| Summary: | 碩士 === 國立清華大學 === 生物科技研究所 === 93 === 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.
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