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系統識別號 U0026-3107201314154600
論文名稱(中文) A群鏈球菌中氧化壓力調控因子PerR如何調控sda1基因
論文名稱(英文) How peroxide stress response regulator regulates sda1 in group A streptococcus
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 101
學期 2
出版年 102
研究生(中文) 王志宏
研究生(英文) Chih-Hung Wang
學號 T36001065
學位類別 碩士
語文別 中文
論文頁數 59頁
口試委員 指導教授-吳俊忠
召集委員-何漣漪
口試委員-鄧景浩
口試委員-江倪全
中文關鍵字 化膿性鏈球菌  A群鏈球菌  氧化壓力調控因子  PerR  sda1 
英文關鍵字 Streptococcus pyogenes  group A streptococcus  peroxide stress response regulator  PerR  sda1 
學科別分類
中文摘要 化膿性鏈球菌 (Streptococcus pyogenes) 又稱為A群鏈球菌 (group A streptococcus) 是一種人類致病菌,可造成輕微的咽喉炎到嚴重的鏈球菌毒素休克症候群等多樣化疾病。在A群鏈球菌中,Peroxide stress response regulator (PerR) 為fur家族成員之一,同時也扮演維持金屬離子平衡及適應氧化壓力之調控子的角色。先前研究指出在動物感染模式中,perR突變株的毒力表現比野生株弱,顯示PerR可能調控某些適應環境相關的毒力因子。過去我們的團隊以Real-time PCR分析野生株與perR突變株在一般培養環境與氧化壓力環境下DNase基因 (sda1, mf3, spd) 的表現,發現野生株在氧化壓力下sda1基因表現量會增加,但perR突變株則不會。因此本論文進一步探討PerR是如何調控細菌噬菌體DNase基因-sda1。首先,我們以cDNA五端快速放大法找到sda1轉錄位點在轉譯位點上游第26個核苷酸位置。以回補野生型perR基因到perR突變株後,發現在氧化壓力環境下可以顯著地提升sda1基因表現,但回補鐵離子調控區突變的PerR H99A,則無;顯示PerR的金屬結合位點對於sda1的調控相當重要。此外,在過氧化氫刺激下,野生株sda1啟動子活性會被提升,但perR突變株則不會,這結果再次佐證當A群鏈球菌處在氧化壓力的環境下PerR可以調控sda1轉錄層次的表現。我們以電泳位移試驗進一步證實PerR能直接與sad1啟動子結合,且具專一性。同時在sda1啟動子-144~ -174與-22~-36找到兩個可能的PerR結合位。這些結果顯示A群鏈球菌在受到氧化壓力的刺激後,PerR具有正向調控sda1表現的能力。此研究對於A群鏈球菌PerR在氧化壓力下調控毒力因子提供重要的證據。
英文摘要 Streptococcus pyogenes (group A streptococcus) is a human pathogen, which can cause various diseases from mild pharyngitis to severe streptococcal toxic shock syndrome. Peroxide stress response regulator (PerR) is the member of ferric uptake repressor (fur) super-family, and is the transcriptional regulator for metal homeostasis and oxidative stress response in S. pyogenes. Previous studies showed that the virulence of perR mutant was attenuated than the wild-type in animal in ection models, and it suggested PerR may regulate virulence factors to adapt the environmental changes. Moreover, our lab analyzed DNase genes (sda1, mf3, spd) expression in the wild-type strain and the perR mutant under normal culture conditions and oxidative stress conditions by Real-time PCR, and found that sda1 expression in the wild-type strain was increased under oxidative stress, but perR mutant did not. The aim of this thesis was to demonstrate how PerR regulate bacteriophage-encoded gene, sda1. First, the rapid amplification of 5’ cDNA ends assay demonstrated that the transcriptional start site of sda1 was located at the 26 nucledtides upstream of the translational start site. PerR and PerR H99A mutation, the Fe2+ binding site of PerR, were complemented in the perR mutant to observe the sda1 expression. The results showed that the sda1 expression was increased under hydroxy peroxide treatment in complemented PerR but not in complemented PerR H99A. These results suggested that the metal binding site was important for PerR to regulate sda1. In addition, the sda1 promoter ability was increased under oxidative stress in the wild-type stain but not in the perR mutant. Further, we found PerR can specifically bind to the sda1 promoter by the electrophoretic mobility shift assay. The two Per boxes were found at sda1 promoter -144~ -174 and -22~-36 regions. Our data suggested that PerR can positively regulate sda1 under oxidative stress conditions. This study provides evidence to support the PerR regulates the virulence factors under oxidative stress in group A streptococcus.
