海水養殖業中弧菌為主要的微生物病原，致死率近於100%。ΦA318 是我們研究室新發現的弧菌噬菌體，其能高效率地溶裂宿主溶藻弧菌 (Vibrio alginolyticus ATCC 17749)。經全基因體分析，推測其所產生 RNA 聚合酶 (RNAP) 的活性有別於 T7、SP6、K11噬菌體。為了大量製備 RNAP 以供研究其生化特性及生技應用，本研究選殖 ΦA318 RNAP，將poly-His 融接在它的N端，以利純化。獲得的選殖株 pEB172-1D 生產 RNAP 可達總蛋白之30% 以上。經過純化後的 RNAP 在最高比活性為 365 U/mg (37oC)。RNAP 之最佳 promoter 為 TTTTCTCCTATAGGGCAACTTTATT，具特異性，因此 RNAP 可供生技應用。同時也篩選 RNAP 的結晶條件，結果顯示有三種晶型，分別是微晶、針狀晶型、斜方晶型。據此，將可進一步解出此 RNAP 的原子結構。

　　In aquaculture Vibrio is a major microbial pathogen, Highest death rate may be 100%. ΦA318 is a newly discovered Vibrio phage in our lab, which is characterized by with high efficiency to lyze its host Vibrio alginolyticus (ATCC 17749). After the whole genome analysis, presumably its RNA polymerase (RNAP) activity differs from the T7, SP6 and K11 phages. In order to express RNAP for biochemical characterization and bio-reagent usage. I cloned ΦA318 RNAP, which was fused with a poly-His at its N-terminus to facilitate protein purification. The purified RNAP has the highest specific activity of 365 U/mg at 37oC. The best promoter for this RNAP was TTTTCTCCTATAGGGCAACTTTATT, which is unique and suitable for bio-reagent market. I also screened RNAP crystallization conditions. The results showed three crystal forms: microcrystalline, needles, and orthorombic crystal form. It will be further used to resolve the atomic structure of this RNAP.

