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不同佐劑對H1亞型流感病毒HA類病毒顆粒免疫原性之影響 = The ef...

國立高雄大學生物科技研究所碩士班

不同佐劑對H1亞型流感病毒HA類病毒顆粒免疫原性之影響 = The effect of different adjuvants on immunogenicity of H1 subtype influenza hemagglutinin virus-like particles

紀錄類型:	書目-語言資料,印刷品 : 單行本
並列題名:	The effect of different adjuvants on immunogenicity of H1 subtype influenza hemagglutinin virus-like particles
作者:	陳宏穎,
其他團體作者:	國立高雄大學
出版地:	高雄市
出版者:	國立高雄大學;
出版年:	2013[民102]
面頁冊數:	144面圖，表格 : 30公分;
標題:	黏膜佐劑
標題:	mucosal adjuvant
電子資源:	https://hdl.handle.net/11296/uez7y3
附註:	107年11月1日公開
附註:	參考書目：面134-144
摘要註:	佐劑是一種具有免疫促進的物質，能夠協助抗原更容易被抗原呈現細胞吞噬，進一步呈現給專一性淋巴細胞辨識，強化免疫反應。本實驗室先前的研究結果顯示，以豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumonia, APP)呼吸道毒素結構蛋白ApxIIA製作的融合蛋白具有黏膜佐劑的潛力。根據已知RTX家族毒素的不同功能性區域，分為F(全長)、N端、C端，經實驗證實ApxIIA-N的毒性最弱，且可刺激IgG1及IgA的提升還有低程度的IgG2a。本研究利用新一代次單位流感疫苗，具有病毒的結構及免疫原性但不具有核酸的類病毒顆粒做為抗原，此類病毒顆粒為H1N1亞型A/PR/8/34流感病毒株的血球凝集素蛋白（HA）所組成，由於流感病毒常藉由上呼吸道的氣管黏膜感染宿主，因此搭配具有黏膜免疫促進的ApxIIA融合蛋白為佐劑。本研究用不同劑量的ApxIIA-N、ApxIIA-C以點鼻注射方或肌肉注射方式免疫小鼠。有添加佐劑組點鼻注射免疫後35天HI抗體力價皆超過40倍，免疫後49天達到最高；肌肉注射免疫後28天HI抗體力價超過40倍、免疫後49天達到最高，且免疫後150天HI抗體力價依舊能超過40倍。添加佐劑組以點鼻免疫後49天IgG抗體效價可達102400；而肌肉射免疫組IgG抗體效價可達204800。進一步分析其誘發之IgG1與IgG2a抗體亞型表現，添加佐劑以點鼻注射免疫組別皆能顯著提升IgG1含量，推測主要誘導Th2路徑；添加佐劑以肌肉注射免疫組別結果相同。在佐劑對體重影響試驗方面，添加佐劑組的小鼠體重變化與未添加佐劑組別無顯著差異，顯示佐劑未對小鼠造成不良的影響。進行脾臟增生試驗與Th1 (IL-12、IFN-γ)、Th2 (IL-4、IL-6)表現量分析結果顯示，添加佐劑皆能刺激脾臟細胞增生，30 μg ApxIIA-N效果最好；ApxIIA-N能有效的誘發Th2免疫路徑。在肺沖洗液IgA抗體力價分析，有添加佐劑的組別確實能有效提升IgA分泌。綜合以上結果ApxIIA蛋白具有佐劑及促進黏膜免疫功效，其中以30 μg ApxIIA-N肌肉注射免疫方式效果最佳。 Adjuvant is an immune stimulator which can assist the antigen presenting cells (APC) to endocytose antigens more easily, and present the antigen to lymphocytes recognized specifically to enhance immune response. Previous studies in our laboratory showed that the structural protein of respiratory tract toxin produced by Actinobacillus pleuropneumonia has the potential of as a mucosal adjuvant. 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. ApxIIA-N has been showed that had the weakest toxicity and could significantly enhance the titer of IgG1 and IgA, but limited in IgG2a. In this study, a new generation of subunit influenza vaccine called virus-like particles (VLPs) which maintained the structure and immunogenicity of virus but