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研究生:蔡君琪
研究生(外文):Tsai, Chun-Chi
論文名稱:外源基因片段長度對重組桿狀病毒的基因穩定性之影響
論文名稱(外文):The Influence of Insert Sizes on the Genetic Stability of Recombinant Baculovirus
指導教授:胡育誠胡育誠引用關係
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:71
中文關鍵詞:桿狀病毒基因穩定性外源片段長效表現系統
外文關鍵詞:Sleeping Beauty systemheterologous capacitygenetic stabilitybaculovirus
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重組桿狀病毒表現系統目前已被廣泛應用在蛋白生產、疫苗開發及基因治療上,其具有可攜帶大片段外源基因的優點,但已有文獻報導重組桿狀病毒隨著不斷繼代過程中會產生大量的干擾缺損顆粒,造成後代病毒的基因不穩定性並導致重組蛋白的產量減少。目前尚未有研究針對桿狀病毒深入地探討外源片段大小對於重組桿狀病毒基因穩定性的影響,以及其所能夾帶的最大外源片段容量(capacity)。因此本研究的主要目的為探討外源基因片段大小與重組桿狀病毒基因穩定性之間的關聯,並進一步推展至實際應用時,載體中各種不同性質的外源片段對於病毒載體基因穩定性是否造成影響。我們以EGFP (enhanced green fluorescent protein)基因表現匣作為偵測外源片段穩定存在的指標,同時添加不同大小的外源填塞片段 (stuffer),建構出多株重組桿狀病毒以進行病毒轉導效價及基因DNA定量等分析,深入探討外源片段大小以及病毒代數對於重組桿狀病毒基因穩定性的影響。另外,本實驗室已將長效表現系統Sleeping Beauty (SB)結合桿狀病毒載體應用於癌症的治療且效果顯著,也證實此混成表現系統的應用潛力所在,因此我們在本研究中以SB系統作為基礎平台,插入各種有利於桿狀病毒載體表現的單元因子,在提升SB系統基因表現的同時探討各種因子以及病毒代數對於SB混成重組桿狀病毒基因穩定性的影響。我們發現當外源片段長度增加,第二代病毒仍能維持穩定的外源基因表現,然而隨著病毒代數的提升,其外源片段遺失的現象越明顯,顯示不同大小的外源片段確實會對於重組桿狀病毒的基因穩定性產生影響。另外在混成SB長效表現系統中,將SB100剪切酶表現匣與其轉位子IR/DR置於同一株病毒的建構方式於第二代病毒皆能良好地攜帶外源片段,但隨著病毒連續繼代則明顯較容易產生基因不穩定的現象,我們推測除了外源片段長度的影響之外, CMV啟動子於昆蟲細胞中微量表現的SB100剪切酶也可能致使IR/DR間的egfp表現匣更容易從重組病毒中遭到剃除。這些結果提供實驗室在建構大片段外源基因及SB長效表現系統的重組桿狀病毒時一個基因穩定性的參考。
摘要 I
Abstract II
目錄 III
第一章 文獻回顧 1
1-1 昆蟲桿狀病毒簡介 1
1-2 桿狀病毒/昆蟲細胞蛋白表現系統 2
1-3 Sleeping Beauty (SB)長效表現系統 3
1-4 病毒基因載體之基因穩定性 5
1-5 重組桿狀病毒基因的不穩定性 7
1-5-1 非同源性DNA複製點(non-homologous region ori)對DIP的生成及病毒基因穩定性之影響 7
1-5-2 同源性DNA複製點(homologous regions ori, hr-ori)密度對於基因穩定性之影響 9
1-5-3 利用gp64作為病毒篩選標記(selective marker)對於基因穩定性之影響 10
1-5-4 外源基因片段大小對於基因穩定性之影響 11
1-6 研究動機 12
第二章 實驗材料與方法 16
2-1 聚合酵素連鎖反應(Polymerase Chain Reaction) 16
2-2 DNA純化和回收方法 17
2-2-1 質體純化(Purification of plasmid DNA) 17
2-2-2 瓊脂凝膠萃取DNA片段 (DNA extraction from agarose gel) 18
2-2-3 病毒DNA純化 (Purification of viral DNA) 18
2-2-4 DNA濃度測量 19
2-3 剪切反應、凝膠電泳法、連接反應 19
2-3-1 剪切反應(Digestion) 19
2-3-2 凝膠電泳法(Gel Electrophoresis) 19
2-3-3 接合反應(Ligation) 20
2-4 轉殖作用(Transformation):化學法大腸桿菌(E. coli)細胞轉殖 20
2-5 細胞培養 21
2-5-1 昆蟲細胞培養 21
2-5-2 哺乳動物細胞培養 21
2-6 建構與放大重組桿狀病毒 21
2-6-1 重組桿狀病毒之Donor plasmid建構 21
2-6-2 重組桿狀病毒之製作與放大 25
2-7 桿狀病毒轉導哺乳動物細胞 25
2-8 轉導效價(transducing titers)的測定與計算 26
2-9 以終點稀釋法滴定病毒力價(infectious titer) 26
2-10 即時偵測同步定量聚合酶連鎖反應分析 27
第三章 結果與討論 37
3-1 探討外源片段長度對於基因穩定性之影響 38
3-2 探討額外表現同源性基因複製點hr1對基因穩定性的影響 40
3-3 探討Sleeping Beauty長效表現系統的建構平台之基因穩定性 43
3-3-1 探討轉位因子IR/DR之基因穩定性 43
3-3-2 置入具功能性外源基因於in cis系統中之基因穩定性 46
3-4 結論 49
第四章 未來展望 64
4-1 外源片段長度對基因穩定性的影響 64
4-2 不同建構系統中基因穩定性的差異 64
4-3 提升大片段外源基因表現匣在重組病毒基因體中的穩定性 66
第五章 參考文獻 67

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