Wei
論文名稱:以益生菌作為蝦類白點病口服疫苗開發之研究
論文名稱(外文):Developing a Probiotics-Based Oral Vaccine for White Spot Syndrome in Prawns
指導教授:黃胤唐
指導教授(外文):Huang,Ying-Tang
口試委員:李明亭呂健宏蔡志明
口試委員(外文):Ming-Ting LeeJi-Ho LeuJyh-Ming Tsai
口試日期:2014-07-03
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:海洋生物技術研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:81
中文關鍵詞:草蝦白點病病毒口服疫苗VP28
外文關鍵詞:Tiger prawnswhite spot syndrome virusorval vaccineVP28
相關次數:
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台灣曾被譽為草蝦王國,產量達全球之冠,但好景不常因爆發嚴重白點病(white spot syndrome)使產量大幅縮減。到目前為止此疾病問題一直無法解決,因此開發疫苗就為首要工作。本實驗目的是希望能以益生菌攜帶白點病病毒(white spot syndrome virus, WSSV)的外套蛋白VP28作為疫苗使用。首先分析Pseudomonas sp.的特性,發現Pseudomonas sp.適合在0%~4%鹽度中生長並且降解亞硝酸力不輸Bacillus sp.。VP28基因選殖使用不須IPTG誘導的pBBR1MCS-2作為載體,利用電穿孔將pMCS-VP28轉形至Pseudomonas sp.。在給予草蝦VP28疫苗後第2天草蝦的腸道中開始出現VP28,第4天後肌肉層也開始出現VP28。將WSSV病毒液稀釋至5x105後進行感染實驗,100ppm和200ppm的疫苗組比對照組別多了10%和20%的存活率,而在免疫參數分析中也發現疫苗組其血細胞、原酚氧化酵素、吞噬能力都比對照組來的高,此實驗證明了使用VP28疫苗能有效提升草蝦的存活率及免疫能力。更改培養基後能以低成本來大量生產疫苗,有利未來推廣應用,更希望未來能以Sia10將疫苗改成分泌型且進行田野測試。
Taiwan, a country renowned for its world-leading production of tiger prawns, has unfortunately been struck with white spot syndrome (WSS), severely reducing production. As the disease is currently incurable, developing a vaccine has become a top priority. This research sought to use probiotics to transport the envelope protein VP28 of the white spot syndrome virus (WSSV) as a type of vaccine. First, the characteristics of Pseudomonas sp. were analyzed, and the results showed that a salinity level of 0%~4% is suitable for the growth of Pseudomonas sp. and that its nitrite degradation ability is better than that of Bacillus sp. VP28 gene was cloned into pBBR1MCS-2 which does not require IPTG to induce, and then used electroporation to transform pMCS-VP28 into Pseudomonas sp. Two days after administering the VP28 vaccine to tiger prawns, VP28 was found in their intestines, and it was also found in their muscles after 4 days. A WSSV virus diluted to 5x105 was then used in the challenge tests. The 100ppm and 200ppm vaccine groups exhibited additional survival rates of 10% and 20%, respectively, compared to the control group. According to the immune parameter analysis, the haemocyte count, prophenoloxidase (proPO) activity, and phagocytosis ability of the vaccinated groups were all higher than those of the control group. This experiment verified that using the VP28 vaccine can improve the survival rate as well as the immunity of tiger prawns. After altering its medium, low-cost mass production of the vaccine can commence, allowing for widespread application in the future. Furthermore, it is hoped that by using Sia10, the vaccine can be converted into a secretable form for which additional field testing will be conducted.
中文摘要………………………………………………………………I
Abstract……………………………………………………………….II
一、 前言………………………………………………………….1
1. 緒論……………………...…………………………………..1
2. 養殖現況………………………………...…………………..2
3. 益生菌…………………………………...…………………..5
4. 草蝦………………………………………...………………..7
5. 白點病病毒…………………………….……………………10
6. 疫苗……………………………………….…………………12
二、 實驗架構…………………………………..…...……………15
三、 材料與方法………………………………..…...……………16
1. 實驗動物………………………………….....………........…16
2. 養殖系統……………………………………..…………….…16
3. WSSV病毒液、pET-VP28和pBBR1MCS-2 plasmids.....16
4. 實驗藥品…………………………………...………............…16
5. 實驗儀器…………………………………...…...................…17
6. 實驗步驟…………………………………...................…....…18
6.1 生長速率測定………………...…………...…….…18
6.2 最適生長溫度分析…………...…………...…….…19
6.3 最適生長鹽度分析…………...……………….…19
6.4 降解亞硝酸能力之分析……...……………….…20
6.5 建構VP28表現載………...…….…………….…21
6.6 電穿孔…………....……………………...…….…24
6.7 DNA電泳………...…….……………….…….…25
6.8 Genomic DNA萃取…….…………………….…25
6.9 蝦組織蛋白質萃取…….……………….…….…27
6.10 蛋白質濃度測定…….………………….…….…27
6.11 西方墨點法…….……………………….…….…28
6.12 疫苗施用…….……………………….……….…30
6.13 免疫化學染色.……………………….……….…30
6.14 WSSV感染劑量實驗………………….….….…32
6.15 WSSV病毒感染實驗……………….…….….…33
6.16 血細胞計算………………….…………….….…34
6.17 proPO活性的分析……….……………….….…34
6.18 吞噬能力分析…………….…………………..…35
6.19 T-VP28量產放大製備…………….…….………36
四、 結果…………….…………………………………………38
1. Pseudomonas sp.生理特性之分析………………..…...…38
2. VP28基因選殖和蛋白質表現…………………….......…40
3. 草蝦WSSV感染檢測和VP28疫苗進入草蝦後的組織分佈的情形……………….............................................................…41
4. WSSV病毒感染劑量測試以及疫苗保護力分析…….…43
5. 草蝦免疫參數的分析.....................................................…45
6. 量產放大配方測試.........................................................…46
五、 討論.................................................................................…48
六、 參考文獻.........................................................................…52
七、 圖.....................................................................................…59
八、 表.....................................................................................…79









