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dc.contributorChiou-Lin Chenen_US
dc.contributor.advisorHappy K. Shiehen_US
dc.contributor.authorWu, Yi-Ruen_US
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dc.description.abstract傳染性可利查病又稱傳染性鼻炎,是由 Avibacterium paragallinarum 所引起之急性呼吸道疾病,在1931年首次為De Blieck所報告,IC主要造成的經濟損害,常為蛋雞產蛋率的下降及肉雞及種雞的生長遲緩等問題,感染此病的雞隻臨床上可見有流鼻水、呼吸困難、發出囉音,及眼窩下竇以及顏面腫脹、下痢等症狀。目前 IC的商用疫苗主要以不同血清型菌株的混合不活化全菌疫苗為主,但培養 Avibacterium paragallinarum 需要添加第 V 因子,且疫苗需要多次注射才有相當的成效,造成此疫苗在商用雞上使用的成本及人力較高,故發展新的疫苗是非常重要的。在本次研究中所確認的蛋白質P17、P40及P30,與Large adhesin蛋白被認為可能是IC的保護性抗原,且這些基因在 A. paragallinarum 中是高保留性的,因此我們利用原核表現系統,大量表現上述的重組蛋白,並製備成次單位疫苗,以鼻腔接種攻毒試驗的動物模式,來評估次單位疫苗對雞隻的保護效力,我們以54隻SPF雞,在6週及9週齡時分別免疫不同的重組蛋白,並於12週齡時以血清A型野外株TW-1攻毒,紀錄其臨床症狀至攻毒後第6天,其中並以西方墨點法檢測其血清抗體力價,結果免疫r-P30、r-P17蛋白可得到平均且高度的抗體力價,此外結果也指出,P30及 P17在自然感染下能被表現,並可引發特異性抗體反應,且在不同血清型間能夠交互作用。而疫苗之保護力測試中,顯示在鼻腔接種109 CFU之血清A型TW-1株攻毒條件下,在12週齡 SPF 雞可達到100 % 發病率,而比較其臨床症狀指數則可發現,使用 r-P17重組 蛋白免疫後的雞隻攻毒後臨床症狀的發生及其嚴重程度都有顯著性的下降,總合血清學及保護性測試,顯示此蛋白可能有交叉保護抗原的特性,作為 IC 的次單位疫苗是有相當的潛力的,故未來仍有必要對r-P17蛋白做進一步的研究,包括表現系統的調整,以求大量提升疫苗內之r-P17重組蛋白濃度或表現更多可溶性部分,及交叉保護性的攻毒測試,以求能確實保護各種血清型菌株的攻擊,此外繼續搜尋並測試新的抗原,若能添加多種具抗原性及保護性之蛋白,或與全菌疫苗合併使用,皆可能提升保護力及降低製作成本,這些研究對新一代的疫苗發展都是有益的。zh_TW
dc.description.abstractAvibacterium paragallinarum is the causative agent of infectious Coryza (IC), an acute upper respiratory tract disease of chickens. This disease was firstly reported by De Blieck in 1931, and consequently found to cause significant economic losses in poultry industry. Infectious Coryza is primarily manifested as a marked (10% to 40%) drop in egg production in layers and retardation of growth in breeders and broilers. Infected chickens usually show clinical signs including nasal discharge, facial edema, lacrimation, anorexia and diarrhea. Commercial vaccines against IC, typically based on inactivated A. paragallinarum, are wildly used around the world. However, A. paragallinarum requires "V" factor, a special growth factor in the media to grow. Moreover, the bacterin requires multiple injections to be effective. These make the administration of the vaccine expensive and labor intensive for commercial flocks. Therefore, a novel vaccine is thought to be important for the control of IC. Three antigenic proteins, P17, P40 and P30, identified in our laboratory, together with the large adhesion, are thought to be the protective antigens against IC. They are highly conserved among different types of A. paragallinarum. These gene were cloned and expressed as recombinant P17 (r-P17), P40 (r-P40), P30 (r-P30) and r-adhesion protein in E. coli. Moreover, r-P30, r-P17, and r-adhesin recombinant proteins were evaluated for the vaccine efficacy using a nasal challenge model. Fifty-four chickens were immunized subcutaneously with these recombinant proteins with Freund's adjuvant twice at the age of 6 and 9-week-old. These animals were challenged intra-nasally with A. paragallinarum field strain TW-1 and clinical signs were measured for 6 days. To determine immune responses of the recombinant proteins, sera were collected a week prior to challenge. The serum samples were analyzed by western blot to determine antibody titers against r-P17, r-P30 and r-adhesin. We also collected the serum samples from natural infected chicken for cross-reaction test. The results of these experiments show that r-P30 and r-P17 can induce highly immune responses. P17 and P30 are able to express in hosts under natural infection. Moreover, the serum from natural infected chicken can react with r-P30 and r-P17, and the serum from vaccinated chicken also reacted with different types of A. paragallinarum. These results indicate that these two antigens may be cross-protective antigens. Moreover, intranasal challenge with 109 CFU A. paragallinarum field strain TW-1 can cause 100 % (6/6) mortality in 12-week-old SPF chicken. The use of r-P17 protein as subunit vaccine can protect 66.6% of chickens and significantly reduce the severity of clinical signs against the challenge of the field strain TW-1. These data indicate that immunized with r-P17 protein may induce systemic immunity and provide some protection against IC in chicken. However, it still needs to modify the overexpression system to increase the productivity and solubility of r-P17. Data presented in this study are useful for developing new vaccine be useful for developing new vaccine against IC in Taiwan.en_US
