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研究生:吳文蓉
研究生(外文):Wendy Wu
論文名稱:以CascadeTM蛋白表現系統表現天花病毒之拓樸異構酶一號
論文名稱(外文):Expression of Vaccinia Virus Topoisomerase I by Using CascadeTM Protein Expression System
指導教授:許宗雄許宗雄引用關係吳文騰
指導教授(外文):Tsung-Xiong HseuWen-Teng Wu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類: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:66
中文關鍵詞:拓樸異構酶大腸桿菌天花病毒Cascade
外文關鍵詞:topoisomeraseE.colvaccinia virusCascade
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本研究藉由Cascade TM蛋白表現系統表現毒蛋白─天花病毒之拓樸異構酶 一號。Cascade TM蛋白表現系統利用調節性的迴路增加蛋白的產量。當使用人工染色體pBeloBAC11作為表現載體時,此調控系統能精密地將誘導前的毒蛋白表現量降到很低,而同時避免突變現象的發生。因此,Cascade TM蛋白表現系統為一適合生產毒蛋白之表現系統。
利用基因選殖技術將含有Cascade TM下游的啟動子Pm及天花病毒之拓樸異構酶 一號(vvt)的重組基因選殖到人工染色體上,即得到質體BAC-vvt。將BAC-vvt送入大腸桿菌表現菌株TAP,由DNA鬆弛反應與SDS-PAGE實驗分析得知,此重組蛋白雖然有活性表現,但未能如預期大量表現。為了克服上述問題而在大腸桿菌中能大量表現天花病毒之拓樸異構酶 一號,我們採用T7蛋白表現系統,以噬菌體λCE6感染含pET-vvt之大腸桿菌BL21來大量生產天花病毒之拓樸異構酶 一號。
In this study, Cascade TM expression system was used to express the toxic protein, vaccinia virus topoisomerase I . Cascade TM system was regulated by two circuits to amplify gene expression levels. The expression system is tightly regulated, and maintains low basal levels prior to induction. The low basal level of the single copy artificial chromosome, pBeloBAC11, allows for expression of toxic proteins and reduces the possibility of mutation.
A recombinant artificial chromosome, BAC-vvt, encoding the second portion of the Cascade TM system and the vvt gene was constructed and transformed into E.coli TAP expression strain. The result from DNA relaxation assay indicated that the recombinant proteins displayed topoisomerase activity. However, the result from the SDS-PAGE suggested that the toxic proteins were not overexpressed. To tackle this problem, the T7 expression system, E.coli BL21 carrying pET-vvt infected with bacteriophageλCE6, should be used to overexpress the toxic proteins.

中文摘要 …………………………………………………………… 1
英文摘要 …………………………………………………………… 2
第一章 文獻回顧 …………………………………………………. 3
一. 前言 …………………………………………………………... 3
二. 大腸桿菌蛋白質表現系統 ………………………………… 4
1.基因的套數 …………………………………………………. 5
2.啟動子 ………………………………………………………. 6
3.mRNA穩定性 ……………………………………………… 7
4.轉譯的起始效能 …………………………………………… 8
三. Cascade TM蛋白表現系統 …………………………………… 9
四. T7蛋白表現系統 …………………………………………… 12
五. 天花病毒之拓樸異構酶 一號 ……………………………… 13
第二章 材料及實驗方法 ………………………………………… 18
一、菌株 ………………………………………………………… 18
二、載體 ………………………………………………………… 18
三、藥品與酵素 ………………………………………………… 18
四、培養基與緩衝溶液 ………………………………………… 18
五、實驗方法 …………………………………………………… 19
(一)、Vaccinia virus topoisomeraseⅠ基因之選殖 …………… 19
1.大腸桿菌質體之少量抽取 ………………………………... 19
2.洋菜膠體電泳分析 ……………………………………… 19
3.Vaccinia virus topoisomeraseⅠ基因片段之取得 …………. 20
4.DNA之回收 ……………………………………………… 21
5.大腸桿菌勝任細胞的製備與轉形作用 ………………… 21
6.大腸桿菌之質體篩選 ……………………………………. 21
7.重組質體TA-vvt、pCAS-vvt、BAC-vvt和pET-vvt之製備 … 22
8.噬菌體λCE6之製備 ……………………………………… 23
9.噬菌體的效價測定法 …………………………………… 24
(二)、Vaccinia virus topoisomeraseⅠ的表現 ………………… 24
1.Cascade TM系統之酵素表現 ……………………………… 24
2.T7系統之酵素大量表現 ………………………………… 24
3.蛋白質電泳分析 …………………………………………. 25
4.天花病毒之拓樸異構酶 一號之活性分析-鬆弛反應 …… 25
第三章 實驗結果與討論 ………………………………………… 27
一、Vaccinia virus topoisomerase I基因的選殖 ………………… 27
1.Cascade蛋白表現系統之vvt基因選殖 …………………… 27
2.T7蛋白表現系統之vvt基因選殖 ………………………… 28
二、Vaccinia virus topoisomerase I基因的表現 ………………… 28
1.Cascade TM蛋白表現系統 …………………………………… 29
2.T7蛋白表現系統 …………………………………………… 29
三、Vaccinia virus topoisomerase I重組蛋白之最適化表現條件 .. 30
1.Cascade TM蛋白表現系統 …………………………………… 30
A.水楊酸濃度對重組大腸桿菌之重組蛋白表現的影響 .… 30
B.生長時期對重組大腸桿菌之重組蛋白表現的影響 …...… 31
2.T7蛋白表現系統 …………………………………………… 31
A.λCE6噬菌體濃度對重組大腸桿菌之重組蛋白表現的影響.. 31
B.生長時期對重組大腸桿菌之重組蛋白表現的影響 ……... 32
四、比較各系統之重組蛋白表現量 …………………………. 33
第四章 結論與未來展望 ………………………………………… 35
圖表 ……………………………………………………………… 38
參考文獻 ………………………………………………………… 53
附錄 …………………………………………………………

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