臺灣博碩士論文加值系統

目前染色體已完全解序的幽門桿菌 (Helicobacter pylori) 菌株為26695、J99、G27及HPAG1，此四株幽門桿菌之中，各菌株間其基因變異性高達7%，造成此結果的原因之一，是幽門桿菌具有相當突出的自然轉型能力，可在無須物理化學作用之下將外界的基因攝入菌體內。在我們之前的研究指出，幽門桿菌中hp1089基因對於幽門桿菌自然轉型能力具有影響。而在我們的實驗中，更確切的証實當幽門桿菌hp1089基因受到破壞時，幽門桿菌自然轉型及以電擊法攝入外界基因的能力有相當程度的下降。此一幽門桿菌臨床菌株 (NTUH-C1) hp1089基因序列與已發表幽門桿菌菌株 (26695) hp1089基因具有93%相似性；兩者HP1089蛋白質胺基酸序列則具有94%相似性。 因此我們將幽門桿菌臨床菌株 (NTUH-C1) HP1089蛋白的胺基酸序列根據資料庫進行比對分析，推測HP1089蛋白可能具有兩種與自然轉型能力相關之酵素活性。經由NCBI及FUGUE資料庫分析後，發現HP1089蛋白N端525個胺基酸之片段，與superfamily I helicase酵素活性的群組COG3893，具有39%的一致性。而HP1089蛋白C端253個胺基酸片段則與具有RecB family exonuclease活性之COG2887群組有40 %一致性。為了証明此蛋白是否具有上述兩種酵素活性，我們嘗試將此基因分為三個不同片段加以選殖；它們分別是hp1089 (2334 bp) 完整基因，包含hp1089基因5’端1800 bpDNA片段與hp1089基因3’端867 bp DNA片段。並將它們轉殖進重組蛋白表現載體上，接著嘗試以大腸桿菌誘導表現出具生物活性之個別蛋白，經純化後分析，此三種片段之蛋白，確實可與DNA結合，並且將DNA分解。另外我們也比較了hp1089基因受到破壞之突變株 (M1-38) 與野生株 (NTUH-C1) 兩者在蛋白表現上的差異，找出哪些基因會受到hp1089基因影響。

Nowadays, the completely sequenced Helicobacter pylori are 26695, J99, G27, and HPAG1, and the gene variation between each other is about 7%. One of the reasons that cause the gene variation is that the Helicobacter pylori has an excellent ability in natural transformation, which can uptake DNA fragment from other bacteria without any chemical treatment or physical effect. According to our preceding research, we found that the gene hp1089 could influence the natural transformation ability of Helicobacter pylori. In this research, we verified that both of the natural transformation ability and the electroporation competence of the Helicobacter pylori would be greatly reduced when the hp1089 gene was knockout. The sequence of the hp1089 gene shares a 93% similarity and the sequence of the HP1089 protein shares a 94% similarity between the Helicobacter pylori (NTUH-C1) and the published one (26695). Therefore, we compared and analyzed the amino acid sequence of HP1089 protein of Helicobacter pylori (NTUH-C1) to the database, and predicted that the HP1089 protein probably has two enzyme activities related to natural transformation ability. 525 amino acid in the N-terminal of HP1089 protein share 39% identity with group COG3893 in the inactivated superfamily I helicase; and 253 amino acid in the C-terminal of HP1089 protein share 40% identity with the COG2887 group in the RecB family exonuclease activity. In order to verify whether the HP1089 protein contain the function such as the helicase and the exonuclease, we attempted to clone three different gene sequence which include the full length, the 1800 bp DNA sequence in the 5’, and the 867 bp DNA sequence in the 3’ of hp1089. Moreover, we transformed them into the recombination vector, and then try to use E. coli to induce the expression of bioactivated protein. After purification, we find that this protein could bind to DNA and possess the exonuclease enzyme activity. Furthermore, we compared the differences in proteins expression profile between the hp1089 gene mutation and wild type to find out which gene would be affected by hp1089.

