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研究生:劉伯政
論文名稱:2010至2012年台灣南部地區雞傳染性貧血病毒株親緣關係研究
指導教授:連一洋
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:獸醫學系所
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:2013
畢業學年度:101
語文別:中文
中文關鍵詞:雞傳染性貧血病毒、聚合酵素鏈鎖反應、親緣樹分析
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雞傳染性貧血症是由雞貧血病毒所引起,屬於環狀病毒科之迴旋病毒屬,常造成雞隻貧血與免疫抑制,對全球各地雞隻皆造成相當大的健康問題與養雞業者的經濟損失,為瞭解近年來台灣南部CAV病毒株的核苷酸變異情形,及與疫苗株及其它國外株之間之相關性,故將2010 年至2012 年於台灣南部雞場所收集之17 株陽性檢體以聚合酶鏈鎖反應(PCR) 增幅全長基因,其產物經定序後長度為2,298 bp,與美國國家生物資訊中心基因庫資料比對分析後,發現與各國分離株之序列相似性達95.1%到99.6%。將台灣CAV病毒株與各國病毒株親緣樹分析顯示,其分群可區分為3 個主要分枝基因型。其中大多數分離株和中國大陸株相近,少數病毒株與日本株、美國株等類似,顯見台灣的CAV的來源多源化且受中國大陸影響甚巨,推測可能因為台灣自不同國家輸入種、雛雞及其他禽類遷移,造成病毒的傳播,亦顯示台灣南部地區CAV病毒株具遺傳變異,可能為受到不同來源的病毒株相互影響所產生之結果。
中文摘要 I
Abstract II
誌謝………………………………………………………………………..... III
目錄 IV
圖表目錄 VI
第1章 前言 1
第2章 文獻回顧 3
2.1 雞傳染性貧血症簡介(chicken infectious anemia, CIA) 3
2.1.1 CIA歷史與背景 3
2.1.2 雞傳染性貧血病毒(chicken anemia virus;CAV)之分佈 3
2.1.3 CAV之症狀與病變 4
2.2 CAV之性狀 5
2.2.1 CAV之分類與結構 5
2.2.2 CAV之理化特性 6
2.2.3 CAV之基因體 6
2.2.4 CAV之蛋白質及其功能 7
2.2.5 CAV之致病機轉 8
2.2.6 CAV之增殖與培養 9
2.2.7 CAV之疾病診斷 9
2.2.8 CAV之預防與控制 10
2.3 各國對CAV序列研究狀況 11
2.3.1 國際研究現況 11
2.3.2 台灣研究現況 12
第3章 材料與方法 14
3.1 CAV病例臟器檢體採集 14
3.1.1 臟器乳劑之製備 14
3.1.2 病毒核酸萃取 14
3.1.3 DNA濃度定量 15
3.1.4 聚合酶鏈鎖反應(polymerase chain reaction,PCR) 15
3.1.5 電泳分析 16
3.2 CAV基因之增幅、選殖與定序 16
3.2.1 CAV基因之增幅 16
3.2.2 PCR增幅產物之純化 17
3.2.3 PCR產物之選殖 18
3.2.4 重組質體DNA之抽取與核酸定序 18
3.3 CAV基因序列親緣分析 19
第4章 結果 21
4.1 CAV病材收集與核酸增幅 21
4.2 CAV基因增幅與選殖 21
4.3 核酸與氨基酸序列比對 21
4.3.1 全長核酸序列比對 21
4.3.2 CAV VP1、VP2及VP3核酸序列比對 22
4.3.3 CAV VP1、VP2及VP3氨基酸序列比對 22
4.4 親緣關係樹建構 22
4.4.1 CAV全長核酸親緣關係樹建構 22
4.4.2 CAV VP1、VP2及VP3親緣關係樹建構 23
第5章 討論 50
參考文獻 54
作者簡介 61
附錄 62














圖表目錄

Fig. 1 The 418 bp nucleotide fragments of chicken anemia virus amplified by PCR 24
Fig. 2 Characterization of Anemia by hematocrit values 24
Fig. 3 Severe hemorrhage in the skeletal muscles of the breast 25
Fig 4. Amplification of chicken anemia virus genome by PCR with primer CAV-G1. 26
Fig 5. Amplification of chicken anemia virus genome by PCR with primer CAV-G2 27
Fig 6. Percentage similarities and divergences of the 2,298 bp nucleotide sequence of full length gene in 38 chicken anemia virus isolates using the DNASTAR software package 28
Fig 7. Percentage similarities and divergences of the 2,298 bp nucleotide sequence of full length gene in 17 chicken anemia virus isolates using the DNASTAR software package 29
Fig 8. Percentage similarities and divergences of the 1,350 bp nucleotide sequence of VP1 gene in 38 chicken infectious anemia virus isolates using the DNASTAR software package 30
Fig 9. Percentage similarities and divergences of the 651 bp nucleotide sequence of VP2 gene in 38 chicken anemia virus isolates using the Clustal program of the DNASTAR software package 31
Fig 10. Percentage similarities and divergences of the 366 bp nucleotide sequence of VP3 gene in 38 chicken infectious anemia virus isolates using the Clustal program of the DNASTAR software package 32
Fig 11. Percentage similarities and divergences of the amino acid sequence of VP1 in 38 chicken anemia virus isolates using the DNASTAR software package 33
Fig 12. Percentage similarities and divergences of the amino acid sequence of VP2 in 38 chicken anemia virus isolates using the DNASTAR software package 34
Fig 13. Percentage similarities and divergences of the amino acid sequence of VP3 in 38 chicken anemia virus isolates using the DNASTAR software package 35
Fig 14. The major genetic determinant of CAV virulence (aa 394) 36
Fig 15. Amino acid alignment of highly variable region of VP1 coding sequences of different CAVs (aa 139-151) 37
Fig 16. Phylogenetic relationship among 17 Taiwanese CAVs and 21 full-length nucleotide sequences from different countries using neighbour joining distance method, with 1,000 bootstrap replications 38
Fig 17. Phylogenetic relationship among 17 Taiwanese CAVs and 21 strain VP1 nucleotide sequences from different countries using neighbour joining distance method, with 1,000 bootstrap replications 39
Fig 18. Phylogenetic relationship among 17 Taiwanese CAVs and 21 strain VP2 nucleotide sequences from different countries using neighbour joining distance method, with 1,000 bootstrap replications 40
Fig 19. Phylogenetic relationship among 17 Taiwanese CAVs and 21 strain VP3 nucleotide sequences from different countries using neighbour joining distance method, with 1,000 bootstrap replications. 41
Fig 20. Phylogenetic analysis of the VP1 amino acid sequence of 17 new complete sequences from Taiwan and 21 sequences currently available in GenBank 42
Fig 21. Phylogenetic analysis of the VP2 amino acid sequence of 17 new complete sequences from Taiwan and 21 sequences currently available in GenBank 43
Fig 22. Phylogenetic analysis of the VP3 amino acid sequence of 17 new complete sequences from Taiwan and 21 sequences currently available in GenBank 44
Table 1. Source of chicken infectious anemia viruses isolated in Taiwan during 2010-2012. 45
Table 2. CAV DNA sequences used for phylogenetic analysis and genetic variation comparisons 47
Table 3. Polymorphisms in predicted amino acid sequence among Taiwanese CAV VP1 squences 48
Table 4. Polymorphisms in predicted amino acid sequence among Taiwanese CAV VP2 and VP3 squences 49

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