lack of viral genetic materials. The virus-like particles was H1 subtype of hemagglutinin (HA) produced from influenza strain A/PR/8/34(H1N1). Influenza viruses usually infect the upper respiratory tract via the tracheal mucosa of host. Therefore, ApxIIA proteins were used as adjuvant to evaluate the adjuvanticity for VLPs subunit vaccine. In this study, mice were administrated with different dosages of ApxIIA-N, ApxIIA-C by intranasal and intramuscular injections. The HI antibody titers of adjuvant-containing groups via intranasal immunization were over 40 on day 35 after immunization. The results showed that the highest HI titers were reached on day 49 after immunization. On the other hand, intramuscular injection results revealed that HI antibody titers over 40 on day 28 after immunization, and the highest HI titers were reached on day 49 after immunization. Furthermore, the HI antibody titers were still over 40 on day 150 after immunization. The IgG antibody titers of adjuvant-containing groups on day 49 after immunization via intranasal and intramuscular route were 102400 and 204800, respectively. Antibody subtype analyses of IgG1 and IgG2a showed that all of the adjuvant-containing groups via intranasal or intramuscular immunization with increasing in IgG1 levels significantly. It indicates the major pathway induced by adjuvant was Th2. The body weight change of mice between adjuvant-containing groups and control group showed no significant difference. It indicates that the adjuvant was safety for mice. The splenocyte proliferation and Th1 (IL-12, IFN-γ), Th2 (IL-4, IL-6) expression analyses showed that the splenocytes of adjuvant- containing goups were proliferated by specific antigen stimulation and 30μg ApxIIA-N immunized group obtained the strongest proliferation. The results showed that ApxIIA-N could induce immune response toward to Th2 pathway. Analysis of IgA titer in bronchoalveolar lavages fluid (BALF) showed that all ApxIIA adjuvant- containing groups could effectively enhance IgA secretion. All of the results indicated that ApxIIA protein has adjuvant function and could stimulate mucosal immune response. The best immune response could be received through intramuscular immunization with 30μg ApxIIA-N.

