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研究生:阮燕華
研究生(外文):Yuan-Hwa Ruan
論文名稱:南美白蝦鐵蛋白分子特性、表現及其參與生理及免疫調控之研究
論文名稱(外文):Molecular cloning and expression of Ferritin, and its involvements in physiology and immunity of the Pacific white shrimp, Litopenaeus vannamei
指導教授:胡清華胡清華引用關係郭欽明郭欽明引用關係
指導教授(外文):Chin-Hwa HuChin-Ming Kuo
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:128
中文關鍵詞:生理指標免疫指標病原攻擊鐵蛋白基因南美白蝦
外文關鍵詞:FerritinLitopenaeus vannameiimmune parametersphysiological parameterpathogen challengeVibrio alginolyticuswhite spot syndrome virus(WSSV)
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本研究探討弧菌(Vibrio alginolyticus)和白斑病毒(White spot syndrome virus, WSSV)攻擊感染或鐵蛋白注射南美白蝦後72 hr內之感染死亡率、免疫及生理反應變化,以及鐵蛋白基因之表現等,藉以瞭解病原感染所引起生理及免疫指標間之相關性,鐵蛋白對生理及免疫之影響,進行全盤性之分析研究。
鐵蛋白(ferritin)為生物體內重要的儲鐵蛋白,由heavy chain和light chain共24個subunits以及4500個鐵原子圍繞著中心所組成,儲鐵蛋白ferritin可以儲存過量的鐵,對於維持體內鐵質平衡扮演了重要的角色,另外亦具有解毒作用。
南美白蝦鐵蛋白(ferritin)基因之cDNA全長序列,有1,249 bp,其中包含有510 bp的轉譯讀窗(open reading frame),相當於170胺基酸序列,5’端未轉譯區域132 bp,以及3’端未轉譯區域607 bp,在GenBank中註冊之序號為AY955373。與Crayfish、Tick及Brine shrimp相似度分別為86%、79% 和78%,且在演化上與Crayfish最相近。組織分布方面,ferritin在白蝦血球中的表現量遠高於其他組織。
白蝦分別受到溶藻弧菌(Vibrio alginolyticus)以及白斑病毒(White spot syndrome virus, WSSV)攻擊,均在12 hr後,出現死亡的現象,且其總血球數(THC)、酚氧化酵素(PO)、呼吸爆(O2-)及超氧歧化酵素(SOD)等免疫指標,均有下降之趨勢,顯著低於控制組。ferritin基因表現會受到病原感染而誘發及促進調節(up-regulation)的現象。該等指標之最低點是因病原而異,弧菌在感染36 hr時,白斑病毒則在12~24 hr的階段。在白蝦血糖量(hemolymph glucose)、血乳酸量(hemolymph lactate)及血脂質量(hemolymph lipid)等受到弧菌或WSSV攻擊後都呈現上揚的趨勢,顯著高於控制組。生理指標之上升最高的時間點也因病原而不同,弧菌攻擊下均出現在36 hr,至於白斑病毒攻擊下,血糖、血乳酸及血脂量分別在24, 12, 24 hr達到最高點,由此得知,白蝦受到病原菌感染之後會造成生理的緊迫之外,也會降低其免疫能力,死亡率升高,再者,病原感染注射處理亦引起生理指標上升之現象,對免疫指標則無。生理與免疫指標間亦呈現顯著之相關性。
白蝦注射0.1、0.5、1 ng g shrimp-1劑量之鐵蛋白後,給予WSSV之攻擊,追蹤分析活存率,生理與免疫機能指標。鐵蛋白之注射增強THC、呼吸爆及PO和SOD活性表現,與病毒感染時引發免疫機能下降之現象確有不同之處。特別是注射0.5 ng ferritin g-1之增強免疫機能效果最佳,白蝦存活率亦達87.5%。由此推知,鐵蛋白是具有免疫刺激物質之功效,可提高白蝦抵抗WSSV感染,降低死亡率。至於注射鐵蛋白處理後,白蝦之生理指標,包括血淋巴液中血糖量、血乳酸量及血脂量指標變動趨勢,較為不顯著,確與未加鐵蛋白注射白蝦,引發高血糖、高乳酸及高血脂現象確有不同之處,證實注射ferritin不但不會造成白蝦的緊迫,卻有穩定生理之功效,也證實鐵蛋白在innate immune防禦機制上扮演重要的角色。

