臺灣博碩士論文加值系統

δ-晶體蛋白(δ-crystallin)，是鳥類及爬蟲類眼睛水晶體中特有的蛋白質，扮演對光線折射的重要角色。在演化上，δ-晶體蛋白經由「基因分享」的過程，將精氨醯丁二酸裂解酶徵召到眼睛水晶體內做為結構性蛋白質。精氨醯丁二酸裂解酶，參與肝臟尿素循環之代謝步驟。α-晶體蛋白(α-crystallin)由αA及αB所形成的聚合體，屬於小熱擊蛋白家族(small heat shock protein family)的一員，具類伴護分子(molecular chaperone -like )的功能，為維持水晶體透明度以及預防白內障的重要蛋白。本論文利用熱敏感度研究重組鵝眼αA及αB晶體蛋白-晶體蛋白與重組鵝眼δ-晶體蛋白及重組人類精氨醯丁二酸裂解酶彼此間的相互作用的關係。於25 ℃人類精氨醯丁二酸裂解酶與αA-晶體蛋白、αB-晶體蛋白混合後活性分別上升103 ％及130 ％。而在溫度效應下，人類精氨醯丁二酸裂解酶最佳活性溫度為40 ℃，至60 ℃後活性只剩下7 ％，與αA或αB晶體蛋白混合之活性可穩定至50 ℃，至60 ℃後仍有17 ％及31 ％活性剩下。常溫下αA-晶體蛋白或αB-晶體蛋白與δ-晶體蛋白或人類精氨醯丁二酸裂解酶混合一小時，分析其色胺酸螢光有減弱的現象，並且αA-晶體蛋白與野生型人類精氨醯丁二酸裂解酶之間的螢光減弱現象最為明顯，約多出30 ％~50 ％。由溫度改變的過程中，觀察蛋白質聚合可以發現αB-晶體蛋白對δ-晶體蛋白或人類精氨醯丁二酸裂解酶的保護作用都比αA-晶體蛋白明顯許多，並且由分子篩層析中可以看到αB-晶體蛋白與δ-晶體蛋白或人類精氨醯丁二酸裂解酶皆會形成穩定的大聚合分子。

Taxon-specific -crystallin, the major soluble protein component of the avian and reptilian eye lens, is considered to play a structural role in maintaining a high refractory index while ensuring transparency. However, the most astounding relationship is an evolutionary strategy of gene sharing that the taxon specific crystallins seems to be present in which the same gene product is utilized in dual function as a lens crystallin and as an enzyme in non-lens tissues. These sequence similarities such as the -crystallins are closely related to argininosuccinate lyase. -Crystallin and ASL are a superfamily of metabolic enzymes that catalyze the reversible cleavage of argininosuccinate to arginine and fumarate. ASL is one of the cytosolic enzymes of the urea cycle in ureagenic animals. -Crystallin is the member of the small heat shock protein family and function as a molecular chaperone-like activity in preventing the non specific thermal aggregation of lens proteins. In addition, -Crystallin consists of two polypeptide chains, A and B. In the present study, we used the temperature-dependent to investigate the effect of recombinant goose A- and B-crystallins on recombinant goose -crystallin and HASL. At 25°C, the addition of A and B led to 103% and 130% increase in the specific activity of HASL, respectively. It is found that the optimal temperature of HASL was at 40°C brought about 149% in the specific activity, whereas the specific activity was about 7% at 60°C. In contrast, HASL is stabled up to approximately 50°C in the presence of A and B, and even has about 17% and 31% at 60°C, respectively. Conditions for incubation of -crystallin and HASL were set at room temperature for 1 hr that is determined through time and temperature dependent assays of ASL activity in the presence of A and B. The tryptophan fluorescence experiments of HASLWT on A show significantly decreased at about 30% ~ 50%. Under the temperature-dependent conditions, the protection effect of B on -crystallin is more remarkable than A. In addition, calibrated S-200 size exclusion chromatography revealed that -crystallin and HASL in the presence of B form a stable complex.

