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研究生:彭懿慧
研究生(外文):Yi Hewi Peng
論文名稱:線狀噬菌體phiLf反股基因orf155、orf137及orf102之研究
指導教授:曾義雄曾義雄引用關係
指導教授(外文):Yi Hsiung Tseng
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
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
論文頁數:84
中文關鍵詞:線狀噬菌體
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摘要
phiLf為本實驗室於導致十字花科植物黑腐病之 Xanthomonas campestris pv. campestris 所純化出來之線狀噬菌體,與感染 E .coli 之 Ff 噬菌體具有相似之基因排列方式。phiLf 與 Ff 差異較大之地方為缺乏 gIV,其基因區間之 1478 bp中並無相似 gIV 之區域,並且 DNA 之複製起始點位於 gII 中。在 Ff 噬菌體中,其 508 bp之基因區間之功能並不是在產生蛋白質產物,並含有正股及互補股之複製起始點,而此區間含有極大程度之二級結構,引導噬菌體包裹之訊號。
分析 Lf 之基因區間,發現於互補股轉錄方向可能具有三個 open reading frames,分別為 orf155 (nt 5721-5254)、orf137 (nt 5338- 4925) 及 orf102 (nt 4858-4550);另外在正股轉錄方向上發現一可能之 orf98 (nt 5720-8)。
本實驗之目的為尋找 phiLf 基因間區中可植入抗藥基因而不影響正常功能,可穩定存在之區域。因為 Ff 噬菌體之基因體 DNA 在長度不受限制較為寬鬆,已廣泛作為載體,而於基因間區可植入一些 DNA 片段仍後可穩定存活。本研究中已構築三株重組噬菌體,其一為刪除 core (nt 5339-5353) 之 phiLfCD,其基因體之 orf155 末端遭到破壞;其次為刪除 IHF binding site (nt 5290-5301) 之 phiLfHD,其 orf137 之 5’ 端及 orf155 之 3’ 端遭到破壞;第三為刪除 orf155 基因中之 EcoRI 切位之 phiLf155G。
由各重組噬菌體之溶菌斑觀察,發現其溶菌斑之亮度依序由 phiLfCD、phiLfHD 及 phiLf155G 減弱。將 ORF155 之胺基酸進行比對,發現與線狀噬菌體 Cf1c 之 ORFII 蛋白具有 62% 之相似性,而 ORFII 在 Cf1c之功能可能與形成溶菌斑之清澈度影響有關。
由比較此三種重組噬菌體轉導 P20H 之實驗,發現 Lf155G 具有最高之轉導效率,且導致感染後之菌體有 60 % 之存活率,而 LfCD 感染後之菌體存活率為 48.6%;LfHD 感染後之菌體存活率為 52%。因此推測 ORF155 可能對噬菌體 Lf 感染 P20H 具有毒性,導致菌體於感染後有部份死亡。
另外,將 orf137 基因構築於蛋白表現質體上,純化大量表現之 ORF137 蛋白,並製備抗體。由 ORF137 之西方墨點法分析,發現在 P20H 及 Xc17 本身可能會產生與 ORF137相似之蛋白區域。而線狀噬菌體是否表現 ORF137 蛋白,由本實驗並不能得知。

Abstract
Lf is a filamentous phage specifically infecting Xanthomonas campestris pv. campestris, the pathogenic bacterium causing black rot in crucifers. The genome of Lf is similar to that of the Escherichia coli filamentous phage Ff (f1, fd, and M13), except that Lf lacks the gene homologous to Ff gene IV (gIV ) which is required for phage export. In addition, the Lf origin of replication (ori) is situated within gII, a location different from the case of Ff phage in which the oris for the replication of both complementary and viral strands as well as the DNA packaging signal are in the 508-bp intergenic region (IR) that is rich in secondary structures and encodes no proteins bigger than 16 amino acids. In Lf, the major IR is between gI and gII containing four open reading frames: one (orf98, nt 5720-8) in viral strand and three in complementary strand (orf155, nt 5721-5254; orf137, nt 5338-4925 and orf102, nt 4858-4550). Due to the property that Ff has no DNA length limit during phage packaging and that an insertion of DNA fragment into the IR region does not interfere with phage viability, the phage has wildly been used as cloning vectors. The purpose of this study was to search for the site(s) in the Lf IR region, 1,480-bp long locating between gI and gII, into which an insertion of or a replacement with an antibiotic-resistance cartridge does not affect its viability and stability. For this purpose, we constructed three recombinant phages: LfCD, which had the integration core region (nt 5339-5353) being replaced by a Gmr cartridge, disrupting the 3’ end of orf155; LfHD, which had the IHF binding site (nt5290-5301) being replaced by a Gmr cartridge, disrupting the 5’ end of orf137 and 3’ end of orf155; Lf155G which had the 114-bp EcoRI fragment (nt 5635-5521) being replaced by a Gmr, disrupting orf155. Comparing to those of the wild-type Lf, the plaques manifested by these recombinant phages were turbid, exhibiting degrees of plaque turbidity in the order Lf155G>LfHD>LfCD. Among the four orfs in the Lf IR, ORF155 shares 62% similarity with the Cf1c ORFII which plays a role in the determination of plaque turbidity. The results of transduction showed that the Lf155G has the highest transduction efficiency. The viability of the transductants of Lf155G, LfCD and LfHD were 60, 52, and 48.6%, respectively. These data suggest that orf155 may be harmful to P20H, resulting in death of part of the host cells after transfection. orf137 was cloned and over-expressed and the protein was purified for the preparation of antiserum. Using the antibody for Western hybridization, a protein of the same size as ORF137 was detected in Lf-infected and the non-infected P20H and Xc17. It is still uncertain whether Lf expresses ORF137 protein or not.

