| Summary: | 碩士 === 國立清華大學 === 生物科技研究所 === 97 === 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. Only microarray data indicated that HP0315 up regulated in H. pylori after 30 min culture at pH≦5.5 (Wen et al., 2003). The recombinant HP0315 protein was cloned, expressed in E. coli strain SG13009, purified by Ni-NTA beads column, and used to prepare antibodies for its functional studies previously in my laboratory (Lin, 2006). Increasing HP0315 mRNA and protein expression was found in bacteria cultured in acid condition for 1 hr and H2O2 exposure by RT-PCR and western blotting recently (Wang, 2007). However, its virulence-associated roles were not yet understood. Further characterization of this protein and its biological roles are currently investigated in this study.
RT-PCR and western blotting results showed that H. pylori have HP0315 mRNA and protein expression (Table 3). Furthermore, more HP0315 mRNA expression showed in samples from 0.5-1 h medium at pH 5.5 (2-fold) or 4.5 (1.6-fold) than that at pH 7.2 (Fig. 6A and Fig. 6B). More HP0315 protein expression was found in bacteria 0.5-h cultured in 4.5 (1.58-fold) medium than in pH 7.2 (Fig. 7A). However, no significant changes (1.2-~1.3-fold) on HP0315 protein expression was observed in bacteria after 1 h cultured in pH 4.5, 5.5, and 7.2 medium (Fig. 8A).
Significant HP0315 mRNA expression was observed in H. pylori after H2O2 exposure. At 20 and 40 mM treatment dose for 40 min, HP0315 mRNA increased 1.5- and 2.0-fold of untreated control (Fig. 9). Exposed bacteria at 0-75 mM H2O2 for 40 min, most HP0315 protein expression (2.43-fold) showed in samples after 45 mM H2O2 exposure (Fig. 10). Dose-response increase (1-~2.43-fold) on HP0315 expression was observed in samples exposed at dose 0-45 mM H2O2 for 40 min (Fig. 10A).
HP0315 mRNA expression was observed in H. pylori after SNP (NO donor) exposure for 1 h. At 400 and 800 uM exposure dose, HP0315 mRNA increased 1.1- and 1.6-fold of untreated control (Fig. 11). Exposed bacteria at 0-1000 uM SNP for 1 hr, most HP0315 protein expression (1.64-fold) showed in samples after 800 uM exposure (Fig. 10). Dose-response increase (1-~1.64-fold) on HP0315 expression was observed in samples exposed at dose 0-800 uM SNP for 1 hr (Fig. 10A). The LD50 dose was approximate 1000 uM SNP for either H. pylori or E. coli (Fig. 13). HP0315 protein expression from all samples treated in acid condition, medium having H2O2 or SNP was also confirmed by means of flow cytometry (Fig. 8B, 10B, 12 B).
Acid pretreated rec-HP0315 protein (37 ℃ for 15 min at pH 3.0; 66 ug/ml) would get into nucleus more quickly than that at pH 8 when co-cultured with AGS cells. About 48% acid pretreated protein reached AGS nuclei after 1 h co-cultured and remained 50-60% of them in nuclei after co-cultured with AGS cells for 2-6 h (Fig. 17). However, rec-HP0315 protein either having pH 3 acid-pretreatment or in pH 8 did not show cytotoxic effects and did not affect cell cycle progression on AGS cells (Fig. 18 and 19).
After first applied co-immunoprecipitation (co-IP) with antibodies against HP0315 and blotted with antibodies against VacA in H. pylori extract, then vice versa, then western blotting results indicated there has interaction between HP0315 and VacA (vacuolating cytotoxin, an important virulent factor in H. pylori, HP0887) within H. pylori lysate (Fig. 16). This suggested that HP0315 might play a VacA-association role in bacteria.
On the other hand, HP0315 protein does actually present in H. pylori bacteria. It was detected from 1/500 of total amount log phase untreated bacterial extract after IP, transferred to PVDF membrane, and coomassie blue staining. The protein sequence of a presumed HP0315 protein band (PDVF/staining) from this conduct was confirmed (Fig. 14 and Fig. 15).
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