利用綠膿桿菌外毒素之接合與轉位區當作蛋白質載體運送抑癌蛋白質進入

TMUIR > College of Medicine > Graduate Institute of Medical Sciences > Dissertation/Thesis > Item 987654321/5202

Please use this identifier to cite or link to this item: http://libir.tmu.edu.tw/handle/987654321/5202

Title:	利用綠膿桿菌外毒素之接合與轉位區當作蛋白質載體運送抑癌蛋白質進入 using the binding and translocation domains of pseudomonas
Authors:	徐怡強 Hsu, Yi-Chiang
Contributors:	醫學研究所
Keywords:	綠膿桿菌外毒素抑癌蛋白質 pseudomonas exotoxin A tumor suppressor protein
Date:	1995
Issue Date:	2009-09-11 15:26:04 (UTC+8)
Abstract:	綠膿桿菌外毒素 A ( Pseudomonas exotoxin A ) 的三個功能區是由一條多鏈所組成的，依結構命名分別為功能區 I ( domain I )、功能區 II ( domain II )、功能區 III ( domain III )，而其作用分別為受體接合區 ( receptor binding domain )、轉位區 ( translocation domain ) 及腺甘二磷酸-核糖化作用區 ( ADP-ribosylation domain )。在本研究中，我們製造一個新的融合蛋白質 PE(DIII)-p21，可藉由大腸桿菌來大量表現及純化，此蛋白質是藉由基因工程的方法，將綠膿桿菌外毒素 A 的受體接合區及轉位區兩個功能區，改造成為蛋白質運送載體 ( protein delivery vehicle )，以運送一種週期素活化激素抑制劑( cyclin dependent kinase inhibitor ) 名為 p21WAF1/Cip1 的蛋白質進入 HL-60 細胞中。加入 PE(DIII)-p21 於培養基時會促使 HL-60 細胞趨向分化 ( differentiation )，可藉細胞分化表面標誌 CD11b ( surface marker CD11b ) 的表現得到證實。而且在重氫胸腺口密啶併入 (3H-thymidine incorporation ) 實驗中亦可以發現 PE(DIII)-p21 會使 HL-60 細胞停止生長。另外在西方墨漬法 ( Western blotting ) 及35S 放射線標示 PE(DIII)-p21 實驗結果中，可以發現若將融合蛋白質加入培養基中，此蛋白質會進入 HL-60 細胞內，並裂解為 PE(II)-p21 及 p21 等片段。由以上的實驗結果我們可發現，利用改造後的綠膿桿菌外毒素 A 的受體接合區及轉位區兩個功能區所組成的蛋白質運送載體 PE(DIII) 確實可將 p21 WAF1/Cip1 送入 HL-60 細胞，不但抑制了 HL-60 細胞增生且促進分化。 Pseudomonas exotoxin A (PE) is a polypeptide chain with three functioal domains.these are receptor-binding ,transloction, and ADP-ribosylation domains, which reside on structural domain Ia, domain II, and domain III,respectively.In this study,we have used the binding and transloction domains of PE as a protein delivery vehicle to target p21WAF/Cip1 ,an inhibitor of cyclin/ cdk complex,into HL-60 cells this recombinant protein, designated as PE(DIII)-p21,was expressed in E.Coli. When HL-60 cells were administered with PE(DIII)-p21, they were undergone differentiation,which wasconfirmed by the induction of cell surface marker CD11b. PE(DIII)-p21 treatment also result in growth arrest as shown by H3-thymidine incorporation experiment. In addition,when PE(DIII)-p21 was incubated with HL-60 cells, the protein was degraded intoPE(II)-p21 and p21 fragments, supporting that PE(DIII)-p21 did enter the cells.
Data Type:	thesis
Appears in Collections:	[Graduate Institute of Medical Sciences] Dissertation/Thesis

Files in This Item:

File	Size	Format
摘要.doc	33Kb	Microsoft Word	184	View/Open
摘要.pdf	61Kb	Adobe PDF	236	View/Open
摘要.ppt	144Kb	Microsoft Powerpoint	207	View/Open
摘要.ps	333Kb	Postscript	88	View/Open

Loading...