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研究生:林家弘
研究生(外文):Jia-HungLin
論文名稱:探討蝦類Dscam於白點症病毒感染時之免疫機制
論文名稱(外文):Dscam-mediated immune response to WSSV infection
指導教授:王涵青王涵青引用關係
指導教授(外文):Han-Ching Wang
學位類別:碩士
校院名稱:國立成功大學
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:2014
畢業學年度:102
語文別:中文
論文頁數:90
中文關鍵詞:南美白對蝦白點症病毒唐氏綜合症細胞黏附分子
外文關鍵詞:Litopenaeus vannameiWhite spot syndrome virusDown syndrome cell adhesion molecule.
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本實驗室發現之蝦類高變異性分子 Dscam 的研究顯示,蝦體免疫具有專一性識別不同病原體的特性。在蝦體中,Dscam 存在兩種形式:具有胞質尾的鑲膜型 Dscam 及不具胞質尾的胞泌型 Dscam;胞外區域可經由選擇性剪切產生多變性以參與病原體辨認,而在胞質尾部分之多變性推測可驅使不同下游訊息反應。由於蝦類 Dscam 對於病原體所誘發的免疫特性及反應過程並未被討論,因此本研究乃以白點症病毒作為刺激物,觀測 Dscam 在感染過程的表現模式。結果顯示,在免疫相關組織所表現的鑲膜型 Dscam,其胞質尾帶有免疫相關功能性區域的比例遠超過其他組織,顯示其參與蝦體免疫功能的可能性。在全蝦體組織之整體 Dscam 的變化情形則顯示,病毒感染可誘發蝦體血細胞產生胞泌型 Dscam,推測胞泌型 Dscam 可被分泌進入血淋巴中以識別病原體。本研究接續以螯蝦作為實驗動物以進行長期觀察,從中發現在感染病毒後的存活者,其血漿中可偵測到較高量的 Dscam。利用膠體過濾分析,顯示其可能具有形成多聚體特性。根據以上結果,本研究推測,蝦類 Dscam 在剪切機制、免疫反應及分子結構上,相似於脊椎動物抗體 IgM,也具有相似的作用機制,並參與在白蝦對抗白點症病毒過程中。
Until now, the mechanism of shrimp immune response still not fully understood. Previously, we found that shrimp has a hypervariable molecule called Down syndrome cell adhesion molecule (Dscam) which can recognize different pathogens. Based on Dscam structure and immune properties, Dscam has been proposed to be an antibody-like molecule in arthropod. In this study we use white spot syndrome virus (WSSV) as pathogen stimulator to observe the Dscam’s cytoplasmic tail variants expression in various tissue after WSSV infection. The results show that cytoplasmic tail element 1A which consist of an endocytosis motif was expressed mainly in immune-related tissues, while element 1B which lack of this motif and expressed mainly in non-immune related tissues.So, cytoplasmic tail element 1A may be involved in Dscam immune function. Furthermore, the membrane-bound and tail-less Dscam expressions were analyzed in various tissue after WSSV infection. In hemocyte, tail-less Dscam can be induced after 36 hour post WSSV infection. Thus, we hypothesized that high amount of tail-less Dscam can be secreted into hemolymph after pathogen challenge. In order to test the hypothesis, hemolymph from two months crayfish survivors after WSSV infection were collected. High level Dscam expression was detected in the WSSV survivor’s hemolymph. To investigate whether Dscam has similar property with IgM. We use gel filtration to analyze the possibility of Dscam forms polymer in hemolymph. The result shows that Dscam may form dimer or heterpolymer in the hemolymph. Taken together, based on Dscam cytoplasmic tail splicing mechanism, the secretion ability of Dscam after pathogen challenge and the polymer structure, this study suggested that Dscam may be an IgM-like moleculer in arthropod..
目錄
中文摘要...............................................I
英文摘要..............................................II
誌謝.................................................VII
目錄..................................................IX
表目錄...............................................XII
圖目錄..............................................XIII
縮寫表................................................XV
一、前言...............................................1
1-1 全球水產養殖現況....................................1
1-2 台灣蝦類養殖面臨之問題...............................1
1-3 蝦類免疫系統........................................3
1-4 無脊椎動物免疫專一性及記憶性.........................4
1-5 免疫印記 (Immune priming) 及免疫相關之多樣性分子......5
1-6 Dscam 結構及功能研究................................6
1-7 Dscam 在白蝦之研究..................................8
1-8 疫苗對蝦類之研究.....................................9
二、研究方法及進行步驟..................................11
2-1 實驗動物及病毒源....................................11
2-2 實驗設計及實驗動物配置...............................11
2-3 Total RNA 萃取.....................................14
2-4 反轉錄聚合酶連鎖反應 (RT-PCR).......................14
2-5 PCR 產物純化.......................................15
2-6 基因選殖確認 (Bule and white screening)............16
2-7 菌落聚合酶連鎖反應 (colony PCR).....................17
2-8 質體 DNA 萃取......................................18
2-9 限制酶切割及定序....................................18
2-10 即時定量聚合酶連鎖反應 (Real-time PCR)..............19
2-11 利用膠體過濾分析 Dscam 之結構.......................19
2-12 利用大腸桿菌 BL-21 (DE3) 表現重組 VP28..............23
2-13 VP28 蛋白表現確認..................................25
2-14 VP28 次單位疫苗注射................................27
三、實驗結果............................................28
3-1 Cytoplasmic tail element 1A/B 具有組織特異性........28
3-2 在白點症病毒感染後 E1A 出現頻率提升...................28
3-3 WSSV感染後血球細胞中 Dscam以 Tail-less 形式為主......29
3-4 分泌型 Dscam 存在於白點症病毒感染後之血淋巴中.........31
3-5 Dscam 會以多聚體形式存在於螯蝦血淋巴中................32
3-6 白蝦在VP28注射後能有效獲得保護力.....................33
3-7 白蝦在 VP28 注射後能誘導 Tail-less Dscam 表現提升....34
四、討論...............................................36
4-1 Cytoplasmic tail element 1A/B在全組織的分布情形.....37
4-2 E1A/B 在病原性病原體白點症病毒刺激後之表現情形........38
4-3 兩種不同型式之 Dscam 在白點症病毒感染後表現情形.......39
4-4 螯蝦 Dscam 在白點症病毒感染後血淋巴的表現情形.........41
4-5 螯蝦血淋巴中 Dscam 之構型分析........................42
4-6 白蝦以 VP28 次單位疫苗注射後之保護效率 ...............43
4-7 白蝦在 VP28 注射後能誘導 Tail-less Dscam 表現提升....43
五、參考文獻............................................46

