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系統識別號 U0026-0812200911585751
論文名稱(中文) 鏈球菌熱原性外毒素B及其交互蛋白S-腺同半胱胺酸水解酶和角質蛋白14之功能研究
論文名稱(英文) Functional studies of streptococcal pyrogenic exotoxin B and its associated proteins S-adenosylhomocysteine hydrolase and keratin 14
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 94
學期 2
出版年 95
研究生(中文) 許茹斐
研究生(英文) Ju-Fei Hsu
學號 t3693105
學位類別 碩士
語文別 中文
論文頁數 78頁
口試委員 口試委員-吳俊忠
口試委員-莊偉哲
口試委員-劉清泉
指導教授-黃溫雅
中文關鍵字 酵母菌雙雜交  鏈球菌熱原性外毒素B 
英文關鍵字 yeast two-hybrid  streptococcal pyrogenic exotoxin B 
學科別分類
中文摘要 化膿性鏈球菌(A群鏈球菌,GAS)為一重要的人類病原菌,會引起許多人類的疾病。例如咽炎,膿痂疹,肌炎。A群鏈球菌也會造成致死率極高的鏈球菌毒性休克症後群。但是到目前為止他們的致病因子,還不甚了解。先前的研究顯示嚴重感染A型鏈球菌感染的病人可能帶有其SPE A、C或F的基因,但是無法與任何特殊的臨床症狀產生其相關性,反而從病人分離出來的菌株常帶有高量蛋白酶的活性,且與鏈球菌毒性休克症後群的臨床症狀及死亡率有關。熱原性外毒素B (SPE B) 的基因存在於所有A型鏈球菌的菌株中,且其所代表的蛋白質功能上類似一個半胱氨酸蛋白酶(cysteine protease)。為了研究SPE B在A型鏈球菌感染的過程當中所扮演的角色,所以我們利用酵母菌雙雜交法去篩選跟SPE B有交互作用的蛋白,進一步釐清這些蛋白在功能上的變化。從酵母菌雙雜交法當中,我們篩選到S-腺同半胱胺酸水解酶會與SPE B進行交互作用,此水解酶在核甘酸代謝當中扮演重要的角色。利用A群鏈球菌NZ131(野生型)和SW510(spe B 突變型)去感染人類腎臟纖維組織母細胞293T細胞,結果顯示在感染A群鏈球菌NZ131(野生型),S-腺同半胱胺酸水解酶的表現量明顯下降,而在SW510(spe B 突變型)與控制組之間則沒有差異。S-腺同半胱胺酸水解酶會水解S-腺同半胱胺酸形成半胱胺酸和腺酸。利用酵素免疫法,於感染A群鏈球菌NZ131(野生型)的老鼠血漿中,發現半胱胺酸的含量有明顯下降,而在SW510(spe B 突變型)與控制組之間則沒有差異。利用串連式質譜儀,在老鼠感染A群鏈球菌NZ131(野生型)的全血當中,發現甲硫胺酸的含量有明顯上升,而在SW510(spe B 突變型)跟控制組之間則沒有差異。也發現S-腺同半胱胺酸水解酶是SPE B的受質,而且沒有辨識特定的切點,呈現快速降解。根據以上的結果,發現SPE B會跟S-腺同半胱胺酸水解酶交互作用,進一步使得上游的甲硫胺酸大量累積,而造成高甲硫胺酸血症。SPE B有一個新的病理機制即藉由抑制S-腺同半胱胺酸水解酶的活性在A群鏈球菌感染當中扮演一個重要的角色。我們也發現角質蛋白14會跟SPE B交互作用,角質蛋白14為角質細胞中一個特別的標誌。利用A群鏈球菌A20 (野生型)和SW507(spe B 突變型)去感染人類角質細胞HaCaT細胞,結果顯示在感染A群鏈球菌A20 (野生型), 角質蛋白14的表現量有明顯下降,而在SW507(spe B 突變型)跟控制組之間則沒有差異。同時也發現角質蛋白14也是SPE B的受質。根據以上的結果,我們推測,被A群鏈球菌感染後所造成的一些皮膚上的病理特徵,可能是藉由SPE B跟角質蛋白14交互作用所導致。總結,SPE B會幫助A群鏈球菌進入宿主細胞裡面,造成嚴重的疾病,並且在這病理機轉上扮演一個重要的角色。



