臺灣博碩士論文加值系統

棘阿米巴角膜炎是一種由棘阿米巴原蟲透過角膜損傷引起的感染性角膜病。一般而言，大部分的棘阿米巴角膜炎高風險群的患者為長期配戴隱形眼鏡者。此外，致病性棘阿米巴會造成嚴重的後果，例如: 角膜受損、失明，最後甚至無法治療導致手術摘除患者眼球。然而目前針對棘阿米巴的診斷及治療方式都非具專一性且效果不佳，因此，我們希望找出該病原體具特異性的致病因子，未來能夠在棘阿米巴角膜炎患者或是潛在危險群的診斷或是治療的開發上提供幫助。為改善目前採集檢體需刮取病人角膜的方式，我們寄望從棘阿米巴分泌於外的蛋白質中找出具潛力的目標，提供未來檢驗及治療試劑開發之用。首先以比較蛋白質體學針對棘阿米巴ATCC標準蟲株與本土蟲株的分泌性蛋白質進行分析，從中挑選出各個不同蟲株間具共通性且表現恆定的蛋白質：M28氨肽酶，作為候選目標。根據棘阿米巴與細胞共同培養的結果顯示：棘阿米巴M28氨肽酶能促使宿主細胞走向細胞凋亡並裂解人體補體分子，我們推測此蛋白酶可能會幫助蟲體逃離宿主的免疫攻擊。在檢驗標的方面，我們已成功製備數株M28氨肽酶單株抗體，並使用西方點墨法及酵素免疫分析法對棘阿米巴進行其專一性及敏感度分析，結果顯示目前檢測方式仍有待改善。總而言之，我們成功地找出棘阿米巴分泌性M28氨肽酶並鑑定出其功能，期望未來能以棘阿米巴M28氨肽酶作為標的發展出對棘阿米巴角膜炎更有效的檢驗及治療方式。

Acanthamoeba keratitis (AK) is a serious disease caused by pathogenic Acanthamoeba through wounds of cornea; by and large, most high-risk patients catching AK wear contact lens generally over a long period of time. Moreover, pathogenic Acanthamoeba causes serious consequence that it makes the cornea turbid and defective to operate such as enucleation of eyeball. However, little is known about the diagnosis and treatment of AK. Hence, we purpose to provide the diagnostic and treatment procedure to AK patients according the specific virulence factor of Acanthamoeba. To improve diagnostic procedure, we purpose to fine the potential candidate from Acanthamoeba-secreted proteins (Asp) for diagnosis and treatment. First, we analyzed the secreted proteins of ATCC_30010 and clinical isolates to select the abundant consensus protein, Acanthamoeba M28 aminopeptidase (M28AP), as a diagnostic target. According to the result of in vitro co-culture model, we showed that the M28AP of Acanthamoeba can cause host cells apoptosis and degrade human complement protein to avoid parasite from human immunity attack. On diagnosis, the M28AP monoclonal antibodies have been produced to detect the sensitivity and specificity of Acanthamoeba isolates through antibody-based diagnostic tests, such as a western blot and an enzyme-linked immunosorbent assay (ELISA). Based on these results, the diagnostic test has yet to be improved. In conclusion, we have investigated successfully the molecular characteristics of M28AP. In the future, we anticipate M28AP to be the target for diagnosis and treatment of AK.

