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研究生:張順福
論文名稱:大腸桿菌Thioredoxin與噬菌體T3/T7DNA聚合酵素之交互作用
論文名稱(外文):Interactions between Escherichia coli thioredoxin and the DNA polymerase of phage T3/T7
指導教授:林苕吟
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
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
中文關鍵詞:大腸桿菌DNA 聚合酵素噬菌體T3/T7
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噬菌體T7之DNA聚合酵素與大腸桿菌Thioredoxin所參與之DNA聚合複製系統,一直以來皆是科學家研究DNA複製作用的一個重要系統,Thioredoxin主要當作T7 DNA聚合酵素之延展因子,可提高T7 DNA聚合酵素延展性達數千倍之高。噬菌體T7 DNA聚合酵素與Thioredoxin之作用三級結構已在1998年被解出並發表於Nature雜誌上。根據前人研究顯示噬菌體T3和T7之基因5蛋白的氨基酸相同性高達96.7%,故本論文之實驗主要在研究T3/T7基因5蛋白與大腸桿菌Thioredoxin之作用的相關性。實驗上所使用之噬菌體稱之為T3/T7,其主要是以T3的DNA序列為主,而序列33332位置至35342位置之間被噬菌體T7的相對基因位置所取代,另噬菌體T3/T7野生株之基因5蛋白在第248、305及421位置的氨基酸與T3野生株之基因5蛋白不相同。本實驗利用實驗室前人所做帶有無法支持噬菌體T3/T7野生株正常生長之Thioredoxin突變株的大腸桿菌來篩選可回復生長作用之噬菌體抑制者,再藉由觀察基因5蛋白上的突變位置,及證實回復感染作用是導因於基因5蛋白的突變因素,來瞭解T3/T7基因5蛋白與thioredoxin間之作用相關位置。結果發現在每株噬菌體抑制者之基因5蛋白上皆存在著數個突變氨基酸,所以我們再挑選數個具重複性出現之突變點在T3/T7野生株基因5上作定點突變,再經由感染作用所得結論為噬菌體抑制者回復感染作用確實需要多個位置氨基酸突變才能達到最佳之感染效率,而在所有突變之氨基酸位置又以305為回復感染之最重要點。
中文摘要 …………………………………………………………………………i
英文摘要 …………………………………………………………………………ii
誌 謝 …………………………………………………………………………iii
目 錄 …………………………………………………………………………iv
表 目 錄 …………………………………………………………………………vi
圖 目 錄 …………………………………………………………………………vii
附表目錄…………………………………………………………………………viii
附圖目錄……………………………………………………………………………ix
縮 寫 表 …………………………………………………………………………xi
壹、 緒論 …………………………………………………………………………1
1.1 Thioredoxin………………………………………………………………1
1.2 大腸桿菌Thioredoxin……………………………………………………2
1.3 噬菌體T7及T7族群……………………………………………………2
1.4 噬菌體T7與大腸桿菌Thioredoxin的關係………………………… 4
1.5 噬菌體T3…………………………………………………………………4
貳、研究目的………………………………………………………………………6
參、 實驗材料
3.1菌株……………………………………………………………7
3.2噬菌體…………………………………………………………7
3.3質體……………………………………………………………9
3.4引子(primer)………………………………………………10
3.5培養基…………………………………………………………12
3.6溶液與緩衝液…………………………………………………12
3.7分析材料………………………………………………………14
3.8試劑組(kit)…………………………………………………15
肆、 實驗方法
4.1質體的抽取………………………………………………………17
4.2勝任細胞(competent cell)的製備……………………………17
4.3轉型作用(transformation)……………………………………17
4.4 DNA電泳…………………………………………………………18
4.5 DNA回收…………………………………………………………18
4.6噬菌體抑制者(phage suppressor)之挑選………………………18
4.7 T3/T7噬菌體DNA的抽取…………………………………………19
4.8 pACYC184-噬菌體T3/T7 gene 5之製備……………………………20
4.9 DNA定序…………………………………………………………21
頁次
4.10噬菌體之感染…………………………………………………22
4.11回復感染作用導因於基因5突變之證明…………………………23
4.12定點突變對回復感染之影響……………………………………23
4.13表現蛋白質噬菌體T3/T7基因5蛋白及大腸桿菌Thioredoxin……24
4.14蛋白質電泳(SDS-PAGE)………………………………………24
伍、 結果
5.1噬菌體T3/T7抑制者之篩選………………………………………26
5.2抑制作用(Suppression)主要來自噬菌體基因5蛋白突變所造成之證明
………………………………………………………………………27
5.3噬菌體抑制者之抑制作用非因蛋白大量表現所導致之證明 …………28
5.4定點突變證實特定點對噬菌體抑制者抑制作用之影響 ……………28
5.5基因5蛋白之表現 ………………………………………………29
陸、 討論 ……………………………………………………………………………31
柒、 結論 ……………………………………………………………………………36
捌、 未來展望 ………………………………………………………………………36
玖、 參考文獻 ………………………………………………………………………37
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