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研究生:吳婉鈴
研究生(外文):Wan-Ling Wu
論文名稱:探討嗜熱菌之UDP-glucose4'-epimerase與生物膜生成的關聯性
論文名稱(外文):Characterization of UDP-glucose 4'-epimerase from thermophilic bacteria involved in biofilm formation
指導教授:林光慧林光慧
指導教授(外文):Guang-Huey LinGuang-Huey Lin
學位類別:碩士
校院名稱:慈濟大學
系所名稱:微免暨分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:95
語文別:中文
論文頁數:66
中文關鍵詞:生物膜生成生物膜生成UDP-glucose 4-epimerase嗜熱菌
外文關鍵詞:thermophilic bacteriaUDP-glucose 4'-epimerasebiofilm formationthermophilic bacteriaUDP-glucose 4-epimerasebiofilm formation
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UDP-glucose 4'-epimerase (EC 5.1.3.2) (GalE)是由galE基因轉錄轉譯而來,主要參與在半乳糖的代謝途徑中,用以催化可逆反應UDP-glucose和UDP-galactose,其另一項功能可用於合成細胞壁上的多種醣化物,例如lipopolysaccharide (LPS)、exopolysaccharide(EPS),此二者為構成生物膜主要成份。本實驗研究目的是要探討galE基因在嗜熱菌所扮演的角色,並觀察GalE是否會影響到生物膜的形成。經比對Thermus thermophilus HB8、Thermus thermophilus HB27、Thermus aquaticus NTU103、Thermus aquaticus YT-1四株嗜熱菌的GalE胺基酸序列,顯示嗜熱菌的galE基因被高度的保留下來,並且帶有三個具高度保留性的催化位置,分別是與NAD+結合的GxxGxxG序列和Ser124、Tyr149-X-X-X-Lys153。我們藉由一段不完整的嗜熱菌galE基因建構在含kanamycin-resistant基因的載體上,使它與染色體DNA上的galE基因發生同源基因重組,將整個載體插入而破壞嗜熱菌的galE基因,藉此得到galE基因缺失的突變株,結果發現galE基因缺失的菌株會減少生物膜的形成能力。另外我們也將嗜熱菌的galE基因送入大腸桿菌,幫助合成大量的重組蛋白質,再以鎳離子親和性管柱層析法來純化GalE,其分子量大小為35 kDa,並利用生化分析法測試GalE的蛋白質活性,證明GalE具有催化功能,其酵素動力參數Km值為79.3 μM及酵素最大反應速率Vmax值為0.017 (μmol NADH/min-1)。
UDP-glucose 4'-epimerase (EC 5.1.3.2) is an obligatory enzyme of the galactose metabolic pathway. It catalyses the reversible epimerization of UDP-glucose and UDP-galactose. The protein encoded by galE gene provides UDP-galactose needed for the synthesis of galactose-containing polysaccharides, including lipopolysacchrides (LPS) and extracellular polysacchride (EPS). Mature biofilm structures are encased in an extracellular polymeric matrix. The aim of this study is to identify the relationship between GalE and biofilm formation in the Thermus spp. By comparison of the GalE amino acid sequences from Thermus thermophilus HB8, Thermus thermophilus HB27, Thermus aquaticus NTU103, Thermus aquaticus YT-1 show high-level homology with each other. The amino acid sequences of GalE also contain three highly region conserved NAD-binding domain (GxxGxxG), Ser124, Tyr149-X-X-X-Lys153. The galE genes of four thermophilic bacteria are disrupted by homologous recombination using a thermostable kanamycin-resistant marker. We demonstrate that the galE mutants are reduced in the ability of biofilm formation about 1.5 to 2 folds.The galE gene encodes 35 kDa protein and recombinant GalE expressed in E. coli is purified by Ni2+ affinity chromatography. The recombinant protein is analysed by a coupled enzymatic activity assay to establish kinetic parameters Km and Vmax about 79.3 μM and 0.017 μmol NADH/min-1,respectively.