目錄
謝誌 i
摘要 ii
Abstract iii
目錄 iv
表目錄 vii
圖目錄 viii
壹、 前言　1
　一、水產養殖的威脅　1
　二、弧菌感染　2
　三、抗生素與抗藥菌　9
　四、噬菌體療法　9
　五、弧菌噬菌體 ΦA318的簡介　13
　六、噬菌體 RNA 聚合酶選殖　14
　七、研究目的　15
貳、 材料與方法　16
　一、 宿主細菌培養　16
　二、 噬菌體效價之測定 (phage titer)　16
　三、單株噬菌體的分離　17
　四、噬菌體與菌種(選殖株)冷凍保存法　18
　五、弧菌生長曲線測試　18
　六、宿主溶裂 (bacteriolysis) 曲線測試　18
　七、弧菌噬菌體之增殖 (amplification)　19
　八、超高速離心噬菌體純化法　19
　九、流式光度計分析　20
　十、穿透式電子顯微鏡觀察負染色弧菌噬菌體樣本　20
　十一、噬菌體核酸萃取　21
　十二、DNA電泳製備與分析（DNA agarose gel electrophoresis)　22
　十三、限制性內切酶之分析 (Restriction endonuclease analysis)　23
　十四、質體的抽取　23
　十五、純化膠體中之DNA　24
　十六、TA cloning　26
　十七、勝任細胞(competent cell) 的製作　26
　十八、 細胞轉型 (transformation) 　27
　十九、Insert聚合酶鏈鎖反應(PCR)　28
　二十、菌落聚合酶鏈鎖反應 (colony-PCR)　28
　二十一、基因定序　30
　二十二、大腸桿菌表現弧菌噬菌體 ΦA318 RNA 聚合酶 (Vibrio phage ΦA318 RNA polymerase)　 30
　二十三、ΦA318 RNA 聚合酶之純化 (墊糖離心法)　32
　二十四、使用 Ni-NTA column 純化 poly-His tagged pEB172-1 BL21(DE3)　32
　二十五、蛋白質定量 (Lowry 法)　33
　二十六、蛋白質電泳　34
　二十七、ΦA318 RNA polymerase 活性分析　36
　二十八、結晶方法 (Crystallization)　37
參、 結果　39
　一、ΦA318 溶裂曲線　39
　二、ΦA318純化條件　39
　三、穿透式電子顯微鏡觀察弧菌噬菌體ΦA318型態　40
　四、pY31 菌落 PCR　40
　五、pY318 定序結果　41
　六、pEB 選殖前準備　41
　七、pEB17 菌落 PCR　42
　八、pEB17 選殖株分析　42
　九、pEB17 蛋白質表現　43
　十、pEB172-1D 誘導表現 ΦA318 RNA 聚合酶及純化　44
　十一、使用 Ni-NTA column 純化 poly-His tagged pEB172-1D　45
　十二、不同 IPTG 濃度對 ΦA318 RNA 聚合酶表現的影響　46
　十三、ΦA318 RNA polymerase 活性分析　46
　十四、ΦA318 RNA polymerase初步結晶結果　48
肆、 討論　49
　一、弧菌噬菌體 ΦA318的增殖最佳化時間　49
　二、ΦA318純化條件　49
　三、ΦA318 RNAP 最佳化的表現條件　49
　四、pEB172-1D 純化條件　50
　五、利用Ni-NTA column 純化 poly-His tagged pEB172-1D　50
　六、活性分析　51
　七、初步結晶探討　51
　八、未來展望　52
伍、 參考文獻　53
陸、圖表　59
附錄　117
　附錄A、選殖質體圖與序列　117
　附錄B、ATP 活性分析之波長　122
　附錄C、ATP 分析之反應機構　123
　附錄D、活性分析之計算公式　125
　附錄E、結晶條件　127
表目錄
　Table 1 The list of primers　59
　Table 2 The conditions for PCR reactions　59
圖目錄
　Fig. 1 Lysis curves of V. alginolyticus by Vibriophage ΦA318　60
　Fig. 2 Sedimentation profiles of vibriophage ΦA318 in a Sucrose gradient centrifugation　61
　Fig. 3 Electron microscopy (EM) analysis of negatively stained preparation of purified vibriophages　62
　Fig. 4 The cloning strategy of pY318　63
　Fig. 5 Colony PCR for pY311-pY3112　64
　Fig. 6A pY318 Sequencing M13F(-40) forward primer　66
　Fig. 6B pY318 Sequencing by M13R(-24) reverse primer　68
　Fig. 7 The cloning strategy of pEB　69
　Fig. 8A Agarose gel electrophoresis for DNA　70
　Fig. 8B Restriction of pET28a(+)　71
　Fig. 9 PCR for ΦA318 RNAP　72
　Fig. 10 Colony PCR for different colonies from pEB171-178 and 171H-178H　73
　Fig. 11A pEB17 plasmid DNA　74
　Fig. 11B pEB17 plasmid DNA was digested with NdeI　75
　Fig. 11C pEB17 plasmid DNA was digested with BamHI and SalI　76
　Fig. 12 pEB sequence detail　77
　Fig. 13A pEB172 Sequencing by T7P forward primer　79
　Fig. 13B pEB172 Sequencing by T7T reverse primer　81
　Fig. 13C pEB172 Sequencing by RNAPF24 forward primer　83
　Fig. 13D pEB172 Sequencing by RNAPR20 reverse primer　86
　Fig. 13E pEB172 Sequencing by TF24 forward primer　88
　Fig. 13F pEB172 Sequencing by TR22 reverse primer　90
　Fig. 14A pEB173 Sequencing by T7P forward primer　92
　Fig. 14B pEB173 Sequencing by T7T reverse primer　94
　Fig. 14C pEB173 Sequencing by RNAPF24 forward primer　96
　Fig. 14D pEB173 Sequencing by RNAPR20 reverse primer　98
　Fig. 14E pEB173 Sequencing by TF24 forward primer　100
　Fig. 14F pEB173 Sequencing by TR22 reverse primer　102
　Fig. 15 The SDS-PAGE analysis of pEB172 expression in BL21 competent cell　103
　Fig. 16 The SDS-PAGE analysis of pEB172 expression in BL21(DE3) competent cell　104
　Fig. 17 The SDS-PAGE analysis of pEB173 expression in BL21 competent cell　105
　Fig. 18 The SDS-PAGE analysis of pEB173 expression in BL21(DE3) competent cell　106
　Fig. 19 The tome course of pEB172 expression in BL21(DE3) competent cell　107
　Fig. 20A Induction of pEB172-1 bearing BL21(DE3) cells before purification　108
　Fig. 20B SDS-PAGE analysis for purification of poly-His tagged RNAP from pEB172-1D bearing BL21(DE3)　109
　Fig. 20C RNAP from 28k suspension was further purified by Ni–NTA column　110
　Fig. 21 Effects of inducer concentration on the expression of ΦA318 RNA polymerase　111
　Fig. 22A Activity assay of the RNAP purification process　112
　Fig. 22B Activity assay of the 609-E6 by different In vitro transcription time　113
　Fig. 22C Activity assay of the 609-E6 by different temperatures in vitro transcription　114
　Fig. 22D Activity assay of the 609-E6 by different ΦA318 promoters in vitro transcription at 37oC, 3 hours　115
　Fig. 23 Crystals of ΦA318 RNA polymerase expressed from construct pEB172-1D　116

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