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研究生:王嚴緒
研究生(外文):Yan-Syu Wang
論文名稱:鵝小病毒活毒疫苗株與田間強毒株之全長序列比較
論文名稱(外文):Comparison of Complete Nucleotide Sequences of Live Vaccine Strains and Virulent Field Strains of Goose Parvovirus
指導教授:張伯俊
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫微生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:48
中文關鍵詞:鵝小病毒末端重複序列位置髮夾狀結構
外文關鍵詞:goose parvovirusinverted terminal repeats regionhairpin structure
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鵝小病毒 (Goose parvovirus, GPV) 之活毒疫苗,目前已被廣泛應用於GPV所引起疾病之防治,然而此GPV活毒疫苗之馴化機制目前仍不清楚。本研究完成二株GPV活毒疫苗株,即台灣82-0321V株與歐洲VG32/1株,以及二株台灣田間分離株 (即82-0321與06-0329株) 之全長基因體序列之定序工作,其中82-0321為82-0321V之親代株,而06-0329為2006年台灣田間分離株。序列之分析結果顯示,如與其親代株比較,疫苗株82-0321V與VG32/1在病毒DNA之末端重複序列位置,含有多處之核酸序列缺損 (deletion) 及取代 (substitution) ,然而這些缺損與取代,並不會影響病毒DNA之末端髮夾狀結構 (hairpin structure) 之形成
以及病毒之複製。另一方面疫苗株82-0321V 與VG32/1在其非結構蛋白 (nonstructural protein, NS) 之胺基酸序列中,也各有5個胺基酸之改變,但這些改變所發生之位置,皆遠離NS蛋白已知之功能區域,應不會影響NS蛋白之功能。相對而言,疫苗株82-0321V與VG32/1在其外殼蛋白 (capsid protein, VP1) 之胺基酸序列上,分別有9個及11個胺基酸序列之改變,而這些改變所發生之位置,大部分與VP1蛋白之受體結合區域 (receptor binding site) 極為接近,可能影響GPV受體之結合能力,而此受體結合區域是依據adeno-associated virus 2之結構來予以推定。總結而言,本研究之結果顯示,VP1受體結合區域之序列改變,應為GPV活毒疫苗株馴化之主要原因,本研究為第一篇探討水禽小病毒活毒疫苗馴化機制之報導。
Live attenuated vaccines have been used for control of the disease caused by goose parvovirus (GPV), but the mechanism involved in attenuation of GPV remains elusive. This report presents the complete nucleotide sequences of two live attenuated strains of GPV (82-0321V and VG32/1) that were independently developed in Taiwan and Europe, together with the parental strain of 82-0321V and a field strain isolated in Taiwan in 2006. Sequence comparisons showed that 82-0321V and VG32/1 had multiple deletions and substitutions in the inverted terminal repeats region when compared with their parental strain or the field virus, but these changes did not affect the formation of the hairpin structure essential for viral replication. Moreover, 82-0321V and VG32/1 had five amino acid changes in the non-structural protein (NS), but these changes were located at positions distant from known functional motifs in the NS protein. In contrast, 82-0321V had nine changes and VG32/1 had eleven changes in their capsid proteins (VP1), and the majority of these changes occurred at positions close to the putative receptor binding sites of VP1, as predicted by using the structure of adeno-associated virus 2 as the model system. Taken together, the results suggest that changes in sequence near the receptor binding sites of VP1 appear to be responsible for attenuation of GPV. This is the first report of complete nucleotide sequences of GPV other than the virulent B strain and suggests a possible mechanism for attenuation of GPV.
目次

中文摘要................................................................................................................... I
英文摘要................................................................................................................... II
目次........................................................................................................................... III
表次........................................................................................................................... V
圖次........................................................................................................................... VI
第一章 緒言............................................................................................................. 1
第二章 文獻探討..................................................................................................... 3
第一節 水禽小病毒歷史背景....................................................................... 3
第二節 水禽小病毒特徵............................................................................... 4
2-1 分類............................................................................................... 4
2-2 形態、結構與理化特性............................................................... 5
2-3 宿主範圍與病毒傳播................................................................... 6
第三節 小病毒之基因體與複製................................................................... 7
3-1 小病毒基因體................................................................................7
3-2 小病毒感染與複製........................................................................7
第四節 小病毒之非結構蛋白與結構蛋白................................................... 9
4-1 小病毒非結構蛋白 (NS) 的功能分析........................................9
4-2 小病毒結構蛋白 (VP1、VP2和VP3) 的功能分析..................9
4-3 台灣地區小病毒核酸序列之比對...............................................10
第五節 小病毒感染症之診斷與預防...........................................................11
5-1 臨床症狀與病理變化....................................................................11
5-2 小病毒感染之診斷方法................................................................11
5-3 小病毒感染之預防方法................................................................13
5-4 疫苗發展之現況............................................................................14
第三章 材料與方法..................................................................................................16
第一節 病毒來源及確認................................................................................16
1-1 病毒來源........................................................................................16
1-2 病毒核酸萃取................................................................................16
第二節 聚合酶連鎖反應 (polymerase chain reaction;PCR)......................16
2-1 引子 (Primers) 設計.....................................................................16
2-2 核酸增幅........................................................................................17
第三節 PCR產物之選殖 (Cloning) 與定序 (Sequencing).........................19
3-1 選殖 (Cloning).................................................................................19
3-1.1 PCR產物之萃取 (Extraction)..............................................19
3-1.2 TA-cloning............................................................................19
3-1.3 Colony PCR..........................................................................19
3-1.4 Isopropanol/ phenol / chloroform 萃取質體.......................20
3-2 定序 (Sequencing).........................................................................21
3-3 DNAStar分析 (DNAStar analysis)...............................................21
第四章 結果............................................................................................................22
第一節 PCR增幅鵝小病毒 (GPV) 之完整基因體...................................22
第二節 完整基因體之核酸序列..................................................................22
第三節 基因體序列之完整比對..................................................................23
第四節 反向末端重覆序列 (ITRs) 之比對...............................................23
第五節 非結構蛋白 (NS) 與VP1蛋白之胺基酸序列比對.....................24
第六節 Promoters與其他調節區域序列之比對.........................................25
第七節 小病毒立體結構圖之比對..............................................................26
第五章 討論............................................................................................................34
參考文獻..................................................................................................................37
附錄..........................................................................................................................49
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