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論文中文名稱:含旋轉酶及拓撲異構酶IV之質體構築 [以論文名稱查詢館藏系統]
論文英文名稱:Construction of Plasmid Containing Gyrase and Topoisomerase IV [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生化與生醫工程研究所
畢業學年度:103
畢業學期:第一學期
中文姓名:林明樞
英文姓名:Lin Ming-Shu
研究生學號:97679024
學位類別:博士
語文別:中文
口試日期:2015/01/26
指導教授中文名:侯劭毅
指導教授英文名:Hou Shao-Yi
口試委員中文名:陳錫圭;王勝仕;黃志宏;劉正哲;江謝正雄
中文關鍵詞:旋轉酶、拓撲異構酶IV、質體、套索
英文關鍵詞:gyrase, topoisomerase IV, plasmid, catenanes
論文中文摘要:在真核細胞中,最常使用的轉染載體為病毒及質體,但是病毒的缺點是DNA會嵌入宿主的染色體上,可能因此產生突變,而且轉染後無法移除。用質體轉染的優點就是不易與染色體產生重組,但重組基因無法長期表達。質體轉染真核細胞後,外源蛋白質表達會在轉染後的24~48小時內達到高峰期。然而在細胞內的質體會藉由細胞分裂或其他因素丟失,其可能原因為質體複製後會形成套索(catenate)的結構,而真核細胞內缺少分離套索所需要之酵素-拓撲異構酶IV與旋轉酶,所以細胞分裂後無法得到質體。
本研究中構築的質體pABGEC含有大腸桿菌的拓撲異構酶IV的基因parE、parC與旋轉酶的基因gyrA、gyrB,以及能夠發出綠色螢光的基因EGFP作為報告基因。每組基因前均加入內部的核醣體進入位(Internal ribosome entry site, IRES)片段,使所有的基因進入真核細胞後都能夠用IRES片段順利轉譯為蛋白質。當蛋白質順利表達,會產生旋轉酶與拓撲異構酶IV,能夠分離質體複製後形成的套索,使質體不需要嵌入染色體上就能穩定表達外源蛋白。在初步的成果顯示構築出的質體pABGEC具有穩定表達蛋白質的效果。
論文英文摘要:Virus and plasmid are the most popular vectors for transfection. However, the most disadvantage for virus vector is that the viral insertion into chromosome may cause serious side effects, such as mutation etc. To avoid these side effects, the alternate is plasmid vector. Plasmid is much easier to be manipulated than virus, but the recombinant plasmid can’t exist in mammal cell in long-term. One of the reasons for the loss of plasmid DNA in mammal cell is that circular plasmids form catenation structure after DNA replication and there is no topoisomerase IV and gyrase to decatenate the plasmids.
In this study, we constructed a plasmid pABGEC containing genes parE and parC for topoisomerase IV, genes gyrA and gyrB for gyrase, and gene EGFP as reporter gene. The ability of gyrase and topoisomerase IV is to resolve catenanes and the plasmid doesn’t integrate into the host’s chromosomes. In preliminary results show the plasmid pABGEC consistently expresses of the exogenous protein.
論文目次:摘 要 i
ABSTRACT ii
目 錄 iv
表目錄 vii
圖目錄 viii
第一章 文獻回顧 1
1.1真核細胞中的轉染與表達蛋白質 1
1.2各種轉染方式以及遭遇的問題 2
1.3獲得穩定表現細胞株的現行方法 5
1.4 DNA套索結構 7
1.5旋轉酶 9
1.6拓撲異構酶IV 10
1.7 氟喹啉酮類抗生素 11
1.8内核糖體進入位 13
1.9穿梭載體pIRES2-EGFP介紹 14
1.10 實驗策略 16
第二章 實驗方法與步驟 17
2.1實驗架構 17
2.2 實驗儀器與設備 18
2.3 實驗藥品 20
2.4實驗材料 22
2.4.1菌種與細胞 22
2.4.2 質體 23
2.5培養基 23
2.6重組質體構築 24
2.6.1 PCR 引子設計 24
2.6.2聚合酶鏈鎖反應 25
2.6.3瓊脂糖膠電泳 26
2.6.3.1膠體製備 26
2.6.3.2凝膠電泳分離 27
2.6.3.3影像分析 27
2.6.4膠體純化 28
2.6.5質體純化 28
2.6.5.1鹼性質體純化法 29
2.6.5.2質體純化套組純化法 29
2.6.6限制酶酶切 30
2.6.7連接酶接合反應 30
2.6.8轉型作用 30
2.6.8.1電轉殖法 31
2.6.8.2化學轉殖法 31
2.7真核細胞轉染 32
2.7.1冷凍細胞活化 32
2.7.2細胞繼代 33
2.7.3細胞計數 33
2.7.4細胞轉染 33
2.7.4.1 轉染前準備 33
2.7.4.2 轉染當天 34
2.7.5螢光顯微鏡觀察 34
第三章 結果與討論 36
3.1質體構築 36
3.1.1質體pEIG的構築 37
3.1.2質體pEIC的構築 40
3.1.3質體pIEIC的構築 43
3.1.4質體pBIG的構築 46
3.1.5質體pIBIG的構築 49
3.1.6質體pAIBIG的構築 52
3.1.7質體pABGEC的構築 55
3.2 質體穩定性分析 58
第四章 結論 62
參考文獻 63
附錄 70
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