臺灣博碩士論文加值系統

本研究分別使用褐藻酸鈉 (Sodium alginate) 包埋 (Alginate組) 及 褐藻酸鈉與幾丁聚醣 (chitosan) 雙重包埋 (Alginate Chitosan組)，分別包覆腸炎弧菌 (Vibrio parahaemolyticus)、創傷弧菌 (Vibrio vulnificus) 及溶藻弧菌 (Vibrio alginolyticus) 製備三價口服疫苗。點帶石斑接種後，藉由分析1免疫基因表現量、2相對活存率、3血清抗體力價，來探討口服疫苗是否具有效性，並比較不同包覆方式之間保護力的差異。

以不同材料: Alginate、Alginate Chitosan、CAP、Agarose、Curdlan 分別包覆口服疫苗，經模擬石斑魚腸胃道環境的抗原釋放量實驗，以 Alginate包埋及以 Alginate Chitosan 雙重包埋之口服疫苗在仿胃液環境 (PBS, pH 3.45) 僅少量釋放抗原，並在仿腸液 (PBS, pH 7.4) 環境時抗原會大量釋放，具有良好的口服疫苗包覆殼材的特性，故以此兩種包覆方式製備口服疫苗進行生物體實驗。

點帶石斑魚於實驗第 1 週及第 5 週接種口服疫苗，於實驗第 0、4、8、12、16、20、24 週採樣及細菌攻擊。基因表現量部分，實驗第 4 週免疫相關基因 IL-1β、 TLR9、 GATA-3、 CD4 表現量均高於 Control組，表示接種後能確實誘導免疫基因表現。其中，GATA-3 為後天記憶型免疫中誘導生成抗體的轉錄因子，實驗第 4 週 GATA-3 基因大量表現並與 Control組有顯著差異 (p＜0.05)；第 8 週時，二次接種雖有誘導免疫基因表現，但對 GATA-3 基因的刺激效果並不如初次接種；第 12 週時因為未再接受抗原，GATA-3 基因的表現量持續下降。

細菌攻擊實驗部分，Alginate組的相對活存率 (RPS) 最高可達 100%，Alginate Chitosan組最高可達 83%。Alginate組保護效力可維持至第 16 週 (RPS 83.3%)，第 20 週對 V. parahaemolyticus、V. vulnificus 及 V. alginolyticus 之 RPS 分別仍有 66.7、100、50%；Alginate Chitosan組相對活存率至第 16 週即下降至 50%，第 20 週僅33.3%。兩口服疫苗於第 24 週已無明顯保護力，Alginate 組對 V. parahaemolyticus、V. vulnificus 及 V. alginolyticus 的相對活存率分別為 33.3、50、16.7%，Alginate Chitosan組的相對活存率皆為 0%。

血清抗體力價部分，Alginate組在每個採樣點的力價均高於 Alginate Chitosan組；Alginate組於第 8 週達到最高後緩慢下降，抗體維持高力價至第 16 週，第 8 週至第 16 週之間無顯著差異 (p＞0.05)，第 20 週下降至與 8至 16 週有顯著差異 (p<0.05)；Alginate Chitosan組於第 8 週達到最高後即大幅下降，對 V. parahaemolyticus 及 V. vulnificus 的抗體力價於第 12 週即與第 8 週有顯著差異 (p<0.05)。

本研究結果顯示，以褐藻酸鈉或褐藻酸鈉與幾丁聚醣之弧菌三價口服疫苗對石斑魚具良好之保護效果，具有進一步商業化之潛力。

This study investigated the protective effectiveness of oral trivalent vaccines of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus encapsulated with alginate and chitosan-alginate complex, respectively, in cultured grouper. Immuno-related gene expression, relative percent survival and serum antibody titer of grouper were determined after vaccination.
Oral vaccine encapsulated with agarose, CAP, curdlan, alginate and chitosan-alginate complex, respectively, were tested under simulation of gastric fluid condition (PBS, pH 3.75) and intestine fluid condition (PBS, pH 7.4). The results showed that alginate encapsulated oral vaccine and chitosan-alginate complex encapsulated oral vaccine released few antigens in gastric acidic condition, indicating that antigens were effectively coated and its destruction might therefore be prevented. In intestine alkaline condition, large quantity of antigens were released, indicating that these two encapsulated vaccines were good candidates to be administered orally.
After vaccination, both alginate encapsulated group and chitosan-alginate complex encapsulated group had stimulated fish immune-genes, including IL-1β, TLR9, GATA-3 and CD4. Of these, GATA-3 was a transcription factor that played an important role in affecting production of antibodies. The GATA-3 genes expressed in both vaccine groups were significantly higher than that of control group.
High relative percent survival (RPS) of grouper could be maintained in alginate encapsulated group until weeks 16 (83.3%), while decreased to 66.7% at weeks 20 after bacterial challenge. In chitosan-alginate complex encapsulated group, relative percent survival of grouper were dropped to 50% at weeks 16, and decreased to 33.3% at weeks 20 after bacterial challenge.
Serum antibody titer in grouper could maintain steadily at high level until weeks 16 and decreased slowly in alginate encapsulated group, while chitosan-alginate complex encapsulated group decreased rapidly after reaching the highest level at weeks 8.
The present results revealed that the oral trivalent vibrio vaccines encapsulated with sodium alginate or chitosan-alginate complex exhibited good protective effectiveness in grouper. It seemed that these two oral vaccines possess good potential for further commercialization.

