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研究生:蔡忠勤
研究生(外文):TSAI , CHUNG CHIN
論文名稱:登革病毒NS1蛋白在沙門氏菌中的表現及分泌
論文名稱(外文):Secretion of recombinant dengue-2 NS1 protein via the Caf1 protein secretion system in Salmonella enterica serovar Typhimurium aroA
指導教授:劉雨田劉雨田引用關係
指導教授(外文):Y. T . Liu
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
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:59
中文關鍵詞:登革病毒NS1蛋白Caf1蛋白沙門氏菌
外文關鍵詞:Dengue virusNS1 proteinCaf1 proteinSalmonella
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鼠疫桿菌可藉由細胞之pFra質體中的caf1 gene cluster 做出Caf1莢膜蛋白。而Caf1蛋白必須在Caf1 M chaperone/Caf1 A usher分泌系統幫助之下才能分泌至鼠疫桿菌的莢膜上甚至菌體之外。本研究欲探討登革病毒NS1蛋白是否可在E. coli及Salmonella enterica serovar Typhimurium aroA中,藉鼠疫桿菌Caf1蛋白的分泌系統分泌到莢膜上,甚至是菌體之外。結果顯示當NS1蛋白及NS1:Caf1融合蛋白之前接上Caf1 signal peptide時,NS1蛋白及NS1:Caf1融合蛋白皆會被轉運至細菌細胞膜上,且形成與膜相連接的不可溶蛋白。若將NS1:Caf1融合蛋白與Caf1 M chaperon蛋白一起表現,便可於細菌的膜腔( periplasmic space )偵測到可溶性之NS1:Caf1融合蛋白。此結果顯示Caf1 M chaperon蛋白可幫助NS1:Caf1融合蛋白從細菌內膜脫落下來,且會促進NS1:Caf1融合蛋白成為可溶性蛋白。當NS1:Caf1融合蛋白與Caf1 M chaperon蛋白及Caf1 A蛋白一起表現時,可在E. coli及Salmonella enterica serovar Typhimurium aroA的莢膜上,甚至是菌體之外,偵測到NS1:Caf1融合蛋白的存在。綜合以上結果,利用Caf1 A,Caf1 M,Caf1蛋白的分泌系統,我們已成功地可在E. coli及Salmonella enterica serovar Typhimurium aroA細胞中表現登革病毒NS1蛋白至細菌莢膜或細胞外

The Caf1 capsule protein is encoded by the Caf1 gene cluster of Yersinia pestis. It is secreted via the Caf1 M chaperone/Caf1 A usher pathway. We investigated the ability of this secretion system to facilitate secretion of dengue virus type 2 NS1 full-length protein fused to the Caf1 subunit in Escherichia coli and Salmonella enterica serovar Typhimurium aroA. The processed product consisting of mature NS1 and Caf1 remained insoluble in spite of correct processing of this chimeric protein. However, co-expression of this chimera with a functional Caf1 M chaperone led to the accumulation of soluble NS1:Caf1 in the periplasm, which can be released by osmotic shock. Souble NS1:Caf1 reacted with monoclonal antibody directed against structural epitopes of NS1 protein. The result suggest that Caf1 M-induced release of NS1:Caf1 from the plasma membrane promotes folding of the NS1 domain. In this system, gene encoding chimeric protein was created in which the NS1 was sandwiched between the Caf1 signal peptide and the mature Caf1 subunit, leaving the C terminus of the nature of the N-terminal heterologous protein, the Caf1 domain of the chimera remained free to interact with Caf1 M and that this interaction enhanced the solubility of the periplasmic NS1 protein. Following co-expressing of NS1:Caf1 with both Caf1 M chaperone and Caf1 A outer membrane protein. NS1:Caf1 fusion protein could be detected on the cell surface and in the culture broth of E. coli and Salmonella enterica serovar Typhimurium aroA . These results indicate the potential application of the Caf1 protein secretion system in the transport of NS1:Caf1 fusion protein.

目錄
Pages
目錄-----------------------------------------------------------I
表目錄--------------------------------------------------------IV
圖目錄---------------------------------------------------------V
中文摘要-----------------------------------------------------VII
英文摘要----------------------------------------------------VIII
第一章 緒論
一、登革熱之流行現況-------------------------------------------1
二、登革病毒之特性---------------------------------------------1
三、登革病毒之防治---------------------------------------------2
四、鼠疫之流行現況----- ---------------------------------------3
五、鼠疫之防治及疫苗的發展-------------------------------------4
六、鼠疫桿菌Caf 1蛋白之分泌系統--------------------------------4
七、沙門氏菌於口服疫苗之應用-----------------------------------5
八、研究目標---------------------------------------------------6
第二章 材料與方法
壹、實驗材料
一、菌株與質體-------------------------------------------------7
二、實驗所採用之、藥品、試劑、培養基、套組等-------------------7
三、主要儀器---------------------------------------------------8
四、各式培養基與緩衝液的配製---------------------------------- 8
貳、實驗方法
一、菌種的準備及儲存------------------------------------------11
二、建構表現DEN-2 NS1及Yersinia pestis Caf1融合蛋白之質體--- -11
三、蛋白質表現之西方墨點分析----------------------------------20
四、NS1及NS1-F1融合蛋白Salmonella enterica erovar Typhimurium
aroA疫苗株中之表現--------------------------------------------26
第三章 結果
DEN-2 NS1與Yersinia pestis Caf 1之重組質體在E. coli JM109 及Salmonella enterica serovar Typhimurium aroA中NS1及 NS1:Caf 1融合蛋白之表現及其分佈位置----------------------------------- 27
第四章 討論-----------------------------------------------31
第五章 結論-----------------------------------------------35
參考資料---------------------------------------------------- 52
附錄---------------------------------------------------------59
表目錄
Pages
表一:本研究所使用之核酸引子及寡核甘酸 -----------------------36
表二:本研究使用之菌株及質體----------------------------------37
圖目錄
Pages
圖 一:登革病毒之基因圖譜-------------------------------------38
圖 二:以PCR方法合成之DNA片段---------------------------------39
圖 三: Spacer之設計------------------------------------------40
圖 四:建構之重組質體-----------------------------------------41
圖 五:E. coli中表現NS1及NS1:Caf 1融合蛋白的Western blot分析--42
圖 六:Caf 1 signal peptide 對NS1及NS1:Caf1融合蛋白運送作用的
影響----------------------------------------------------------43
圖 七:Caf1 M chaperon 蛋白對NS1:Caf1融合蛋白folding之影響----44
圖 八:含不同重組質體E. coli JM109之免疫螢光染色分析----------45
圖 九:免疫電鏡圖---------------------------------------------46
圖 十:Caf1 A對NS1及NS1:Caf1 融合蛋白分泌的影響---------------47
圖 十一:Salmonella enterica serovar Typhimurium aroA /p18AMSNF1 的免疫螢光染色分析---------------------------48
圖 十二:NS1:Caf 1融合蛋白在Salmonella enterica serovar Typhimurium aroA中的表現和分泌--- ----------------------------49
圖 十三:NS1:Caf 1 融合蛋白之聚合(polymerization)作用- -------50
圖 十四:NS1:Caf1融合蛋白之分泌機轉假想圖---------------------51

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