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以豬胸膜肺炎放射桿菌之ApxII A蛋白開發新型疫苗佐劑 = Devel...

國立高雄大學生物科技研究所

以豬胸膜肺炎放射桿菌之ApxII A蛋白開發新型疫苗佐劑 = Development of novel vaccine adjuvant using ApxIIA protein of Actinobacillus pleuropneumoniae

紀錄類型:	書目-語言資料,印刷品 : 單行本
並列題名:	Development of novel vaccine adjuvant using ApxIIA protein of Actinobacillus pleuropneumoniae
作者:	蔣庭彬,
其他團體作者:	國立高雄大學
出版地:	[高雄市]
出版者:	撰者;
出版年:	2010[民99]
面頁冊數:	97面圖，表 : 30公分;
標題:	黏膜佐劑
標題:	Mucosal adjuvant
電子資源:	http://handle.ncl.edu.tw/11296/ndltd/86781696373864751617
附註:	104年10月31日公開
附註:	參考書目：面67-74
摘要註:	佐劑是一種用以增加抗原免疫原性的免疫促進劑，藉此增加疫苗保護效力或改變疫苗免疫反應類型。許多研究主要以大腸桿菌毒素及霍亂毒素次單位蛋白進行黏膜佐劑之開發，本研究嘗試以豬胸膜肺炎放線桿菌產生的呼吸道毒素結構蛋白為標的，開發新型佐劑。ApxII在已知豬胸膜肺炎放線桿菌的毒素中，具有較弱的溶血能力及細胞毒性，ApxIIA為其結構蛋白。依據已知RTX家族毒素的不同功能性區域，將ApxIIA基因分為全長(F)、N端(N)及C端(C)片段進行選殖、表達與純化。此等重組蛋白質對巨噬細胞的進行毒性分析，其中以ApxIIA-N的毒性最弱，在100 μg/ml仍有45 %細胞存活率，而ApxIIA-F及ApxIIA-C二者於50 μg/ml時，造成所有細胞死亡，其毒性低於大腸桿菌腸毒素LTB約10倍，低於豬胸膜肺炎放線桿菌脂多醣約200倍。以此等蛋白開發為疫苗佐劑，在安全性的考量上更具有優勢。依據此等重組蛋白誘導細胞激素表現情形，推測?徙xIIA-N能誘導Th1路徑，而ApxIIA-F與 ApxIIA-C能同時誘導Th1與Th2路徑。以卵白蛋白為抗原，搭配上述重組蛋白免疫小鼠進行佐劑功能測試，其中含有ApxIIA之佐劑免疫組抗體效價可達51200-102400，為僅施打卵白蛋白抗原的8-16倍。進一步分析其誘發之IgG1與IgG2a抗體亞型表現，結果顯示ApxIIA-F、ApxIIA-N及ApxIIA-C組皆能顯著提升IgG1含量及部份提升IgG2a含量，推測主要能誘導Th2免疫途徑。分析肺沖洗液IgA抗體效價，顯示添加ApxIIA蛋白組的IgA效價可高於僅施打卵白蛋白抗原的10倍以上。在佐劑對體重影響試驗方面，在第三次免疫後，ApxIIA-F及ApxIIA-C組體重沒有如無佐劑組正常地增加，由先前細胞實驗結果推測可能是由於ApxIIA-F及ApxIIA-C蛋白較具有細胞毒性。綜合上述結果，證實ApxIIA蛋白確實有發展為黏膜佐劑之潛力，能有效增強抗體效價。但由於ApxIIA-F與ApxIIA-C較具有毒性，因此以ApxIIA-N最適合發展為新型的黏膜佐劑。 Adjuvant is an immune stimulator which can enhance the immunogenicity of vaccine. The function of adjuvant is to enhance the protective immunity of vaccine or to change the type of immune response. The development of mucosal adjuvant was major focused in the subunit protein of E. coli heat labile toxin (LT) and cholera toxin (CT). In this study, a novel mucosal adjuvant was developed using the structural protein of respiratory track toxin produced by Actinobacillus pleuropneumoniae. It was known that ApxII has the lower hemolytic and cytotoxic activity in all Apx toxins of A. pleuropneumoniae, and ApxIIA is the structural protein of ApxII. According to the functional domain of RTX (repeats in toxin) family toxin, the full length, N terminal and C terminal fragment of ApxIIA gene was cloned, expressed and purified, respectively. In the MTT assay for the toxicity analysis of these recombinant proteins to macrophage, revealed that ApxIIA-N has lowest cytotoxic activity. The viability of 100µg/ml ApxIIA-N treatment is still about 45 %. In the ApxIIA-F or ApxIIA-C treatment, all cells could not survive in the concentration of 50µg/ml. The toxicity is 10-fold less than E. coli heat labile toxin B subunit (LTB) and 200-fold less than LPS of A. pleuropneumoniae. These recombinant proteins have the superiority to develop as adjuvant in safety concerned issue. According to the cytokine expression profile induced by these recombinant proteins, the data indicated that ApxIIA-N could induce Th1 pathway, and both Th1 and Th2 pathway could be induced by ApxIIA-F and ApxIIA-C. Using ovalbumin as antigen and combined with these recombinant proteins to evaluate their adjuvant effect in mice. The antibody titers specific to ovalbumin of ApxIIA adjuvant-containing groups could reach 51200-102400, which are 8- to 16-fold than adjuvant-free group. Analysis the expression of IgG1 and IgG2a subclass revealed that all ApxIIA adjuvant-containing groups could significantly enhance IgG1 and limited IgG2a titer. It indicates that the major induced pathway was Th2. The analysis of IgA titer in bronchoalveolar lavages fluid (BALF) of all ApxIIA adjuvant-containing groups showed that was 10-fold than adjuvant-free group. The body weight of mice was measured to evaluate the influence of adjuvant administered. After third immunization, the mice body weight of ApxIIA-F and ApxIIA-C groups did not increase normally as adjuvant-free group. It indicates that ApxIIA-F and ApxIIA-C may have higher cytotoxic activity as previously in vitro results shown. In summary, ApxIIA protein could enhance the antibody titer and has the potential for the development of mucosal adjuvant. However, ApxIIA-F and ApxIIA-C have higher cytotoxic activity. Therefore, ApxIIA-N is thebest candidate for the development of novel mucosal adjuvant.

