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研究生:林政賢
研究生(外文):Cheng-Shien Lin
論文名稱:創傷弧菌中引發巨噬細胞死亡的毒殺基因之研究
論文名稱(外文):Study of the genes required for macrophage cytotoxicity from Vibrio vulnificus
指導教授:陳昱仲陳昱仲引用關係
指導教授(外文):Yu-Chung Chen
學位類別:碩士
校院名稱:嘉南藥理科技大學
系所名稱:生物科技系暨研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:65
中文關鍵詞:創傷弧菌巨噬細胞跳躍子細胞毒殺
外文關鍵詞:Vibrio vulnificusmacrophagetransposoncytotoxicity
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創傷弧菌是一種嗜鹽性的革蘭氏陰性菌,普遍存在於海洋、河口交界處,亦稱海洋弧菌。此菌為人體致病菌,當人體受到感染時,常常引發兩種的臨床病症分別是原發性敗血症和傷口感染,其中原發性敗血症的病患往往在到院後的48小時內死亡,致死率高達50%。然而,創傷弧菌致病機制和致病因子,至今日仍尚未清楚,但目前研究報告證實的致病因子有莢膜、磷脂酶、細胞溶解毒素、金屬蛋白酶及RTX毒素等。
巨噬細胞是身體防禦外來病原體侵略的重要成員之一,也是人體先天性免疫的第一道防線,然而,當人體遭受創傷弧菌感染而造成嚴重的病症,其主要的原因是由於創傷弧菌會毒殺巨噬細胞,導致人體免疫系統的功能降低,造成人體急劇死亡。因此,本論文目的是要選殖出創傷弧菌對巨噬細胞的毒殺基因,進而分析該毒殺基因的特性及其與其他基因的相關聯性,並提供往後探討創傷弧菌與人體免疫統系統之間關聯性的依據。
為了要選殖創傷弧菌對巨噬細胞的毒殺基因,我們以顯微鏡和細胞毒性分析,由867株創傷弧菌跳躍子 (transposon5, Tn5) 突變株中篩選出7株 (29D2、1E5、35F11、34C7、47C9、45E8及3F1) 對巨噬細胞毒殺能力下降的跳躍子突變株,其中5株突變株 (29D2、1E5、35F11、34C7及47C9),經由基因的選殖與序列比對的分析,得知對巨噬細胞具有毒殺能力之毒殺基因是rtxA、rtxE、argD及VV3093。rtxA的核苷酸序列長度為15,621 bp,RtxA由5,206個氨基酸構成,其分子大小為~572 kDa,是一種cytotoxic toxin,屬於RTX家族的一員,而rtxE的核苷酸序列長度為2,169 bp,RtxE 由722個氨基酸組成,其分子大小為~79 kDa,RtxE的功能為ABC轉運蛋白 (ABC transporter),與RtxB和 RtxD共同形成ABC transporter,是屬於第一型蛋白質分泌系統,幫助毒殺蛋白RtxA由菌體內運送至胞外,造成對巨噬細胞的毒殺作用,argD的核苷酸序列長度為1,212 bp,ArgD由403個氨基酸構成的蛋白質,其分子大小為~44 kDa,是一個與尿素循環及胺基酸代謝 (urea cycle and metabolison of amino acid) 有關的基因,VV3093的核苷酸序列長度為636 bp,由211個氨基酸組成的,其分子大小為~23 kDa,其功能為一種轉錄作用的調節子 (transcriptional regulator)。
綜合上述結果,我們由創傷弧菌跳躍子突變株中,共篩選出7株對巨噬細胞毒殺能力下降的突變株,經由基因選殖與序列比對分析,顯示這些毒殺巨噬細胞,分別是RTX家族的毒殺蛋白RtxA和負責轉運毒殺蛋白RtxA的RtxE、尿素循環及胺基酸代謝有關的ArgD及轉錄作用調節子的VV3093。
Vibrio vulnificus a halophilic gram-negative marine bacterium that is survied in ocean, estuarine water. V. vulnificus causes both fatal primary septicemia and wound infection in human, mortality was up to 50% in septic patients, with most of them dying within 48 h. Recently, Several putatie virulence factors of V. vulnificus are demonstrated, such as capsule, metalloprotease, phospholipase, cytolysin, iron acquisition systems and RTX toxin. To identify the genes required for killing of macrophage, we screened an insertional mutant library of V. vulnificus generated by transposon mutagenesis for reduced cytotoxicity toward macrophage. Here we reported that among 867 Tn5 mutants screened, 7 Tn5 mutants exhibited decreased cytotoxic activity toward marcrophage. Four genes inserted by transposon were cloned and sequenced. Sequence analysis revealed that the transposon was inserted into the genes : rtxA, rtxE, argD and VV3039. RtxA was composed of 5,206 amino acids with a theoretical molecular mass of ~572 kDa and is a member of RTX family. RtxE was composed of 722 amino acids with a theoretical molecular mass of ~79 kDa. The RtxE is an ABC transporter (ATP-binding cassette transporter) that belongs to a type I secretion system. ArgD was composed of 403 amino acids with a theoretical molecular mass of ~44 kDa and was involved in an urea cycle pathway and amino acid metabolism. VV3093 was composed of 636 amino acids with a theoretical molecular mass of ~23 kDa and probably was a transcription regulator. In this study, we cloned and sequenced four genes: rtxA, rtxE, argD and VV3093 and these genes probably were involved in cytotoxicity towards macrophages.
