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研究生:林怡欣
研究生(外文):Yi-hsin Lin
論文名稱:克雷白氏菌溶血基因與鹼性磷酸酉每基因突變株構築與特性分析
論文名稱(外文):Characterization of Klebsiella pneumoniae CG43 hlyK and phoA mutants
指導教授:彭慧玲彭慧玲引用關係
指導教授(外文):Hwei-Ling Peng
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
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:70
中文關鍵詞:克雷白氏肺炎桿菌溶血素鹼性磷酸酉每
外文關鍵詞:Klebsiella pneumoniaehemolysinalkaline phosphatase
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克雷白氏肺炎桿菌(Klebsiella pneumoniae) 是一種伺機性的病原菌,主要在免疫不全的病人身上引起尿道感染及呼吸感染,並且造成僅次於大腸桿菌的院內感染。我們實驗室先前經由分析比對華盛頓大學已近完成的克雷白氏肺炎桿菌MGH78578基因體序列後,在K. pneumoniae CG43中以PCR選殖一可能與致病相關的核酸片段,這個片段包含一開讀股架與Bacillus cereus的溶血素( Hly-III )胺基酸序列有37﹪的一致性(identity),因此命名為hlyK。我們曾將這段序列構築在pET30c和pET32c表現載體中,卻無法藉此大腸桿菌表現系統在蛋白質電泳膠偵測到重組蛋白HlyK的產生,而進一步利用anti-His-tag抗體也無法偵測得到。經轉換GST表現載體,藉由蛋白質電泳膠分析再利用GST多株抗體做西方墨點法均偵測不到HlyK溶血蛋白的表現。我們進一步利用slot-blot分析hlyK mRNA的表現,但可能因此基因表現量過低或未找出適當刺激其表現因子,無法測得訊號。最後,我們利用基因取代法藉同源互換取得了hlyK基因的突變株。藉由小鼠毒性試驗,我們發現克雷白氏菌野生株和HlyK突變株的小鼠的半致死率沒有顯著的差異。
由於細菌的致病因子有一大部分表現在細胞外膜或分泌性的蛋白,我們希望藉TnphoA突變系統來篩選表現在克雷白氏菌CG43細菌表面或分泌到胞外的分子,進一步研究這些分子和致病的相關性。由於克雷白氏肺炎桿菌CG43有明顯鹼性磷酸酉每活性,為了降低鹼性磷酸酉每的背景值,我們以基因取代方式建構phoA的突變株CG43-S3P1,而卻意外的發現此240 bp的缺損並不影響其鹼性磷酸酉每活性,我們將此帶有缺損的磷酸酉每基因的質體,轉形入PhoA缺損的大腸桿菌,卻發現此質體不能彌補PhoA缺損的大腸桿菌鹼性磷酸酉每的活性。而建構第二個較大phoA缺損的突變株CG43-S3P2,也不影響其鹼性磷酸酉每活性,因此我們推測克雷白氏菌可能還有另一個磷酸酉每的存在。

Klebsiella pneumoniae is an opportunistic pathogen that often causes urinary and respiratory tract infections in immunocompromised patients. Next to Escherichia coli, it is the second leading cause of nosocomial infections in Taiwan. We have previously used BLASTN analysis and isolated from a virulent K. pneumoniae strain CG43 a hemolysin-like gene hlyK, which has a 37% amino acid sequence identity with the Bacillus cereus hemolysin (Hly-III). In order to study the functional role of hlyK, we have previously cloned hlyK into plasmids pET30c and pET32c, respectively. None of them were found to be expressed in E. coli, however the anti-His tag monoclonal antibody still could not detect the His-Tag fusion proteins either. Thus, a GST fusion expression system was used and a GST polyclonal antibody was prepared by a immunizing mice with the purified GST. Nevertheless, no GST-HlyK fusion protein was observed using SDS-PAGE analysis or western blot analysis with the polyclonal antibody. No signal detected in the slot-blot used to measure the hlyK mRNA expression in K. pneumoniae CG43 suggesting an appropriate condition for hlyK mRNA expression is required. In the meantime, a hlyK mutant CG43-S3H was constructed by allelic exchange through homologous recombination, no significant changes of the LD50 of CG43-S3H comparing to that of the wild type CG43-S3 was measured using mouse peritonitis model.
Most virulence factors involved in bacterial pathogenesis are either secretory or cell-surface molecules. We intend to use TnphoA system to isolate the membrane-associated and secretory proteins from K. pneumoniae CG43. In order to remove the endogenous PhoA activity in K. pneumoniae, a phoA gene deletion mutant CG43-S3P1 using gene replacement method was constructed. Interestingly, the truncated phoA in K. pneumoniae still carried an enzyme activity. However, the transformation of E. coli phoA mutant CC188 with the plasmid carrying truncated phoA could not confer the transformants a phosphatase activity. Furthermore, the phosphatase activity remained in the second phoA mutant CG43-S3P2, which has most of the phoA gene deleted, suggesting the presence of a phosphatase in addition to PhoA.

1. 中文摘要....... .........1
2. 英文摘要....... .........2
3. 縮寫表 ..................4
4. 序論........... .........5
5. 實驗材料與方法..........12
6. 結果.. ..................27
7. 討論........... .........35
8. 參考文獻....... .........40
9. 圖表........... .........45
10. 附圖................... 70

沈惠齡,國立交通大學碩士論文,中華民國八十九年,綠膿桿菌尿嘧啶雙磷酸葡萄糖去氫酵素缺損株致病性之探討。
蔡易芊,國立交通大學碩士論文,中華民國八十九年,克雷白氏肺炎桿菌參與致病的基因--鑑定與特性分析。
黃盈蓉,國立交通大學碩士論文,中華民國九十年,克雷白氏菌侵入細胞相關蛋白IalK的功能研究。
賴怡琪,國立清華大學博士論文,中華民國九十一年,克雷白氏肺炎桿菌CG43致病基因相關的搜尋( RmpA2調控夾膜生合成的機轉)。
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