以豬胸膜肺炎放射桿菌之ApxII A蛋白開發新型疫苗佐劑 = Development of novel vaccine adjuvant using ApxIIA protein of Actinobacillus pleuropneumoniae
蔣, 庭彬

以豬胸膜肺炎放射桿菌之ApxII A蛋白開發新型疫苗佐劑 = Development of novel vaccine adjuvant using ApxIIA protein of Actinobacillus pleuropneumoniae / 蔣庭彬撰 - [高雄市] : 撰者, 2010[民99]. - 97面 ; 圖，表 ; 30公分.
104年10月31日公開參考書目：面67-74.
黏膜佐劑Mucosal adjuvant

以豬胸膜肺炎放射桿菌之ApxII A蛋白開發新型疫苗佐劑 = Development of novel vaccine adjuvant using ApxIIA protein of Actinobacillus pleuropneumoniae
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328 $a 碩士論文--國立高雄大學生物科技研究所
330 $a 佐劑是一種用以增加抗原免疫原性的免疫促進劑，藉此增加疫苗保護效力或改變疫苗免疫反應類型。許多研究主要以大腸桿菌毒素及霍亂毒素次單位蛋白進行黏膜佐劑之開發，本研究嘗試以豬胸膜肺炎放線桿菌產生的呼吸道毒素結構蛋白為標的，開發新型佐劑。ApxII在已知豬胸膜肺炎放線桿菌的毒素中，具有較弱的溶血能力及細胞毒性，ApxIIA為其結構蛋白。依據已知RTX家族毒素的不同功能性區域，將ApxIIA基因分為全長(F)、N端(N)及C端(C)片段進行選殖、表達與純化。此等重組蛋白質對巨噬細胞的進行毒性分析，其中以ApxIIA-N的毒性最弱，在100 μg/ml仍有45 %細胞存活率，而ApxIIA-F及ApxIIA-C二者於50 μg/ml時，造成所有細胞死亡，其毒性低於大腸桿菌腸毒素LTB約10倍，低於豬胸膜肺炎放線桿菌脂多醣約200倍。以此等蛋白開發為疫苗佐劑，在安全性的考量上更具有優勢。依據此等重組蛋白誘導細胞激素表現情形，推測?徙xIIA-N能誘導Th1路徑，而ApxIIA-F與 ApxIIA-C能同時誘導Th1與Th2路徑。以卵白蛋白為抗原，搭配上述重組蛋白免疫小鼠進行佐劑功能測試，其中含有ApxIIA之佐劑免疫組抗體效價可達51200-102400，為僅施打卵白蛋白抗原的8-16倍。進一步分析其誘發之IgG1與IgG2a抗體亞型表現，結果顯示ApxIIA-F、ApxIIA-N及ApxIIA-C組皆能顯著提升IgG1含量及部份提升IgG2a含量，推測主要能誘導Th2免疫途徑。分析肺沖洗液IgA抗體效價，顯示添加ApxIIA蛋白組的IgA效價可高於僅施打卵白蛋白抗原的10倍以上。在佐劑對體重影響試驗方面，在第三次免疫後，ApxIIA-F及ApxIIA-C組體重沒有如無佐劑組正常地增加，由先前細胞實驗結果推測可能是由於ApxIIA-F及ApxIIA-C蛋白較具有細胞毒性。綜合上述結果，證實ApxIIA蛋白確實有發展為黏膜佐劑之潛力，能有效增強抗體效價。但由於ApxIIA-F與ApxIIA-C較具有毒性，因此以ApxIIA-N最適合發展為新型的黏膜佐劑。 Adjuvant is an immune stimulator which can enhance the immunogenicity of vaccine. The function of adjuvant is to enhance the protective immunity of vaccine or to change the type of immune response. The development of mucosal adjuvant was major focused in the subunit protein of E. coli heat labile toxin (LT) and cholera toxin (CT). In this study, a novel mucosal adjuvant was developed using the structural protein of respiratory track toxin produced by Actinobacillus pleuropneumoniae. It was known that ApxII has the lower hemolytic and cytotoxic activity in all Apx toxins of A. pleuropneumoniae, and ApxIIA is the structural protein of ApxII. According to the functional domain of RTX (repeats in toxin) family toxin, the full length, N terminal and C terminal fragment of ApxIIA gene was cloned, expressed and purified, respectively. In the MTT assay for the toxicity analysis of these recombinant proteins to macrophage, revealed that ApxIIA-N has lowest cytotoxic activity. The viability of 100µg/ml ApxIIA-N treatment is still about 45 %. In the ApxIIA-F or ApxIIA-C treatment, all cells could not survive in the concentration of 50µg/ml. The toxicity is 10-fold less than E. coli heat labile toxin B subunit (LTB) and 200-fold less than LPS of A. pleuropneumoniae. These recombinant proteins have the superiority to develop as adjuvant in safety concerned issue. According to the cytokine expression profile induced by these recombinant proteins, the data indicated that ApxIIA-N could induce Th1 pathway, and both Th1 and Th2 pathway could be induced by ApxIIA-F and ApxIIA-C. Using ovalbumin as antigen and combined with these recombinant proteins to evaluate their adjuvant effect in mice. The antibody titers specific to ovalbumin of ApxIIA adjuvant-containing groups could reach 51200-102400, which are 8- to 16-fold than adjuvant-free group. Analysis the expression of IgG1 and IgG2a subclass revealed that all ApxIIA adjuvant-containing groups could significantly enhance IgG1 and limited IgG2a titer. It indicates that the major induced pathway was Th2. The analysis of IgA titer in bronchoalveolar lavages fluid (BALF) of all ApxIIA adjuvant-containing groups showed that was 10-fold than adjuvant-free group. The body weight of mice was measured to evaluate the influence of adjuvant administered. After third immunization, the mice body weight of ApxIIA-F and ApxIIA-C groups did not increase normally as adjuvant-free group. It indicates that ApxIIA-F and ApxIIA-C may have higher cytotoxic activity as previously in vitro results shown. In summary, ApxIIA protein could enhance the antibody titer and has the potential for the development of mucosal adjuvant. However, ApxIIA-F and ApxIIA-C have higher cytotoxic activity. Therefore, ApxIIA-N is thebest candidate for the development of novel mucosal adjuvant.
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