論文口試合格證明 I
中文摘要 II
Abstract III
致謝 IV
Abbreviation V
Contents VII
Table list XII
Figure list XIII
CHAPTER 1 Introduction 1
1.1 Acanthamoeba 1
1.1.1 Life cycle of Acanthamoeba 1
1.1.2 Genotyping 1
1.2 Acanthamoeba-associated human diseases 2
1.2.1 Granulomatous Amebic Encephalitis (GAE) 2
1.2.2 Acanthamoeba keratitis (AK) 2
1.3 The treatment and diagnosis of Acanthamoeba-associated human disease 3
1.3.1 Diagnosis of AK 3
1.3.2 Treatment of AK 3
1.4 The pathogenesis of Acanthamoeba 4
1.4.1 The virulence factor of Acanthamoeba 4
1.4.2 Acanthamoeba secreted extracellular proteases 6
1.4.3 Acanthamoeba secreted extracellular proteases in innate immunity 6
1.4.4 Acanthamoeba secreted extracellular proteases in complement 7
1.4.5 Aminopeptidases (metalloproteases) 8
CHAPTER 2 Rationale and experimental designs 10
2.1 Rationale 10
2.2 Experimental designs 11
CHAPTER 3 Materials and methods 12
3.1 Culture of Acanthamoeba Protozoa 12
3.1.1 Peptone-Yeast Extract-Glucose (PYG) medium 12
3.1.2 Page's modified Neff's amoeba saline (PAS) buffer 12
3.1.3 Passaging Acanthamoeba Cells 12
3.1.4 Acanthamoeba freezing and thawing 13
3.2 Culture of C6 glioma Rat cell line, A549 human cell line and human primary corneal epithelial cells 13
3.2.1 Dulbecco's Modified Eagle Medium (DMEM) High Glucose medium 14
3.2.2 Phosphate buffered saline (PBS) buffer 14
3.2.3 Passaging cell line 14
3.2.4 Cell freezing and thawing 14
3.3 Isolation of secreted proteins 15
3.3.1 Two-dimensional gel electrophoresis (2-DE) 15
3.3.2 Silver staining of proteins in polyacrylamide gels 16
3.3.3 Protein identification via MALDI TOF/TOF MS 17
3.3.4 Bioinformatic analysis 18
3.4 Cytopathic effect assay (CPE) 18
3.4.1 Giemsa stain 18
3.5 Total DNA extraction 18
3.6 Total RNA Isolation 19
3.7 cDNA Synthesis 20
3.8 Polymerase chain reaction (PCR) 20
3.9 Expression and purification of recombinant protein from E. coli 21
3.9.1 SDS-PAGE and Coomassie Blue staining 22
3.9.2 Western blot analysis 22
3.9.3 Enzyme-linked immunosorbent assay (ELISA) 22
3.10 DNA construction, cell culture and transient transfection 23
3.10.1 Biochemical Properties of recombinant protein M28AP 24
3.10.2 Ni-NTA-Atto Conjugates for recombinant protein M28AP and host cell interaction 24
3.10.3 Annexin V apoptosis assay 25
3.11 Amoebolysis activity of normal human serum (NHS) 25
3.12 Complement protein C3b and iC3b degradation assay 25
3.13 Immunofluorescence assay (IFA) 26
3.14 M28 mutant strain constructed by sgRNA and the Cas9 protein 26
CHAPTER 4 Results 28
4.1 Analysis of secreted protein profiles of non-pathogenic and pathogenic Acanthamoeba 28
4.2 Protein identification by MALDI TOF/TOF MS 28
4.3 Functional analysis of specific proteins using GO terms 29
4.4 Immunoblot and RT-PCR analysis of the secreted proteins 30
4.5 The novel genomic M28 aminopeptidase (M28AP) on Acanthamoeba 30
4.6 Expression and purification of M28AP 31
4.7 The rabbit polyclonal antibody production and detection of M28AP 32
4.8 The mouse polyclonal and monoclonal antibody production and detection of M28AP 32
4.9 The detection of anti-M28AP antibody on different antigens 33
4.10 The virulence of Asp induces cell damage in human primary corneal epithelial cells 33
4.11 The biochemical characterization of M28AP 34
4.12 The virulence of M28AP induces host cell damage 34
4.13 Acanthamoeba secretes protein to prevent human complement-mediated amoebolysis 35
4.14 Acanthamoeba secretes M28AP to degrade human complement C3b and iC3b 36
4.15 Acanthamoeba M28 mutant strains constructed by CRISPR-Cas9 cannot prevent complement-mediated lysis 37
CHAPTER 5 Discussion and future perspective 39
5.1 Secreted protein profiles on non-pathogenic and pathogenic Acanthamoeba strains 39
5.2 Treatment and virulence on the secreted protein of Acanthamoeba 40
5.3 Diagnosis using the M28AP antibody 41
5.4 The biochemical characterization of M28AP 43
5.5 The host cell apoptosis by M28AP 44
5.6 The innate immunity of M28AP 45
5.7 Future perspective 47
CHAPTER 6 References 49
APPENDIX I The DNA sequence of M28AP 90
APPENDIX II The RNA sequence of M28AP 94
APPENDIX III The protein sequence of M28AP 96
APPENDIX IV The pET-21b vector map 97
APPENDIX V The pcDNA 3.1/Zeo(+) vector map 98
APPENDIX VI The recombinant M28AP protein from E.coli identification report 99
APPENDIX VII The recombinant M28AP protein from CHO cell identification report 101
APPENDIX VIII The DNA sequence of M28AP mutant strains 103
APPENDIX IX The journal publication 104

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