- 學位論文
哈維氏弧菌鞭毛基因重組蛋白對石斑魚之佐劑效力研究
Development of recombinant protein adjuvant based on Vibrio harveyi flagellin gene in Epinephelus coioides
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摘要
哈維弧菌(Vibrio harveyi)是一種革蘭氏陰性桿菌，具有鞭毛可助於運動。這種細菌是引起蝦和一些海洋魚類弧菌病(vibriosis)的重要致病原病原，包括橙點石斑魚(Epinephelus coioides)和錦鯉(Cyprinus carpio)都是易感受的宿主。在哈維弧菌中，已鑑定出多種毒力基因，包含幾丁質酶(chitinase)、外毒素(exotoxin)、金屬蛋白酶(metalloprotease)、絲氨酸蛋白酶(serine protease)、溶血素(hemolysin)和鞭毛蛋白(flagellin)基因等，哈維弧菌鞭毛產生的毒素也可能是造成魚類感染的原因。本研究的目的是研究橙點石斑魚 (Epinephelus coioides) 和錦鯉 (Cyprinus carpio) 的基因表現反應。VhFliA 與 TLR5 的結合可以刺激細胞激素產生進而激活先天性和適應性免疫系統。本研究使用哈維弧菌鞭毛蛋白 (VhFliA) 和其突變基因蛋白注射石斑魚和錦鯉，並分析脾臟及頭腎中細胞激素(IFNγ、IL-1β、IL-6、IL-8 和 IL-10)以及 IFNγ 下游基因(MHC class I 、MHC class II 、CD4、CD8)等免疫基因表現。從脾臟和頭腎抽取RNA，並使用qRT-PCR對基因表現進行分析。本實驗中發現以每隻魚注射 5 µg 蛋白是產生最佳免疫反應的劑量，並於腹腔注射蛋白後6h、12h、24h分離脾、腎臟器抽取RNA。將以三種不同之實驗組及EGFP 作為對照組、野生型 VhFliA (WT-VhFliA) 和兩種 VhFliA 突變(ΔMH-VhFliA 和 ΔD0MH-VhFliA)。結果顯示TLR5、IFNγ及某些細胞因子下游基因上調。基因表現的增加約從 12 小時到 24 小時不等。在此研究中，我們鑑定並表現了哈維弧菌鞭毛蛋白 A (VhFliA) 基因。我們研究了重組 VhFliA 蛋白及其突變形式(ΔMH-VhFliA 和 ΔD0MH-VhFliA)在石斑魚和錦鯉中的體內活性。本研究將有助於利用重組 VhFliA 及其突變蛋白研製水產養殖物種的疫苗防疫策略。
並列摘要
Vibrio harveyi is a gram-negative bacterium that has flagella which are useful for locomotion. This bacterium is an important pathogen causing vibriosis in shrimp and some marine fish, including the orange-spot grouper (Epinephelus coioides) and koi carp (Cyprinus carpio), can become infected with these pathogens. In Vibrio harveyi virulent genes such as chitinase, exotoxin, metalloprotease, serine protease, hemolysin and flagellin genes have been identified. The toxin produced by Vibrio harveyi flagella can be a source of infections in fish. The purpose of this study was to investigate the immune response after injection flagellin and its mutant in orange-spot grouper (Epinephelus coioides) and koi carp (Cyprinus carpio). Binding of VhFliA to TLR5 could activate the innate and adaptative immune system to stimulate production of cytokines. This study used VhFliA and mutants gene and analyzed cytokine (IFNγ, IL-1β, IL-6, IL-8, and IL-10) and also IFNγ downstream gene (MHC class I, MHC class II, CD4, CD8) immune response gene. Determine the responsiveness of Vibrio harveyi flagellin (VhFliA) gene expression in the spleen and head kidney of grouper and carp. Total RNA was extracted from the spleen and head kidney, and qRT-PCR was used to analyze it. The dose utilized in this experiment was 5 µg/fish, which is the best level for determining immunological response. The dose utilized had already performed a preliminary test to ensure dose optimization. At 6h, 12h, and 24h after intraperitoneal injection, the spleen and kidney organs were isolated. There are four different treatments: EGFP as a protein control, wild-type VhFliA (WT-VhFliA), and two mutagenesis of VhFliA (∆MH-VhFliA and ∆D0MH-VhFliA). In the result, we've found an upregulation in TLR5, cytokines and IFNγ downstream genes. The time it took for gene expression to increase varied from 12 to 24 hours. In this study, we identified and characterized Vibrio harveyi flagellin A (VhFliA) gene. We investigated the in vivo activities of recombinant VhFliA protein and its mutant forms (∆MH-VhFliA and ∆D0MH-VhFliA) in grouper and carp. This study will provide the development of epidemic prevention strategies in aquaculture species using recombinant VhFliA and its mutants protein
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