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研究生:陳宗嶽
研究生(外文):Chen, Tzong-Yueh
論文名稱:發展綠膿桿菌外毒素為具醫療用途之蛋白
論文名稱(外文):Development of Pseudomonas exotoxin A into useful proteins for disease treatment
指導教授:許祖法黃昭蓮
指導教授(外文):Cho-Fat Hui, Ph. D.Jaulang Hwang, Ph. D.
學位類別:博士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:英文
論文頁數:138
中文關鍵詞:綠膿桿菌外毒素疫苗基因輸送遺傳工程基因治療蛋白工程生物技術
外文關鍵詞:Pseudomonas exotoxin AvaccineDNA deliverygenetic engineeringgene therapyprotein engineeringbiotechnology
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綠膿桿菌為革蘭氏陰性桿菌,在自然界中具廣泛分佈,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,極具發展潛力。由於綠膿桿菌會分泌多種有毒物質,其中又以綠膿桿菌外毒素A (PE)之毒性最強,我們以遺傳工程技術刪除此毒素羧基端三十八個胺基酸,使綠膿桿菌外毒素之毒性喪失,但仍保有大部份抗原性,使其成為無毒性之PE(D576-613)類毒素蛋白。動物實驗的結果証明PE(D576-613)類毒素蛋白可有效刺激動物產生中和綠膿桿菌外毒素毒性之抗體,但無法阻止此菌在病人體內繁殖,與破壞組織細胞。因此進一步將二種綠膿桿菌外膜蛋白OprI、OprF,和無毒性之PE(1-405)結合,此一融合蛋白PEIF不但可刺激免疫後之動物產生中和綠膿桿菌外毒素毒性的抗體,且可產生具調理作用之抗體,能與侵入之不同種血清型綠膿桿菌的細胞表面結合,進而加強巨噬細胞之吞噬作用,多管齊下,達到較佳的免疫效果。另一方面,受體調節的基因輸送系統,能高效率的攜帶目標基因至細胞中表現。此一新方法除適用於癌病治療也可廣泛應用在基因治療上。所以我們希望能建立以綠膿桿菌外毒素為基礎的基因輸送系統。首先我們利用遺傳工程技術重組基因,將人類第一型拓樸異構酵素去氧核糖核酸結合區確定出來(人類第一型拓樸異構酵素之胺基酸序列 51-200、271-375、422-596 和 651-696)。再利用遺傳工程技術重組基因,將人類第一型拓樸異構酵素去氧核糖核酸結合區 (人類第一型拓樸異構酵素之胺基酸序列 3-200) 的去氧核糖核酸序列置換掉綠膿桿菌外毒素的腺核甘二磷酸核糖化區(ADP-ribosylation domain) 的去氧核糖核酸序列。此一融合蛋白 PE(DIII)-TOPN ,經由瓊膠電泳阻延試驗(agarose gel retardation assay)的實驗證明PE(DIII)-TOPN-DNA複合體能有效結合,再經細胞實驗證明PE(DIII)-TOPN-DNA複合體有效輸送綠色螢光蛋白質體至細胞中,並表現出綠色螢光蛋白。這些實驗証明新的基因輸送系統能有效輸送DNA,將可應用在基因治療及基礎生命科學研究上。
Pseudomonas aeruginosa is an opportunistic pathogen that 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 carboxy1 terminus, designated as PE(D576-613). This nontoxic PE had lost its cytotoxicity but still retained its 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. Therefore, we have designed a more potent vaccine containing the receptor binding, membrane translocation domains of PE and the outer membrane proteins I and F (OprF, OprI) of P. aeruginosa. We demonstrated that this chimeric protein PEIF could induce antibodies that neutralize the toxicity of PE and also preventing the colonization of P. aeruginosa by promoting the opsonophagocytic activity of macrophages. In the second part of the project, since receptor-mediated gene delivery can offer high efficiency in gene transfer for gene therapy. We developed a novel receptor-mediated gene delivery system, we first examined the DNA binding regions of the human DNA topoisomerase I (Topo I), using agarose gel mobility shift assay, in order to identify sites of noncovalent binding of human DNA Topo I to plasmid DNA. We identified four DNA binding regions in human DNA Topo I. They resided in 51-200, 271-375, 422-596, and 651-696 a.a of the human DNA Topo I. We then used it as a DNA binding protein fragment in the construction of a DNA delivery vehicle. Based on the known functional property of each Pseudomonas exotoxin A (PE) domain and human DNA Topo I, we fused the receptor binding and membrane translocation domains of PE with a highly positively charged DNA binding region of the N-terminal 198 amino acid residues of human DNA Topo I. The resulting recombinant protein was examined for DNA binding in vitro and transfer efficiency in cultured cells. The results show that this DNA delivery protein is a general DNA delivery vehicle without DNA sequence, topology, and cell-type specificity. The DNA delivery protein could be used to target gene of interest into cells for genetic and biochemical studies.
封面
目錄
附圖目錄
附表目錄
縮寫表
中文摘要
英文摘要
第一章 緒論
一、綠膿桿菌
二、綠膿桿菌感染方式與症狀
三、綠膿桿菌的致病機制
四、綠膿桿菌外毒素A
五、綠膿桿菌外毒素A的應用
第二章、發展綠膿桿菌疫苗
一、前言
二、材料與實驗方法
三、實驗結果
四、討論
第三章 以綠膿桿菌外毒素A開發基因輸送系統
一、前言
二、材料與實驗方法
三、實驗結果
四、討論
圖表
參考文獻
英文附錄
Appendix 1 A Nontoxic Pseudomonas Exotoxin A Induces Active Immunity and Passively Protective Antibody Against Pseudomonas Exotoxin A Intoxication
Appendix 2 Recombinant Protein Composed of Psudomonas Exotoxin A, Outer Membrane Proteins I and F as Vaccine Against P. aeruginasa Infection
Appendix 3 Development of DNA Delivery System Using Pseudomonas Exotoxin A and a DNA Binding Region of Human DNA Topoisomerase I
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