論文目次 目錄
中文摘要 I
Abstract II
致謝 IV
目錄 V
表目錄 IX
圖目錄 X
符號與縮寫 XI
緒論 1
一、 A群鏈球菌背景介紹 1
I A群鏈球菌所引起的疾病 1
II 流行病學調查 2
二、 A群鏈球菌的致病機轉與毒力因子 3
I 黏附與聚集 3
II 毒力因子 3
三、 A群鏈球菌對於氧化壓力的調控 6
I 氧化壓力 6
II 氧化壓力調控因子:peroxide response regulator (PerR)7
四、 研究動機 9
材料與方法 10
一、 菌種、質體與實驗動物來源 10
二、 藥品與溶液配方 10
三、 菌種培養與保存 10
四、 DNA實驗操作 10
I DNA萃取 10
I.a 大腸桿菌質體DNA萃取 10
I.b A群鏈球菌質體DNA萃取 11
I.c A群鏈球菌染色體DNA萃取 11
II 分子選殖技術 (molecular cloning) 12
II.a 限制酶切割及DNA 接合作用 12
II.b 大腸桿菌勝任細胞的製作 (全程置於冰上操作) 12
II.c 大腸桿菌轉型作用 (Transformation) 12
II.d A群鏈球菌勝任細胞製備 13
II.e A群鏈球菌電穿孔轉型作用 (Electroporation) 13
II.f 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 13
II.g 定點突變 (Site-directed mutagenesis) 14
五、 RNA實驗操作 14
I A群鏈球菌RNA萃取 (for Northern blot) 14
II 北方墨點法 (Northern blot) 15
III A群鏈球菌RNA萃取 (for Real-Time PCR) 16
IV Real-time PCR 17
V cDNA五端快速放大法 (Rapid amplification of 5’ cDNA ends) 17
六、 蛋白質實驗操作 18
I 蛋白質濃度定量 18
II 蛋白質膠體電泳 (SDS-PAGE) 19
III 西方點墨法 19
IV 重組蛋白質製作 19
IV.a 誘導重組蛋白質載體 20
IV.b 純化重組蛋白質 (全程4℃或冰上操作) 20
V 小鼠多株抗體備製 20
VI 酵素免疫分析法 (Enzyme-Linked Immunosorbent Assay, ELISA) 21
VII 電泳遷移試驗 (Electrophoretic mobility shift assay, EMSA) 21
VII.a EMSA 21
VII.b EMSA for competition 22
結果 23
一、 在氧化壓力的環境PerR 能調控sda1表現 23
二、 PerR金屬結合位點H99在調控sda1具有重要角色 23
三、 了解sda1轉錄位點與啟動子區域 24
四、 野生株受到氧化壓力刺激後會提升sda1啟動子活性 24
I 建構啟動子報導系統 24
II 定量cat報導基因mRNA表現量 25
五、 PerR重組蛋白質與sda1啟動子結合 25
I PerR重組蛋白質會和sda1啟動子結合 25
II PerR重組蛋白質與sda1啟動子結合具有專一性 26
六、 探討不同片段的sda1啟動子與重組蛋白質PerR結合能力 26
七、 純化Sda1 H188R重組蛋白質並製備anti-Sda1 H188R多株抗體 27
八、 測定anti-Sda1 H188R多株抗體效價 28
九、 在野生株A20中構築perR與sda1雙基因突變菌株 28
討論 30
二、 PerR調控sda1在A群鏈球菌中扮演的角色 30
I PerR在轉錄層次調控sda1表現 30
II PerR對於Sda1蛋白質的調控 31
三、 PerR在sda1啟動子的結合位 32
四、 A群鏈球菌中PerR調控sda1可能的致病機轉 32
五、 總結 34
參考文獻 35
圖表 41
附錄I 54
附錄II 55

表目錄
Table 1. Bacterial strains used in this study. 41
Table 2. Plasmids used in this study. 42
Table 3. Primers used in this study. 43

圖目錄
Figure 1. H2O2刺激野生株A20與perR突變株 (SW612) 後sda1 mRNA表現量 44
Figure 2. 分析A群鏈球菌中PerR的金屬結合位點H99對於調控sda1表現的重要性 45
Figure 3. cDNA五端快速放大法識別sda1轉錄位點流程圖 46
Figure 4. sda1基因的轉錄位點及可能的Per box序列 47
Figure 5. PerR於氧化壓力下調控sda1啟動子活性 48
Figure 6. PerR重組蛋白質與sda1啟動子結合能力之分析 49
Figure 7. 不同片段sda1啟動子與重組蛋白質PerR之結合能力 50
Figure 8. 純化Sda1 H188R重組蛋白 51
Figure 9. 不同小鼠打入重組蛋白質Sda1 H188R所產生的多株抗體效價 52
Figure 10. 構築A20 perR與sda1雙基因突變株 53
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