中文摘要 I
Abstract II
目錄 III
圖表目錄 VI
第一章 前言 1
1.1 幽門桿菌 (Helicobacter pylori) 之簡介 1
1.2 幽門桿菌的致病因子 2
1.2.1 鞭毛 (Flagella) 2
1.2.2 脂多醣體 (Lipopolysaccharide, LPS) 2
1.2.3 尿素分解酵素 (Urease) 3
1.2.4 黏附因子 (Adhesin) 3
1.2.5 Neutrophil activating protein (NAP) 4
1.2.6 空泡性細胞毒素 (Vacuolating cytotoxin) 4
1.2.7 Cag pathogenicity island (cag-PAI) 4
1.3 幽門桿菌自然轉型作用機制 5
1.4 解旋�� (Helicase) 簡介 7
1.5 外切�� (Exonulcease) 簡介 8
1.6 實驗目的 9
第二章 材料 10
2.1 幽門桿菌菌株 10
2.2 大腸桿菌菌株 10
2.3 質體 11
2.4 培養基 11
2.5 引子 12
2.6 限制�� 13
2.7 抗生素 13
第三章 方法 14
3.1 幽門桿菌 (H. pylori) 的培養條件 14
3.2 HP1089 蛋白胺基酸序列比對 14
3.3 自然轉型作用 (Natural transformation) 14
3.4 以electroporation進行DNA轉型作用 15
3.4.1 勝任細胞 (competent cell) 之製備 15
3.4.2 Electroporation條件 15
3.5 幽門桿菌染色體DNA (chromosomal DNA) 之抽取與純化 16
3.6 質體DNA (plasmid DNA) 的抽取與純化 16
3.7 洋菜膠的電泳 (Agarose gel electrophoresis)及DNA回收 (Gel extraction) 17
3.7.1 洋菜膠製備、電泳條件及DNA顯像 17
3.7.2 DNA回收 17
3.8 大腸桿菌化學轉型作用 (chemical transformation) 18
3.8.1 勝任細胞 (competent cell) 之製備 18
3.8.2 熱休克 (heat shock) 18
3.9 聚合��鏈鎖反應 (Polymerase chain reaction, PCR) 18
3.10 TA選殖 (TA cloning) 19
3.11 接合反應 (Ligation) 19
3.12 去氧核醣核酸定序 (DNA sequencing) 19
3.13 表現蛋白 20
3.14 純化HP1089、HP1089_1-1800、HP1089_1466-2334蛋白 20
3.15 純化蛋白之透析 (dialysis) 與濃縮 (concentration) 21
3.16 外切�� (exonuclease) 酵素活性分析 22
3.17 Gel retardation功能分析 22
3.18 二維電泳 (2-dimensional electrophoresis) 23
3.18.1 樣品的前處理 23
3.18.2 一維電泳，等電點的聚焦電泳 （Isoelectric Focusing） 24
3.18.3 二維電泳，聚丙烯醯胺凝膠電泳 (SDS-Polyacrylamide gel electrophoresis、SDS-PAGE) 24
3.18.4 染色， coomassie blue 25
3.18.5 比對 25
3.18.6 膠內水解反應 (In gel digestion) 25
第四章 結果 27
4.1 野生株 (NTUH-C1) 與hp1089基因遭破壞之突變株 (M 1-38) 生長 狀態之比較 27
4.2 幽門桿菌自然轉型與electroporation效率分析結果 27
4.3 幽門桿菌HP1089蛋白胺基酸序列分析結果 28
4.4 幽門桿菌HP1089蛋白之誘導表現與純化 29
4.5 幽門桿菌HP1089蛋白中C端，HP1089_1466-2334蛋白之誘導 表現與純化 29
4.6 幽門桿菌HP1089蛋白中N端，HP1089_1-1800蛋白之誘導表現 30
4.7 外切�� (exonuclease) 功能分析結果 30
4.8 HP1089、HP1089_1466-2334、HP1089_1-1800蛋白與DNA結合能力 之Gel rotardation功能分析結果 31
4.9 二維電泳 (2-dimensional electrophoresis) 分析結果 31
第五章 討論 32
第六章 附圖表 34
第七章 參考文獻 53

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