臺灣博碩士論文加值系統

在革蘭氏陰性菌中，克雷伯氏肺炎桿菌（Klebsiella pneumoniae）是常見引起院內感染的重要病原菌。其常易引起菌血症、腦膜炎、肺炎及尿路系統之感染，而且即使投予抗生素之治療後，仍有相當高的死亡率。另外，由K. pneumoniae引起之化膿性肝膿瘍在外國早期文獻中並不常見，然在1960年代開始發現K. pneumoniae肝膿瘍之發生有逐年增加趨勢。據國外文獻報導，K. pneumoniae化膿性肝膿瘍發生率佔所有化膿性肝膿瘍之27﹪。相對於此，近年來在台灣地區，K. pneumoniae肝膿瘍之發生率由1980年代之30%至1990年代之82.1%。更重要的，國內許多K. pneumoniae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. pneumoniae致病機轉的研究主要是分成下列五種因子來探討：(1) capsule（莢膜）; (2) LPS（脂多醣體）;(3) serum resistance（血清抗性）; (4) siderophores; (5) adhesins 。而在台灣地區 K. pneumoniae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。故本實驗室積極地希望能從 K. pneumoniae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。
之前已由實驗室所構築好的 K. pneumoniae（K.p.w.t.）基因庫中篩選到三個蛋白質，分別為溶血酵素、外膜蛋白質（OMP1）、核酸分解酵素（D1）。在其他報告中也陸續提到此三種蛋白質是某些致病菌之致病因子，且溶血酵素、D1在細胞毒力試驗中，也展現出其毒性，因此促使我們去探討此三種蛋白質是否與 K. pneumoniae的致病力有相關。首先，利用酵素聯結免疫分析法（enzyme-linked immunosorbent assay ; ELISA）的方式，發現在 K. pneumoniae感染之病人身上，其吸光值有高於正常人的現象，所以我們更進一步以動物模式的實驗，來比較此三種蛋白質主動及被動免疫的效果，由結果看來，OMP1似乎與 K. pneumoniae的致病力有著密切的關係，D1次之，溶血酵素則無顯著差異。
然而，在它們基因保留性之探討中，意外發現 K.p.w.t. OMP1和 D1無法在臨床分離出的其它 K. pneumoniae菌株中以聚合酵素鏈反應（PCR）找到，而且原始選殖基因之 K.p.w.t.是一隻低致病力菌株（LD50＞106 c.f.u.），其野生株的核酸分解酵素活性為本實驗收集可分型的 K. pneumoniae菌株最低的。因此我們便猜想，是否因外膜蛋白或核酸分解酵素基因的變異，導致其致病力如此之低。所以，我們便重新建立一高致病力 K.p.129（LD50＜102 c.f.u.）之基因庫，積極尋找核酸分解酵素與外膜蛋白質之正確核酸序列。我們一共獲得三段DNA序列，一是由 K.p.129基因庫重新選殖出的核酸分解酵素（D2），和原始選殖之D1並無任何相似性；二是由NCBI中未完成 K. pneumoniae 基因庫中所找到與 D1有相似性的一段基因，但是，仍無法看出此段基因的保留性；三是以菌落雜交法（colony hybridization）所找到的外膜蛋白 OMP2。且由 OMP1與 OMP2在胺基酸序列上的保留區，所設計之前、後端引子，可在其它 K. pneumoniae菌屬中擴增出預期中之DNA片段。
另外莢膜（CPS）一直被認為與 K. pneumoniae之致病力有著密切關係，它可幫助K. pneumoniae在寄主細胞的colonization，adhesion，maintenance及 proliferation。近二十年來的臨床研究報告指出，K. pneumoniae肝膿瘍最常見的伴隨疾病是糖尿病（53%）。雖然尚未證明 K. pneumoniae肝膿瘍與糖尿病有直接關係，但是糖尿病患者很容易罹患 K. pneumoniae肝膿瘍是無庸置疑的，或許因其免疫力降低，且體內高血糖之環境，提供 K. pneumoniae充足之碳源以利其莢膜合成，因此相較於一般人，糖尿病患者更易遭受 K. pneumoniae的侵襲。也因此我們利用穿透式電子顯微鏡（TEM）來觀察不同糖濃度下所培養出的 K. pneumoniae，其莢膜厚度是否有差異性。由初步結果看來，在高糖濃度（0.5%Glu/ M9）的條件下，所產生的莢膜厚度較低濃度（0.1%Glu/ M9）或以甘油取代糖來源（0.1%Gly/ M9）來的厚。
本文亦根據動物實驗，基因保留性探討及糖濃度對莢膜厚度之影響等結果，討論外膜蛋白、核酸分解酵素及莢膜厚度在 K. pneumoniae致病過程中可能扮演的角色。

Klebsiella pneumoniae is the common pathogen of nosocomial and community-acquired infections, especially bacteremia, pneumonia, and urinary tract infections. Althogh the development of new and potent antibiotics, K. pneumoniae infections are still associated with a high mortality rate up to 50%. In Taiwan, the high incidence of K. pneumoniae pyogenic liver abscess during the period from 1980 (30%) to 1990 (82.1%) was never reported in English literature. In addition, extrahepatic metastases, such as septic endophthalmitis, are often occurred with serious complications. The high mortality rate and high incidence of K. pneumoniae pyogenic liver abscess in Taiwan make further investigation is necessary.
