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研究生:陳瑾妤
研究生(外文):Chin-Yu Chen
論文名稱:探討BIG-3蛋白質在細胞內的位置及性質
論文名稱(外文):Characterization of the cellular location and properties of the recombination BIG-3 protein
指導教授:蘇建國
指導教授(外文):Jyan-Gwo Su
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:63
中文關鍵詞:融合蛋白
外文關鍵詞:WD-repeat
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探討BIG-3蛋白質在細胞內的位置及性質
摘要
BIG-3是一種新發現的蛋白質,其會被BMP-2所誘導表現;BIG-3屬於WD重複(WD-repeat)的成員,WD重複的序列的功能目前尚未完全明白,至目前含有此部分重複序列的蛋白質已被發現的不同功能中,還未有促使骨頭發育的功能,於是擬對此蛋白質做進一步的分析研究。
首先利用綠螢光蛋白觀察細胞位置,將綠螢光蛋白的基因接在BIG-3的後面使之形成一個融合蛋白,如此可藉由綠螢光的表現位置觀察蛋白質的位置。結果顯示蛋白質的表現位置集中於細胞中的某處,另外也以間接免疫螢光法觀察細胞位置,發現BIG-3所在位置與DNA相同,所以BIG-3是一在細胞核表現的蛋白質。
此外,也以此蛋白質建立一套實驗室中可利用的桿狀病毒在哺乳類細胞表現系統,以使我們所要表現的哺乳類動物蛋白質有完整的後修飾作用,避免影響到其功能的表現。本實驗同時將在哺乳類動物細胞表現的CMV啟動子及在昆蟲細胞表現的P10啟動子放在一起,所以首先構築的質體就是在pEGFP-N1中將CMV啟動子的後面加上桿狀病毒所含有的啟動子P10,再將此質體送入哺乳類細胞中表現,藉由綠螢光的表現,觀察其蛋白質的表現量。結果顯示在CMV啟動子後面加上P10啟動子並不會抑制CMV啟動子的表現,所以未來再將含有CMV及P10啟動子的片段送入桿狀病毒表現質體中,經過同源重組互換後所得到的桿狀病毒因含有哺乳類可用的啟動子,所以將可在哺乳類細胞中表現蛋白質。除了藉由這套系統可以來表現BIG-3蛋白質進行更進一步的探討外,更方便於未來對哺乳類蛋白質表現的應用,並可進一步作為基因療法的工具。

Characterization of the cellular location and properties of the recombination BIG-3 protein
Abstract
BIG-3 is a new protein that can be induced by BMP-2, and it contains WD-repeat. The functions of WD-repeat proteins is not clear yet. WD-repeat proteins perform a wide range of cellular function. However, none of the WD-repeat proteins has been shown to play a role in the regulation of endochondral bone formation. Therefore, we would like to analyze the function of BIG-3 protein.
The first step is to analyze the cellular location of BIG-3. Green fluorescence protein, linked to the carboxyl terminal of BIG-3, has been used to reveal the localization of BIG-3 by observing its fluorescence. The result has shown that green fluorescence protein as produced and gathered in one small area in cells. We also used indirect immunofluorescence staining to confirm the cellular location of BIG-3. The experimental results demonstrated that BIG-3 and DNA localized in the same place. Accordingly, BIG-3 is located in nucleus.
In addition, we would like to build a system, which can express recombination proteins in mammalian cells by infection of recombinant baculovirus. Thus, the recombinant proteins will be produced with completed post-translation modification. In this study, attempt to link the CMV promoter, driving the expression in mammalian cells, and P10 promoter, driving the expression in insect cells together. The P10 promoter was link to the 3’end of CMV promoter in the pEGFP-N1. This recombinant plasmid was transfected into mammalian cells to monitor the function of these promoters by observing the expression of enhancer green fluorescent protein (EGFP). According to the quantity of green fluorescence, P10 promoter does not inhibit the expression of EGFP, driven by CMV promoter. Therefore, both CMV promoter and P10 promoter can be used together in baculoviral transfer vector and, in turn, transferred into baculovirus genome. This recombinant promoter will be used for expression of BIG-3 in both baculovirus-infected mammalian and insect cells, which can be applied in further studies of BIG-3 and as a tool for gene therapy.

目錄
項目 頁數
中文摘要………………………………………………………………………Ⅰ
英文摘要………………………………………..……………………….…... Ⅲ
目錄………………………………………………………………………..…..Ⅴ
圖目錄………………………….…………………..……….…………………Ⅶ
壹、緒論………………………………………………..………………..……..1
一、研究起源及目的………………………………..……….………….…..1
二、綠螢光蛋白……...…………………………………………...…………5
三、重組蛋白質...……………………………………………….…..………6
貳、材料與方法……………………………………………………..…..….…. 10
一、儀器……………………………………………………….…..….…..….10
二、材料……………..………………………………………………………11
三、方法……………………..……………………………….….……..…....13
(一)、細胞培養………………………………………….……………..….13(二)、構築質體…………………………………….………….………..…14
(三)、Transformation for DNA plasmid ………..…..……….….…….…..18
(四)、PCR…………………………………..………..………...…….……19
(五)、抽取 DNA…………….………………………….…......……….…20
(六)、轉染哺乳類細胞…………………………………….……….….…..22
(七)、間接免疫螢光法……………………………………...……………..23
(八)、西方點墨法…………………………………………………….….24
參、結果…………………………………………………..………...….…...…27
一、構築質體…………………..…………………..………………...……..27
二、轉染哺乳類細胞(transfection of mammalian cell)蛋白質所在位置之觀察……………………………………...……...………………...…....29
三、利用螢光觀察不同質體在哺乳細胞的表現情形……...…..….……....31
肆、討論………………………………………………………………..…....…33
伍、參考文獻………………………………………………………….…….…36
陸、附錄……………………………………………………………….……..…57

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