英文摘要 Streptococcus pyogenes is a common pathogen for pharyngitis, impetigo and myositis. It also causes severe streptococcal toxic shock syndrome (STSS), which is lethal in humans. GAS secrets a number of exotoxins, among which the streptococcal pyrogenic exotoxin B (SPE B) is highly associated with the disease severities. SPE B is a cysteine protease and matures itself by autocalalysis. To study the in vivo function of SPE B, we screened for the proteins that interact with it by yeast two-hybrid assays. We found that S-adenosylhomocysteine hydrolase (AdoHcyase), an essential factor for nucleotide metabolism, is associated with SPE B. Protein level of AdoHcyase was reduced in the human kidney fibroblast 293T cells infected with S. pyogenes NZ131 strain (wild-type); whereas they were not reduced in the cells infected with SW510 strain (spe B mutant). AdoHcyase has been shown to cleave the AdoHcy into the products of adenosine and homocysteine. By enzyme immunoassay, we found that the homocysteine level was significantly reduced in plasma of mice infected with NZ131, but not with SW510. By tandem mass spectrometry, we found that methionine was accumulated in blood of mice infected with NZ131, but not with SW510. We also found that AdoHcyase was a substrate for SPE B cysteine protease and SPE B could not cleave at specific sites. Based on our results, we indicated that SPE B induced host hyper-methioninemia through interaction with AdoHcyase. The SPE B has a novel pathogenic mechanism by group A streptococcal infection through suppressing AdoHcyase activity. We also found that SPE B was associated with keratin 14, a tissue-specific marker for keratinocytes. Protein level of keratin 14 was reduced in the keratinocyte HaCaT cells infected with S. pyogenes A20 strain (wild-type); whereas they were not reduced in the cells infected with SW507 strain (spe B mutant). By in vitro cleavage assay, we also found that keratin 14 appears to be a substrate of SPE B enzyme. Based on our results, we suggest that skin pathogenesis caused by S. pyogenes infection was mediated through keratin 14. Future work will focus on studies of functional relationships of SPE B with keratin 14 in mouse models. Thus, SPE B increases the ability of S. pyogenes to cause severe invasive disease and therefore is an important virulence factor for this organism.



論文目次 目錄
中文摘要---------------------------------------------------------------------Ⅰ
英文摘要---------------------------------------------------------------------Ⅱ
誌謝-------------------------------------------------------------------------Ⅲ
目錄-------------------------------------------------------------------------Ⅳ
表目錄-----------------------------------------------------------------------Ⅶ
圖目錄-----------------------------------------------------------------------Ⅶ
附錄-------------------------------------------------------------------------Ⅷ
縮 寫 檢 索 表---------------------------------------------------------------Ⅸ

第一章 緒論 ----------------------------------------------------------------1
1.1 化膿性鏈球菌之介紹--------------------------------------------------------1
1.2 熱原性鏈球菌釋放免疫調節相關酵素之介紹----------------------------------- 4
1.3 熱原性鏈球菌外毒素B (SPE B)之介紹-----------------------------------------7
1.4 研究動機-----------------------------------------------------------------10
1.5 酵母菌雙雜交法 (Yeast two-hybrid) 的原理及應用---------------------------13
1.6 S-腺同半胱胺酸水解酶和角質蛋白14之簡介-----------------------------------15
1.7 研究內容之介紹-----------------------------------------------------------18

