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研究生:蔡國威
研究生(外文):Kuo-Wei Tsai
論文名稱:表現牛蛙α-Crystallin水晶體蛋白對大腸桿菌的紫外線耐受性之影響及機制探討
論文名稱(外文):Expression of Bullfrog α-Crystallin in E. coli Enhances Viability of Recombinant Cells under UV Stress
指導教授:吳世雄
指導教授(外文):Shih-Hsiung Wu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
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:2001
畢業學年度:89
語文別:英文
論文頁數:88
中文關鍵詞:水晶體蛋白、紫外線傷害、微生物細胞死亡、小熱休克蛋白
外文關鍵詞:alpha-crystallin、UV stress、bacteria cell death、small heat shock protein
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  • 被引用被引用:0
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Alpha-crystallin為水晶體蛋白中的主要成分蛋白,包含αA與αB兩種類型,與小熱休克蛋白有相似的氨基酸序列,同時具有molecular chaperon活性,與維持眼球水晶體蛋白的穩定有關,其中αB於眼球水晶體表皮細胞以外的各類組織中均有表現,主要分布於心肌、骨骼肌、皮膚、大腦及脊隨神經及肺臟等,而αA則僅於脾臟及胸腺亦見其分布。在水晶體以外的組織中αB的功能可能在細胞骨架cytoskeleton聚合時扮演molecular chaperon協助纖維蛋白的形成,近年來的研究指出其亦能抑制細胞apoptosis的進程,在調節細胞分化與細胞骨架重組扮演重要的角色。
因其主要分布位置在眼球水晶體表皮細胞,此處細胞的主要環境逆境為電磁輻射(日光/紫外線)的照射,同時亦有文獻報告於水晶體表皮細胞表現Alpha水晶體蛋白能抑制紫外線照射引發的細胞程式死亡,因此此類蛋白不僅能維持其他蛋白質穩定,同時亦能藉由抑制apoptosis強化細胞在逆境下的存活。
我們在研究過程中意外的發現轉殖此基因的大腸桿菌在誘導基因表現後,對紫外線照射後的存活率有10~30倍以上的提昇,突顯此類蛋白在紫外線保護上的功能在演化上如此遙遠的原核生物上亦相當明確,依據我們目前的實驗結果,我們大膽的推論可能在原核生物上的細胞程式死亡與真核生物上有共同的機制存在,使的表現此類基因在原核與真核細胞上均見相同功能展現。當然尚需近一步的研究結果證實我們目前的推論。原核生物的細胞程式死亡是個尚待開發的領域,我們的研究資料顯示我們可以利用現有實驗材料-牛蛙晶體蛋白,以免疫沉澱及蛋白質體等方法來找出與Alpha水晶體交互作用而與執行微生物細胞程式死亡有關的基因,以探索此一尚待開發的研究領域。

Alpha-crystallins, the major components of lens proteins, contribute to lens proteins stability and confer UV resistance potency to lens epithelial cells. We introduced bullfrog αA and αB-crystallin genes to E.coli and induced their expression, these recombinant cells showed significant UV resistance up to more then 10 times survival ratios relative to control cells, which were consistent with those of previous reports in lens epithelial cells. UV resistance of E.coli transformed by truncated form of alpha-crystallins gene constructs showed that the N-terminal 60 residues also confer significant UV resistance to E.coli. The N-terminal domain showed no molecular chaperone activity in our classic chaperone activity assay. These results suggest that the UV resistance of E.coli was not due to the trivial chaperone activity but rather there might exist conserved cell death pathways in prokaryotics and mammalian cells such that similar experimental outcomes were obtained. Or α-crystallin might interrupted UV triggered E.coli cell death by unknown mechanisms awaiting further study. Our results demonstrated that α-crystallins might be promising tools to unveil the mysterious prokaryotics cell suicide pathways.

中文摘要………………………………………………………………………………1
Abstract……………………………………………………………………………..…2
List of Abbreviations…………………………………………………………………..3
Introduction……………………………………………………………………………4
Distribution of α-Crystallin…………………………………………………….4
Physical—chemical properties of alpha-crystallin………………………………..5
Chaperone-like function of alpha-crystallin……………………………………..9
The function of alpha-crystallin in the lens……………………………………..10
The function of alpha-crystallin in other parts of the visual system……………14
Diseases involving alpha-crystallin……………………………………………..15
Heat shock proteins as death determinants……………………………….……..19
Figures…………………………………………………………………………..20
Material and Methods………………………………………………………………...32
Isolation of Frog α-Crystallin……………………………………………….…..32
Characterization of Frog α-Crystallin…………………………………………..32
Gene Construct of Truncated From of Alpha-A and Alpha-B Crystallin……….33
Growth Curve Determination and Expression of α-crystallin in E.coli………...34
UV Irradiated Cell-Viability Experiments……………………………………...35
Protein Purification and Characterization………………………………………35
Protein refolding………………………………………………………….……..36
Figures..……………………………………………………………………...….37
Results and discussion………………………………………………………………..46
Induction of Recombinated Protein Expression in E. coli Cells………………..46
UV resistance of recombinant E. coli cells……………………………………..47
UV protection domain determination…………………………………………..49
E.coli survival ratio of co-incubation exogenous α-crystallin in culture broth...52
Conclusion…………………………………………………………….…….…..53
Figures…….…………………………………………………………………….54
Reference.…………………………………………………………………….………73

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