臺灣博碩士論文加值系統

雞傳染性鼻炎是一種雞的急性上呼吸道疾病，由雞傳染性鼻炎菌(Avibacterium paragallinarum) 感染所導致，病徵包括產蛋率下降及生長遲緩等問題，對家禽業造成嚴重的經濟損失。莢膜是一層多醣類或蛋白質外殼，由細菌產生並包覆在其細胞壁外圍，有助於保護細菌免受外界環境的損害，也有助於細菌對抗抗生素或宿主免疫系統的攻擊。早期之研究發現，Av. paragallinarum分離株之莢膜生合成基因群可分為兩種基因型，即莢膜第一基因型(Genotype I)與莢膜第二基因型(Genotype Ⅱ)。然而自2016年起，本實驗室透過細菌全基因體定序，發現台灣Av. paragallinarum田間分離株，除莢膜第一、二基因型外，還存在了一個新的莢膜基因型，被命名為莢膜第三基因型(Genotype Ⅲ）。第三基因型在序列上與第二基因型相似，但第三基因型序列中額外嵌入了一個源自鴨源雞桿菌(Gallibacterium anatis)的gcbC基因，其功能不明。本研究之目標分為四個部分，第一部分為進行疫苗保護力測試，探討疫苗對於莢膜第二、三基因型菌株之保護效力是否有差異，結果發現疫苗之保護力與攻毒株之莢膜基因型無相關性。本研究第二部分則進行菌株致病力之相關性分析，發現莢膜基因型與菌株致病力並無明顯相關性。本研究第三部分則是萃取莢膜並分析其化學成份，結果發現第二、三基因型莢膜之主成分皆為肝素醣。本研究第四部分透過NCBI-blast和序列比對，預測GcbC蛋白的功能。結果顯示GcbC與AsnS和CarA具有序列相似性。以上發現有助於對Av.paragallinarum致病機轉與GcbC功能之了解。

Infectious coryza is an acute respiratory disease in chickens caused by Avibacterium paragallinarum infection, which can lead to a decrease in egg production and growth retardation, causing significant economic losses to the poultry industry. Capsule is a layer of polysaccharides or protein shell produced by bacteria and enveloped in their cell walls. Capsule protects bacteria from damage by the external environment and aids in bacterial resistance against antibiotics or host immune responses. Early studies found that the capsule biosynthesis gene clusters of Av. paragallinarum were classified into two genotypes: capsule genotype I and capsule genotype II. However, since 2016, through whole-genome sequencing, our laboratory has discovered a new capsule genotype in Taiwanese field isolates of Av. paragallinarum, named capsule genotype III. Genotype III has sequences similar to genotype II, but genotype III contains an inserted gcbC gene derived from Gallibacterium anatis.The aims of this study is divided into four parts. The first part involves vaccine efficacy testing to investigate whether there are differences in vaccine protection efficacy between capsule genotypes II and III strains. The results show that the vaccine efficacy has no correlation with the capsule genotypes. The second part of this study analyzes the pathogenicity of genotypes II and III strains and finds no significant correlation between capsule genotypes and bacterial pathogenicity. The third part of the study involves the extraction of capsules and analysis of their chemical composition. The result shows that the main component of capsules from genotypes II and III strains is heparosan. In the fourth part, the function of GcbC protein is predicted by Blast search and sequence alignment. the result shows that GcbC have sequence similarity to AsnS and CarA. These findings could be useful for understanding the pathogenic mechanism of Av. paragallinarum and the function of gcbC.

摘要i
Abstractii
目次iii
表次vi
圖次vii
第一章 緒論1
第二章 文獻探討3
第一節Av. paragallinarum之歷史背景3
第二節 Av. paragallinarum 之生物學特性3
第三節 Av. paragallinarum之生化學特性4
第四節 Av. paragallinarum之血清型分型4
第五節 Av. paragallinarum之流行病學5
第六節 Av. paragallinarum之傳染6
6-1 感染與傳播途徑6
6-2 臨床症狀及病理變化7
6-3 診斷方法與鑑定7
6-4 防治策略8
第七節 Av. paragallinarum之毒力因子9
7-1 血球凝集蛋白 (Hemagglutinin, HA)9
7-2 莢膜10
7-3 其它毒力因子11
第八節 Av. paragallinarum疫苗開發12
第三章 材料與方法14
第一節 Av. paragallinarum 菌株收集14
1-1菌株來源14
1-2菌株增殖與型態鑑定14
第二節 菌株保存15
2-1 Av. paragallinarum 菌株保存15
2-2 Av. paragallinarum 菌株增殖15
2-3 E. coli 菌株保存15
第三節 Genomic DNA及Plasmid DNA 之萃取16
3-1 Av. paragallinarum 菌株Genomic DNA 之萃取16
3-2 E. coli 菌株Plasmid DNA 之萃取16
第四節 以E. coli原核系統表現HA重組蛋白之片段18
4-1 以PCR擴增HA蛋白高度變異區之片段18
4-2 HA蛋白高度變異區片段重組表現載體之構築19
4-3 轉型作用20
4-4 篩選構築成功之質體20
4-5 勝任細胞之製備21
4-6 以原核系統表現重組蛋白21
4-7 重組蛋白之聚丙烯醯胺凝膠電泳 (SDS-PAGE) 分析22
4-8 以西方墨點法 (Western Blot) 確認重組蛋白23
4-9 可溶性重組蛋白之純化 (Purification)24
第五節 以重組Hemagglutinin (HA) 蛋白作為次單位疫苗之保護效力分析24
5-1 實驗動物來源25
5-2 重組次單位疫苗置備25
5-3 攻毒株之製備26
5-4 雞隻之免疫計畫26
5-5 攻毒計畫27
5-6 病變指數評估27
5-7 利用Western blot multi-screen法檢測免疫重組HA蛋白後雞隻血清中的抗體反應28
5-8 固定雞隻紅血球之製備28
5-9 血球凝集試驗抗原製備29
5-10 血球凝集抑制試驗之血清前吸附處理29
5-11 血球凝集試驗 (Hemagglutination Test, HA Test) 及4 HAU定量30
5-12 血球凝集抑制試驗 (Hemagglutination inhibition Test, HI Test)30
第六節 Av. paragallinarum菌株之莢膜分析31
6-1 莢膜之萃取與分析31
第七節 透過核酸及蛋白質序列分析推測GcbC之潛在功能32
7-1 NCBI-Blast分析GcbC蛋白質序列32
7-2 GcbC蛋白質序列之官能基特性比對32
7-3 利用InterPro軟體分析GcbC是否含有已知的蛋白質功能區32
第四章 結果34
第一節Av. paragallinarum 菌株34
第二節 四價純化重組HA疫苗之製備34
2-1 PCR擴增目標基因片段34
2-2 膠體電泳分析確認重組表現載體之目標片段34
2-3 E. coli表現重組蛋白35
2-4 重組蛋白之純化35
第三節 以A+B+C+TW08純化重組HA蛋白作為疫苗之效力評估36
3-1 免疫四價純化重組HA蛋白疫苗前後之雞隻血清抗體檢測36
3-2 以血球凝集抑制試驗檢測免疫後雞隻之HI抗體力價36
第四節 A+B+C+TW08四價純化重組HA疫苗對莢膜第三基因型菌株之保護力、菌株毒力及莢膜分析37
4-1 免疫及雞隻攻毒試驗結果37
4-2 菌株之致病力分析39
4-3 抽取菌株莢膜並分析其化學成分39
4-4 以蛋白質序列分析比對推測GcbC之的可能功能40
第五章 討論78
第六章 參考文獻81

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