中文摘要 ---------------------------------------------------------------- I
英文摘要 --------------------------------------------------------------- II
縮寫對照表------------------------------------------------------------- III
前言
一、嗜熱性細菌 --------------------------------------------------------------- 1
二、生物膜----------------------------------------------------------------------- 3
三、GalE的角色與功能------------------------------------------------------- 5
四、GalE蛋白質與生物膜之相關性-------------------------------------------7
五、研究目的--------------------------------------------------------------------- 7
材料及方法-------------------------------------------------------------------------- 9
一、材料-----------------------------------------------------------------------------9
1.菌種、質體、及引子-------------------------------------------------------------9
2.試藥----------------------------------------------------------------------------------9
3.儀器----------------------------------------------------------------------------------9
二、實驗方法
1.製備細菌培養基-----------------------------------------------------------------10
2.微量質體DNA的萃取 ( Miniprep )------------------------------- ------- -11
3.質體DNA的快速篩選------------------------------------------------------------11
4.利用化學法製備勝任細胞 ( competent cell )---------------------- ------------12
5.大腸桿菌的轉形作用 ( transformation )---------------------------- -----------12
6.細菌染色體DNA的萃取--------------------------------------------------------12
7.聚合酶連鎖反應 ( Polymerase chain reaction,PCR )----------- -----------13
8.酒精沉澱純化DNA----------------------------------------------------------------13
9.從電泳凝膠中回收DNA ( Gel extraction )------------------------ -------------14
10.接合反應 ( Ligation )----------------------------------------------- ----------14
11.嗜熱菌的轉形作用--------------------------------------------------------------15
12.大腸桿菌表現蛋白的誘導-----------------------------------------------------15
13.製備細菌蛋白質均質液-------------------------------------------------------16
14.蛋白質電泳分析 (SDS-PAGE)-------------------------------------------------16
15.電泳膠的固定及染色 ( Coomossie blue stain )---------------- ------------------17
16.西方墨點法(Western blotting)---------------------------------------------------17
17. GalE的蛋白質純化-------------------------------------------------------------------18
18.蛋白質濃縮及冷凍乾燥----------------------------------------------------------------19
19.蛋白質濃度定量-------------------------------------------------------------------------19
20.GalE蛋白質抗體製備------------------------------------------------------------------19
21.GalE蛋白質in vitro活性測試-------------------------------------------------------20
22.Kinetic參數的計算-------------------------------------------------------------------20
23.生物膜活性測試----------------------------------------------------------------------21
24.嗜熱菌的生物膜與浮游菌體收集--------------------------------------------------21
25.螢光染色法-----------------------------------------------------------------------------22
26.互補測試galE基因缺失的大腸桿菌---------------------------------------------24
結果
一、galE被高度的保留在嗜熱菌------------------------------------------------------- 25
二、GalE胺基酸序列與演化的關係---------------------------------------------------26
三、建構galE基因缺失的突變株-----------------------------------------------------26
四、確認galE基因缺失的突變株------------------------------------------------------- 27
五、galE突變株的生物膜測試------------------------------------------------------------ 28
六、建構GalE的表達質體與蛋白質純化---------------------------------------------- 29
七、比較生物膜與浮游菌體的GalE蛋白質消長------------------------------------- 30
八、以螢光染色觀察嗜熱菌的GalE蛋白質在菌體分布情形--------------------- 31
九、大腸桿菌galE突變株的互補測試------------------------------------------------ 31
十、GalE的 in vitro生化活性測試-----------------------------------------------------32
十一、總結-----------------------------------------------------------------------------------33
討論
一、GalE在嗜熱菌參與半乳糖的代謝------------------------------------------------ 34
二、GalE在嗜熱菌參與生物膜形成-------------------------------------------------- 35
三、GalE在浮游狀態菌體表現量較高以促使生物膜形成----------------------- 36
四、GalE的生化活性測試------------------------------------------------------------- 38
參考文獻--------------------------------------------------------------- ------------------39
附表、圖-------------------------------------------------------------------------------------46
表一、本論文所使用的質體及菌種---------------------------------------------------46
表二、本論文中所使用的引子------------------------------------------------------ 47
表三、比較不同菌種間galETK三個基因在染色體上的相對位置------------48
圖一、生物膜形成的過程----------------------------------------------------------------49
圖二、The Leloir pathway-----------------------------------------------------------------50
圖三、二維式電泳分析培養 8小時的Thermus aquaticus NTU 103總蛋白質-51
圖四、不同菌種間的GalE胺基酸序列比對--------------------------------------52
圖五、利用GalE胺基酸序列作親緣關係樹狀圖--------------------------------54
圖六、建構具缺失的galE基因載體及發生同源基因重組的機制------------55
圖七、利用PCR分析galE基因缺失的突變株之電泳圖-----------------------57
圖八、galE突變株的生物膜活性測試----------------------------------------------59
圖九、建構GalE的表達質體與蛋白質純化---------------------------------------60
圖十、比較生物膜與浮游菌體的GalE蛋白質消長------------------------------61
圖十一、以螢光染色觀察嗜熱菌的GalE蛋白質分布----------------------------62
圖十二、大腸桿菌GalE突變株的互補測試---------------------------------------63
圖十三、化學反應途徑及雙倒數圖( Lineweaver-Burk plot ) --------------------64
圖十四、GalE蛋白質所需最適當溫度條件-----------------------------------------65
圖十五、分析GalE所需輔酶及輔因子最適當的量------------------------------66
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