臺灣博碩士論文加值系統

重金屬鉛是一個有毒物質，會對人體造成嚴重的傷害。 研究Pb2+的細胞毒理現象，必須要有適合的Pb2+指示劑，才能觀察到細胞中Pb2+的堆積。 本實驗利用螢光共振能量轉移(FRET)的策略建構出Pb2+生物感測器，對Pb2+的專一性高而且不受Ca2+和其它二價金屬離子的影響，可用於研究Pb2+進入細胞的機制。 使用螢光蛋白ECFP(△C11)和cp173Venus做為FRET的螢光配對，以及利用C. metallidurans CH34中的Pb2+結合蛋白PbrA、PbrD和PbrR當做Pb2+結合結構，建構出不同系列的Pb2+生物感測器。 其中以PbrR第4個到第6個a螺旋結構的蛋白片段所建構的Pb2+生物感測器Met-lead1.59和Pb2+結合產生FRET的反應最佳。 Met-lead1.59的螢光比值動態範圍是3.29~5.84，解離常數 Kd1和Kd2分別為68.87 ± 48.35 nM和22.08 ± 5.18 uM。 在短時間(240秒)和長時間(三小時)的鉛處理下，Met-lead 1.59可以偵測到Pb2+進入和累積在人類胚胎腎臟細胞中。 即使環境只有0.5 uM的Pb2+，Met-lead1.59仍然可以偵測到細胞內的Pb2+。 Met-lead1.59是一個蛋白質生物感測器，未來可以接上不同的訊號胜肽，讓Met-lead1.59標定在重要的胞器像是內質網和粒線體中，以了解Pb2+是否會進入以及累積在胞器內，並且造成胞器功能的傷害。 Met-lead1.59確實是一個有力的工具，可幫助研究者進一步了解Pb2+如何進入細胞內並且造成細胞的毒性。

Lead (Pb) is a toxic substance that would cause serious damage to the human body. Cytotoxicity assays of Pb2+ must have a suitable Pb2+ indicator to monitor intracellular Pb2+ accumulation. The present study aimed to construct a Pb2+ biosensor by using fluorescence resonance energy transfer (FRET) strategy. It is highly specific for Pb2+ and does not interfere with Ca2+ or other divalent metal ions. Pb2+ biosensor can be used to study the mechanism of intracellular Pb2+ entry. We used the fluorescent protein ECFP(△C11) and cp173Venus as FRET pairs, and Pb2+-binding protein PbrA, PbrD and PbrA of C. metallidurans CH34 as Pb2+-binding domain to construct a series of Pb2+ biosensors. Met-lead1.59 constructed by using the 4th to 6th a-helix of PbrR, which has better FRET signal when senses with Pb2+. The dynamic range of emission ratio of Met-lead1.59 is 3.29 to 5.84, and dissociation constant Kd1 and Kd2 of Met-lead1.59 are 68.87 ± 48.35 nM and 22.08 ± 5.18 uM. The intracellular Pb2+ entry and accumulation in human embryonic kidney cells was successfully monitored using Met-lead1.59 under both short- and long-term Pb2+ treatments. Under low concentration of Pb2+ treatment (0.5 uM), Met-lead1.59 can still detect intracellular Pb2+ accumulation. Met-lead1.59 is a protein-based biosensor. In order to understand whether Pb2+ will enter or accumulate in organelles such as endoplasmic reticulum (ER) and mitochondria then cause abnormal function, Met-lead1.59 can be fused with signal peptides and target to organelles in the future. Met-lead1.59 is a powerful tool, can help researchers to study more about the mechanism of Pb2 + entry that cause cytotoxicity.