總目錄
總目錄………..…………………………………………………………...……Ⅰ目錄.....................................................................................................................Ⅱ
圖目錄………..………………………………………………………………...Ⅳ
圖目錄………..………………………………………………………………...Ⅶ
名詞縮寫對照表……..…………………………………………………….…..Ⅷ
中文摘要………..………………………………………………………...……Ⅸ
英文摘要…………………..………………………………………….……….XI











目錄
序 論………………………………………………………………………………………….1
實驗材料……………………………………………………………………………………….6
一、藥品試劑………………………………………………………………………….....6
二、主要儀器及器材………………………………………………………………….....8
三、試劑的配製………………………………………………………………………..….9
實驗方法……………………………………………………………………………………...12
一、轉殖作用 (Transformation)……………………………………………………...…12
二、重疊聚合酶聯鎖反應 (Overlapping PCR)………………………………………...13
三、檢視PCR之成品......................................................................................................14
四、限制酶 (restriction enzyme)切目標DNA和載體 (vector)…………………….….15
五、DNA連結反應 (Ligation)………………………………………………………….16
六、勝任細胞 (Competent Cell；DH5α、BL21)的製備………………………………....16
七、菌體培養………………………………………………………………………….…17
八、蛋白質純化……….……………………………………………………..…………..18
九、蛋白質純化(His-tag)…………………………………………………………..……20
十、活性的測定…………………………………………………………..………..…….22
十一、蛋白質濃度測定………………………………………………………...………..22
十二、SDS-PAGE鑑定蛋白質純度和分子量……………………………………...…..23
十三、圓偏振二色旋(Circular Dichroism)分析光譜……………………….…………..24
十四、分子篩色層分析(Superdex gel filtration)S-200HX(10 mm/30 cm)……………..24
十五、螢光光譜實驗………………………………………………….…………………26
十六、溫度效應下蛋白質聚集散射分析……………………………….………………27
結果…………………………………………………………………………………...………28
一、蛋白質純化………...……………………………………………………………..…28
二、含His-tag蛋白質之純化……………………………………………………………31
三、圓偏振二旋 (CD)光譜分析………………………………………………………..31
四、αA-晶體蛋白、αB-晶體蛋白對人類精氨醯丁二酸裂解酶之活性影響………..…32
五、色胺酸螢光分析不同比例之α-晶體蛋白與δ-晶體蛋白以及人類精氨醯丁二
酸裂解酶在25℃之作用關係…………………………………………..…………32
六、熱效應下α-晶體蛋白對δ-晶體蛋白以及人類精氨醯丁二酸裂解酶保護作用….34
七、δ-晶體蛋白與人類精氨醯丁二酸裂解酶於溫度影響下的四級結構…………….36
討論……………………………………………………………………………...……………38
一、不同溫度下αA-晶體蛋白、αB-晶體蛋白對人類精氨醯丁二酸裂解酶之活性
影響………………………………………………………………...………………38
二、室溫下αA-晶體蛋白、αB-晶體蛋白對人類精氨醯丁二酸裂解酶及δ-晶體
蛋白之相互影響…………………………………………………………..……….39
三、熱效應下αA-晶體蛋白、αB-晶體蛋白對人類精氨醯丁二酸裂解酶及
δ-晶體蛋白之相互影響………………………………………………………..….40
參考文獻……………………………………………………………………………………...74



































