首先是利用資料庫進行氨基酸序列的比對及結合 Ang,Wen及Allan所著的文獻中得知，胃幽門螺旋桿菌在酸性環境下會誘導或抑制某些基因的mRNA的表現量,所以我們根據蛋白質序列的相似度和酸性調控的機制從中挑選5個基因來進行研究，分別是本論文中HP0406, HP0751, HP0753, HP0979, HP1399.。首先將這5個基因分別建構入在5’含有6個Histidine的pQE30載體中。接下來將建構好的載體轉形(transformation)至SG13009菌株中，並使用相對應的引子做PCR分析與IPTG誘導細菌在37℃,3個小時作用下做蛋白質表現。之後發現在所選擇的5個基因中，皆能成功表現出可溶性的蛋白質； HP0751在別的文獻中被認為可能與形成極性鞭毛有關，然而HP0406卻沒有任何功能性的介紹。帶有P0406及HP0751H基因的質體，皆可穩定表現出大量蛋白質，所以接下便以P0406和HP0751基因為我研究的目標基因。
轉型成功後的菌株，使用0.5 mM IPTG在20℃下誘導20個小時，使用Ni-NTA親和力管柱純化細菌內所表現的重組蛋白質HP0406及HP0751,一升的菌液最後分別地可得到10mg和25 mg。然而在pH等於8時，可穩定地被儲存並持續數週，並可進一步去對蛋白質結構與蛋白質功能的研究與探討。
重組蛋白HP0406及HP0751可以當做抗原(antigen)並選用兔子來進行免役以製備多株抗體.從第5次注射後所得到的兔子血清經過處理後，得到的抗體（Anti-HP0406）及(Anti-HP0751)經過校價的測試後發現，可以分別偵測到2 ng及10ng的重組蛋白HP0406和HP0751。因此，便可利用所製作的抗體與西方點墨法分析胃幽門螺旋桿菌在不同環境下，HP0406和HP0751蛋白質的表現量。
發現胃幽門螺旋桿菌在pH 5.5與pH 7.2的Brucella agar plates培養48小時後，以HP0406蛋白質在pH5.5的表現量比在pH7.2的環境多了1.5倍，符合於Ang所發表的文獻；然而在HP0751蛋白質在pH5.5的表現量和在pH7.2的環境下卻幾乎沒有什麼差別，這個現象與Wen所著的文獻有所不同，但Wen所用的是液狀方式培養在0∼120min，培養方式及時間不同，所以可能會有所不同。
HP0751蛋白質在資料庫中被預測為Polar flagellar G，在過去的研究認為它可能參與鞭毛的形成，但實際上在胃幽門螺旋桿菌的功能還不清楚。所以首先利用Circular Dichroism這個儀器針對其HP0751蛋白質的二級結構做探討，發現此HP0751蛋白質可穩定存在於pH值5到8的範圍，並且在溫度超過82℃才開始劇烈地改變二級結構，而尿素變性劑實驗，也說明其蛋白質可忍受尿素濃度至4.68M，
才開始明顯改變結構，比較於其他蛋白質，HP0751蛋白質是較為穩定的。
針對HP0751可能參與細胞附著的現象，我們利用分別利用HP0751蛋白質和Anti-HP0751 serum配合胃幽門螺旋桿菌附著細胞會分泌尿素分解脢的特性，與AGS細胞Co-culture，由urea assay buffer由酸性變成鹼性（由黃變紅）的特性，
來分辨胃幽門螺旋桿菌附著AGS細胞的多寡，從數據來看似乎HP0751與胃幽門螺旋桿菌附著AGS細胞並無直接的關連。在未來也可利用基因交換的技術將flaG gene置換，來觀察野生型和突變型的差別，或許可以瞭解HP0751是否參與附著細胞的過程。
HP0406蛋白質資料庫中被預測為假設性蛋白質，並無任何相關這個蛋白質功能性的研究，然而我們利用NCBI的比對系統找出HP0406蛋白質與Methanocaldococcus jannaschii DSM 2661的Purine NTPase有13.2% identity。依此，我們設計了三種方式想要去證明HP0406蛋白質是否具ATPase活性？首先我們從Circular Dichroism觀察到鎂離子可改變其二級結構，並發現不論是否存在於鎂離子其ATP亦可改變HP0406蛋白質的二級結構；我們還進一步的發現存在於鎂離子的HP0406蛋白質降低了其Tm值（melting temperature），而ATP卻成功增加了存在於鎂離子的HP0406蛋白質的Tm值，由此可知ATP應可增加HP0406蛋白質的熱穩定性。接著我們想要知道HP0406蛋白質是否能水解ATP釋放出磷酸根離子，由Malachite-Green ATPase assay我們得知其HP0406蛋白質水解ATP的活性是隨著鎂離子濃度上升而增加（5mM上升到200mM）；並且當鎂離子濃度降為零的時候，HP0406蛋白質就不能水解ATP釋放出磷酸根。除了觀察磷酸根以判斷HP0406蛋白質水解ATP的能力之外，我們設計利用HPLC分離HP0406蛋白質存在於鎂離子的狀態時水解的作用反應物及產物，正如我們所期望的，存在於5mM鎂離子的實驗組明顯地被發現的確有ADP的產生，而且在鎂離子濃度為零的另一實驗組則沒有任何的ADP產生，這個結果呼應了Malachite-Green ATPase assay的數據，說明了存在於鎂離子的狀態下的HP0406蛋白質才能明確地執行水解ATP的功能，最後我們想知道其他二價離子是否也會促進HP0406的酵素功能？將結果以相對活性來表示，其活性的順序為Mg2+＞Ca2+＞Mn2+＞Ni2+。總而言之我們可以從上述實驗的結果重新定義HP0406的功能為一個具有ATPase-like的蛋白質，並以此為出發點找出HP0406蛋白質在胃幽門螺旋桿菌中其他的生物功能。