第二章 材料與方法
2.1 酵母菌雙雜交試驗---------------------------------------------------------17
2.1.1 Clone製備--------------------------------------------------------------20
2.1.2 大腸桿菌的質體轉化-----------------------------------------------------20
2.1.3 小量質體抽取-----------------------------------------------------------21
2.1.4 酵母菌的質體轉化------------------------------------------------------ 22
2.1.5 酵母菌蛋白質的萃取與定量---------------------------------------------- 23
2.1.6 西方墨點-------------------------------------------------------------- 23
2.1.7 b-galactosidase濾紙試驗----------------------------------------------- 25
2.1.8 玻璃磁珠快速萃取酵母菌核酸法-------------------------------------------26
2.1.9 大腸桿菌的質體轉化-----------------------------------------------------26
2.1.10 限制酶分群----------------------------------------------------------- 27
2.2 利用親和性管柱篩選SPE B之交互蛋白--------------------------------------- 27
2.3 S-腺同半胱胺酸水解酶蛋白表現與純化---------------------------------------28
2.3.1 clone的製備------------------------------------------------------------28
2.3.2 宿主種類,基因種類,載體,生長培養基和試劑-----------------------------28
2.3.3 S-腺同半胱胺酸水解酶蛋白表現與純化------------------------------------ 29
2.4 熱原性鏈球菌外毒素B對於S-腺同半胱胺酸水解酶,角質14重組蛋白之活性測定----30
2.5 S-腺同半胱胺酸水解酶活性測定---------------------------------------------31
2.6 類胱胺酸的測定方法-------------------------------------------------------32
2.7 甲硫胺酸的測量方法-------------------------------------------------------32
2.8 細胞培養方法------------------------------------------------------------ 33
2.9 免疫螢光染色-------------------------------------------------------------33
2.10 萃取細胞之蛋白質--------------------------------------------------------34
2.11 化膿性鏈球菌之細胞感染模式----------------------------------------------34
2.12 化膿性鏈球菌之小鼠感染模式 ---------------------------------------------35

第三章 實驗結果
3.1 酵母菌雙雜交法篩選跟熱原性鏈球菌外毒素有交互作用的蛋白-------------------36
3.2 熱原性鏈球菌外毒素B和S-腺同半胱胺酸水解酶的交互作------------------------37
3.3 熱原性鏈球菌外毒素B造成S-腺同半胱胺酸水解酶蛋白表現和活性下降------------38
3.4 熱原性鏈球菌外毒素B降低類胱胺酸的含量以及造成甲硫胺酸的累積--------------39
3.5 熱原性鏈球菌外毒素B切割S-腺同半胱胺酸水解酶------------------------------40
3.6 熱原性鏈球菌外毒素B與角質蛋白14的交互作用--------------------------------42
3.7 熱原性鏈球菌外毒素B造成角質蛋白14蛋白表現量下降--------------------------42
3.8 熱原性鏈球菌外毒素B造成細胞骨架不完整------------------------------------43
3.9 熱原性鏈球菌外毒素B切割角質蛋白14----------------------------------------45

第四章 結論與討論------------------------------------------------------------47
參考文獻---------------------------------------------------------------------52

表目錄
表1 酵母菌雙雜交法篩選出的結果與基因分群-------------------------------------58
表2 酵母菌雙雜交法篩選出會與熱原性鏈球菌外毒素B結合的蛋白--------------------59

圖目錄
圖1 酵母菌雙雜交法的原理-----------------------------------------------------60
圖2 AH109 酵母菌-------------------------------------------------------------61
圖3 酵母菌雙雜交法的流程-----------------------------------------------------62
圖4 酵母菌雙雜交法篩選過程的結果---------------------------------------------63
圖5 熱原性鏈球菌外毒素B與S-腺同半胱胺酸水解酶蛋白與交互作用------------------64
圖6 熱原性鏈球菌外毒素B影響S-腺同半胱胺酸水解酶蛋白活性和蛋白表現量----------65
圖7 熱原性鏈球菌外毒素B影響胱胺酸和甲硫胺酸----------------------------------66
圖8 S-腺同半胱胺酸水解酶為熱原性鏈球菌外毒素B的受質--------------------------67
圖9 熱原性鏈球菌外毒素B與角質蛋白14交互作用----------------------------------68
圖10 熱原性鏈球菌外毒素B影響角質蛋白14蛋白表現量-----------------------------69
圖11 熱原性鏈球菌外毒素B造成細胞骨架不完整S ---------------------------------70
圖12 熱原性鏈球菌外毒素B引起嚴重的發炎反應和組織壞死(air pouch)--------------71
圖13 熱原性鏈球菌外毒素B引起嚴重的發炎反應和組織壞死(subcutaneous)-----------72
圖14 角質蛋白14為熱原性鏈球菌外毒素B的受質-----------------------------------73
圖15 S-腺同半胱胺酸水解酶和角質蛋白14 與化膿性鏈球菌致病機轉之相關性-------- 74

附錄
附錄1 載體圖譜 --------------------------------------------------------------75
附錄2 S-adenosylmethionine-dependent transmethylation reactions--------------76
附錄3 角質蛋白表現的位置和疾病的相關性---------------------------------------77
附錄4 宿主種類,基因型和載體;HPLC的條件-------------------------------------78
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