中文摘要 .............................................. I
英文摘要 .............................................. III
本文目錄 .............................................. V
圖目錄 .............................................. IX
表目錄 .............................................. X
附錄目錄 .............................................. XI
縮寫表 .............................................. XII
第一章 緒論 .......................................... 1
1.1 創傷弧菌 ......................................... 1
1.2 創傷弧菌毒力因子 ................................. 4
1.3 創傷弧菌跳躍子突變株的建構 ....................... 8
1.4 研究動機 ......................................... 9
第二章 材料與方法 .................................... 11
2.1 材料與藥品 ....................................... 11
2.1.1 菌株與質體 ..................................... 11
2.1.2 細胞 ........................................... 11
2.1.3 培養基 ......................................... 11
2.1.3.1 細菌培養基 ................................... 11
2.1.3.2 細胞培養基 ................................... 12
2.1.4 抗生素 ......................................... 13
2.1.5 溶液 .......................................... 13
2.1.6 DNA標準品 ...................................... 15
2.1.7 酵素 ........................................... 16
2.1.8 套組 ........................................... 16
2.2 實驗方法 ......................................... 17
2.2.1 菌株培養技術 ................................... 17
2.2.1.1 菌株的培養條件 ............................... 17
2.2.1.2 菌株的保存 ................................... 17
2.2.1.3 跳躍子突變株於96孔盤的保存 ................... 17
2.2.1.4 細菌生長曲線之測定 ........................... 18
2.2.2 細胞培養技術 ................................... 18
2.2.2.1 冷凍細胞之解凍活化 ........................... 18
2.2.2.2 細胞的保存方法 ............................... 18
2.2.2.3 細胞的繼代培養 ............................... 19
2.2.2.4 細胞計數的方法 ............................... 19
2.2.2.5 細胞培養於96孔培養盤的方法 ................... 19
2.2.2.6 細胞毒性分析 ................................. 20
2.2.3 基本的分生技術 ................................. 21
2.2.3.1 套組純化質體DNA .............................. 21
2.2.3.2 鹼裂解法純化質體DNA .......................... 22
2.2.3.3 套組純化細菌染色體DNA ........................ 22
2.2.3.4 DNA電泳 ...................................... 23
2.2.3.5 聚合酶連鎖反應 ............................... 23
2.2.3.6 限制酵素切割DNA .............................. 24
2.2.3.7 DNA片段膠體分離回收及純化 .................... 24
2.2.3.8 接合反應 ..................................... 25
2.2.3.9 勝任細胞製備 ................................. 25
2.2.3.10 轉形作用 ..................................... 25
2.2.4 創傷弧菌中巨噬細胞毒殺基因之選殖 ............... 26
2.2.4.1 對巨噬細胞毒性降低的跳躍子突變株之篩選 ....... 26
2.2.4.2 跳躍子突變株對巨噬細胞毒性測試 ............... 26
2.2.4.3 選殖巨噬細胞毒殺基因 ......................... 26
第三章 結果 .......................................... 28
3.1 創傷弧菌對巨噬細胞毒殺基因之選殖 .................. 28
3.1.1 篩選創傷弧菌對巨噬細胞毒殺能力下降之突變株 ...... 28
3.1.2 創傷弧菌跳躍子突變株於LB broth培養基之生長情形 .. 30
3.1.3 選殖創傷弧菌對巨噬細胞之毒殺基因 ................ 31
3.1.4 創傷弧菌對巨噬細胞之毒殺基因序列分析 ............ 32
第四章 討論 .......................................... 34
第五章 結論 .......................................... 37
參考文獻 .............................................. 38
圖 .................................................... 46
表 .................................................... 52
附錄 .................................................. 56
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