First, we utilized NCBI database to align protein sequences and referred to Ang,Wen,and Allan studies, when the Helicobacter pylori were cultured on low pH medium, some gene expression in mRNA level would be induced or reduced. So the five genes were selected according to above-mentioned two
terms, they were HP0406, HP0751, HP0753, HP0979, and HP1399 genes in this thesis. These genes of H. pylori 26695 were cloned into the 5’ position of histidine-tag site in pQE30 vector, respectively. After transformation individual recombinant vector into E. coli SG13009, antibiotic resistant clones were screened with corresponding primers of various target gene by PCR and 1 mM IPTG-induction in individual culture at 37C for 3 h. All candidates expressed out soluble protein, however ,the HP0751 function was announced on acting as polar flagellar formation, but the HP0406 had a unknown function as a hypothetical protein from TIGR published. Individually, since #3 and clone#9 contains right sequence of HP0406 and HP0751genes in vector and could produce more soluble and stable protein than the others, the HP0406 and HP0751 were used in the rest of my study.
More recombinant (rec)- HP0406 and HP0751 proteins were induced from bacteria culture after 0.5 mM IPTG addition at 20℃ for 20 h. From 1 liter containing bacteria culture, almost 10 mg HP0406 protein and 25mg HP0751 protein could be purified through Ni2+-NTA affinity column. Newly prepared protein were stable easily in Tris, imidazole, and NaCl elution buffer at pH 8.0 for several weeks. Therefore, HP0406 and HP0751 proteins were stored in this buffer for further functional and structural studies in this research.
Rec-HP0406 and HP0751 proteins were used as antigen to produce antibodies against HP0406 and HP0751 protein in rabbits, individually. Anti-HP0406 and HP0751 antibodies from rabbit sera were prepared and tittered, respectively. About 2 ng HP0406 protein and 10ng HP0751 protein could be detected in 1:4000 and 1:3000 of antisera from rabbits after 5th boosted injection, separately. Using antibodies and western analysis, HP0406 and HP0751 proteins expression were recognized in H. pylori culture at different pH conditions.
