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研究生:林正隆
研究生(外文):Cheng-Long Lin
論文名稱:對龍膽石斑神經壞死病毒似病毒顆粒具高親和力之peptides之篩選研究
論文名稱(外文):Screening fragment peptides with high affinity bound to Dragon Grouper Nervous Necrosis Virus-like particles
指導教授:林全信林全信引用關係
指導教授(外文):Chan-Shing Lin
學位類別:碩士
校院名稱:國立中山大學
系所名稱:海洋生物科技暨資源學系研究所
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:2010
畢業學年度:98
語文別:中文
論文頁數:103
中文關鍵詞:噬菌體呈現胜肽技術
外文關鍵詞:phage display peptide technology
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魚類神經壞死病毒會感染多種魚類的仔稚魚並造成近乎 100%
的死亡率,造成養殖魚業大量的損失。本研究以龍膽石斑神經壞死病毒(DGNNV)之似病毒顆粒(VLPs)作為材料,使用噬菌體呈現胜肽技術來篩選抗體決定位的一致性序列。分別以兩種淘選方法大量篩選噬菌體,以酵素連結免疫分析(ELISA)確認其親和力。測試不同的Blocking Buffer 與不同的 96 孔盤之對 ELISA 分析的影響,另外還測試 96 孔盤照射 UV 光之影響。最後挑選高親和力的噬菌體進行定序,將序列以 NCBI 的蛋白質序列比對軟體 BLAST 比對後,12 株的胜肽與 claudin 之間互相比對,結果顯示其 EL1 和 EL2 兩區域與所定序得到的胜肽之相似程度較高,而有部分不是相似於 claudin 的序列,要
進行更多 receptor 的比對,才能更確定此 paratope 胜肽的性質。
Piscine nodaviruses are members of genus Betanodavirus, which infects a large number of species of fish and causes massive mortality in larvae and juveniles. The disease causes great economic losses in aquaculture and sea-ranching. Virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were employed to screen the consensus sequences of paratopes by the phage display peptide
technology. Using two biopanning methods to screen phages, their binding efficiency was examined by ELISA. The effects of treatment by different blocking buffers on the ELISA assays and the effects of the ELISA plates exposed to UV light were tested. The display phages with
high binding efficiency were sequenced after the inserted fragment by PCR amplification. With NCBI BLAST, they were closely related to the claudin family and the consensus sequences were on the region of EL1 or
EL2. Few non-claudin sequences demand further comparison with other kinds of receptors to ascertain the characters of these sequences.
中文摘要 I
Abstract II
目錄 III
表目錄 V
圖目錄 VI
壹、前言
一、台灣石斑養殖現況 1
二、魚類神經壞死病毒(fish nervous necrosis virus) 3
三、病毒性神經壞死症(viral nervous necrosis)之臨床表徵 5
四、Betanodavirus 之基因體研究 5
五、外殼蛋白之特性分析 7
六、魚類神經壞死病毒之防疫 10
七、噬菌體呈現胜肽基因庫技術 14
八、研究目的 19
貳;材料與方法
一、似病毒顆粒(viruslike particle, VLPs)之製備與純化 20
二、SDSPAGE 蛋白質電泳 21
三、蛋白質定量 (Lowry method) 22
四、噬菌體呈現胜肽基因庫 23
五、淘選(Biopanning) 23
六、噬菌體效價測試(Phage titering) 25
七、酵素聯結免疫分析(ELISA) 25
八、不同blocking buffer 對於ELISA Binding Assay 的影響 26
九、以UV 光照射ELISA plate 27
十、蔗糖梯度離心淘選法及連續式分光光度計分析 29
十一、DNA 定序反應 30
(1) 噬菌體DNA 的製備 30
(2) PCR 反應及定序 31
十二、龍膽石斑神經壞死病毒之抗體決定位圖譜分析 31
参、結果
一、淘選之效價測試結果 33
二、不同ELISA plate 與不同Blocking Buffer 的影響親和力變化 33
三、以UV 光照射ELISA plate 之影響 35
四、噬菌體之ELISA 結果 36
五、Epitope 及paratope 型之噬菌體比較 37
六、蔗糖梯度離心淘選法之ELISA 結果 38
七、挑選高親和力價噬菌體之定序 39
八、胺基酸序列的比對與整理 40
肆、討論 42
伍、參考文獻 54
陸、圖表 66
附錄 A 86
附錄 B 87
附錄 C 88
附錄 D 89
附錄 E 90
附錄 F 92
附錄 G 93
附錄 H 94
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