在許多物種體內，毒性相關蛋白 (virulence-associated protein) 基因已廣泛地被發現在染色體及載體中。 現今在許多物種中也正在研究其功能。 Hp0315在胃幽門螺旋桿菌中並未被完整地研究。 而重組Hp0315蛋白已被clone和表現在大腸桿菌（SG13009）中並加以純化。此蛋白在此研究中可由以下實驗來加以探討。
根據超高速離心之結果，在pH 3之環境下較趨向5聚體，而在pH 8下趨向於24聚體。 Hp0315蛋白在pH 3~8 (25oC) 之二級結構變化在圓二光圖譜（Circular Dichroism）分析下，當蛋白處在較酸性條件下其β-sheet比率由4 % （pH 8）上升到20 % （pH 3）。 而當蛋白先放在37 oC下15分鐘之後，其β-sheet組成會明顯地由19 % （pH 8）增加到36 %（pH 3）。 相對地，其α-helix之比率卻無太大改變。
Hp0315進入AGS細胞可藉由流式細胞儀和免疫螢光顯微鏡來觀察。 有酸前處理之蛋白(37 oC for 15 min at pH 3.0) 進入細胞速度比沒處理的蛋白(pH 8.0)快上許多。 AGS細胞與有酸前處理的蛋白共同培養兩小時後，細胞中Hp0315的螢光強度約是對照組的兩倍。 而有酸前處理的Hp0315蛋白和細胞培養在4、6和8小時的時候，相對於對照組在細胞中有很明顯的Hp0315的螢光強度(9到13倍)。 直到24小時，有酸前處理的Hp0315蛋白在AGS細胞裡的量仍為沒前處理的兩倍。 而此現象在免疫螢光顯微鏡下也可見到相同現象。 SRB細胞存活分析顯示300 μg/ml之Hp0315蛋白與AGS細胞共同培養24小時後，約可引起30 %的細胞死亡。 但是有酸前處理之Hp0315蛋白卻沒有發現明顯的細胞毒性。
RT-PCR及西方點墨法結果顯示胃幽門螺旋桿菌有Hp0315 mRNA和蛋白表現。 此外，Hp0315 mRNA在pH 5.5 (1.48~1.26倍)或pH 4.5 (2.2~1.6倍)中培養1~2小時表現會高於在pH 7.2中。 胃幽門螺旋桿菌培養在pH 5.5和pH 4.5下一小時後， 其Hp0315蛋白表現量會高於培養在pH 7.2。 然而，當胃幽門螺旋桿菌培養在pH 7.2、5.5和4.5二和三小時後，並沒有明顯的Hp0315蛋白表現變化。
Hp0315 mRNA表現量在胃幽門螺旋桿菌經過H2O2處理後有明顯上升現象。 在25 & 50 mM H2O2所引起之氧化環境下30 min後，其Hp0315 mRNA之表現相對於未處理組上升了2.75到3.89倍。 而暴露在25 mM H2O2下0到60分鐘內，其Hp0315蛋白表現量在約40分鐘時表現量較多。 而在劑量上Hp0315蛋白表現量約在0到50 mM H2O2下40分鐘相對於未處理組有著1到1.5倍的上升變化。

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 □g/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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