https://scholars.lib.ntu.edu.tw/handle/123456789/188192
標題: | 克雷白氏肺炎桿菌 DNA adenine methylase (Dam) 與致病力相關性的研究(1/2) | 作者: | 方啟泰 | 關鍵字: | 克雷白氏肺炎桿菌;致病力;去氧核醣核酸腺核;甲基化;Klebsiella pneumoniae;virulence;DNA adenine methylase | 公開日期: | 2003 | 出版社: | 臺北市:國立臺灣大學醫學院內科 | 摘要: | 克雷白氏肺炎桿菌是一種革蘭氏陰性的腸內菌。在台灣,克雷白氏肺炎桿菌是最 重要的人類致病菌之一。常在有糖尿病的成人,甚至健康人,引起致命性的化膿 性肝膿瘍、菌血症、轉移性眼內炎及腦膜炎,此外克雷白氏肺炎桿菌也是一種重 要的院內感染病原菌,可造成肺炎,泌尿道感染。其致病力的調控機轉目前完全 不清楚。去氧核醣核酸腺核?甲基化?可把細菌基因體上核酸序列GATC 的 adenine 甲基化,從而控制細菌基因表現,在細菌基因調控機轉上扮演重要角 色。在沙門氏桿菌-另一種革蘭氏陰性的腸內菌,去氧核醣核酸腺核?甲基化? 在致病力調控機轉上的重要性已被證實,去氧核醣核酸腺核?甲基化?基因突變 之沙門氏桿菌菌株,致病力顯著下降。但去氧核醣核酸腺核?甲基化?在克雷白 氏肺炎桿菌致病力調控機轉的角色則尚未有人研究。本研究的目的即為探討去氧 核醣核酸腺核?甲基化?與克雷白氏肺炎桿菌致病力的相關性。我們目前已成功 的選殖出克雷白氏肺炎桿菌的去氧核醣核酸腺核?甲基化?基因。先利用由 NCBI (National Center for Biotechnology Information) 網站所搜尋到的E. coli K12 的去氧核醣核酸腺核?甲基化?基因序列為模本,將其基因的5 ’及3 ’端序列設 計為引子對。以在臺灣已知為高毒力的克雷白氏肺炎桿菌原始型菌株 NTUH-2044 的基因體DNA 為模板,用PCR 的方式順利增幅出一段669 個鹼基 的開放讀架片段。將此開放讀架片段定序後,與E. coli K12 的去氧核醣核酸腺核 ?甲基化?基因序列作比對,發現有99 ﹪的相似度。選殖出克雷白氏肺炎桿菌 菌株NTUH-2044 的去氧核醣核酸腺核?甲基化?基因之後,我們進一步建構鑲 嵌有chloramphenicol acetyltransferase (CAT) cassette 的去氧核醣核酸核?甲基化 ?基因,以conjugation 的方法送回NTUH-2044 ,篩選出4 株dam - 基因突變菌株。 測試突變菌株的血清抗性,與野生型比較發現有明顯的降低。未來的研究將進而 以動物實驗測定其50%致死量的變化,來量化去氧核醣核酸腺核?甲基化?基因 與克雷白氏肺炎桿菌致病力的相關性。進一步,將用微陣列晶片技術,比較原始 型菌株與突變型菌株在基因表現上的差異,來找出受到去氧核醣核酸腺核?甲基 化?基因控制的下游基因群。最後,將以動物實驗測試去氧核醣核酸腺核?甲基 化?基因突變菌株作為疫苗菌株的可行性。 Klebsiella pneumoniae, an enteric Gram-negative bacillus, is one of the most important human pathogen in Taiwan. K. pneumoniae causes life-threatening pyogenic liver abscess, bacteremia, septic endophthalmitis and meningitis in both diabetic persons and otherwise healthy people. K. pneumoniae is also an important pathogen of nosocomial pneumonia and urinary tract infection. The regulation of virulence gene expression in K. pneumoniae has been poorly understood. DNA adenine methylase (Dam) can add methyl group to the adenine of sequence GATC, and thus play an important role in the control of bacterial gene expression. In Salmonella, another enteric Gram- negative bacillus, the role of Dam in the regulation of virulence has been well documented. dam - Salmonella mutants significantly loss the virulence potential. The role of Dam in K. pneumoniae has not yet been studied. The purpose of this proposal is to investigate the relationship between Dam and bacterial virulence in K. pneumoniae. We have successfully cloned the dam gene in K. pneumoniae. We used the nucleotides sequence of dam gene in E. coli K12, available on NCBI website, to design PCR primers. Using designed primers, we successfully amplify a DNA fragment containing a 669 bps open reading frame from the genomic DNA of clinical invasive strain K. pneumoniae NTUH-2044. We further constructed dam - mutant by inserting chloramphenicol acetyltransferase (CAT) cassette into dam and sending the mutated dam into wild type NTUH-2044 for homologous recombination. Four dam - K. pneumoniae mutants were obtained, and all of them had much reduced serum resistance in comparison with the wild type. In the second year, animal study will be used to determine the difference in LD50 to quantify the correlation between Dam and bacterial virulence in K. pneumoniae. Microarray technique will be used to identify the downstream genes regulated by dam. Finally, the possibility of dam - K. pneumoniae mutants as vaccine strains will be explored through animal experiments. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/23591 | 其他識別: | 912314B002178 | Rights: | 國立臺灣大學醫學院內科 |
顯示於: | 醫學系 |
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912314B002178.pdf | 252.08 kB | Adobe PDF | 檢視/開啟 |
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