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研究生:李韋佑
研究生(外文):Wei-yu Lee
論文名稱:偽產鹼假單胞菌F-111脂解酶之分子伴護基因選殖及特性研究
論文名稱(外文):Gene cloning and characterization of lipase chaperone from Pseudomonas pseudoalcaligenes F-111
指導教授:楊文仁楊文仁引用關係林順富林順富引用關係
指導教授(外文):Wen-Jen YangShuen-Fuh Lin
學位類別:碩士
校院名稱:國立高雄大學
系所名稱:生命科學系碩士班
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:2015
畢業學年度:103
語文別:中文
論文頁數:64
中文關鍵詞:分子伴護脂解酶偽產鹼假單胞菌
外文關鍵詞:ChaperonelipasePseudomonas pseudoalcaligenes
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大腸桿菌(Escherichia coli)在現代生物工程中經常被用作基因選殖以及生產目標蛋白質的宿主。然而,由於格蘭氏陰性菌細胞膜的內、外膜雙層結構,致使目標蛋白質往往會堆積於雙層膜之間的周質(periplasm)空間,形成不具生物活性的包涵體(inclusion body)。本篇研究期望能以Pseudomonas pseudoalcaligenes F-111菌株的脂解酶分子伴護,來研究其對於F-111脂解酶能否分泌至細胞外所扮演的角色。

以PCR技術將F-111的脂解酶以及脂解酶分子伴護基因片段選殖到載體pGEM-3zf(+),電腦軟體預測顯示所選殖的脂解酶基因具備有胞外分泌所需的信號肽;並以同源模擬的方式預測所選殖的脂解酶分子伴護基因的蛋白質結構。將目標基因以E. coli表現系統進行蛋白質表現之後發現,具備完整脂解酶以及脂解酶分子伴護基因片段的重組質體能表現出脂解酶活性;然而,若將此重組質體上的脂解酶分子伴護基因剔除,儘管SDS-PAGE證實仍有脂解酶分子的表現,但是在進行活性分析時卻無法如原先的實驗結果觀察測得脂解酶活性。綜合實驗結果,研究所選殖出的脂解酶分子伴護基因,對於脂解酶的活性確實有著決定性的影響。期望此項發現能於未來建構帶有此段分子伴護基因之DNA表現載體,使大腸桿菌表現系統原本目標蛋白容易形成包涵體的問題,能在脂解酶分子伴護的協助下,便能順利送出細胞外,以增進蛋白質純化的方便性。
Escherichia coli is a commonly used gene transformation and protein production host in modern bioengineering. However, its Gram-negative double layer membrane structure also causes target protein aggregation in its periplasm space, leading to a result of inactive inclusion body formation. The aim of this study is to utilize a bacterial strain Pseudomonas pseudoalcaligenes F-111 to study its lipase chaperone’s characteristics of assisting lipase secretion.

By utilizing PCR, both the lipase and lipase chaperone genes from F-111 were cloned and ligated with vector pGEM-3zf(+). The computer bioinformatics prediction showed the lipase polypeptide contains a signal peptide, a necessary for secretion. Meanwhile, the structure of lipase chaperone was predicted by homology modeling. Protein analysis showed the E. coli strain transformed with complete lipase and lipase chaperone genes express lipase activities. However, if the lipase chaperone gene is knocked out, despite SDS-PAGE still shows lipase expression, it did not express its activity as the E. coli strain transformed with complete lipase and lipase chaperone genes. In conclusion, the lipase chaperone gene cloned in this study plays a crucial role in lipase activity. The study expects the discovery would lead to constructing an expression vector with the lipase chaperone gene attached for it assisting target protein secreting, to improve the convenience of protein purification in future.
目錄
頁次
目錄····················································································· I
中文摘要 ············································································ 1
英文摘要 ············································································ 2
第一章 前言········································································· 3
1.1偽產鹼假單胞菌 (Pseudomonas pseudoalcaligenes)··················3
1.2 脂解酶 (lipase)····························································3
1.3 分子伴護 (molecular chaperone)········································4
1.3.1分子伴護歷史與定義···················································4
1.3.2分子伴護功能····························································6
1.4 脂解酶分子伴護···························································6
1.4.1 脂解酶分類······························································6
1.4.2 脂解酶分子伴護作用機制·············································7
1.5 研究背景與實驗設計·····················································8
第二章 材料與方法·································································10
2.1 偽產鹼假單胞菌 F-111目標蛋白基因之選殖······················10
2.1.1 偽產鹼假單胞菌F-111菌株來源····································10
2.1.2 偽產鹼假單胞菌 F-111菌體培養···································10
2.1.3 偽產鹼假單胞菌F-111基因體DNA之抽取·······················10
2.1.4 聚合酶連鎖反應 (polymerase chain reaction, PCR)··············11
2.1.5 瓊膠電泳 (agarose gel electrophoresis)························11
2.1.6 目標DNA純化·························································12
2.2 偽產鹼假單胞菌F-111目標基因之重組質體建構·················12
2.2.1 勝任細胞製備···························································12
2.2.2 以pGEM-T Easy載體接合目標DNA·······························13
2.2.3 重組質體轉殖···························································13
2.2.4 質體DNA正確性之確認··············································13
2.2.4.1 以Colony PCR確認質體DNA正確性···························13
2.2.4.2 質體DNA之抽取····················································14
2.2.4.3 以限制酶確認質體DNA正確性··································15
2.2.5 質體DNA之分子伴護基因剔除·····································15
2.3 偽產鹼假單胞菌F-111目標基因之重組質體次選殖············16
2.3.1 次選殖目標基因純化··················································16
2.3.2 次選殖載體放大與製備···············································16
2.3.3 次選殖重組質體製備··················································17
2.3.4 次選殖重組質體轉殖以及特性分析································17
2.4 蛋白質表現以及活性分析··············································18
2.4.1 Glycerol tributyrate LB agar培養基分析····························18
2.4.2 目標蛋白誘導···························································18
2.4.3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate
polyacrylamide gel electrophoresis, SDS-PAGE)················19
2.4.3.1 蛋白質樣本製備······················································19
2.4.3.2 十二烷基硫酸鈉聚丙烯醯胺凝膠之製備························19
2.4.4 p-nitrophenyl laurate呈色反應分析蛋白質活性···················20
第三章 結果··········································································21
3.1基因選殖····································································21
3.1.1偽產鹼假單胞菌F-111基因體DNA之抽取························21
3.1.2偽產鹼假單胞菌 F-111目標蛋白基因之選殖·····················21
3.1.3偽產鹼假單胞菌F-111目標基因之重組質體建構················21
3.2重組質體生物資訊分析··················································22
3.2.1 序列比對(BLAST)·····················································22
3.2.2 脂解酶基因信號肽分析···············································22
3.2.3 分子伴護蛋白質結構同源模擬······································23
3.3蛋白質表現以及活性分析···············································23
3.3.1分子伴護協助脂解酶分泌之分析····································23
3.3.2目標蛋白誘導····························································23
3.3.3 SDS-PAGE總蛋白分析················································24
3.3.4總蛋白PNL呈色分析··················································24
圖表··············································································25
第四章 討論··········································································41
參考文獻··············································································44
附錄····················································································51
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