臺灣博碩士論文加值系統

嗜水性產氣單胞桿菌Aeromonas hydrophila是一種革蘭氏陰性之兼性厭氣性菌，廣泛地生長於各類水域之中。根據過去的文獻或臨床病例報告，嗜水性產氣單胞桿菌對人類及多種動物具有致病能力。在許多致病因子當中除了較廣為人知的溶血素 (haemolysin) 外，蛋白酶亦扮演著相當重要的角色，包括了會破壞宿主組織蛋白質、改變宿主免疫系統、修飾其他致病毒素及幫助菌株抵抗環境壓力等。PrtS1為本研究室先前自嗜水性產氣單胞桿菌臨床株CKH-29中所選殖的絲胺酸蛋白酶基因，其可轉譯出453個胺基酸，並且可以在E. coli中表現出蛋白酶活性。為了解prtS1基因是否也普遍存在於同類菌株中，本研究篩選了30株A. hydrophila環境菌株。針對prtS1基因序列製作引子並以各菌株染色體作為模板進行PCR放大反應，結果發現絕大多數的環境菌株都含有相同的prtS1基因片段。
為了進一步了解prtS1在A. hydrophila CKH-29的表現，本研究利用部分prtS1的結構基因，及含有His-tag序列之表現載體pQE-30來建構生產免疫用抗原的重組質體pPrtS16。以親和層析管柱Ni-NTA純化此重組蛋白，並將所純化出的蛋白質免疫Balb/c小鼠以產生PrtS1多株抗體。利用得到之多株抗體以免疫點漬法分析PrtS1在A. hydrophlia CKH-29的表現情形。結果發現PrtS1會受到幫助氧化作用之二價鐵離子、蔗糖造成的高滲透壓、44℃高溫的誘導而增加其表現量。而且可於菌株培養之對數期分泌到胞膜間隙部位，當接近定常期時就無法於此部位觀察到PrtS1的存在，在胞內部分則是當培養時間進入後定常期時PrtS1的表現會明顯減少。根據prtS1基因及其上游DNA序列之比對加以PrtS1於免疫分析之結果，推測PrtS1在A. hydrophila CKH-29中之功用甚至於與致病性之關係應類似於E. coli及病原菌Salmonella typhimurium、Yersinia enterocolitica等之stress response protease HtrA。

Aeromonas hydrophila is a gram negative, facultatively anaerobic freshwater bacterium. In according to many literatures and clinical reports, the strains are pathogens of humans and animals. In many virulent factors, proteases play key roles among them including high level proteolysis of host tissue, modulation of host immune system, adapting to environmental stresses and modification of other toxins. During previous study, we have cloned and sequenced a serine protease gene prtS1 from clinical strain A. hydrophila CKH-29, which encoding 453 a.a. and has protease activity in recombinant E. coli. In order to find out if prtS1 distributes among A. hydrophila strains, we had isolated 30 environmental bacteria, and then identified by VITEK instrument. After analysis of PCR, the same fragment of prtS1 was amplified from most environmental strains’ genomic DNA. It’s suggested that prtS1 is a common gene among A. hydrophila.
To understand the expression of prtS1 in A. hydrophila CKH-29, we used restriction enzyme RsaI, PstI to generate a fragment of prtS1 structure gene and cloned it into the expression vector, pQE-30 and named this plasmid pPrtS16. After purification of this recombinant protein with affinity, ion-exchange and gel filtration chromatography, it’s injected into Balb/c mouse to produce polyclone anti-serum, and then applied it to the immunoblotting. The results of immunoassay indicated that PrtS1 could be induced by Fe2+, high concentration sucrose and high temperature (44℃). Additionally, PrtS1 was translocated to periplasmic fraction in log phase, and expressed in cytoplasma during incubation except late stationary phase. To summarize, PrtS1, which is similar with E. coli, Salmonella typhimurium and Yersinia enterocolitica HtrA protease, may protectively response environmental stresses from infection.

壹、前言 1
一、Aeromonas hydrophila之生理性質 1
(1) 分類 1
(2) 生長環境 1二、Aeromonas hydrophila的致病性 3
三、Aeromonas hydrophila的致病機制 4
(1) 菌體的附著 5
A. Surface adhesins/lectins 5
B. Fimbriae (pili) 5
(2) 細胞表層構造 5
A. OMP (outer membrane protein) 6
B. S-Layer 6
C. LPS (lipopolysaccharide endotoxin) 6
(3) 外泌性因子 6
A. Haemolysin (aerolysin) 7
B. GCAT(glycerophospholipid:cholesterol
acyltransferase) 7
C. Protease 9
D. Enterotoxin 10
四、蛋白酶的致病機制 10
(1) 直接或間接地破壞宿主組織蛋白質 11
(2) 引發Kallikrein-kinin cascade 11
(3) 改變宿主免疫系統、降低宿主防禦能力 13
(4) 修飾、活化其他毒素 13
(5) 幫助菌體在不良環境下生長 14
五、研究動機與目的 14
貳、研究材料 16
一、菌株 16
二、質體 16
三、培養基 16
(1) LB medium 16
(2) LA medium 20
(3) NB medium 20
(4) NA medium 20
(5) T.C.B.S. Cholera medium 20
(6) SA medium 20
(7) MacConkey medium 20
(8) AH medium 20
(9) LA + 2 % skim milk 21
四、藥品 21
五、儀器 21
參、研究方法 23
一、菌種篩選與鑑定 23
(1) 篩選培養基 23
(2) 生化測試 23
(3) 菌種鑑定 23
二、DNA操作 23
(1) 小規模抽取染色體 23
(2) 小規模質體製備 25
(3) 限制酶的作用 26
(4) DNA鏈5'端磷酸基之去除 26
(5) 製作平端切點DNA 27
(6) 接合反應 27
(7) 受容細胞(competent cells)的製作 28
(8) 細胞轉形 28
(9) 質體快速檢驗法 29
(10) DNA片段的回收 30
(11) DNA電泳 30
(12) PCR放大反應 31
(13) DNA序列分析 32
三、菌株培養 32
(1) A. hydrophila CKH-29生長曲線之操作 32
(2) 重組蛋白質之誘導表現 33
四、蛋白質操作 33
(1) 粗蛋白質的製備 33
(2) 各細胞分劃蛋白質之分離 (Osmotic shock法) 34
(3) 蛋白質定量 34
(4) Azocasein分解活性(caseinase標準活性測試)測定 35
五、蛋白酶的純化 35
(1) 親和管柱層析法 36
(2) 離子交換層析 36
(3) 膠體過濾層析 37
六、蛋白質電泳 37
(1) SDS-PAGE 37
(2) Native-PAGE 40
七、免疫點漬法 41
八、小鼠免疫 43
九、胺基酸序列分析方法 44
肆、結果與討論 45
一、嗜水性產氣單胞桿菌環境菌株之篩選與PCR放大分析 45
(1) 環境菌株之篩選 45
(2) PCR放大反應 46
二、PrtS1多株抗體之製備 53
(1) 表現質體之構築 53
(2) PrtS16之表現 54
(3) PrtS16之純化 57
(4) 小鼠免疫 65
(5) 免疫點漬法分析PrtS16之修飾或降解產物 65
三、Aeromonas hydrophila CKH-29中PrtS1之表現 67
(1) prtS1啟動子之預測與比較 67
(2) PrtS1之誘導表現 69
(3) PrtS1之分泌 70
(4) PrtS1之角色定位及其與致病性之關係 78
四、PrtS1 PDZ domain之分析 81
(1) PDZ domain之預測 81
(2) PDZ domain之分析 84
伍、結論 87
陸、參考文獻 90

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