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研究生:謝彩凡
研究生(外文):Chai-Fan Hsieh
論文名稱:創傷弧菌致病因子之基因研究
論文名稱(外文):Genetic Study of the Virulent Factor from Vibrio vulnificus
指導教授:張敏政張敏政引用關係
指導教授(外文):Ming C. Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:72
中文關鍵詞:創傷弧菌致病因子脂解酵素活化因子基因54轉錄活化因子
外文關鍵詞:Vibrio vulnificusvirulent factorlipaseactivator protein54 transcriptional activator
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創傷弧菌(Vibrio vulnificus)是一種嗜鹽性的革蘭氏陰性菌,此菌為伺機性致病菌。其致死率引起臨床醫師相當的重視。臨床病徵包括敗血症,組織潰爛、急性下痢等。臨床治療乃採用抗生素(cefotaxime和minocycline)來治療;必要時需施以外科手術(擴創手術、嚴重時需要截肢)。抗生素的濫用會導致病菌產生抗藥性是現在醫療界的重大問題,故抗生素治療並非根本之道。再加上此菌感染在南台灣病例居多的地緣關係,創傷弧菌其他治療方法的開發和致病機轉之研究,我們是當仁不讓的。
創傷弧菌外泌的許多產物和因子被認為與致病有關,包括磷酸脂解酵素(phospholipase)和脂解酵素(lipase)。於是我們由先前所建立的基因庫(gene bank)中成功的選殖出對蛋黃培養基有活性的菌株(命名為V4P)。並取細胞萃取液來做細胞毒性試驗,發現對人類膀胱癌細胞株TSGH8301有明顯的毒性。核酸序列分析顯示它是由906-bp之脂解酵素基因(lipA)和840-bp之活化因子基因(lipB)所組成的。如果將lipB從載體中刪除,lipA單獨存在時,將喪失脂解酵素的活性。另外,在西方點漬法中證實LipA的確是外泌性的蛋白質,它的外泌過程可能是需要LipB參與的。
另外,在核酸序列分析中意外地發現一段有趣的基因:54轉錄活化類似因子(54 transcriptional activator,CT4)。利用聚合酵素鏈合反應(PCR)的方式夾出全長1341-bp open reading frame的CT4。一般而言,54轉錄活化因子並非細胞常存性的蛋白質,只有在外在環境改變時才會表現,以應付緊急的環境變化。例如:致病菌感染至宿主而表現致病因子,就需要此蛋白質。由於CT4的氨基酸序列,亦具有一般54轉錄活化因子的特徵,即N端具有磷酸化位置;中心區域有ATP結合位置;而C端的Helix-turn-Helix為DNA結合位置。是否當外在訊息刺激細胞時,也會導致一連串的磷酸化,而將CT4 N端的Aspartic acid 磷酸化,並在ATP參與下,使下游所控制的基因表現。這一連串的訊息傳導課題,是我們相當感興趣的。故將CT4架構於載體 pET21-(b)中,表現其重組蛋白質,並經由鎳管柱(Nickel column)純化出分子量約為49.8 kDa之蛋白質。經由西方點漬法的分析中發現,創傷弧菌在一般生長條件下,的確不能表現此蛋白質。
而什麼外在環境的變化,CT4才會表現?經各種外在環境改變的實驗中,我們發現,創傷弧菌培養在綿羊血液態培養基4.5小時會表現CT4。故我們推測CT4與創傷弧菌侵入血液系統,並使之有利在外在環境生存有莫大的關係。
Vibrio vulnificus is a marine Gram-negative bacillus, that is recognized as a cause of fulminant primary septicemia, wound infections and acute self-limiting diarrhea. Vibrio vulnificus septicemia is associated with a mortality greater than 50%. With septic shock, mortality approaches 100%. Aggressive wound care must include administration of antibiotics (cefotaxime and minocycline), supportive care, debridement of nonviable or necrotic tissues, as well as consideration of amputation of affected extremities.
Many extracellular proteins and virulent factors of V. vulnificus, including phospholipase and lipase, are associated with pathogenic mechanism. In this study, a clone (V4P), which exhibited a clear zone on egg yolk plate, was isolated from a genomic library of V. vulnificus. The crude cell extract obtained from V4P is toxic to bladder cancer cell line TSGH8301. Nucleotide sequence analysis of the cloned DNA fragment in V4P predicted that a gene (lipA) encoding extracellular lipase consists of an open reading frame (ORF) of 906-bp. Downstream from the lipA gene an ORF was identified and the corresponding was named lipB. Further experments revealed that no lipase activity is produced by lipA in E. coli unless the lipB is also present. Immunoblot analysis confirmed that LipA is an extracellular lipase.
