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研究生:商惠芳
研究生(外文):Shang, Huey-Fang
論文名稱:發展對抗綠膿桿菌感染之免疫療法
論文名稱(外文):Development of immunotherapy against pseudomonas aeruginosa infection
指導教授:黃昭蓮
指導教授(外文):Jaulang Hwang
學位類別:博士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:147
中文關鍵詞:微生物學免疫學綠膿桿菌
外文關鍵詞:MICROBIOLOGYIMMUNOLOGY
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綠膿桿菌為革蘭氏陰性桿菌,在自然界中分佈很廣,是目前院內感染之主要致病菌。但此菌極易產生抗藥性引起治療上之困難,故發展免疫治療法來預防、治療綠膿桿菌感染,以取代傳統的抗生素療法,是本論文的主要目的。已知綠膿桿菌會分泌多種有毒物質,其中以綠膿桿菌外毒素A (PE)之毒性最強,我們曾以遺傳工程技術刪除此毒素羧基端三十八個胺基酸,使PE之毒性完全喪失,但仍保有大部份抗原性,使其成為無毒性之PE類毒素(toxoid)。動物實驗証明此類毒素可有效刺激動物產生中和PE毒性之抗體,但無法阻止此菌在病人體內繁殖,破壞組織細胞。本論文將二種綠膿桿菌外膜蛋白I、F和無毒性之PE結合,此融合蛋白不但可刺激免疫後之動物產生中和PE毒性的抗體且可產生具調理作用之抗體,會和侵入之多種血清型綠膿桿菌細胞表面結合,以加強巨噬細胞之吞噬作用,多管齊下,達到最好之免疫效果。此外為了發展被動免疫療法,以保護免疫力有缺失的病人,在被綠膿桿菌感染早期,避免PE毒素導致肝細胞壞死,本論文再利用融合瘤技術製備抗PE之單源抗體,並篩選出一株可中和PE毒性之單源抗體定名為MAbB7。MAbB7所辨識之抗原決定位在PE羧基端之第575-595氨基酸片段,它可有效抑制PE之酵素活性,但不干擾PE和細胞表面受體之結合,亦不影響PE在細胞內之分解過程,以酵素免疫吸附試驗,可証明在pH4之酸性環境下,MAbB7仍可和PE結合,此結果顯示在內膜體酸性環境中,MAbB7仍可和PE羧基端結合,因而干擾PE羧基端5個重要氨基酸REDLK(第609-613氨基酸)和細胞內某些未知蛋白結合,繼而阻止PE的第三區城無法移位至細胞質;但實驗結果不排除MAbB7亦可能隨PE之第三區域移位至細胞質後,直接抑制PE之酵素活性,而使其失去了殺死細胞的能力。深入瞭解單源抗體B7中和PE毒性之機制將有助於進一步瞭解PE羧基端5個胺基酸REDLK對PE殺細胞毒性之重要性。
Pseudomonas aeruginosa is an opportunistic pathogen which has become a major cause of nosocomial infections. Therapy for P. aeruginosa infection is hindered by its well-known antibiotic resistance. Thus, there is considerable interest in the development of immunotherapy through either passive or active immunization. The most potent cytotoxic agents produced by P. aeruginosa is Pseudomonas exotoxin A (PE). We have engineered a nontoxic PE with a deletion of 38 amino acids from the carboxyl terminus, designated as PE(Δ576-613). This nontoxic PE had lost its cytotoxicity but still retained the most antigenicity, so it can induce protective immunity against the intoxication of PE on mice. However, anti-PE antibody alone cannot block the colonization and multiplication after P. aeruginosa infection. In this thesis, we have designed a more potent vaccine containing the receptor binding, membrane translocation domains of PE and the outer membrane proteins I and F (Opr F, Opr I) of P. aeruginosa. We demonstrated that this chimeric protein will induce antibodies not only neutralizing the toxicity of PE but also preventing the colonization of P. aeruginosa by promoting the opsonophagocytic activity of macrophages. For passive immunization, a series of monoclonal antibodies recognized different domains of PE have been produced. MAb B7 recognized the carboxyl terminal amino acid residues 575 to 595 of PE has strong neutralizing activity against the PE cytotoxicity in cell culture and in BALB/c mice. In consistence with the result of epitope mapping, MAb B7 did not block PE receptor-binding activity nor the cellular processing of PE but strongly inhibited the ADP-ribosylating activity of PE. In addition, MAb B7 retained strong binding activity to PE even at pH 4.0, indicating that the complex of MAb B7 and PE is stable in the phagolysosome. Based on these observations, the neutralization of PE cytotoxicity by MAb B7 could be due to its binding to the carboxyl- terminal of PE. This binding may interfere with the interaction of the carboxyl-end amino acid residues REDLK of PE with some unknown cellular factor, so that it may block the translocation of PE. However, we could not rule out the possibility that MAb B7 may directly blocks the ADP-ribosylation activity of PE in the cytosol. This study on the neutralizing mechanism of MAb B7 will help us to further understand the importance of the carboxyl end amino acid residues REDLK for the cytotoxicity of PE.
封面
目錄
中文摘要
英文摘要
附圖目錄
附表目錄
第一章 緒論
一、綠膿桿菌的形態、構造與生長之基本特性
二、綠膿桿菌的感染
三、綠膿桿菌所分泌的毒素及與致病性有關之構造
四、綠膿桿菌的致病機制
五、圖表
六、參考文獻
第二章 單源抗體B7中和綠膿桿菌外毒素A作用機制之研究
摘要
前言
實驗材料與方法
實驗結果
討論
圖表
參考文獻
第三章 綠膿桿菌新疫苗之開發-去毒性綠膿桿菌外毒素A和外膜蛋I、F融合蛋之免疫抗菌作用
摘要
前言
實驗材料與方法
實驗結果
討論
圖表
參考文獻
英文附錄
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