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論文中文名稱:構築含旋轉酶,拓撲異構酶IV及核定位序列片段之質體於真核細胞中長期表達 [以論文名稱查詢館藏系統]
論文英文名稱:Construction of plasmid containing Gyrase, Topoisomerase IV and Nuclear Localization Signal and its long term expression in CHO cell [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:104
畢業學期:第一學期
中文姓名:曾冠慈
英文姓名:Guan-Ci ,Tseng
研究生學號:102688015
學位類別:碩士
語文別:中文
口試日期:2016/01/29
指導教授中文名:侯劭毅
口試委員中文名:黃志宏;楊俊仁;王勝仕
中文關鍵詞:轉染旋轉酶拓撲異構酶IV核定位序列套索質體
英文關鍵詞:transfectiongyrasetopoisomerase IVNLScatenanesplasmid
論文中文摘要:轉染(Transfection)是將外源基因(DNA或RNA)轉入真核細胞中,讓基因在細胞內表達的一種過程,然而外源基因並不能主動進入細胞,要進入細胞內必須藉由載體(Vector)運輸,而最常用的兩種載體是病毒的 DNA 和細菌質體,病毒載體的優點在於其轉染效率佳,而缺點是外源基因的隨機插入,可能會破壞原本基因組序列,造成基因突變的風險;質體則安全性較高,不會發生基因重組,但其轉染效率及表現基因方面可能都有很大的限制,然而基因無法持續表達的原因可能為質體在複製時會形成套索(Catenate)結構,在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,導致細胞分裂後無法得到質體,使細胞表現外源基因量下降。
本研究改良本實驗室林明樞學長構築的質體pABGEC,該質體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,及綠色螢光蛋白(Enhanced Green Fluorescent Protein, EGFP)基因作為報告基因,且在每組基因前均接上內部核醣體進入位(Internal ribosome entry site, IRES)片段,使轉染到真核細胞後都能夠用順利轉譯為蛋白質。先前實驗結果顯示構築出的質體pABGEC 雖有穩定表達蛋白質,但效果不明顯。探討其原因可能為拓撲異構酶IV與旋轉酶無法進入細胞核內作用,因此改良質體 pABGEC 在每組基因3’端均接上核定位序列(Nuclear Localization Sequence, NLS)。藉由 NLS 協助拓撲異構酶IV與旋轉酶進入細胞核內發揮作用,進而提升質體持續表達之效果。
論文英文摘要:Introduction of foreign DNA into eukaryotic cells is often called "transfection". In general, the vectors of transfection are viral vector and bacterial plasmid. Although viral vectors have high transfection efficiency, it will insert into the native chromosome. That may cause the mutation of the host cell. The plasmid is more stable than viral vector. However the plasmid shows lower transfection efficiency and gene expression than virus vector. The reason could be that lacking of topoisomerase IV and gyrase which decatenate the plasmid causes the cell cannot get the plasmid after cell division.
In this study, we constructed a plasmid pABGEC-n which contains genes parE and parC for topoisomerase IV, genes gyrA and gyrB for gyrase, gene EGFP as reporter gene and each gene was added a nuclear localization sequence which tags the follow gene product for import into the cell nucleus. The expression of exogenous protein increased via adding the nuclear localization sequence which assists topoisomerase IV and gyrase import into the cell nucleus.
論文目次:目錄
摘 要 i
ABSTRACT iii
目錄 iv
表目錄 vii
圖目錄 viii
第一章 緒論 1
1.1轉染與蛋白質表達 1
1.2各種轉染方法 2
1.3遺傳黴素的機制(Geneticin,G418) 4
1.4 核定位序列 5
1.5旋轉酶 6
1.6拓樸異構酶 IV 7
1.7內核醣體進入位 8
1.8穿梭載體pIRES-EGFP 9
1.9研究策略 12
第二章 實驗方法與流程 13
2.1實驗架構 13
2.2實驗儀器與設備 14
2.3實驗藥品 18
2.4實驗材料 21
2.4.1菌種與細胞 21
2.4.2質體 22
2.4.3培養基 23
2.5實驗流程 25
2.5.1質體pABGEC-n構築 26
2.5.2細胞轉染實驗 35
2.5.2.1細胞培養 35
2.5.2.2細胞轉染 37
2.6.2.3螢光顯微鏡觀察 38
第三章 結果與討論 39
3.1質體構築 40
3.1.1質體pEIG-n的構築 40
3.1.2質體pEIC-n的構築 42
3.1.3質體pIEIC-n的構築 44
3.1.4質體pBIG-n的構築 46
3.1.5質體pIBIG-n的構築 48
3.1.6質體pAIBIG-n的構築 50
3.1.7質體pABGEC-n的構築 52
3.2轉染條件的最佳化 54
3.2.1 Lipofectamine 2000使用量 54
3.2.2 轉染質體使用量 57
3.2.3 Lipoplex放置時間 61
3.2.4 G418篩選濃度測試 62
3.3質體轉染的效率與穩定性 63
第四章結論 73
參考文獻 74
附錄 77
一、pEIG的parE定序 77
二、pEIC的parC定序 81
三、pIEIC的IRES定序 86
四、pBIG的gyrB定序 88
五、pIBIG的IRES定序 93
六、pAIBIG的gyrA定序 95
七、pABGEC的定序 101
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