臺灣博碩士論文加值系統

從古細菌至人類的所有物種的細胞中皆存在的thioredoxin，是一個具有熱穩定性的蛋白質，分子量一般約為12 kDa。在大腸桿菌中除了可以參與去氧核糖核酸的形成，並且在噬菌體T7感染時能作為T7 DNA聚合酵素(DNA polymerase)的次單元，幫助聚合酵素與單股DNA結合；在線形噬菌體感染時可以與pI 蛋白結合，幫助脫去pV蛋白，為噬菌體組合的必要成份。
本實驗中利用定點突變改變大腸桿菌thioredoxin活性區裡第33,34位置的氨基酸，並將基因置於不同載體中表現。突變株P34Y與G33V/P34Y分別會讓噬菌體T7/T3的感染平盤效率（E.O.P.）值下降109倍與77倍；這兩個突變株也分別在線形噬菌體的E.O.P值下降1.2x105倍與140倍。突變株P34G只能讓T7/T3的E.O.P.值下降43倍，但是對線形噬菌體的感染能力無明顯影響。除此之外，大量表現突變株thioredoxin時也會影響上述結果，讓噬菌體的感染能力恢復到與野生株類似。
根據本實驗室過去的實驗中發現pET32c上所攜帶的f1 複製原點可能會使線形噬菌體的感染能力下降。因此將基因接至與pET32c類似但不攜帶f1 origin的pET12a，發現以pET12a為載體表現thioredoxin時，被噬菌體f1所感染的E.O.P.會較pET32高19倍；被M13噬菌體所感染時會高3倍。

Thioredoxin is a 12-kDa-heat-stable protein, which present in all living orgnisms ranging from archaebacteria to humans. Escherichia coli thioredoxin is required for the DNA replication of bacteriophage T7 and the assembly of the filamentous phage.
We use site-directed mutagnesis to construct three mutants of E.coli thioredoxin to understand the effects of the different amino acid substitutions. The mutants were characterized using an in vivo assay based on the ability of cell to support growth of T7 and filamentous phage. In ordor to express mutant thioredoxin, these mutated trxA genes were constructed in different vectors. P34Y and G33V/P34Y decrease the efficient of plating (E.O.P.) of both kinds of phage. The E.O.P. decreased by 109 and 77 folds for P34Y and G33V/P34Y respectively infected by phage T7/T3. and 1.2x10 9 and 140 folds for P34Y and G33V/P34Y infected by filamentous phage. P34G only decreases the infection ability of T7 slightly. High level expression of the mutated thioredoxin increased infection ability of phages. Wild-type trxA gene was inserted into pET12a, a plasmid does not carry f1 origin. The results indicate that the f1 origin of plasmids can decrease the infection ability of phages. The changes of E.O.P. are 19 and 3 folds in the infection of phage f1 and M13.

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖目錄 vii
表目錄 ix
附圖目錄 xi
縮寫表 xii
一﹑ 緒論 1
1.1Thioredoxin簡介1
1.2大腸桿菌thioredoxin簡介2
1.3噬菌體T7簡介3
1.4Thioredoxin與噬菌體T7的關係3
1.5線形噬菌體簡介4
1.6Thioredoxin與線形噬菌體的關係5
1.7表現量不同對噬菌體感染的影響7
1.8質體f1 origin 對線形噬菌體感染的影響7
1.9T7 RNA 聚合酵素與啟動子表現系統7
1.10乳糖（Lac）啟動子表現系統8
1.11研究目的8
二﹑ 研究材料與方法 9
2. 1材料 9
2.1.1菌株9
2.1.2噬菌體10
1 線形噬菌體10
2 T7 噬菌體10
2.1.3質體11
2.1.4引導子15
2.1.5培養基16
2.1.6溶液與緩衝液17
2.1.7分析材料18
2.1.8酵素19
2.1.9試劑組20
2.1.10Marker20
2.1.11儀器20
2.2方法 21
2.2.1 點突變之製作21
2.2.2 大腸桿菌勝任細胞的製備與轉型作用 22
2.2.3 質體之製備 22
2.2.4DNA定量 23
2.2.5 瓊酯糖膠體電泳23
2.2.6 由瓊酯糖膠體分離DNA片段 23
2.2.7 聚合酵素連鎖反應 23
2.2.8黏接反應 24
2.2.9DNA定序 24
2.2.10菌株基因表現 25
2.2.11 噬菌斑的製作26
2.2.12 噬菌體f1感染作用 26
2.2.13 噬菌體M13感染作用 27
2.2.14噬菌體T7與T7/T3感染作用 27
三﹑ 結果 29
3.1野生與突變株質體的建構 29
3.1.1野生株質體的建構29
3.1.2 以pKs#5trxA進行定點突變 29
3.1.3 質體pKs#5野生與突變株去除f1 origin 30
3.1.4將突變之trxA接入pET32c(+)-stop 30
3.1.5將野生與突變之trxA接入pET12a31
3.1.6 將野生與突變之trxA基因接入pKs(-)並去除f1 origin 31
3.1.7 將野生與突變之trxA基因接入pACYC184 32
3.1.8將野生與突變之trxA基因接入KsN 33
3.2不同載體thioredoxin表現 34
3.2.1T7啟動子表現系統34
3.2.2 Lac啟動子表現系統 34
3.2.3 Thioredoxin啟動子表現系統 34
3.2.4比較不同載體於SK3967中的thioredoxin表現量 35
3.2.5比較不同載體於A179中的thioredoxin表現量35
3.3噬菌體感染作用 36
3.3.1噬菌體T7與T7/T3感染36
1 載體pET32c(+)-stop之野生與突變株感染結果 36
2 載體pACYC184帶有啟動子之野生與突變株感染結果36
3載體pKs(-)之野生與突變株感染結果 36
4載體pKs#5之野生與突變株感染結果37
5 載體pKsN之野生與突變株感染結果 37
3.3.2線形噬菌體感染 37
1 載體pET32c(+)-stop之野生與突變株感染結果 37
2 載體pACYC184帶有啟動子之野生與突變株感染結果38
3載體pKs(-)與pKs#5之野生與突變株感染結果 38
4質體f1 origin的影響38
四﹑ 討論 40
五﹑ 結論 45
六﹑ 未來的展望 46
七﹑ 參考文獻 92

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