| Summary: | 碩士 === 國立清華大學 === 生物科技研究所 === 95 === Virulence-associated protein gene has been extensively discovered in the chromosome or plasmid from many species. Its function has been studied in several species recently. Hp0315, annotated as virulence-associated protein D, was not yet completely studied in H. pylori. The recombinant Hp0315 protein was cloned, expressed in E. coli strain SG13009 and purified by Ni-NTA beads column recently. This protein was further characterized in current study.
Rec-Hp0315 protein in solution pH 3 appeared as a 5-mer but in solution pH 8 appeared to have 24-mer state according to analytical ultracentrifugation. The secondary structural change of rec-Hp0315 protein was measured with circular dichroism at pH 3~8 at 25 oC. More beta sheet content from 4 % (pH 8.0) to 20 % (pH 3.0) was observed when protein stayed in more acid environment (pH 3.0). More significant increase on beta sheet content showed from 19 % (pH 8.0) to 36 % (pH 3.0) in samples pre-incubated at 37 oC for 15 min. In contrast, no obvious change on its alpha helix content was observed on above mentioned conditions.
Hp0315 entrance into AGS cells was measured by flow cytometry and Immuno-fluorescence microscopy. Acid-pre-incubated Hp0315 protein (37 oC for 15 min at pH 3.0) got into cells much quicker than that from normal condition (pH 8.0). Two-fold of mean fluorescence intensity (MFI) was observed in AGS cells co-cultured for 2 h with acid pre-treated protein. After 4, 6 and 8 h co-culture with acid pretreated protein, significant MFI (9~13-fold) were found in AGS cells. Up to 24 h co-culture, the amount of pre-acid treated protein remained 2-fold of that normal protein in AGS cells. These phenomena were also observed in samples by means of immuno-fluorescent (IF) microscopy. SRB survival assays showed that 30 % cytotoxicity (or 70 % survival fraction) was observed in AGS cells after 24 h co-cultured with 300 �慊/ml rec-Hp0315 protein. No significant cytotoxicity was observed in same co-culture protocol with acid-pre-incubated rec-Hp0315 protein.
RT-PCR and western blotting results showed that H. pylori have Hp0315 mRNA and protein expression. Furthermore, more Hp0315 mRNA expression showed in samples from 1-2 h medium at pH 5.5 (1.48~1.26-fold) or 4.5 (2.2~1.6-fold) than that at pH 7.2. More Hp0315 protein expression was found in bacteria 1-h cultured in pH 5.5 (1.27-fold) and 4.5 (1.66-fold) medium than in pH 7.2. However, no significant changes on Hp0315 protein expression was observed in bacteria after 2 or 3 h cultured in pH 4.5, 5.5, and 7.2 medium.
Significant Hp0315 mRNA expression was observed in H. pylori after H2O2 exposure. At 25 and 50 mM treatment for 30 min, Hp0315 mRNA increased 2.75- and 3.89-fold of untreated control. Exposed to 25 mM H2O2 between 0 to 60 min, most Hp0315 protein expression (1.32-fold) showed in samples after 40 min exposure. Dose-response increase (1-1.5-fold) on Hp0315 expression was observed in samples exposed at dose of 0-50 mM H2O2 for 40 min.
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