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National Chung Hsing University Institutional Repository - NCHUIR > 獸醫學院 > 獸醫病理生物學所 > 依資料類型分類 > 碩博士論文 >  Actinobacillus pleuropneumoniae Apx重組次單位蛋白在豬隻之免疫保護效力

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/121700

標題: Actinobacillus pleuropneumoniae Apx重組次單位蛋白在豬隻之免疫保護效力
Protective Efficacy of Actinobacillus pleuropneumoniae Recombinant Subunit Apx Proteins in Swine
作者: 劉冠志
Liou, Guan-Jhih
Contributors: 宣詩玲;葉光勝
Shin-Ling Hsuan;Kuang-Sheng YEH
簡茂盛
Maw-Sheng Chien
中興大學
關鍵字: 次單位蛋白
APP
日期: 2009
Issue Date: 2012-09-11 14:00:24 (UTC+8)
Publisher: 獸醫病理生物學研究所
摘要: 胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae; APP)為造成豬隻壞死性、纖維素性肺炎及胸膜與肋膜炎的主要病原。由於APP血清型別眾多,且目前市面上APP的疫苗大多仍是以死菌疫苗型態為主,但在不同的血清型間因無足夠的交叉免疫保護性,且所誘發之抗體對不同毒素中和能力的差異性,導致傳統疫苗在現場保護效力上仍有其限制性。因此利用基因重組技術來表現重組次單位抗原為現今疫苗發展的另一策略。在免疫交叉保護實驗中,經以rsApx次單位毒素蛋白添加細菌的lysate作為免疫抗原,發現經免疫後之小鼠,除了可產生具中和毒素之抗體外也可耐過APP1、2、5及10等不同血清型致死劑量的攻毒,證實重組次單位Apx毒素(rsApx)蛋白於小鼠模式中具有對不同APP血清型之交叉保護效力。本實驗亦進一步構築並表現Apx毒素的次單位蛋白,經由最佳化表現條件之測定後,製備此兩種rsApx蛋白並免疫兔子,所產生的抗體能夠辨識到APP野外分離株所分泌的Apx的毒素。進一步以混合三種次單位蛋白進行豬隻之免疫效力試驗,結果顯示所誘發之抗體可有效中和authentic Apx毒素之溶血活性。經利用氣管內接種的方式進行不同劑量與APP1及APP2不同血清之攻毒以評估其免疫保護效力。結果顯示,免疫混合三種次單位Apx毒素蛋白與添加細菌lysate的豬隻於攻毒後無論在臨床症狀、肺臟肉眼病變、組織病理學病變以及肺臟病變區面積的分析及評分,均比只免疫三種次單位Apx毒素蛋白而無細菌lysate豬隻輕微。此外,於攻毒後具中和溶血毒素及抑制細胞毒性的抗體力價也有明顯揚升之情形,顯示經豬隻免疫後一旦暴露於APP感染時可有效誘發具保護之中和抗體。上述結果顯示不具毒性之次單位Apx表現蛋白混合細菌lysate作為免疫抗原可保護豬隻耐過APP不同血清型別和Apx溶血毒素及細胞毒素之攻擊。
Actinobacillus pleuropneumoniae (APP) is the etiological agent of porcine pleuropneumonia, a severe respiratory disease that is characterized by a necrotizing fibrinous pneumonia and pleuritis. It often causes large economic losses in swine industry. The Apx (ApxI to IV) toxins, having cytotoxic and hemolytic activities, secreted by APP are major virulence factors resulting in the typical lung lesions. Because Apx toxins can induce swine's specific antibodies, so they are considered as the particular importance components of immunogens. In cross-protection trials, mice immunized with rsApx protein and bacterins could survive the LD50 dose of APP2, APP5 and APP10 challenge. We constructed and expressed two recombinant subunit Apx proteins using the E. coli expression system. Rabbit immunized with rsApx proteins elicited antibodies which could recognize Apx toxins in western blotting assay. It was also revealed in the experiments that swines immunized with rsApx proteins elicited antibodies which could neutralize the hemolytic activity of authentic Apx toxins. Furthermore, after immunization, the immunized swines were intratracheally challenged and the pathological scores were evaluated. Swines immunized with rsApxs and bacterins could protect swines from APP1 challenge of which had lower clinical signs scores, lung lesion scores and percentage area of lung lesions than the other immunized groups of swine. After challenge, the specific neutralizing antibodies could be elicited to neutralize against the hemolytic and cytotoxic activities of authentic Apx toxin in immunized swines. Whether the rsApx proteins could be developed to an successful APP subunit vaccine, further evaluation is required.
Appears in Collections:[依資料類型分類] 碩博士論文

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