圖目錄
圖一、αA-晶體蛋白經Q-Sepharose管柱純化SDS-PAGE分析圖................................43
圖二、αB-晶體蛋白經Q-Sepharose管柱純化SDS-PAGE分析圖................................43
圖三、野生型δ-晶體蛋白經Q-Sepharose管柱純化SDS-PAGE分析圖......................44
圖四、野生型人類精氨醯丁二酸裂解酶經Q-Sepharose管柱純化SDS-PAGE
分析圖..................................................................................................................................44
圖五、αB-晶體蛋白經疏水性層析管柱純化SDS-PAGE分析圖...................................45
圖六、野生型δ-晶體蛋白經疏水性層析管柱純化SDS-PAGE分析圖.........................45
圖七、野生型人類精氨醯丁二酸裂解酶經疏水性層析管柱純化SDS-PAGE
分析圖..................................................................................................................................46
圖八、αA-晶體蛋白經S-300管柱純化SDS-PAGE分析圖...........................................46
圖九、野生型δ-晶體蛋白經S-300管柱純化SDS-PAGE分析圖.................................47
圖十、野生型δ-晶體蛋白經S-300管柱純化SDS-PAGE分析圖.................................47
圖十一、野生型人類精氨醯丁二酸裂解酶經S-300管柱純化SDS-PAGE分析圖.....48
圖十二、C端含有His-tag之δ-晶體蛋白經鎳離子管柱純化SDS-PAGE分析圖.......49
圖十三、C端含有His-tag之人類精氨醯丁二酸裂解酶經鎳離子管柱純化
SDS-PAGE分析圖...............................................................................................................50
圖十四、純化後之蛋白質..................................................................................................51
圖十五、人類精氨醯丁二酸裂解酶圓偏振二色旋(CD)光譜分析二級結構變化比較..52
圖十六、δ-晶體蛋白圓偏振二色旋(CD)光譜分析二級結構變化比較...........................52
圖十七、溫度變化下野生型人類精氨醯丁二酸裂解酶活性之改變..............................53
圖十八、於冰上不同時間野生型人類精氨醯丁二酸裂解酶活性之改變......................54
圖十九、色胺酸螢光分析野生型人類精氨醯丁二酸裂解酶與αA-晶體蛋白在25℃
之作用關係..........................................................................................................................55
圖二十、野生型人類精氨醯丁二酸裂解酶與不同比例之αA-晶體蛋白之色胺酸
螢光改變..............................................................................................................................56
圖二十一、野生型人類精氨醯丁二酸裂解酶與不同比例之αB-晶體蛋白之色胺酸
螢光改變..............................................................................................................................56
圖二十二、野生型δ-晶體蛋白與不同比例之αA-晶體蛋白之色胺酸螢光改變..........57
圖二十三、野生型δ-晶體蛋白與不同比例之αB-晶體蛋白之色胺酸螢光改變..........57
圖二十四、C端含有His-tag之人類精氨醯丁二酸裂解酶與不同比例之αA-晶體
蛋白之色胺酸螢光改變......................................................................................................58
圖二十五、C端含有His-tag之人類精氨醯丁二酸裂解酶與不同比例之αB-晶體
蛋白之色胺酸螢光改變......................................................................................................58
圖二十六、C端有His-tag之δ-晶體蛋白與不同比例之αA-晶體蛋白之色胺酸螢光
改變......................................................................................................................................59
圖二十七、C端有His-tag之δ-晶體蛋白與不同比例之αB-晶體蛋白之色胺酸螢光
改變......................................................................................................................................59
圖二十八、比較四種蛋白質與αA-晶體蛋白之色胺酸螢光改變...................................60
圖二十九、比較四種蛋白質與αB-晶體蛋白之色胺酸螢光改變...................................61
圖三十、熱效應下人類精氨醯丁二酸裂解酶與αB-晶體蛋白於360 nm散射光
變化......................................................................................................................................62
圖三十一、熱效應下δ-晶體蛋白與αB-晶體蛋白於360 nm散射光變化.....................63
圖三十二、熱效應下人類精氨醯丁二酸裂解酶與αA-晶體蛋白於360 nm散射光
變化......................................................................................................................................64
圖三十三、熱效應下δ-晶體蛋白與αA-晶體蛋白於360 nm散射光變化.....................65
圖三十四、野生型人類精氨醯丁二酸裂解酶與αB-晶體蛋白於不同溫度下之
分子篩層析圖......................................................................................................................66
圖三十五、C端有His-tag之人類精氨醯丁二酸裂解酶與αB-晶體蛋白於不同溫度
下之分子篩層析圖..............................................................................................................67
圖三十六、野生型δ-晶體蛋白之與αB-晶體蛋白於不同溫度下之分子篩層析圖.......68
圖三十七、C端有His-tag之δ-晶體蛋白與αB-晶體蛋白於不同溫度下之分子篩
層析圖..................................................................................................................................69
圖三十八、熱效應下離心取上清液SDS-PAGE分析圖.................................................70
圖三十九、野生型δ-晶體蛋白四聚體結構圖..................................................................71


表目錄
表一、Crystallins in Celluar……………..…………………………………...72
表二、單位a-晶體蛋白對δ-晶體蛋白及人類精氨醯丁二酸裂解酶所減弱的螢光量.........................................................................................................73

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