H. pylori were cultured on Brucella agar plates at pH 5.5 and pH 7.2 for 48 h growth. The 1.5 fold HP0406 proteins were more at pH 5.5 than pH 7.0 from samples cultured, this result conformed to Ang’s study. However, HP0751 will not induce in acid medium. No significant different hp0751 from bacteria grown in different pH medium. This event did not correspond with Wen’study, but H. pylori were cultured on broth medium for 0 to 120min in Wen’s report condition. So cultured methods and times were different from our conditions, the result maybe showed difference.
HP0751 protein was predicted as polar flagellar G from TIGR database, it maybe associated the flagellar formation, in fact, the role was not clear in H.pylori. First, we seeked out the secondary structure of HP0751 protein by Circular Dichroism measurement. From the CD spectra, the HP0751 protein could be stored stably at pH5 to 8, and it explained the HP0751 protein was as a typical α-helix protein. In temperature resistance analysis, the violent change of secondary structure appeared over 82℃, and in urea denaturant experiment, we also obtained the Cm (transition point) value was in 4.68M.
To investigate the HP0751 protein whether it played a part in cell adherence.We took advantage of HP0751 protein and anti-HP0751 serum to co-culture with H.pylori and AGS cells by H.pylori dependence on urea assay measurement. But the data indicated the HP0751 seemed no direct relation for H.pylori adherence to AGS cells. In the future study, the isogenic mutant will be constructed to knock out fla G with kanamycin-cassette insertion and transformed into H. pylori cells. The adherence assay of wild type and mutant bacteria to AGS cells would be compared to analyze the real role of flaG in H. pylori.
HP0406 was annotated as a hypothetical protein, and had no functional study. We exploited alignment tools of protein sequence to find HP0406 protein possessed 13.2% identity to Purine NTPase from Methanocaldococcus jannaschii DSM 2661. Three methods were designed to prove HP0406 protein ATPase activity. First, we pointed out that the Mg2+ and ATP could change secondary structure of HP0406 protein in Circular Dichroism spectra, then ATP and Mg2+ also induced change of thermal stability. Following, HP0406 protein was examined the ATPase activity to observe whether it could release phosphate by Malachite-Green ATPase assay. The result indicated the HP0406 protein possessed ATPase activity and depended on Mg2+ concentration. Finally, we capitalized on separating from ATP and ADP by HPLC, this method could judge whether producing ADP. Just as our expecting, when the reaction incubated in the presence of 5mM Mg2+, the obvious peak appeared in the same as ADP standard position. Additionally, we found out ATPase activity of the HP0406 was induced by other divalent ions. The relative activity was as following : Mg2+＞Ca2+＞Mn2+＞Ni2+. In conclusion, these three methods were applied to prove that the HP0406 protein possessed ATPase activity in this study. The HP0406 protein was regarded as ATPase-like protein.

Reference:
Allan, E., C. L. Clayton, et al. (2001). "Characterization of the low-pH responses of Helicobacter pylori using genomic DNA arrays." Microbiology 147(Pt 8): 2285-92.
Ang, S., C. Z. Lee, et al. (2001). "Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray." Infect Immun 69(3): 1679-86.
Arora, S. K., B. W. Ritchings, et al. (1998). "The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible for mucin adhesion." Infect Immun 66(3): 1000-7.
Capdevila, S., F. M. Martinez-Granero, et al. (2004). "Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization." Microbiology 150(Pt 11): 3889-97.
Cascio, M., J. A. Mayor, et al. (2004). "Analysis of the secondary structure of the cys-less yeast mitochondrial citrate transport protein and four single-cys variants by circular dichroism." J Bioenerg Biomembr 36(5): 429-38.
Davey, M. J. and B. E. Funnell (1997). "Modulation of the P1 plasmid partition protein ParA by ATP, ADP, and P1 ParB." J Biol Chem 272(24): 15286-92.
Diamant, S., A. Azem, et al. (1995). "Effect of free and ATP-bound magnesium and manganese ions on the ATPase activity of chaperonin GroEL14." Biochemistry 34(1): 273-7.