目錄
中文摘要
英文摘要
前言 1
文獻研討 8
材料與方法
I. 材料
一、 菌種、噬菌體及質體 13
二、 藥品 13
三、 培養液及緩衝液 13
四、 引子 14
II. 實驗方法
一、 染色體 DNA 之抽取 16
二、 小量質體 DNA 之抽取 16
三、 洋菜膠體電泳分析 17
四、 DNA 片段之回收 17
五、 DNA 補齊反應 18
六、 DNA 之粘接反應 19
七、 勝任細胞之製備 19
八、 轉形作用 19
九、 質體快速篩選法 20
十、 DNA 探針之製備 20
十一、南方墨點雜交反應 21
十二、蛋白樣本之製備 21
十三、蛋白質凝膠電泳分析 22
十四、線狀噬菌體 phiLf 之 ORF137、ORF155 及 ORF102
蛋白之表現與純化 22
十五、蛋白純化後之透析 24
十六、蛋白質濃度測定 24
十七、西方墨點法 24
十八、生長曲線之測定 25
十九、聚合脢鏈鎖反應 25
結果與討論
一、phiLf 基因間區之 open reading frames 分析 27
二、重組噬菌體 phiLfCD、phiLfHD 之構築 28
三、重組噬菌體 phiLf155G 之構築 29
四、重組噬菌體 phiLf137 G 之構築 30
五、重組噬菌體 phiLfCD之點測試試驗及噬菌體濃度測定 31
六、重組噬菌體 phiLfCD 轉導效率之測定 32
七、重組噬菌體 phiLfCD 與 helper phage 關係之探討 33
八、phiLf 感染 P20H 之效率測定 33
九、線狀噬菌體 phiLf 生長曲線之測定 34
十、重組噬菌體 phiLfCD 於不同寄主之生長曲線測定 35
十一、重組噬菌體 phiLfHD之點測試試驗及噬菌體濃度測定 36
十二、線狀噬菌體 phiLfHD 之純化 37
十三、重組噬菌體 phiLfHD 轉導效率之測定 37
十四、重組噬菌體 phiLfHD 感染 P20H 之生長曲線之測定 39
十五、重組噬菌體 phiLf155G 溶菌斑型態之觀察 39
十六、重組噬菌體 phiLf155G 轉導效率之測定 40
十七、噬菌體 phiLf 對 P20H 之感染造成大部分之菌體死亡 41
十八、重組噬菌體 phiLf、phiLfHD、phiLf155G單一溶菌斑
含約 105 ~ 106 PFU/ml 42
十九、重組噬菌體phiLfCD、phiLfHD、phiLf155G 互補質
體之構築 43
二十、重組噬菌體phiLfCD、phiLfHD、phiLf155G 之互補
試驗 44
二十一、蛋白表現載體 pET137、pET155 及 pET102 之構築 45
二十二、pET137 、pET155 及 pET102 蛋白之大量表現情形 45
二十三、線狀噬菌體phiLf 之 ORF137 蛋白之純化及抗體之
製備 46
二十四、線狀噬菌體 phiLf 之 ORF155 大量表現之蛋白無法
與His- bind 親和性管柱結合 47
二十五、線狀噬菌體 phiLf 之ORF137 之西方墨點法分析 48
參考文獻 50
圖表 59
附錄 81

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