關鍵字:生理指標、免疫指標、病原攻擊、鐵蛋白基因、南美白蝦
The objective of this study is aimed to comprehend the molecular characterization and expression of ferritin gene, and to monitor the survival, immune and physiological responses, and the expression of haemocyte ferritin of the white shrimp, Litopenaeus vannamei under Vibrio and WSSV challenge for 72 hr period. Correlations between physiological and immunological parameters, involvements and physiological roles of ferritin in the immunological compensation process were further attempted.
Ferritin, a cytosolic iron storage protein composed of 24 subunits of heavy chain and light chain, is an intracellular protein primarily involved in iron metabolism and detoxification. A full-length ferritin cDNA was cloned from the haemocyte of the Pacific white shrimp. It comprises 1249 bp, including 132 bp in the 5’-untranslated region, 510 bp in the open reading frame which encodes 170 amino acid residues, and 607 bp in the 3’-untranslated region. The ferritin sequence has been registered in the GenBank (AY955373). Alignments of the deduced amino acid sequence further showed that the Pacific white shrimp ferritin is evolutionally closer to crayfish, tick and brine shrimp than others, sharing 86%, 79% and 78% identity, respectively. The tissue-specific expression pattern was examined by reverse transcript polymerase chain reaction and real-time quantitative PCR. The ferritin mRNA is expressed in various tissues of the shrimp, highest in haemocyte.
The susceptibilities of the white shrimp to the challenge of Vibrio alginolyticus and white spot syndrome virus (WSSV) were determined, and the physiological and immune responses of these shrimp were also monitored for 72 hr period. The immune parameters measured include total haemocyte count, phenoloxidase and superoxide dimutase activities and respiratory burst, while the physiological parameters cover hemolymph glucose, lactate and lipid contents.
The mortality of the white shrimp started to appear at 12 hr after Vibrio and WSSV challenge, and depression of the immune parameters (total haemocyte count, phenoloxidase and superoxide dimutase activities, and respiratory burst) were also observed. The expression of ferritin gene was up-regulated in both cases. Besides, occurrence of the minimum level of immune parameters under the challenge varied with types of pathogens, 36 hr under Vibrio and 12~24 hr under WSSV. The physiological parameters (hemolymph glucose, lactate and lipid) all notably elevated under Vibrio and WSSV challenge, and the time of highest increases varied also with pathogens. These parameters were all peaked at 36 hr under Vibrio infection, while under WSSV challenge, hyperglycemia occurred at 24 hr, hyperlacticemia at 12 hr and hyperlipidemia at 24 hr. These observations suggested that the pathogen challenge imposed physiological stress and immune depression of the white shrimp, resulted in increased mortality. In addition, the handling and injection stress do induce increasing trends of the physiological parameters, and the challenge from the pathogens augmented the magnitude of elevation, but this was not true for the immune parameters.
Effects of a single ferritin injection at the doses of 0.1, 0.5 and 1 ng ferritin/g shrimp on the physiological and immune responses were also monitored for 72 hr period. The experiment with WSSV challenge was also attempted. The administration of ferritin improved the immunity of the white shrimp, indicated by significant elevation of immune parameters to the control level or even higher, and the survival was improved as well. The best survival of 87.5% was obtained by a single injection of 0.5 ng ferritin/g shrimp, at this dose, the immunity was notably enhanced. It is therefore concluded that ferritin possess the immunostimulant functions, indicated by improved resistance to WSSV and increased survival under the challenge. Furthermore, the physiological parameters were not affected by ferritin injection, even in the case that the shrimp were challenged with WSSV. It is evidently demonstrated that ferritin is able to reduce stress response and is consequently capable to maintain physiological homeostasis. The ferritin does play important roles in the innate immunity of the white shrimp.

Key words: Ferritin, Litopenaeus vannamei, immune parameters, physiological parameter, pathogen challenge, Vibrio alginolyticus, white spot syndrome virus (WSSV)
目錄

頁次
中文摘要……………………………………………………………….…i
英文摘要………………………………………………………………..iii
目錄……………………………………………………………………..vi
圖目錄………………………………………………………………......vii
表目錄………………………………………………………………. …xi
第一章 前言…………………………………………………………….1
第二章 文獻整理………………………………………………………11
第三章 材料與方法……………………………………………………24
第四章 結果……………………………………………………………37
第五章 討論……………………………………………………………54
第六章 結論……………………………………………………………63
第七章 參考文獻………………………………………………………64
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