中英文縮寫對照表………………………………………………………………………………………………I
中文摘要…………………………………………..…………………………………………………………………II
Abstract….………………….………………………………………………………………………….……………III
目錄………………………………………………………………………………………………………………………IV
圖目錄………………………………………………………………………………………………….………………VI
表目錄………………………………………………………………………………………………….………….…VII
第一章 、 背景介紹………………………………………………………………….………………………1
一、 重金屬鉛…………………………………………………………………..…………………………1
二、 離子指示劑…………………………………………………………………………….……………2
三、 螢光共振能量轉移………………………………………………………………………………3
四、 綠色螢光蛋白………………………………………………………………………………………3
五、 螢光共振能量轉移方式的生物感測器……………….………………………………4
六、 鉛離子結合蛋白……………………………………………………………….…………………5
(1) PbrA………………………………………………………………………………………..…………6
(2) PbrD……………………………………………………………..……………………………………7
(3) PbrR………………………………………………………..…………………………………………7
第二章 、 實驗目的和策略………………………………………………………………………………9
第三章 、 實驗材料和方法……………………………………………………………………………10
一、 化學試劑………………………………………………………………………..…………………10
二、 Pb2+生物感測器的建構……………………….……………………………………………10
三、 蛋白質表現和純化……………………………………….……………………………………11
四、 螢光光譜分析Pb2+反應、離子滴定和離子選擇性……………….……………12
五、 細胞培養及細胞轉染…………………………………………………..……………………12
六、 雙螢光影像擷取系統及細胞內鉛反應實驗…………………..…………………13
七、 統計方法……………………………………………………………………………………………14
第四章 、 實驗結果………………………………………………………….………………………………15
一、 以PbrA建構的Pb2+生物感測器………..………………………………………………15
二、 以PbrD建構的Pb2+生物感測器………………..………………………………………16
三、 以PbrR建構的Pb2+生物感測器…………………………..……………………………16
四、 離子滴定和離子選擇性的測試…………………………………………………………18
五、 細胞內Met-lead1.59對Pb2+的反應…………………………………..………………19
六、 Met-lead1.59偵測Pb2+進入到細胞內………………………………….……………20
第五章 、 討論…………………………………………………………………………..………………………22
一、 以Pb2+結合蛋白建構Pb2+生物感測器………………………………………………22
二、 Met-Lead1.59的Pb2+解離常數……………………………….…………………………23
三、 Met-Lead1.59對Zn2+的選擇性……………………………………….…………………23
四、 Cu2+對Met-Lead1.59的影響……………………………………..………………………24
五、 雙聚體對FRET的影響……………………………………………………..………………25
六、 結論……………………………………………..……………………………………………………25
第六章 、 文獻參考……………………………………………………………………………….…………27
附錄……………………………………………………………………………………………………..…………..55
附件………………………………………………………………………………………………………..…………….58

圖一、利用PbrA建構Pb2+生物感測器……………………………………..……………………………38
圖二、Pb2+生物感測器YCPbrA對Pb2+的反應….…………………………………………………39
圖三、利用PbrD建構Pb2+生物感測器……………………………………..……………………………40
圖四、Pb2+生物感測器YCPbrD對Pb2+的反應…………………………………..………………..41
圖五、利用PbrR建構Pb2+生物感測器……………………………………..……………………………42
圖六、Pb2+生物感測器YCPbrR (由第三到第六個螺旋結構組成)對Pb2+的反
應…………..…………………………………..………………………………………………….……………43
圖七、Pb2+生物感測器YCPbrR (由第四到第六個螺旋結構組成)對Pb2+的反
應…………..…………………………………..………………………………………………….……………44
圖八、Pb2+生物感測器的Pb2+滴定測試…………………………………..……………………………45
圖九、Pb2+生物感測器的二價金屬離子選擇性測試………………..……………………………46
圖十、測試Met-lead1.59對不同濃度的二價金屬離子的反應………………………………47
圖十一、細胞內Met-lead1.59對Pb2+的反應………………………………………….………………48
圖十二、細胞內Pb2+生物感測器對Pb2+的反應……………………………………………..………49
圖十三、細胞內Met-lead1.59對Pb2+和Zn2+的反應………………………………………………50
圖十四、Met-lead1.59偵測Pb2+透過鈣池調控鈣離子通道進入細胞內………..………51
圖十五、長時間處理Pb2+，Met-lead1.59偵測細胞內的Pb2+含量………………….………52

表一、生物感測器對Pb2+的反應……………………………………………...……………………………53
表二、Pb2+生物感測器的反應動態範圍和解離常數………………..……………………………54

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