Dunn, B. E., H. Cohen, et al. (1997). "Helicobacter pylori." Clin Microbiol Rev 10(4): 720-41.
Eichelberg, K., C. C. Ginocchio, et al. (1994). "Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins." J Bacteriol 176(15): 4501-10.
Hansen, T., C. Urbanke, et al. (1999). "The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization." Arch Biochem Biophys 363(1): 135-47.
Hsiao, N. W., D. Samuel, et al. (2003). "Mutagenesis study on the zebra fish SOX9 high-mobility group: comparison of sequence and non-sequence specific HMG domains." Biochemistry 42(38): 11183-93.
Kim, J. S., J. H. Chang, et al. (1999). "Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility." J Bacteriol 181(22): 6969-76.
Korchazhkina, O., G. Wright, et al. (1999). "No effect of aluminium upon the hydrolysis of ATP in the coronary circulation of the isolated working rat heart." J Inorg Biochem 76(2): 121-6.
Lanzetta, P. A., L. J. Alvarez, et al. (1979). "An improved assay for nanomole amounts of inorganic phosphate." Anal Biochem 100(1): 95-7.
Lin, H. J., C. L. Perng, et al. (2004). "Helicobacter pylori cagA, iceA and vacA genotypes in patients with gastric cancer in Taiwan." World J Gastroenterol 10(17): 2493-7.
Marshall, B. J. and J. R. Warren (1984). "Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration." Lancet 1(8390): 1311-5.
Martin, B. L., B. Li, et al. (2000). "Differences between Mg(2+) and transition metal ions in the activation of calcineurin." Arch Biochem Biophys 380(1): 71-7.
McGee, K., P. Horstedt, et al. (1996). "Identification and characterization of additional flagellin genes from Vibrio anguillarum." J Bacteriol 178(17): 5188-98.
McNamara, D. and C. O'Morain (1998). "Helicobacter pylori and gastric cancer." Ital J Gastroenterol Hepatol 30 Suppl 3: S294-8.
Moss, S. F. and S. Sood (2003). "Helicobacter pylori." Curr Opin Infect Dis 16(5): 445-51.
Namsaraev, E. A. and P. Berg (1998). "Interaction of Rad51 with ATP and Mg2+ induces a conformational change in Rad51." Biochemistry 37(34): 11932-9.
Niehus, E., H. Gressmann, et al. (2004). "Genome-wide analysis of transcriptional hierarchy and feedback regulation in the flagellar system of Helicobacter pylori." Mol Microbiol 52(4): 947-61.
Pawlowski, K., B. Zhang, et al. (1999). "The Helicobacter pylori genome: from sequence analysis to structural and functional predictions." Proteins 36(1): 20-30.
Rabaan, A. A., I. Gryllos, et al. (2001). "Motility and the polar flagellum are required for Aeromonas caviae adherence to HEp-2 cells." Infect Immun 69(7): 4257-67.
Razzera, G., J. Vernal, et al. (2004). "Expression, purification, and initial structural characterization of rat orphan nuclear receptor NOR-1 LBD domain." Protein Expr Purif 37(2): 443-9.
Rosberg, K., R. Hubinette, et al. (1991). "Studies of Helicobacter pylori in a gastric mucosa in vitro animal model." Scand J Gastroenterol 26(1): 43-8.
Sheng, Y., H. Ip, et al. (2003). "Binding of ATP as well as tetrahydrofolate induces conformational changes in Lactobacillus casei folylpolyglutamate synthetase in solution." Biochemistry 42(6): 1537-43.
Tomb, J. F., O. White, et al. (1997). "The complete genome sequence of the gastric pathogen Helicobacter pylori." Nature 388(6642): 539-47.
Wang, H. T., Z. H. Li, et al. (2005). "Effect of Helicobacter pylori VacA on gene expression of gastric cancer cells." World J Gastroenterol 11(1): 109-13.
Wen, Y., E. A. Marcus, et al. (2003). "Acid-adaptive genes of Helicobacter pylori." Infect Immun 71(10): 5921-39.