dc.description.tableofcontents頁次 中文摘要------------------------------------------------- i 英文摘要------------------------------------------------ ii 目錄 --------------------------------------------------- iv 表次 ---------------------------------------------------vii 圖次 --------------------------------------------------viii 第一章 緒言-----------------------------------------------1 第二章 文獻探討-------------------------------------------3 第一節 A. paragallinarum 之相關研究----------------------3 2.1.1 A. paragallinarum 之歷史背景----------------------3 2.1.2 A. paragallinarum 之結構與生長條件----------------3 2.1.3 A. paragallinarum 之生化特性----------------------4 2.1.4 A. paragallinarum 之基因體------------------------4 2.1.5 A. paragallinarum 之血清學分類--------------------4 2.1.6 A. paragallinarum 致病因子之研究------------------5 第二節 A. paragallinarum 所造成之傳染性可利查病相關研究---7 2.2.1 A. paragallinarum 之流行病學 --------------------7 2.2.2 感染傳播及致病機轉-------------------------------8 2.2.3 臨床症狀與病理變化-------------------------------9 2.2.4 診斷方法及鑑定-----------------------------------9 2.2.5 預防與治療 -------------------------------------10 第三節 A. paragallinarum 所造成傳染性可利查病之疫苗研發--11 第三章 材料與方法----------------------------------------13 第一節 A. paragallinarum 之分離 -------------------------13 第二節 A. paragallinarum 之鑑定 -------------------------13 3.2.1 生化鑑定---------------------------------------13 3.2.2 PCR 鑑定---------------------------------------13 第三節 分離株之毒力測試 -------------------------------14 第四節 重組蛋白 r-P30 、r-P17、r-P40及 r-adhesin 之製備--14 3.4.1 A. paragallinarum之核酸萃取---------------------14 3.4.2 以PCR增幅p30,p17與P40之部分基因片段 -----------15 3.4.3 以Inverse PCR完成基因片段之全長定序與序列分析---16 3.4.4 以 PCR 增幅基因之全長---------------------------16 3.4.5 pTP30、pTP17、pTP40及pTAdhesin重組質體之構築----17 3.4.6 pP30、pP17、pP40及pAdhesin重組蛋白表現載體之構築------18 3.4.7 pP30、pP17、pP40及pAdhesin重組表現載體之確認----19 3.4.8 pP30、pP17、pP40及pAdhesin於大腸桿菌之表現------19 3.4.9 r-P30表現蛋白之純化-----------------------------20 3.4.10 r-P30、r-P17、r-P40及r-adhesin重組蛋白之電泳分析------------------------20 3.4.11 r-P30、r-P17、r-P40及r-adhesin重組蛋白之確認----21 第五節 r-P30、r-P17及 r-adhesin次單位疫苗之保護效力評估----------------------------22 3.5.1 在SPF雞以108 CFU 菌量 A. paragallinarum之攻毒試驗---------------------------22 3.5.2 在SPF雞以109 CFU 菌量 A. paragallinarum之攻毒試驗-------------------23 3.5.3 檢測免疫後雞隻血清抗體對菌體蛋白之抗體反應-----------------------------23 3.5.4 檢測野外血清對各重組蛋白之抗體反應---------------- ------------------------------------------------23 3.5.5 統計分析----------------------------------------24 第四章 結果---------------------------------------------25 第一節 A. paragallinarum 之分離--------------------------25 第二節 A. paragallinarum 之鑑定--------------------------25 3.2.3 生化鑑定----------------------------------------25 3.2.4 PCR 鑑定----------------------------------------25 第三節 分離株之毒力測試----------------------------------25 第四節 重組蛋白 r-P30、r-P17、r-P40及r-adhesin之製備-----26 4.4.1 以 PCR 增幅p30、p17與P40之部分基因片段----------26 4.4.2 以 Inverse PCR完成P30、P17與P40基因全長定序-----26 4.4.3 以PCR增幅P30、P17、P40與large adhesion 之基因全長------------------------------26 4.4.4 pTP30、pTP17、pTP40及pTAdhesin重組載體之構築----------------------26 4.4.5 pP30、pP17、pP40及pAdhesin重組蛋白表現載體之構築----------------27 4.4.6 以原核表現系統製備r-P30、r-P17、r-P40及r-adhesin重組蛋白與確認--------------27 4.4.7 r-P30 重組表現蛋白之純化------------------------27 第五節 以人工感染之動物試驗評估次單位疫苗效益-----------27 4.5.1 在 SPF 雞以108 CFU 菌量 A. paragallinarum 之攻毒試驗----------------------------------------------27 4.5.2 在 SPF 雞以109 CFU 菌量 A. paragallinarum 之攻毒試驗---------------------------------28 4.5.3 檢測免疫後雞隻血清抗體對菌體蛋白之抗體反應-----------------------------------------28 4.5.4 檢測野外血清對各重組蛋白之抗體反應--------------------------------28 第五章 討論----------------------------------------------50 參考文獻 ------------------------------------------------55zh_TW
dc.subjectinfectious coryzaen_US
dc.titleDevelopment of Subunit Vaccine against Chicken Infectious Coryzaen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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