在許多物種體內之毒性相關(或稱為毒性攜帶)蛋白(virulence associated protein)已經被研究，而胃幽門螺旋桿菌26695株中的HP0315和HP0967基因，在TIGR網站上被預測為毒性相關蛋白D類。然而，HP0315和HP0967之間的關係以及生物功能仍然不清楚，目前並未證實這類蛋白的功能。因此，非常值得去研究與探討這兩個毒性相關蛋白的功能與彼此之間的關係。為了研究這兩個蛋白質的功能，重組的HP0315、HP0967蛋白分別被建構、純化及功能分析。HP0315和HP0967表現了和其他物種所含毒性相關蛋白(VapD)的序列約35%的同源性, 例如: Neisseria meningitidis和Haemophilus influenzae。而HP0315和HP0967彼此之間則存在了49.5%的同源性。HP0315和HP096的開放讀碼區(285bp和288)分別被選殖入pQE30表現載体，並送入大腸桿菌(SG13009)中表現該重組蛋白。利用Ni-NTA親和性色層分析法將N端接有六個Histidine的HP0315和HP0967蛋白純化出來的產量大約每升菌可以得到10毫克的蛋白質。根據超高速離心分析的結果顯示，HP0315 和HP0967 兩個蛋白質如果存在於pH 3 的環境下，皆會趨向於形成單體。HP0315在接近等電點(pI= 6.7)的時候，非常容易沉澱，反觀HP0967則相對的比較穩定。在pH 8的環境下，HP0315 和HP0967的alpha-螺旋組成比例分別為35.3%和52.7%。由CD的結果指出，在不同的pH環境下， HP0315在pH 3 的二級結構和在其他pH比較下，所呈現的改變比HP0967在同樣情況下來的多。在pH 3的環境下，HP0315 和HP0967的alpha-螺旋組成比例分別為66.3%和50.7%。在和AGS 細胞共同培養48小時中，當HP0315(經過30分鐘的活化處理)的蛋白質濃度達到150~300μg/ml，，可以造成大約20~40%的細胞死亡。HP0315 和HP0967兩個重組蛋白分別加入AGS細胞培養48小時後，再用流式細胞儀及螢光顯微鏡觀察。發現兩個重組蛋白的抗體可以互認對方的抗原。HP0315影響細胞生長的機制需要更進ㄧ步的去研究。HP0315蛋白在胃幽門螺旋桿菌中的表現量可由西方墨點法來偵測。當胃幽門螺旋桿菌呈現螺旋狀時，HP0315的表現量比其在呈現球狀時多(5:1)。當胃幽門螺旋桿菌培養在pH 5.5的環境時，HP0315的蛋白質表現會比培養在pH 7.2時的表現量多(10:1)。除此之外，在mRNA的表現上，HP0315 和HP0967皆為酸活化基因，但是在蛋白質表現上，只有HP0315會表現更多的蛋白質。

Virulence associated protein D has been studied in the chromosome or plasmid of many species, and two different putative virulence associated protein D, HP0315 and HP0967, in Helicobacter pylori 26695 were reported in TIGR. However, the relationship and bio-functions between HP0315 and HP0967 remained largely unknown elements, and there were no published data to support their functions. Therefore, it was worth to investigate the functions and study the interaction between each other or other virulence factors.
To study their functions, recombinant HP0315 and HP0967 were constructed, purified and assayed. The deduced amino acid sequence of either HP0315 or HP0967 shares less than 35.5% sequence identity with that of virulence associated protein D from several other species, for example Neisseria meningitidis MC58 and Haemophilus influenzae. About 49.5% identity was found in deduced amino acid sequence between HP0315 and HP0967.
The HP0315 and HP0967 open reading frame (285 bp and 288bp) were individually cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The results of N-terminally 6xHis-tagged HP0315 protein (12.58 kDa) and HP0967 (12.17 kDa) were purified by Ni–NTA affinity chromatography at a yield of 10 mg/L of bacteria culture, respectively.
Analytical ultracentrifugation has shown that the HP0315 protein exists as a monomer in solution pH 3 and HP0967 appeared as a monomer, too. The HP0315 is easy to precipitate below its pI (at pH 6.7), and the HP0967 is more stable at this pH. Both recombinant HP0315 and HP0967 protein dominated with alpha-helix structure (35.3% and 52.7%) at pH 8, respectively. At pH 3, more apparent change in secondary structure was observed in Circular Dichroism spectra for HP0315 than for HP0967. Both recombinant HP0315 and HP0967 protein dominated with alpha-helix structure (66.3% and 50.7%) at pH 3, respectively.
About 20 and 60 % cytotoxicity (corresponding to 80 and 40% survival fraction, respectively) were observed in Adenocarcinoma (AGS) cells after 48 hr co-culture with 150 and 300 μg/ml HP0315 protein (30 min acid-activation and neutralization previously) for 48 hr. Recombinant HP0315 (rec-HP0315) and recombinant HP0967 (rec-HP0967) proteins in AGS cells after co-culture were detected by Flow cytometry or immuno-fluorescence staining with antibodies against rec-HP0315 and rec-HP0967, respectively. Cross reaction to antibodies against (protocol a) HP0315 or (protocol b) HP0967 was also observed when AGS cells exposed to (protocol a) HP0967 or (protocol b) HP0315 for 24-48 hr. The mechanisms to affect cell proliferation of the virulence associated protein HP0315 remained to be further investigated.
HP0315 protein could be detected in various stages H. pylori bacteria by means of western analysis. More HP0315 protein in the 24 hr spiral form than 48 hr coccoid form from suspension at pH 7.2 (ratio = 5:1) was observed. Acid-inducible phenomenon (10-fold) was observed in bacteria cultured on agar plates for 48 h at initial pH 5.5, compared to those at initial pH 7.2. In addition, acid-inducible HP0315 mRNA or HP0967 was identified also from log stage H. pylori after 2 hr culture on brucella agar plates at pH 5.5.

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