摘要 I
Abstract III
圖目錄 VII
表目錄 VIII
第一章 前言 1
第二章 文獻整理 3
2.1點帶石斑魚 3
2.2石斑魚之弧菌症 3
2.3腸炎弧菌 (Vibrio parahaemolyticus) 4
2.4創傷弧菌 (Vibrio vulnificus) 5
2.5溶藻弧菌 (Vibrio alginolyticus) 6
2.6魚類免疫系統 6
2.6.1魚類免疫器官 7
2.6.2先天性免疫 7
2.6.3適應性免疫 7
2.7魚類疫苗 8
2.8口服疫苗 9
2.9卡德蘭膠 (Curdlan) 9
2.10鄰苯二甲酸乙酸纖維素 (Cellulose acetate phthalate，CAP) 10
2.11褐藻酸鈉 (Sodium Alginate) 10
2.12幾丁聚醣 (Chitosan) 11
第三章 材料方法 13
3.1實驗菌株 13
3.1.1菌株來源 13
3.1.2菌株確認 13
3.1.3 API 20E system 13
3.1.4聚合酶連鎖反應 (Polymerase Chain Raction；PCR) 13
3.1.4.1菌株DNA萃取 13
3.1.4.2 Vibrio parahaemolyticus 聚合酶連鎖反應 14
3.1.4.3 Vibrio vulnificus 聚合酶連鎖反應 14
3.1.4.4 Vibrio alginolyticus 聚合酶連鎖反應 15
3.1.4.5洋菜膠膠體電泳 15
3.1.5菌株強化 15
3.1.6菌株毒力試驗 16
3.2抗原製備 16
3.3口服疫苗製備 16
3.3.1鄰苯二甲酸乙酸纖維素 (CAP) 包覆製備 17
3.3.2卡德蘭膠 (Curdlan) 包覆製備 17
3.3.3洋菜膠 (Agarose) 包覆製備 17
3.3.4褐藻酸鈉 (Sodium Alginate) 包覆製備 17
3.3.5褐藻酸鈉 (Sodium Alginate) 與幾丁聚醣 (chitosan) 雙重包埋包覆製備 17
3.4口服疫苗於模擬石斑腸胃道環境中的抗體釋放量測試 18
3.5血清採集與Rabbit anti-Grouper血清製備 18
3.6主動免疫實驗 18
3.6.1實驗設計 18
3.6.2核糖核酸萃取 19
3.6.3反轉錄聚合酶連鎖反應 (Reverse transcription-polymerase chain reaction, RT-PCR) 19
3.6.4及時定量聚合酶連鎖反應 (Real-time quantitative PCR) 20
3.6.5蛋白質含量檢測 20
3.6.6攻擊試驗 21
3.6.7血清抗體力價分析 21
3.7統計分析 21
第四章 結果 23
4.1實驗菌株 23
4.1.1菌株確認及強化 23
4.1.2菌株毒力試驗 23
4.2疫苗於模擬石斑腸胃環境中的抗體釋放量測試 23
4.3主動免疫實驗 24
4.3.1接種後三個月內免疫基因表現量 24
4.3.1.1 IL-1β 基因在免疫後石斑魚頭腎之表現情形 24
4.3.1.2 TLR9 基因在免疫後石斑魚頭腎之表現情形 24
4.3.1.3 T-bet 基因在免疫後石斑魚頭腎之表現情形 25
4.3.1.4 GATA-3 基因在免疫後石斑魚頭腎之表現情形 25
4.3.1.5 CD4 基因在免疫後石斑魚頭腎之表現情形 25
4.3.2攻擊試驗 25
4.3.3接種疫苗後之血清抗體力價分析 26
4.3.3.1接種疫苗後石斑血清對 V. parahaemolyticus 之抗體力價分析 27
4.3.3.2接種疫苗後石斑血清對 V. vulnificus 之抗體力價分析 28
4.3.3.3接種疫苗後石斑血清對 V. alginolyticus 之抗體力價分析 29
第五章 討論 30
5.1口服疫苗抗體釋放量 30
5.2接種口服疫苗對免疫基因的影響 31
5.3口服疫苗之保護力 32
第六章 結論 35
第七章 參考文獻 36

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