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系統識別號 U0026-2801201303245100
論文名稱(中文) 布魯氏錐蟲鞭毛蛋白PF20的原核表達及其在哺乳動物細胞內蛋白質交互作用之研究
論文名稱(英文) Sdudy of TbPF20 in prokaryotic expression and protein-protein interaction in mammalian epithelial cell
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 101
學期 1
出版年 102
研究生(中文) 林祐丞
研究生(英文) Yu-Cheng Lin
電子信箱 s46981177@mail.ncku.edu.tw
學號 s46981177
學位類別 碩士
語文別 中文
論文頁數 84頁
口試委員 指導教授-胥直利
口試委員-戴榮湘
口試委員-洪良宜
中文關鍵字 布魯氏錐蟲  鞭毛  鞭毛相關蛋白  胞質分裂  微管  中心體  細胞骨架 
英文關鍵字 Trypanosoma brucei  flagellum  PF20  cytokinesis  microtubule  centrosome  cytoskeleton 
學科別分類
中文摘要 鞭毛相關蛋白PF20 (Paralyzed flagella protein)是存於細胞纖毛與鞭毛的蛋白質,在具有這些運動胞器生物中,它的基因在演化上呈現高度的保守性。對單細胞寄生蟲布魯氏錐蟲(Trypanosoma brucei)來說,基因TbPF20缺失,不僅移動性失能,細胞也因而無法正常進行胞質分裂,讓此基因和錐蟲存活有決定性的關係。TbPF20共有589個胺基酸,經過序列比對預測出SMC (structure maintenance of chromosome) domain與WD-repeats domain兩個高保守性的功能區,而在SMC domain之前的序列與其他物種的PF20比較下呈現低相似度,稱為變異區。先前的研究發現含有SMC domain及WD-repeats domain的截短化TbPF20片段對哺乳動物上皮細胞造成細胞週期停滯在G2/M期,成對中心體(centrosome)無法分離到細胞兩極,不能順利進行細胞分裂而產生細胞毒殺效應。另外,SMC片段刪除的TbPF20(簡稱SacII)及單獨WD-repeats片段(WDN)也造成細胞週期停滯在G2/M期,中心體無法分離,甚至細胞移動性降低以及微管系統出現捲曲、束狀及被剪切的不正常現象。特別的是,許多SacII蛋白在細胞中的分布大多形成斑點狀聚集在細胞的周圍邊緣。為了探討各個片段毒殺細胞的貢獻與原因,以及驗證先前在影像實驗上的發現,本研究將TbPF20截成不同的重要片段,接到GST蛋白形成融合蛋白,並且利用原核表現系統與親和純化法得到各片段蛋白,而其中發現SMC刪除片段(GST-Sac)、WD-repeats(GST-WD)蛋白會進入到包含體(inclusion body)內,必須以尿素純化法純化。接著將這些純化的融合蛋白再做為餌,進行GST pull down 實驗,以西方點墨法分析與餌交互作用的細胞內蛋白。實驗結果顯示與GST控制組比較, GST-Sac、GST-WD及WD前含SMC片段(GST-272)三種融合蛋白能與某些細胞內的蛋白質交互作用,且GST-Sac能夠結合微管切解蛋白劍蛋白p60次單元,驗證影像實驗中與SacII共存在的結果。此外,實驗結果也顯示Sac、WD及272蛋白會結合γ-tubulin及β-actin,其它片段則無,且所有片段都不結合微管蛋白,證明此GST pull down實驗所釣到的交互作用蛋白具有專一性。或許因TbPF20結合了細胞骨架相關蛋白及中心體相關蛋白影響有絲分裂而毒殺細胞,然而更詳細的機制以及其它交互作用的蛋白則需再進一步研究。
英文摘要 PF20 (Paralyzed flagella protein) is an essential and conserved gene of organism with motile cilium/flagellum. For protozoan Trypanosoma brucei , a parasite living in mammalian bloodsteam, TbPF20 is essential for cytokinesis in addition to motility. Within 589 residues, bioinformatic method indicated TbPF20 containing SMC (structure maintenance of chromosome) domain and WD-repeats domain. Previous studies showed that truncated TbPF20 bearing SMC and WD-repeats domains confered cytotoxicity in mammalian epithelial cells due to G2/M arrest and centrosome disjunction failure. Furthermore, SMC domain deleted TbPF20 (SacII) construct and construct bearing WD repeat domain only (WDN) both not only conferred cytotoxicity due to G2/M arrest and