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研究生:蔡雨蓁
研究生(外文):Yu-Chen Tsai
論文名稱:多殺型巴斯德桿菌脂多醣體抗原合成基因之研究與快速分類之應用
論文名稱(外文):Characterization of The Biosynthesis Genes for Lipopolysaccharide Somatic Antigens of Pasteurella multocida and Application to Rapid-typing
指導教授:張伯俊
指導教授(外文):Poa-Chun Chang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:微生物暨公共衛生學研究所
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:2010
畢業學年度:98
語文別:中文
論文頁數:69
中文關鍵詞:多殺型巴斯德桿菌脂多醣體抗原合成基因分類
外文關鍵詞:Pasteurella multocidalipopolysaccharidesomatic antigenbiosynthesis genetyping
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Pasteurella multocida (多殺性巴斯德桿菌)為能引起多種重要動物疾病的病原菌,其脂多醣體抗原 (lipopolysaccharide somatic antigen)是主要的毒力因子之一,傳統上可用瓊膠擴散沉澱反應 (AGP)將P. multocida分為16種不同體抗原血清型,但此方法需製備高免血清且操作時間需1-2天,另一方面,目前在文獻中只有少數幾種P. multocida之體抗原之成份與其生合成基因序列被發表。本研究以PCR技術,成功增幅18株P. multocida標準株之體抗原合成基因序列,定序結果發現,依其序列可將P. multocida之體抗原合成基因分為5種 (G1-G5)不同型別,其中G1為能引起家禽霍亂之體抗原1型之禽源P. multocida (A:1);G2為體抗原2型與5型之菌株;G3為體抗原3型及4型之禽源菌株;G4為能引起豬萎縮性鼻炎之體抗原3型菌株及其他體抗原血清型10、11、12、15和16菌株;G5則為分離自人類之體抗原13型菌株。本研究亦利用PCR增幅與限制&;#37238;切割圖譜多型性 (restriction fragment length polymorphism,RFLP)來快速區分P. multocida標準株之體抗原型別,此技術並可應用於田間禽源與豬源P. multocida體抗原型別之快速鑑定,本研究為第一篇利用PCR/RFLP來鑑定P. multocida體抗原型別之報導。

Pasteurella multocida is the causative agent of many diseases in a wide range of domestic animals. Its lipopolysaccharide (LPS) somatic antigen is one of key virulence factors. Conventionally, P. multocida can be divided into 16 serotypes based on LPS somatic antigens by using the agar gel diffusion precipitation test (AGP). However, this method needs preparation of serotype-specific antisera, and takes few days to complete the work. To date, only a few of LPS structures and genome sequences for biosynthesis of LPS in P. multocida have been determined. In this study, we used PCR to amplify the biosynthesis genes of lipopolysaccharide somatic antigen of 18 reference strains of P. multocida. Based on sequencing results of the amplified genes, the biosynthesis genes of LPS of P. multocida could be divided into 5 groups (G1-G5). Strains belong to G1 are serotype A:1 which cause fowl cholera. Stains belong to G2 are LPS serotype 2 and 5. Strains belong to G3 are LPS serotype 3 and 4 that cause fowl cholera. Strains belong to G4 are LPS serotype 3 which causes atrophic rhinitis in pigs and other LPS serotypes such as 10, 11, 12, 15, and 16. Strains belong to G5 are LPS serotype 13 that infects humans. This study also develops a PCR/RFLP method for rapid identification of the LPS serotypes of reference strains of P. multocida quickly. Moreover, this method can be used to identify types of somatic antigen of field strains of P. multocida. This is the first report on development of a PCR/PFLP method for rapid identification of the somatic antigen serotypes of P. multocida.

中文摘要…………………………………………………………………ⅠI
英文摘要…………………………………………………………………ⅡI
目次………………………………………………………………………IV
表次………………………………………………………………………Ⅵ
圖次………………………………………………………………………Ⅶ
第一章 緒言……………………………………………………………1
第二章 文獻探討………………………………………………………2
 第一節 P. multocida之歷史背景…………………………………2
 第二節 P. multocida之生物學特性………………………………2
 第三節 P. multocida之生化特性…………………………………3
 第四節 P. multocida之血清學分型………………………………3
 第五節 P. multocida之傳染性疾病………………………………4
  5.1 感染與傳播途……………………………………………… 4
  5.2 臨床症狀及組織病理變化………………………………… 5
  5.3 診斷方法與鑑定…………………………………………… 7
   5.3.1 P. multocida之分離及鑑定………………………… 7
   5.3.2 聚合酶連鎖反應 (PCR)在診斷上的應用…………… 8
   5.3.3 血清學上對P. multocida之診斷…………………… 9
   5.3.4 其他診斷方法…………………………………………10
  5.4 疾病治療與控制……………………………………………11
 第六節 P. multocida之毒力因子……………………………… 11
  6.1 莢膜 (Capsule)……………………………………………11
  6.2 脂多醣 (Lipopolysaccharide)………………………… 11
  6.3 纖毛與黏附素 (Fimbriae and adhesins)………………14
  6.4 毒素 (Toxin)………………………………………………15
第三章 材料與方法………………………………………………… 16
 第一節 P. multocida之分離…………………………………… 16
 第二節 LPS生合成基因定序與其化學成分之分析………………16
  2.1 P. multocida之核酸萃取…………………………………16
  2.2 以PCR增幅P. multocida標準株LPS生合成基因群………17
  2.3 LPS生合成基因定序與序列分析………………………… 18
  2.4 限制酶切割圖譜多型性……………………………………18
 第三節 以PCR/RFLP方法分析P. multocida野外田間株……… 19
  3.1 以PCR增幅LPS生合成基因之特定片段……………………19
  3.2 限制酶切割圖譜……………………………………………20
  3.3 LPS生合成基因定序與序列分析………………………… 20
第四章 結果………………………………………………………… 22
 第一節 P. multocida之分離…………………………………… 22
 第二節 LPS生合成基因定序與基因結構分析……………………22
  2.1 LPS生合成基因定序……………………………………… 22
  2.2 限制酶切割圖譜……………………………………………22
  2.3  LPS生合成基因之序列分析………………………………23
 第三節 LPS生合成基因之演化親緣樹狀圖之分析………………25
  3.1 LPS生合成基因全長之演化親緣樹狀圖分析…………… 25
  3.2 LPS生合成基因pm1138之演化親緣樹狀分析…………… 25
  3.3 LPS生合成基因pm1139之演化親緣樹狀分析…………… 26
  3.4 LPS生合成基因pm1140之演化親緣樹狀分析…………… 26
  3.5 LPS生合成基因pm1141之演化親緣樹狀分析…………… 27
  3.6 LPS生合成基因rpL31_2之演化親緣樹狀分析……………27
  3.7 LPS生合成基因losA之演化親緣樹狀分析……………… 28
  3.8 LPS生合成基因pm1144之演化親緣樹狀分析…………… 28
 第四節 利用PCR/PFLP技術分析P. multocida野外田間株…… 28
 第五節 田間株Kobe-5之LPS生合成基因定序與基因結構分析…29
  5.1 LPS生合成基因定序……………………………………… 29
  5.2 LPS生合成基因之序列分析……………………………… 29
第五章 討論………………………………………………………… 60
參考文獻……………………………………………………………… 64


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