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研究生:于寶琳
研究生(外文):Yu, Pauline
論文名稱:篩選能與石斑魚神經壞死病毒外殼蛋白產生交互作用之蛋白質
論文名稱(外文):Screening for the proteins that can interact with grouper nervous necrosis virus coat protein (GNNVCP)
指導教授:陳歷歷
指導教授(外文):Chen, Li-Li
口試委員:賴裕順呂健宏
口試委員(外文):Lai, Yu-ShenLeu, Ji-Ho
口試日期:2018-07-06
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:海洋生物研究所
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:2018
畢業學年度:106
語文別:中文
論文頁數:70
中文關鍵詞:神經壞死病毒酵母菌雙雜合法His-tag pull-down分析蛋白交互作用
外文關鍵詞:Nervous necrosis virusYeast two-hybrid systemHis-tag pull-down analysisInteract proteins
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神經壞死病毒是感染多種海水硬骨魚類的主要病原體,會引起魚類的神經壞死,並造成大量的死亡。目前神經壞死病毒的基因體與結構已有許多文獻可以參考,但對於病毒的致病機制卻尚未清楚。由於神經壞死病毒的構造上僅有外殼蛋白一種結構性蛋白,本研究使用兩種方法最為測試蛋白之間交互作用初步篩選。我們參考Chen 等人 2015年研究,將外殼蛋白兩大結構體基因片段作為釣餌分別進行酵母菌雙雜合法實驗,以及利用完整重組的外殼蛋白做His-tag pull-down 分析。初步篩選結果發現有幾個蛋白質會與外殼蛋白產生交互作用,這些分子主要可歸類為受器或共同受器、結合或分解細胞骨架、糖類代謝與ATP 生成、免疫相關、粒線體離子調節以及與核醣體蛋白結合等多種類型。本研究中鑑定的蛋白質特徵表明它們在病毒入侵和復製過程中起作用。病毒必須入侵寄主細胞才能完成複製程序,又因神經壞死病毒會引發誘導寄主產生細胞凋亡與細胞壞死現象,糖類代謝途徑受到影響與ATP 能量失衡也可能是造成細胞凋亡與細胞壞死的原因之一。這次結果中的蛋白質與神經壞死病毒之間的交互作用仍需要更多實驗去驗證其強弱及真偽。
Nervous necrosis virus (NNV) is the main pathogen that infects a variety of cultured marine fish, causing nerve necrosis in fish and causing high mortality rate. At present, there are many references for the gene and structure of nervous necrosis virus, but the pathogenic mechanism of the virus is not clear. To further investigate this mechanism this study investigated the interaction of the structural proteins by using two methods. First, the yeast two-hybrid system used the two large domains of coat proteins as bait; then the His-tag pull-down analysis used intact recombinant coat proteins to screen for protein interaction. Initial results indicate a list of proteins that can interact with coat proteins that may contribute to putative receptor or co-receptor, cytoskeleton, glucose metabolism and ATP generation, immunity, mitochondrial ion regulation and ribosomal proteins. Previous research has shown that the virus must invade the host cell to complete the replication process. The characteristics of the proteins identified in this research suggest that they function in virus invasion and replication process. Further indication of their function is that nerve necrosis virus has also been shown to induce the host to produce apoptosis and cell necrosis. Identified proteins function in metabolic pathways of sugar and calcium, which can affect ATP energy imbalance and may also be the cause of cell apoptosis and cell necrosis. Further experiments are needed to verify the strength and authenticity of the protein interaction with nervous necrosis virus found in this study.
壹、前言(文獻回顧) 1
1. 石斑魚之分類與習性 (Taxonomic and ecological of grouper) 1
2. 臺灣石斑魚養殖概況 (Aquaculture grouper in Taiwan) 1
3. 神經壞死病毒 3
4. 酵母菌雙雜合法 (Yeast two-hybrid system) 3
5. His-tag pull-down 分析 (His-tag pull-down assay) 4
貳、研究動機與目的 5
參、實驗架構 6
肆、實驗材料與方法 7
1. 製備與確認酵母菌雙雜合法材料 7
1.1. 製備NNVCP-pGBKT7重組質體 7
1.2. 將NNVCP-pGBKT7轉形至Y2HG酵母菌 - Bait 8
1.3. 製備與確認酵母菌雙雜合法Library材料 - Pery 10
1.4. Mating 12
1.5. Yeast two-hybrid mating之篩選與定序 12
1.6. Yeast two-hybrid mating之分析 13
2. His-tag pull-down 分析 14
2.1. NNVCP重組質體轉型至 BL21 大腸桿菌 14
2.2. NNVCP重組蛋白誘導測試 14
2.3. NNVCP 重組蛋白小量純化 14
2.4. NNVCP 重組蛋白大量純化 15
2.5. 石斑魚組織溶解液與NNVCP重組蛋白pull-down 分析 15
2.6. Pull-down分析樣本的西方墨點法 15
伍、研究結果 17
1. 酵母菌雙雜合法 17
2. His-tag pull-down 分析 17
陸、討論 20
柒、參考文獻 26
圖次
圖一、DDO/X-α-gal/AbA 培養皿 33
圖二、QDO/X-α-gal/AbA 培養皿 34
圖三、NNVCP重組蛋白誘導測試 35
圖四、NNVCP重組蛋白純化 36
圖五、全魚苗與重組 NNVCP 蛋白進行 Western Blot 37
圖六、全魚苗與重組 NNVCP 蛋白進行 His-tag pull-down 分析圓餅圖 38
圖七、成魚腦與重組 NNVCP 蛋白進行 Western Blot 39
圖八、成魚腦組織與重組 NNVCP 蛋白進行 His-tag pull-down 分析圓餅圖 40
圖九、成魚頭腎與重組 NNVCP 蛋白進行 Western Blot 41
圖十、成魚頭腎組織與重組 NNVCP 蛋白進行 His-tag pull-down 分析圓餅圖 42
圖十一、成魚肝臟與重組 NNVCP 蛋白進行 Western Blot 43
圖十二、成魚肝臟組織與重組 NNVCP 蛋白進行 His-tag pull-down 分析圓餅圖 44
圖十三、成魚視網膜與重組 NNVCP 蛋白進行 Western Blot 45
圖十四、成魚視網膜與重組 NNVCP 蛋白進行 His-tag pull-down 分析圓餅圖 46
表次
表一、酵母菌雙雜合法引子列表 47
表二、全魚苗與重組 NNVCP 蛋白進行 His-tag pull-down 分析 48
表三、成魚腦組織與重組 NNVCP 蛋白進行 His-tag pull-down 分析 50
表四、成魚頭腎組織與重組 NNVCP 蛋白進行 His-tag pull-down 分析 53
表五、成魚肝臟組織與重組 NNVCP 蛋白進行 His-tag pull-down 分析 57
表六、成魚視網膜與重組 NNVCP 蛋白進行 His-tag pull-down 分析 62
表七、魚組織與重組 NNVCP 蛋白進行 His-tag pull-down 分析統整 65
附錄
附錄一、台灣養殖的石斑魚種類 67
附錄二、石斑魚繁養殖過程中易受神經死病毒感染的階段 68
附錄三、石斑魚神經壞死病毒外殼蛋白結構 69
附錄四、酵母菌雙雜合系統 (Yeast two-hybrid system) 70
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盧子喬(2008)點帶石斑魚RNF2基因之選殖及鑑別RNF2蛋白與神經壞死性病毒殼體蛋白之交互作用。國立中正大學分子生物研究所學位論文。
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