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研究生:賴韻如
研究生(外文):Yun-Ru Lai
論文名稱:老鼠細胞中熱休克蛋白八十六的基因選殖與蛋白質功能分析
論文名稱(外文):Molecular Cloning and Functional Analysis of Rat 86-kDa Heat Shock Protein
指導教授:張大慈
指導教授(外文):Margaret Dah-Tsyr Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
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:56
中文關鍵詞:熱休克蛋白
外文關鍵詞:heat shock protein
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熱休克蛋白九十 (HSP90) 為一種廣泛存在於真核生物細胞質中的保護子,可使與細胞生長相關之蛋白質折疊以調控其正常生長;而當哺乳動物細胞受到高溫、藥物或是病毒的刺激時會誘發出逆境蛋白的合成,使變性的蛋白質重新折疊,以維持正常的生理功能。由目前已知的HSP90研究當中,可知HSP90是由兩組基因來負責調控,分別為HSP86和HSP84。因此我們利用物種間的DNA序列比對來設計PCR的引子,由大鼠基因組中夾擊出小片段的hsp86之保留區段,並以 [-32P] dCTP標定為探針後從基因組庫中篩選出完整的 hsp86 cDNA。
目前已定序完成的大鼠hsp86 cDNA共有2,795 bp,而其編碼區 (coding region) 包含有2,202 bp。HSP90可分為三個區段,為了分析HSP90的功能,將全長的hsp90 cDNA、ATPase區段以及含有突變的ATPase 區段分別建構到pET23(a)載體內,以ITPG誘導於大腸桿菌BL21內進行蛋白質的大量表現。表現完後將細胞收下可以12 % SDS-PAGE分析蛋白質的產量、純度與溶解度,最後再以鎳親和性管柱將重組蛋白質純化。
將純化完成的蛋白質利用[-32P] ATP進行ATPase的功能分析,由實驗結果可知,全長HSP86之ATPase活性為ATPase區段的十五倍,而加入ADP競爭亦可抑制蛋白質的ATPase活性。我們也發現鎂離子的存在可增加HSP86對ATP的水解能力;但在突變的ATPase 區段並無觀察到明顯的ATPase活性差異。

Heat shock protein 90 (HSP90), an abundant molecular chaperone in the eukaryotic cytosol, is involved the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. In vertebrates, hsp90 has two isoforms, hsp84 and hsp86. Here we report the cDNA cloning and functional analysis of the rat hsp86. Based on the homology among human hsp90, horse hsp90, porcine hsp90 and mouse hsp86, the conserved region in rat hsp86 was amplified by RT-PCR and used as the probe. The full-length rat hsp86 cDNA was obtained from cDNA library screening. The cDNA sequence data contains 2,795 bp and the length of coding region was 2,202 bp (GeneBank accession number AJ428213). Based on a crystal structure of the N-terminal domain of human HSP90 with bound ADP-Mg, several mutants were made in the rat HSP86 ATPase domain. Our results show that the full-length HSP86 has 15-fold higher ATPase activity than the N-termunal ATPase domain only, but the ATPase activity of HSP84 ATPase domain was similar to HSP86 ATPase domain. In addition, we found that the presence of magnesium can increase the ability of ATP hydrolysis of HSP86F Therefore, the results establish that magnesium binding, ATP binding and hydrolysis are required for HSP90 function in vitro.

Introduction..............1
Materials and Methods.....5
Results..................13
Discussion...............18
Figure and Legend........21
Table....................41
Appendix.................44
References...............52

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