The current research on the pathogenicity of K. pneumoniae focuses on the group of five factors: (1) capsules; (2) lipopolysaccharides (LPS); (3) serum resistance; (4) siderophores; (5) adhesins. The exact mechanism of increasing incidence of K. pneumoniae liver abscess in Taiwan is unclear, so our purposes will look for the proteins involving in the pathogenesis of K. pneumoniae from its genomic library.
Hemolysin, outer membrane protein (OMP1) and DNase (D1) have been cloned from K. pneumoniae (K.p.w.t.) genebank with formerly effort. Several reporters cite these three proteins playing some roles in K. pneumoniae pathogenesis; furthermore, the cytotoxicity of hemolysin and D1 provide the clues to study the correlation of these three proteins with respect to the virulence of K. pneumoniae. Our results indicate that higher absorbance by ELISA detection in patients with K. pneumoniae infection. However, OMP1 shows better effects in active and passive immunization experiments. In the research on genetic conservation, K.p.w.t. OMP1 and D1 can not be amplified by PCR reaction in other clinical K. pneumoniae isolates, and the original cloned DNase activity (D1) from K.p.w.t. with low pathogenesis (LD50>106 c.f.u.) is the lowest compared to the other typeable serologically K. pneumoniae in this study. We suppose the high LD50 value in K.p.w.t. because of the variations in DNase or outer membrane protein, so we create K. pneumoniae 129 (LD50<102 c.f.u.) genomic library to clone the exact DNase and outer membrane protein. We obtain D2, OMP2 from K.p.129 genebank, and D3 from unfinished K. pneumoniae genomic database in NCBI. OMP2 shows amino acid sequence conservation with OMP1, and can be detected in the clinical K. pneumoniae isolates by PCR reaction except for K.p.w.t..
Among the virulence factors of K. pneumoniae, the capsular polysaccharides (CPS) protects the organisms against the bactericidal effect of serum, ingestion, and killing by phagocytes. The association between K. pneumoniae liver abscess and diabetes mellitus (D.M.) is closely in clinical statistic from 1980 to recently research, implying that the high blood glucose concentration in D.M. patients facilitates CPS biosynthesis. In preliminary data, we observe thicker CPS in high glucose media (0.5%Glu/ M9) than in 0.1%Glu/ M9 or substitution glucose for glycerol.
The roles of outer membrane protein, DNase, and the thickness of CPS in the virulence of K. pneumoniae are discussed according to the results from animal experiments, genetic conservation, and the influence in the CPS thickness in different glucose concentration.

目錄
目錄……………………………………………………………………I
圖目錄……………………………………………………………… III
表目錄……………………………………………………………… V
附錄………………………………………………………………… V
中文摘要…………………………………………………………… VI
英文摘要…………………………………………………………… IX
縮寫檢索表……………………………………………………… XII
第一章 克雷伯氏肺炎桿菌致病因子於血清反應與動物實驗之研究 1