不同佐劑對H1亞型流感病毒HA類病毒顆粒免疫原性之影響 = The effect of different adjuvants on immunogenicity of H1 subtype influenza hemagglutinin virus-like particles
陳, 宏穎

不同佐劑對H1亞型流感病毒HA類病毒顆粒免疫原性之影響 = The effect of different adjuvants on immunogenicity of H1 subtype influenza hemagglutinin virus-like particles / 陳宏穎撰 - 高雄市 : 國立高雄大學, 2013[民102]. - 144面 ; 圖，表格 ; 30公分.
107年11月1日公開參考書目：面134-144.
黏膜佐劑mucosal adjuvant

不同佐劑對H1亞型流感病毒HA類病毒顆粒免疫原性之影響 = The effect of different adjuvants on immunogenicity of H1 subtype influenza hemagglutinin virus-like particles
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330 $a 佐劑是一種具有免疫促進的物質，能夠協助抗原更容易被抗原呈現細胞吞噬，進一步呈現給專一性淋巴細胞辨識，強化免疫反應。本實驗室先前的研究結果顯示，以豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumonia, APP)呼吸道毒素結構蛋白ApxIIA製作的融合蛋白具有黏膜佐劑的潛力。根據已知RTX家族毒素的不同功能性區域，分為F(全長)、N端、C端，經實驗證實ApxIIA-N的毒性最弱，且可刺激IgG1及IgA的提升還有低程度的IgG2a。本研究利用新一代次單位流感疫苗，具有病毒的結構及免疫原性但不具有核酸的類病毒顆粒做為抗原，此類病毒顆粒為H1N1亞型A/PR/8/34流感病毒株的血球凝集素蛋白（HA）所組成，由於流感病毒常藉由上呼吸道的氣管黏膜感染宿主，因此搭配具有黏膜免疫促進的ApxIIA融合蛋白為佐劑。本研究用不同劑量的ApxIIA-N、ApxIIA-C以點鼻注射方或肌肉注射方式免疫小鼠。有添加佐劑組點鼻注射免疫後35天HI抗體力價皆超過40倍，免疫後49天達到最高；肌肉注射免疫後28天HI抗體力價超過40倍、免疫後49天達到最高，且免疫後150天HI抗體力價依舊能超過40倍。添加佐劑組以點鼻免疫後49天IgG抗體效價可達102400；而肌肉射免疫組IgG抗體效價可達204800。進一步分析其誘發之IgG1與IgG2a抗體亞型表現，添加佐劑以點鼻注射免疫組別皆能顯著提升IgG1含量，推測主要誘導Th2路徑；添加佐劑以肌肉注射免疫組別結果相同。在佐劑對體重影響試驗方面，添加佐劑組的小鼠體重變化與未添加佐劑組別無顯著差異，顯示佐劑未對小鼠造成不良的影響。進行脾臟增生試驗與Th1 (IL-12、IFN-γ)、Th2 (IL-4、IL-6)表現量分析結果顯示，添加佐劑皆能刺激脾臟細胞增生，30 μg ApxIIA-N效果最好；ApxIIA-N能有效的誘發Th2免疫路徑。在肺沖洗液IgA抗體力價分析，有添加佐劑的組別確實能有效提升IgA分泌。綜合以上結果ApxIIA蛋白具有佐劑及促進黏膜免疫功效，其中以30 μg ApxIIA-N肌肉注射免疫方式效果最佳。 Adjuvant is an immune stimulator which can assist the antigen presenting cells (APC) to endocytose antigens more easily, and present the antigen to lymphocytes recognized specifically to enhance immune response. Previous studies in our laboratory showed that the structural protein of respiratory tract toxin produced by Actinobacillus pleuropneumonia has the potential of as a mucosal adjuvant. 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. ApxIIA-N has been showed that had the weakest toxicity and could significantly enhance the titer of IgG1 and IgA, but limited in IgG2a. In this study, a new generation of subunit influenza vaccine called virus-like particles (VLPs) which maintained the structure and immunogenicity of virus but lack of viral genetic materials. The virus-like particles was H1 subtype of hemagglutinin (HA) produced from influenza strain A/PR/8/34(H1N1). Influenza viruses usually infect the upper respiratory tract via the tracheal mucosa of host. Therefore, ApxIIA proteins were used as adjuvant to evaluate the adjuvanticity for VLPs subunit vaccine. In this study, mice were administrated with different dosages of ApxIIA-N, ApxIIA-C by intranasal and intramuscular injections. The HI antibody titers of adjuvant-containing groups via intranasal immunization were over 40 on day 35 after immunization. The results showed that the highest HI titers were reached on day 49 after immunization. On the other hand, intramuscular injection results revealed that HI antibody titers over 40 on day 28 after immunization, and the highest HI titers were reached on day 49 after immunization. Furthermore, the HI antibody titers were still over 40 on day 150 after immunization. The IgG antibody titers of adjuvant-containing groups on day 49 after immunization via intranasal and intramuscular route were 102400 and 204800, respectively. Antibody subtype analyses of IgG1 and IgG2a showed that all of the adjuvant-containing groups via intranasal or intramuscular immunization with increasing in IgG1 levels significantly. It indicates the major pathway induced by adjuvant was Th2. The body weight change of mice between adjuvant-containing groups and control group showed no significant difference. It indicates that the adjuvant was safety for mice. The splenocyte proliferation and Th1 (IL-12, IFN-γ), Th2 (IL-4, IL-6) expression analyses showed that the splenocytes of adjuvant- containing goups were proliferated by specific antigen stimulation and 30μg ApxIIA-N immunized group obtained the strongest proliferation. The results showed that ApxIIA-N could induce immune response toward to Th2 pathway. Analysis of IgA titer in bronchoalveolar lavages fluid (BALF) showed that all ApxIIA adjuvant- containing groups could effectively enhance IgA secretion. All of the results indicated that ApxIIA protein has adjuvant function and could stimulate mucosal immune response. The best immune response could be received through intramuscular immunization with 30μg ApxIIA-N.
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