In the course of determing the nucleotide sequence of the lipB region, an additional ORF was found. This ORF is 1341-bp in length and would encode a polypeptide, that showed high homology to 54 transcriptional activator of V. cholerea, and therefore named CT4. Transcritional activity of the non-housekeeping protein 54 transcriptional activator is usually modulated in response to environmental signals. A number of important virulent genes are co-ordinately regulated through the action of the 54 transcriptional activator. There are three functional different domains in 54 transcriptional activator:COOH-terminal domain contains a helix-turn-helix DNA binding motif involved in binding to the enhancer-like element, a conserved central domain contains an ATP binding site responsible for the activation of transcription, and a nonconserved NH2-terminal domain presumably consists of phosphorylation site to be the target for certain regulatory signals. To investigate whether the CT4 of V. vulnificus is responsive to enviromental signals or not, the CT4 PCR product was inserted into pET system and the 49.8 kDa CT4 protein was purified, and then anti-CT4 polyclonal antibodies were prepared. The results of immunoblot analysis showed that no CT4 protein was detected when V. vulnificus was clutured in the general growth condotion, however, when the organism was clutured in the sheep blood medium after 4.5 hours, significant amount of CT4 was detected. The data suggest that CT4 seems to be associated with V. vulnificus invasion to human blood system.
目錄I
圖目錄III
中文摘要IV
英文摘要VI
縮寫檢索表VIII
第一章 創傷弧菌致病基因之選殖及研究1
緒論2
材料與方法6
使用之菌株、載體及培養基6
製備少量質體DNA8
細胞培養(cell culture)9
限制酵素切割質體DNA10
鹼性去磷酸酵素(BAP)處理載體10
接合反應(ligation)11
大腸桿菌之形質轉換 (Transformation)12
lipA和lipB序列與pET載體融合基因之構築13
LipA和LipB融合蛋白之表現14
SDS-PAGE之蛋白質分子量分析15
融合蛋白之純化17
滲透壓震盪法(Osmotic shock)取得膜間隙蛋白質18
抗體製備19
西方點漬法20
蛋白質濃度的定量21
脂解酵素活性測定22
結果23
選殖脂解酵素之相關基因23
細胞毒性試驗23
核酸序列分析及氨基酸分析24
野生株LipA、LipB分泌模式26
lipB缺失對脂解酵素之影響27
野生株脂解酵素之特性分析27
討論29
第二章 創傷弧菌54轉錄活化因子與致病機轉之探討32
緒論33
材料與方法35
染色體DNA的抽取35
南方點漬法 (Southern blotting)35
CT4序列與pET載體融合基因之構築37
54轉錄活化因子CT4之表現條件39
結果40
核酸序列分析及氨基酸分析40
南方點漬法確定CT4基因存在創傷弧菌野生株染色體41
54轉錄活化因子CT4之蛋白質純化及抗體製備42
正常環境生長之野生株並未表現54轉錄活化因子CT442
54轉錄活化因子CT4在血液培養中表現42
討論44
參考文獻48
圖目錄
圖1脂解酵素在蛋黃培養基之活性表現52
圖2細胞毒性試驗53
圖3lipA、lipB之核酸序列及氨基酸序列54
圖4來自不同菌種之脂解酵素氨基酸序列比對55
圖5來自不同菌種之脂解酵素活化因子氨基酸序列比對55
圖6利用鎳親和性管柱純化LipA重組蛋白57
圖7利用鎳親和性管柱純化LipB重組蛋白58
圖8V. vulnificus野生株LipA各區間之西方點漬分析59
圖9V. vulnificus野生株LipB各區間之西方點漬分析60
圖10lipB缺失在橄欖油平板培養基上之活性表現61
圖11V. vulnificus野生株對於不同p-nitrophenyl esters受質之活性分析62
圖12Serine、Histidine、Aspartate形成之活化中心(catalytic triad)63
圖13LipA由細胞內運送至細胞外可能的機制64
圖14細菌訊息傳導機制圖65
圖15CT4之核酸序列及氨基酸序列66
圖16來自不同菌種之54轉錄活化因子氨基酸序列比對67
圖17南方點漬法鑑定CT4存在V. vulnificus野生株染色體68
圖18利用鎳親和性管柱純化CT4重組蛋白69
圖19西方點漬法確定CT4為非細胞常存性的蛋白質70
圖20西方點漬法確定CT4在綿羊血液培養中表現71
圖21V. vulnificus野生株綿羊血液培養之生長曲線72
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