緒論………………………………………………………… 2
材料與方法………………………………………………… 12
一、使用之菌株、載體及培養基………………………… 12
二、製備少量質體DNA………………………………… 13
三、限制酵素切割質體DNA…………………………… 14
四、鹼性去磷酸酵素處理載體…………………………… 15
五、接合反應……………………………………………… 15
六、大腸桿菌之形質轉換………………………………… 16
七、D1和OMP1序列與pET載體融合基因之構築…………… 17
八、D1和OMP1重組蛋白之表現……………………………… 19
九、SDS-PAGE之蛋白質分子量分析…………………………… 19
十、重組蛋白之純化……………………………………………… 21
十一、抗體製備…………………………………………………… 23
十二、西方點漬法………………………………………………… 23
十三、蛋白質濃度的定量………………………………………… 25
十四、硫酸銨鹽沈澱免疫球蛋白………………………………… 25
十五、Protein A-conjugated resin 純化免疫球蛋白…………… 26
十六、實驗動物模式之主動、被動免疫………………………… 27
十七、酵素聯結免疫分析法……………………………………… 29
結果………………………………………………………………… 31
第二章 克雷伯氏肺炎桿菌致病因子之再選殖及基因保留性之探討…… 40
緒論………………………………………………………………… 41
材料與方法………………………………………………………… 43
一、使用之菌株、載體及培養基………………………………… 43
二、DNase 活性之測試…………………………………………… 44
三、染色體DNA之抽取………………………………………… 45
四、蔗糖密度梯度離心…………………………………………… 46
五、染色體DNA以Sau3AI作部分切割……………………… 47
六、CsCl密度梯度離心法………………………………………… 48
七、菌落雜交實驗………………………………………………… 49
結果………………………………………………………………… 53
第三章 糖濃度及碳源對克雷伯氏肺炎桿菌莢膜厚度之影響 69
緒論………………………………………………………………… 70
實驗條件與流程…………………………………………………… 72
結果與討論………………………………………………………… 74
總結……………………………………………………………… 77
參考文獻………………………………………………………… 78
圖目錄
圖一、利用鎳親和性管柱純化溶血酵素(Hemolysin)重組蛋白 89
圖二、利用鎳親和性管柱純化核酸分解酵素(D1)重組蛋白 90
圖三、利用鎳親和性管柱純化外膜蛋白(OMP1)重組蛋白 91
圖四、酵素連結免疫分析法(ELISA)之流程圖 92
圖五、以純化後之溶血酵素為抗原，在不同血清稀釋倍數下所獲得之ELISA分析結果 93
圖六、以純化後之核酸分解酵素(D1)為抗原，在不同血清稀釋倍數下所獲得之ELISA分析結果 94
圖七、以純化後之外膜蛋白質(OMP1)為抗原，在不同血清稀釋倍數下所獲得之ELISA分析結果 95
圖八、溶血酵素、核酸分解酵素D1、外膜蛋白OMP1主動免疫之結果 96
圖九、溶血酵素、核酸分解酵素D1、外膜蛋白OMP1抗體於被動免疫之結果 97
圖十、不同 K. pneumoniae菌株之核酸分解酵素D1 PCR之擴增結果 98
圖十一、K.p. 129基因庫構築之流程圖 99
圖十二、利用Sau3AI將染色體進行部分切割 100
圖十三、以蔗糖梯度離心法回收Sau3AI部分切割後之染色體DNA 101
圖十四、重組質體之電泳分析(隨機挑選K.p. 129基因庫之菌落) 102
圖十五、不同次選殖片段於核酸分解酵素測試培養基之活性分析 103
圖十六、核酸分解酵素D2完整核酸序列及胺基酸序列 104
圖十七、不同K. pneumoniae菌株之核酸分解酵素D2 PCR之擴增結果 105
圖十八、不同Klebsiella菌屬間核酸分解酵素D2 PCR之擴增結果 106
圖十九、核酸分解酵素D1完整核酸序列及胺基酸序列 107
圖二十、核酸分解酵素D3完整核酸序列及胺基酸序列 108
圖二十一、D1與D3胺基酸序列比較 109
圖二十二、不同K. pneumoniae菌株之核酸分解酵素D3 PCR之擴增結果 110
圖二十三、核酸分解酵素D1、D2、D3在K.p.w.t.及K.p.129 PCR擴增結果 111
圖二十四、 不同K. pneumoniae菌株之核酸分解酵素D1保留區PCR之擴增結果 112
圖二十五、不同K. pneumoniae菌株之外膜蛋白OMP1 PCR之擴增結果 113
圖二十六、OMP1完整核酸序列及胺基酸序列 114
圖二十七、t-OMP之核酸序列及胺基酸序列 115
圖二十八、OMP1與t-OMP胺基酸序列比對 116
圖二十九、OMP菌落雜交法探針之PCR擴增 117
圖三十、OMP菌落雜交法所獲得有訊號(signal)之菌落位置 118
圖三十一、以PCR方式擴增探針區域篩選OMP菌落雜交法所獲得之candidates 119
圖三十二、OMP2之完整核酸序列及胺基酸序列 120
圖三十三、不同K. pneumoniae菌株之外膜蛋白保留區間PCR擴增結果 121
圖三十四、OMP1、OMP2與Coxiella burnetii OMP胺基酸序列比對 122
圖三十五、穿透式電子顯微鏡(TEM)觀察0.5% Glucose/M9條件下K.p.129莢膜厚度 123
圖三十六、穿透式電子顯微鏡(TEM)觀察0.1% Glucose/M9條件下K.p.129莢膜厚度 124
圖三十七、穿透式電子顯微鏡(TEM)觀察0.1% Glycrol/M9條件下K.p.129莢膜厚度 125
表目錄
表一、克雷伯氏菌屬之各種不同命名法 126
表二、克雷伯氏菌屬之生化反應統整表 127
表三、已分型之克雷伯氏肺炎桿菌核酸分解酵素活性大小及臨床病例對照表 128
表四、K.p.129 基因庫完整性評估（一） 129
K.p.129 基因庫完整性評估（二） 130
表五、D1、D2、D3 NCBI胺基酸序列比對結果 131
表六、OMP1 與OMP2 NCBI胺基酸序列比對結果分析 132
表七、在台灣地區近二十年克雷伯氏肺炎桿菌肝膿瘍之臨床報告 133
附錄
一、 載體pET-21(b)圖譜 134
二、 載體pBR322圖譜 135
三、 載體pGEM圖譜 136

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