臺灣博碩士論文加值系統

本實驗室先前選殖出屬於小熱休克蛋白家族中的一員HspB3，並利用融合瘤技術製得H469E2G單株抗體。藉由免疫轉漬法測得HspB3主要表現在大白鼠的骨骼肌與心臟，其中以心臟的表現量較高。隨後利用酵母菌雙雜交系統的方法，對人類心臟cDNA基因庫進行篩選，確定HspB3能夠與actin相結合作用。本實驗的目的即在於尋找HspB3與actin交互作用的區域，經由胺基酸點突變與一系列胺基酸序列刪除分析，發現在HspB3主要是利用第25到36個胺基酸的部位與actin作用，在actin主要是利用第322到337個胺基酸的部位與HspB3作用。我們隨後利用共軛焦顯微鏡偵測細胞內HspB3與actin結合情形，在RD細胞中分別轉染野生型HspB3與突變型HspB3 (HspB3/△25~36aa) 36~48小時後，發現野生型HspB3能與actin細胞骨架完整的結合在一起，而突變型HspB3與actin細胞骨架結合的程度受到影響。另外在細胞型態上的觀察，當大量表現野生型與突變型的HspB3在RD細胞中，突變型HspB3的表現確實影響到RD細胞膜的完整性。所以，藉由本論文所得到之資訊，我們除了能夠提供HspB3與actin結合的訊息外，利用共軛焦顯微鏡偵測肌肉細胞中兩個蛋白質作用，推斷其具有獨特的生理功能。

Our laboratory has previously identified a cDNA clone encoding the small heat shock protein HspB3. A hybridoma that produces a monoclonal antibody specific to HspB3 was also generated. By using Western blot analysis, HspB3 could be detected in rat skeletal muscle and most abundantly in the heart. Further screening human heart cDNA library using HspB3 as a bait with the yeast two-hybrid system . We have identified that HspB3 is capable of interacting with actin. The goal of this project is to identify the domains required for the interaction between these two proteins. Through serial deletion of these two genes followed by yeast two hybrid analysis, we have identified the region between amino acid position 25-36 of HspB3 is critical for interacting with actin. In actin, amino acid region 322-337, which is near the C-terminus is necessary for its interaction with HspB3. To confirm the interaction between HspB3 and actin in vivo, confocal microcopy was used. When a wild type HspB3 and a truncated mutant of HspB3 (HspB3/△25~36aa) were over-expressed in RD (Human embryonal rhabdomyosarcoma) cell line, the wild type of HspB3 could be co-localized with actin, but the truncated HspB3 could not . Moreover, unlike the wild type HspB3 that did not affect cell morphology, overexpression of truncated HspB3 in RD cells resulted in loss of cell membrane integrity. We concluded that HspB3 is a true actin binding protein. Our results have provided basic information concerning the function role of the small heat shock protein HspB3.

目 錄 頁次
中文摘要-------------------------------------------Ⅰ
英文摘要-------------------------------------------Ⅱ
縮寫表---------------------------------------------Ⅲ
目錄-----------------------------------------------Ⅴ
一、前言-------------------------------------------1
二、材料與方法-------------------------------------10
三、實驗結果---------------------------------------29
四、討論-------------------------------------------39
五、參考文獻---------------------------------------46
圖表-----------------------------------------------53
附錄-----------------------------------------------71

五、參考文獻
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羅明德，私立長庚大學碩士論文，類小熱休克蛋白質cDNA的定序及其在細胞中的表現，中華民國八十七年。

吳欣蓉，國立清華大學碩士論文，SHSPL及RLP-1單株抗體之製備與應用，中華民國八十八年。

蔡靜宜，國立清華大學碩士論文，人類小熱休克蛋白質HSPB3之特性與功能分析，中華民國八十九年。

張家瑗，國立清華大學碩士論文，人類小熱休克蛋白質HSPB3與Actin交互作用的探討，中華民國八十九年。