centrosome disjunction failure but also reduced cell motility. These cells also showed abnormal microtubule patterns. Interestingly, SacII cells displayed delicate puncta, thin thread-like beads along cell edges. To delineate contribution of each domain on the cytotoxicity, and to confirm the discovery of image data, various deletion mutants of TbPF20 were tagged with GST protein, expressed in prokaryotic expression system. Two constructs, SMC deleted fragment (GST-Sac) and WD-repeats only fragment (GST-WD), were harvested as inclusion bodies and renatured back with urea. Through GST affinity method these TbPF20 segments as baits were used to fish proteins from MDCK lysate. The results reported that GST-Sac, GST-WD and pre-WD SMC containing fragment (GST-272) would bind some proteins in MDCK cells. Western blot revealed that katanin p60, a microtubule-severing enzyme, could be pulled down only through SacII bait confirming the image data that SacII colocalized with katanin p60. Moreover, GST-Sac, GST-WD and GST-272 also specificly bound γ-tubulin and β-actin, but not bound β-tubulin. The possibility of cytotoxicity of TbPF20 in blocking cell mitosis may due to interaction with cytoskeleton related proteins and centrosome related proteins. The detail mechanisms and other possible interacted proteins of TbPF20 await further investigation.
論文目次 摘要 I
Abstract II
致謝 III
目錄 VI
表目錄 VIII
圖目錄 IX
附錄目錄 X
第一章 緒論 1
A 布魯氏錐蟲(Trypanosoma brucei)與鞭毛(Flagellum) 1
B 鞭毛相關蛋白PF20(Paralyzed flagella protein 20) 2
C WD重複蛋白(WD repeats protein) 4
D SMC(Structure maintenance of chromosome)蛋白 5
E TbPF20毒殺哺乳動物細胞並影響其細胞分裂與細胞骨架 6
F 細胞骨架(Cytoskeleton) 7
G 微管系統(Microtubule system) 8
H 微管切解酵素(Microtubule-severing enzymes)與劍蛋白(Katanin) 10
I 研究目的(Specific aim) 13
第二章 材料與方法 14
A 實驗材料 14
I 實驗之質體、菌種與培養基 14
II 實驗溶劑 16
III 限制酵素 19
IV 抗體 19
B 實驗方法 19
I 構築TbPF20各個功能區於pGex-4T-3 vector 19
II 利用大腸桿菌系統表現TbPF20重組蛋白及純化 23
III 利用agarose beads純化的蛋白進行GST pull down assay 24
第三章 實驗結果 27
A PF20的序列在演化上的保守性與變異性 27
B 原核表現TbPF20之Pre-SMC domain片段GST重組蛋白構建與純化 30
C 原核表現TbPF20之Pre-WD domain片段GST重組蛋白構建與純化 31
D 原核表現TbPF20之含有WD domain片段GST重組蛋白構建與純化 31
E TbPF20各蛋白片段與MDCK細胞內蛋白有交互作用 33
F Western blot分析Sac蛋白與MDCK細胞katanin p60有交互作用 34
G Western blot分析Sac、WD及272蛋白與MDCK細胞γ-tubulin有交互作用 34
H Western blot分析TbPF20幾乎不與微管蛋白有直接交互作用 35
I Western blot分析Sac、WD及272蛋白與MDCK細胞β-actin有交互作用 35
第四章 討論 37
參考文獻 43
表 50
圖 52
附錄 73
自述 84
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