表目錄
表一、本論文中使用之引子.................................54
表二、Dscam 與抗體特性之比較.............................55


圖目錄
圖一、分析 Cytoplasmic Element 1A/B 在全組織的分布情形...56
圖二、分析在白點症病毒或PBS 注射後,鰓在胞質尾區域E1A 及 E1B
的變化情形..............................................57
圖三、分析在白點症病毒或 PBS 注射後,淋巴器官在胞質尾區域 E1A
及 E1B 的變化情形 .......................................58
圖四、Tail-less Dscam 在白點症病毒感染之後於不同時間點表現情形之絕對值..............................................59
圖五、Membrane-bound Dscam 在白點症病毒感染之後於不同時間點表現情形之絕對值........................................62
圖六、Tail-less Dscam 在白點症病毒感染之後於不同時間點表現情形之改變倍數..........................................65
圖七、Membrane-bound Dscam 在白點症病毒感染之後於不同時間點表現情形之改變倍數......................................68
圖八、利用Western blot 檢測澳洲螯蝦之血淋巴中確實存在分泌Dscam 分子.............................................71
圖九、不同 Dscam 分子量及異構型..........................72
圖十、VP28 疫苗實驗設計及保護效力........................74
圖十一、 Tail-less Dscam 在 VP28 次單位疫苗注射後於不同時間點之表現情形..............................................75
圖十二、 VP28 在白點症病毒感染之後於不同時間點表現情形之改變倍數.....................................................77
圖十三、 利用 Western blot 檢測澳洲螯蝦之血淋巴中不同分子量Dscam 分子.............................................80
圖十四、 LvDscam isoform 20 (2) 藉由 LC-MS/MS 分析後所得之胺基酸片段..............................................81
圖十五、 LvDscam isoform 20 (3) 藉由 LC-MS/MS 分析後所得之胺基酸片段..............................................83
圖十六、 W4 (4) 藉由 LC-MS/MS 分析後所得之胺基酸片段......85
圖十七、 W4 (5) 藉由 LC-MS/MS 分析後所得之胺基酸片段......87
圖十八、 W4 (6) 藉由 LC-MS/MS 分析後所得之胺基酸片段......89

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