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研究生:鄭則琦
研究生(外文):Tse-Chi Cheng
論文名稱:克雷伯氏肺炎桿菌致病因子之研究
論文名稱(外文):Study on the virulence factors of Klebsiella pneumoniae
指導教授:張敏政張敏政引用關係
指導教授(外文):Ming-Chung Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
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:2002
畢業學年度:90
語文別:中文
論文頁數:82
中文關鍵詞:致病因子克雷博氏肺炎桿菌核酸分解酵素
外文關鍵詞:dnasevirulence factorsklebsiella pneumoniae
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在革蘭氏陰性菌中,克雷伯氏肺炎桿菌(Klebsiella pneumoniae)是一種常引起院內感染的重要病原菌。其常易引起全身性感染,而且在經由抗生素治療後,仍然有相當高的死亡率。近年來,由K. p.引起之化膿性肝膿瘍的病例無論國內外,都有逐漸增加的趨勢,在台灣地區甚至高達82.1%。更重要的是,國內許多K. p.肝膿瘍患者易發生肝外轉移病灶,而造成很嚴重的合併症。最麻煩之合併症乃是發生敗血性內眼炎導致失明,而國外則很少有此種病例報告。如此大的差異與高死亡率,頗受國內感染專家學者的重視。
目前,對於K. p.致病因子的研究方面,capsule(莢膜)及LPS(脂多醣體)是已經證實與其致病能力有直接關係,而其他因子則正在進一步研究中。而在台灣地區K. p.肝膿瘍之發生率增加,其原因可能與尚未發現或尚未明暸之某些致病機轉有關。所以本實驗室希望能從K. p.基因庫中找出與致病力有關的蛋白質,期望能對於臨床治療有所幫助。
之前實驗室由兩株致病力相差甚多的臨床K. p.菌株(K.p.w.t.及K.p.129)基因庫中分別篩選到兩種核酸分解酵素基因,分別命名為D1及D2,進一步針對臨床K. p.菌株分析此兩段基因的保留性。在分析病人病歷後發現,D1容易存在於帶有尿道感染病歷的菌株中;而感染帶有D2的菌株之患者,有極高的比例已經死亡。於是推測此段D2基因的存在可能與菌株的高致病力有關,便進行此段基因的構築及表現,並進一步以動物實驗模式觀察其主動及被動免疫的效果,結果顯示分別有60%及40%的保護效果。另一方面,為了進一步確認此段基因對於K. p.菌株感染能力的影響,我們在菌株交配過程中,利用同源染色體重組方式改造野生株(K.p.129)中的D2基因,接著再觀察野生株及突變株的半致死率(LD50),結果發現兩者並無明顯差異,另外我們發現此D2蛋白質位於菌體的膜間隙部位,因此我們推測D2可能並非其主要的致病因子之一,而是在K.p.感染過程中具有協助菌體感染的功能。
Klebsiella pneumoniae is the common pathogen of nosocomial and community-acquired infections. K.p. usually causes systemic infection and the high mortality even if antibiotic treated. The incidence of K.p. pyogenic liver abscess was higher and higher recently, there even was 82.1% in Taiwan. In addition, extrahepatic metastases, such as septic endophthalmitis, are often occurred with serious complications. The high mortality rate and high incidence of K.p. pyogenic liver abscess in Taiwan make further investigation is necessary.
The current research had been evidenced that capsules and lipopolysaccharide were the virulence factor of K.p.. The other virulence factors are currently being studied. The exact mechanism of increasing incidence of K.p. liver abscess in Taiwan is unclear, so we want to look for other proteins involved in the pathogenesis of K.p. from its genomic library.
dnase1 (D1) and dnase2 (D2) cloned from two strain K.p. had different LD50 value, K.p.w.t. (LD50= 106) and K.p.129 (LD50<102) genebank. In the research on genetic conservation, D1 can be amplified by PCR in clinical K.p. isolated from patients with urinary tract; D2 gene detected in K.p. isolated from patient, the ratio of death is high. To investigate the correlation of D2 with respect to the virulence of K. p., we constructed and expressed the D2 protein, and observated the protection effect of active immunization and passive immunization in animal model. These results of protect effect was 60% in active immunization and 40% in passive immunization.
In order to study the virulence roles of dnase2 in the clinical infection of K.p., several isogenic dnase2-deficient strains were constructed by using integrated plasmid to disrupt the orginal dnase2 gene. The 50% lethal does of isogenic dnase2-deficient was equal to wild type (K.p.129). According to the localized experiment, D2 protein located in periplasm region of K.p.129. We supposed dnase2 may not be a very important virulence factor of K.p.129, it maybe help K.p.129 to attack host during infection process.
考試合格證明 II
中文摘要 III
英文摘要 IV
致謝 VI
表 目 錄 IX
圖 目 錄 X
縮寫檢索表 XI
第一章 核酸分解酵素對克雷伯氏肺炎桿菌致病能力影響之動物實驗 1
緒論 2
材料與方法 5
一、使用之菌株、載體及培養基 5
二、製備少量質體DNA 6
三、限制酵素切割質體DNA 7
四、鹼性去磷酸酵素處理載體 7
五、接合反應(ligation) 8
六、大腸桿菌之形質轉換 (Transformation) 8
七、D2序列與pGEX載體融合基因之構築 9
八、D2融合蛋白之表現 10
九、SDS-PAGE之蛋白質分子量分析 11
十、融合蛋白之純化 12
十一、抗體製備 13
十二、西方點漬法 13
十三、蛋白質濃度的定量 15
十四、硫酸銨鹽(ammonium sulfate)沈澱免疫球蛋白 15
十五、Protein A-conjuated resin 純化免疫球蛋白 16
十六、細胞區分法(cell fractionation) 16
十七、實驗動物模式之主動、被動免疫 18
結果與討論 20
第二章 克雷伯氏肺炎桿菌之核酸分解酵素缺失對其致病能力之影響 26
緒論 27
材料與方法 28
一、D2之同源基因與Trk載體之構築 28
二、基因突變株之構築與篩選 29
三、染色體DNA之抽取 29
四、聚合酵素連鎖反應 30
五、南方吸漬法(Southern blotting) 31
六、Klebsiella pneumoniae 129與突變株LD50之測試 35
結果與討論 37
總結: 41
附錄 42
P0持續表現細胞株之建立 42
緒論 43
材料與方法 45
一、使用之菌株、細胞株、載體及培養基 45
二、細胞繼代培養(附著型細胞adherent cell) 46
三、細胞冷凍 46
四、哺乳類細胞之形質轉換 47
五、篩選及建立持續表現P0蛋白質之細胞株 48
結果與討論 49
參考文獻 74
自述 82
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洪瑞陽(2000)與U型環狀RNA鍵結的核糖蛋白P0之研究,國立成功大學理學院生物科技研究所碩士論文
侯玥秀(2000)克雷伯氏肺炎桿菌致病因子之研究,國立成功大學醫學院生物化學研究所碩士論文
楊薇儒(2001)篩選與P0交互作用之蛋白質及其研究,國立成功大學理學院生物科技研究所碩士論文
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