圖目錄
圖 1. Pseudomonas sp.生長速率………………………………………59
圖 2. 鹽濃度對於Pseudomonas sp.的生長之影響………………...…60
圖 3. 溫度對Pseudomonas sp.的生長速度和降解亞硝酸能力之影響……………….……………………………………………………….61
圖 4. Pseudomonas sp.和Bacillus sp.的生長速度及對降解亞硝酸能力之比較……………………………………….………………………….62
圖 5. 不同養殖池的池水對Pseudomonas sp.降解亞硝酸的能力之影響………………………………………………….…………………….63
圖 6. VP28基因選殖策略…………………………………….……….64
圖 7. VP28蛋白質表現分析………………………………….……….65
圖 8. 草蝦感染WSSV之檢測……………………………….…….….66
圖 9. VP28疫苗於蝦體肌肉組織表現之情形……………….……….67
圖 10. VP28疫苗於草蝦的腸道和肌肉組織表現之情形….…..…….68
圖 11. 草蝦的腸道組織之VP28表現及其變化情形.……...…..…….69
圖 12. 草蝦的肌肉組織之VP28表現及其變化情形….…….....…….70
圖 13. WSSV病毒感染劑量測試……………………………….…….71
圖 14. 草蝦感染之分析……………………………………...….…….72
圖 15. 草蝦感染WSSV後形態之分析………………………...…….73
圖 16. 疫苗保護力之分析…..………………………….……….…….74
圖 17. VP28疫苗對草蝦總血球數之影響……….…….……….…….75
圖 18. VP28疫苗對原酚氧化酵素活性之影響...…….………...…….76
圖 19. VP28疫苗對吞噬能力的影響………….….…………….…….77
圖 20. 放大培養基之分析………..…....………...…..………….…….78


表目錄
表.1 PCR用引子……………………………………………………….79
表.2 T-VP28疫苗給予的流程圖……………………………………..80
表.3 感染之流程………………………………………………..……..81

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