臺灣博碩士論文加值系統

克雷白氏肺炎菌是一種伺機性感染細菌，經常在免疫有缺陷的病人造成尿道感染、肺炎及敗血症。而在台灣，這種菌被發現與糖尿病病人引起的肝膿瘍併發症有很高的關聯性。細菌感染的第一步驟，就是藉由細菌表面的纖毛附著寄主細胞。本論文藉由探討台灣克雷白氏肺炎菌的纖毛及纖毛黏附蛋白的種類和特異性，來了解其致病之過程。首先，以Dot blot hybridization及RFLP等技術，分析約一百株臨床分離菌株。結果顯示，第一型纖毛黏附蛋白FimH幾乎存在所有的菌株中。除了已發表的第三型纖毛黏附蛋白之外，另外得到3個新的黏附蛋白基因，mrkD-L1，mrkD-L2和mrkD-L3。其中 mrkD-L1基因在收集的臨床菌株中佔有約28﹪的比例。同時，以南方點墨法，在克雷白氏肺炎菌的噬菌體基因庫中篩選，得到包含有mrkD-L1基因群的噬菌體，並進一步的次選殖和基因定序。經核酸序列分析，與已知的mrk基因群比對，發現分離的基因群中，除了少了調控蛋白mrkE外，還和fim基因群相連。再經由南方點墨法分析，證實此基因組位於染色體上。另外，也成功的構築了結構基因mrkA缺損的突變株，其功能差異仍有待檢測。最後，以大腸桿菌表現系統來大量表現mrkD-L1的基因，純化此重組蛋白來進行實驗測試。經西方點墨法和ELISA分析，在小白鼠測得抗體產生，再以致死劑量的細菌接種，但並無顯著的保護效果。所以，第三型纖毛黏附蛋白在克雷白氏肺炎菌致病過程中，其扮演的角色應再佐以更多的研究。

Klebsiella pneumoniae is an important opportunistic pathogen that often causes urinary infection, pneumonia and septicemia in immunocompromised patients. In Taiwan, K. pneumoniae infection is closely associated with liver abscess among patients with diabetes mellitus, which may cause serious complications. As generally known, attachment of pathogens to their host is a prerequisite step of infection. In order to understand the pathogenesis of K. pneumoniae, I investigate the fimbriae and fimbrial adhesins of the clinical isolates of K. pneumoniae in Taiwan. About 100 isolates were analyzed by Dot blot hybridization and RFLP analysis. The results showed that, almost all the isolates analyzed possessed a gene relating to type 1 fimbrial adhesin. In addition to the type 3 fimbrial genes that have been reported previously, I identified several new adhesin genes, herein, namely mrkD-L1, 2, 3. Clones with the entire mrkD-L1 gene cluster were then isolated from the K. pneumoniae CG43 genomic DNA phage library. Sequence analysis showed that, the arrangement of the mrk genes was similar to that reported previously in another strain, except that the regulator, mrkE, was absent in K. pneumoniae CG43. The result of Southern hybridization indicated that, the mrk genes were chromosomally located by strain CG43. Mutations of the structural gene, mrkA, were subsequently generated. However, the effects of these mutants have not yet determined. Finally, the mrkD-L1 gene was over-expressed in E. coli, and the recombinant protein was purified and used to immunize mice. The production of MrkD-L1 specific antibody in mice has been confirmed by western analysis and ELISA. No protection was observed when the MrkD-L1 immunized mice were challenged with a lethal dose of K. pneumoniae CG43. However, lack of protection can not rule out the importance of type 3 fimbrial adhesin in K. pneumoniae pathogenesis.
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Introduction --------------------------------------------------------- 5
Material and Methods -------------------------------------------- 10
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Discussion --------------------------------------------------------- 25
Reference ---------------------------------------------------------- 28
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