胃幽門螺旋桿菌嗜中性白血球激活蛋白，是胃幽門螺旋桿菌的一個重要致病因子，為了研究胃幽門螺旋桿菌嗜中性白血球激活蛋白是如何活化嗜中性白血球，此蛋白在大腸桿菌內表達，以兩次凝膠過濾層析純化，並且使用液相層析質譜儀，檢測單體的分子量。為了確認胃幽門螺旋桿菌嗜中性白血球激活蛋白的功能性，將藉由此蛋白處理的人類嗜中性白血球，以NBT染劑還原法和胺基苯二醯井化學冷光法分析，發現嗜中性白血球可以增加活性氧的釋放。此外我還發現一個新的功用，胃幽門螺旋桿菌嗜中性白血球激活蛋白能促進骨髓過氧化酶分泌。這些結果顯示，重組胃幽門螺旋桿菌嗜中性白血球激活蛋白，有能力活化人類嗜中性白血球。此結果可以應用在研究胃幽門螺旋桿菌感染時，胃幽門螺旋桿菌嗜中性白血球激活蛋白所扮演的角色，以及研發抗胃幽門螺旋桿菌的疫苗。

Helicobacter pylori neutrophil-activating protein (HP-NAP) is an important virulence factor of Helicobacter pylori (H. pylori) for pathogenesis. To investigate how HP-NAP activates human neutrophils, HP-NAP was expressed in Escherichia coli (E. coli) and purified by the two sequential gel filtration chromatographies. The molecular weight of HP-NAP monomer was confirmed by liquid chromatography/mass spectrometry (LC/MS). HP-NAP was used to stimulate human neutrophils for characterization of the purified recombinant HP-NAP. HP-NAP-stimulated neutrophils increased the production of reactive oxygen species (ROS) which was measured by using nitro blue tetrazolium chloride (NBT) dye reduction assay and luminol-dependent chemiluminescence assay. Interestingly, recombinant HP-NAP promoted the secretion of myeloperoxidase (MPO) in human neutrophils, which was a novel finding for the function of HP-NAP. These results indicate that recombinant HP-NAP may have ability to activate human neutrophils. Furthermore, recombinant HP-NAP in this expression system can be used to investigate the role of HP-NAP in H. pylori infection and to develop the vaccine against H. pylori.

Abstract vi
中文摘要 vii
誌謝 viii
Content ix
List of Figures xi
List of Tables xiii
Abbreviations xiv
Introduction 1
Materials and Methods 6
Reagents 6
Expression of Helicobacter pylori neutrophil-activating protein (HP-NAP) in Escherichia coli (E. coli) 7
Extraction and purification of HP-NAP 8
Determination of protein concentration 9
Liquid chromatography/mass spectrometry (LC/MS) 10
Isolation of human neutrophils 10
Chemiluminescence measurement of reactive oxygen species (ROS) 11
Nitro blue tetrazolium chloride (NBT) dye reduction assay 11
Measurement of myeloperoxidase (MPO) activity 12
Limulus amebocyte lysate (LAL) test 13
Statistical analysis 13
Results 15
Expression of Helicobacter pylori neutrophil-activating protein (HP-NAP) in Escherichia coli (E. coli) 15
Purification of recombinant HP-NAP 15
Molecular weight of recombinant HP-NAP monomer 22
Generation of reactive oxygen radicals (ROS) in neutrophils stimulated by recombinant HP-NAP 25
Release of myeloperoxidase (MPO) from neutrophils stimulated by HP-NAP 28
Discussion 34
References 39

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