臺灣博碩士論文加值系統

Table of Contents
指導教授推薦書 i
口委審定書 ii
致謝 iii
中文摘要 iv
Abstract v
Table of Contents vi
Content of Tables xi
Content of Figures xii

CHAPTER I. Introduction of Angiostrongylus cantonensis 1
1.1 History 1
1.2 Epidemiology 4
1.3 Life cycle in definitive host 5
1.4 Morphology 7
1.4.1 The first-stage larva 7
1.4.2 The second-stage larva 7
1.4.3 The third-stage larva 8
1.4.4 The fourth-stage larva 8
1.4.5 The fifth-stage larva (young adults) 9
1.4.6 Adults 10
1.5 Transmission and Symptoms 11
1.6 Diagnosis and Treatment 12

CHAPTER II. Transcriptomic study on genes expressions in the third- and fifth-stage larvae of Angiostrongylus cantonensis 15
2.1 Abstract 15
2.2 Introduction 16
2.3 Material and methods 16
2.3.1 Parasite and Animals 16
2.3.2 Total RNA Extraction 18
2.3.3 Construction of cDNA libraries and nucleotide sequencing 18
2.3.4 Sequences analysis and annotation 19
2.3.5 Statistical analysis 20
2.4 Results 20
2.4.1 cDNA libraries characteristics and cluster analysis from the third- or fifth-stage larvae 20
2.4.2 BLAST analyses of sequences from the third- or fifth-stage larvae 22
2.4.3 Pathway analysis of the third- or fifth-stage larvae 22
2.4.4 Comparative characteristics and analysis of cDNA libraries from the third- and fifth-stage larvae 23
2.4.5. Comparative BLAST homologies 23
2.4.6 Comparative functional annotation 24
2.5 Discussion 27

CHAPTER III. Identification and characterization of proteases from Angiostrongylus cantonensis 30
3.1 Abstract 30
3.2 Introduction 31
3.3 Material and methods 32
3.3.1 Cloning of an aspartic protease and an asparaginyl endopeptidase genes 32
3.3.2 Bioinformatic analysis 33
3.3.3 Quantitative Real-time PCR 34
3.3.4 Construction of the P. pastoris expression system for Ac-AEP 36
3.3.5 Expression and Purification rAc-AEP 37
3.3.6 SDS-PAGE electrophoresis and Western blot 38
3.3.7 In-gel trypsin digestion and MALDI-TOF MS 39
3.3.8 Gelatin zymography 40
3.3.9 Preparation of mice sera 41
3.3.10 Enzyme-linked immunosorbent assay 41
3.4 Results 42
3.4.1 Molecular cloning an aspartic protease from A. cantonensis 42
3.4.2 Molecular cloning an asparaginyl endopeptidase protease from A. cantonensis 43
3.4.3 Stage-specific distribution of Ac-asp 44
3.4.4 Stage-specific distribution of Ac-aep 45
3.4.5 Heterologous expression of the recombinant Ac-AEP 46
3.4.6 Proteolytic activity of rAc-AEP 47
3.4.7 Antigenic properties of rAc-AEP 47
3.5 Discussion 48

CHAPTER IV. Identification and characterization of microRNAs from the fifth-stage larvae of Angiostrongylus cantonensis 52
4.1 Absract 52
4.2 Introduction 53
4.3 Material and methods 55
4.3.1 Small RNA Extraction 55
4.3.2 Small RNA library construction and high-throughput sequencing 55
4.3.3 Bioinformatic analysis of small RNAs 56
4.3.4 Stem-loop reverse-transcription and Quantitative Real-time PCR 57
4.4 Results 59
4.4.1 Analysis of short RNAs 59
4.4.2 Identification of conserved miRNAs 60
4.4.3 Nucleotide bias of miRNAs 61
4.4.4 Phylogenic distribution 61
4.4.5 miRNAs expression at different developmental stages 62
4.5 Discussion 62

References 69
Tables 91
Figures 101
Appendices 117
Appendix 1: Buffers, media and electrophoresis gel 117
Appendix 2: The transcripts from the third-stage larvae (L3) and fifth-stage larvae (L5) of A. cantonensis 130
Appendix 3: The conserved mature miRNAs in the fifth-stage larvae of A. cantonensis 156
List of publications 162

Content of Tables
Content Page No.
Table 1-1. 23 species of the genus Angiostrongylus. 91
Table 2-1. The most abundant clusters comprised of transcripts from the L3 of A. cantonensis. 92
Table 2-2. The most abundant clusters comprised of transcripts from the L5 of A. cantonensis 93
Table 2-3. The top 20 metabolic pathways in the L3 of A. cantonensis mapped by Kyoto Encyclopedia of Genes and Genomes 94
Table 2-4. The top 20 metabolic pathways in the L5 of A. cantonensis mapped by Kyoto Encyclopedia of Genes and Genomes 95
Table 2-5. Transcripts from A. cantonensis encoding proteolytic enzymes 96
Table 2-6. Gene Ontology (GO) annotations of the clusters from A. cantonen 97
Table 2-7. Comparison the Gene Ontology (GO) annotations of the clusters from the L3 and L5 of A. cantonensis 98
Table 4-1. Identification of conserved miRNAs from the L5of A. cantonensis with 50 or more reads 99
Table 4-2. Phylogenic distributions of conserved miRNA families from the L5 of A. cantonensis with 50 or more read 100

Content of Figures
Content Page No.
Figure 1-1. Morphology of larvae of A. cantonensis 101
Figure 1-2. Morphology of adults of A. cantonensis 102
Figure 2-1. Distribution of the length of expressed sequence tags from the L3 and L5 of A. cantonensis 103
Figure 2-2. G+C contents of the expressed sequence tags from theL3 and L5 of A. cantonensis 103
Figure 2-3. Functional classification of the L3 and L5 of A. cantonensis showing the percentages of predicted genes according to their putative biological functions 104
Figure 3-1. The full length cDNA and deduced amino acid sequence of an aspartic protease from the L5 of A. cantonensis 105
Figure 3-2. Multiple amino acid sequence alignment of an aspartic protease of A. cantonensis with homologues from other nematodes 106
Figure 3-3. The full length cDNA and deduced amino acid sequence of an asparaginyl endopeptidase from the L5 of A. cantonensis 107
Figure 3-4. Multiple amino acid sequence alignment of an asparaginyl endopeptidase of A. cantonensis with homologues from other parasitic helminthes 108
Figure 3-5. Expression levels of an aspartic protease of A. cantonens in different developmental stages 109
Figure 3-6. Expression levels of an asparaginyl endopeptidase of A. cantonensis in different developmental stages 110
Figure 3-7. Purification and de-glycasylation of a recombinant asparaginyl endopeptidase from A. cantonensis in Pichia pastoris 111
Figure 3-8. Detection of proteolytic activities of a recombinant asparaginyl endopeptidase by gelatin zymography and different inhibitors 112
Figure 3-9. Comparison of levels of IgG against a recombinant asparaginyl endopeptidase in serum samples from BALB/c and ICR mice 113
Figure 4-1. Length distribution and classification of small RNAs from the L5 of A. cantonensis identified by a deep-sequencing approach 114
Figure 4-2. Nucleotide bias of conserved mature miRNAs from the L5 of A. cantonensis 115
Figure 4-3. Expression profiles of selected miRNAs in different developmental stages of A. cantonensis 116

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