免疫球蛋白A1 是黏膜層上含量最多的抗體，同時也是人體阻止微生
物入侵的第一道防線。然而有些致病菌，例如腦膜炎雙球菌、流行性
感冒嗜血桿菌，分泌IgA1 蛋白脢水解免疫球蛋白A1，破壞抗體的結
構及其原有的生物功能。因此在過去的研究中，IgA 蛋白脢被認為是
可能的致病因子。在此篇論文中，分別從非分型流行感冒嗜血桿菌及
腦膜炎雙球菌克隆IgA1 蛋白脢之基因並且完成全長的定序，和其它
已發表的IgA1 蛋白脢序列進行比對之後顯示有極高的相似度。同時
為了更加了解IgA1 蛋白脢對於非分型流行性感冒嗜血桿菌在宿主細
胞的黏附、移生及致病性有什麼影響，純化大量的重組IgA1 蛋白脢，
以A549 細胞株為實驗模組，利用adherence assay 探討IgA1 蛋白脢
是否能助於流行性感冒嗜血桿菌貼附至上皮細胞。實驗結果顯示，在
適當IgA1 蛋白脢作用濃度下有助於流行性感冒嗜血桿菌貼附至宿主
上皮細胞，但是其效應會因不同品系(strain)而改變。

IgA1 (immunoglobulin A1), a predominant immunoglobulin, is at the first defense line against microbial pathogens infection and invasion, to neutralize pathogenic antigens. Some bacterial pathogens, such as Neisseria meningitidis and Haemophilus influenzae, however, secrete site-specific IgA1 proteases to counteract with the human defense system. The protease is capable of cleaving at the hinge region of immunoglobulin A1 to destroy the structure and function of human IgA1, impairing the role of the immunoglobulin from the host defense. The protease has therefore been implicated as a putative virulence factor that contributes to bacterial colonization, but bacterial isolates from patients with invasive diseases contain both positive and negative IgA1 proteases. To clarify the role of IgA1 protease in bacterial infection, this project is designed to reveal the molecular mechanism of the protease in bacterial infection and colonization. To do this, iga genes encoding non-typable H. influenzae type 1, type 3 and Neisseria meningitidis type 3 IgA1 proteases were isolated, sequenced and then expressed in IgA1 protease-negative E. coli BL21 (DE3). The recombinant proteases have been purified to homogeneity using ion exchange chromatography. Comparison of the deduced amino acid sequences from non-typable H. influenzae IgA1 proteases with other published H. influenzae IgA1 protease revealed a high degree of homology. Sequence analysis indicates that both type 1 and type 3 non-typable H. influenzae IgA1 proteases lack α-protein in comparison with the iga from N. meningitidis. The role of IgA1 protease in relation to deposition and invasion has also been evaluated in human lung carcinoma cell (A549) model. The results suggest that the IgA1 protease plays a role in the adherence of H. influenzae on epithelial cell surface though the best effectiveness varies upon different pathogenic bacterial strains at different concentrations.

Contents
Abstract •••••••••••••••••••••••••••• 1
摘要••••••••••••••••••••••••••••• 3
Chapter 1 Introduction•••••••••••••••••••••• 4
1.1 Role of IgA1 protease••••••••••••••••••••• 4
1.2 Cleavage sites of IgA1 protease••••••••••••••••• 6
1.3 Structure of IgA1 proteases••••••••••••••••••• 6
1.4 Non-typable Haemophilus influenzae (NTHi) •••••••••••7
Chapter 2 Material and Methods••••••••••••••••• 11
2.1 Bacterial strains, plasmids, and growth conditions••••••••• 11
2.2 Preparation of genomic DNA•••••••••••••••••• 13
2.3 Cloning of non-typable H. influenzae and N. meningitidis iga gene••• 13
2.4 Preparation and transformation of competent E. coli cells••••••• 16
2.5 DNA sequencing of iga gene and sequence analysis••••••••• 17
2.6 Expression and purification of recombinant IgA1 protease•••••• 18
2.7 Purification of human IgG••••••••••••••••••• 18
2.8 IgA1 protease activity assay•••••••••••••••••• 19
2.9 Adherence assay••••••••••••••••••••••• 20
Chapter 3 Results•••••••••••••••••••••••• 22
3.1 Polymerase chain reaction amplification of non-typable H. influenzae and N. meningitidis iga gene••••••••••••••••••••• 22
3.2 Cloning of non-typable H. influenzae and N. meningitidis iga gene•• 22
3.3 DNA sequencing of iga gene and sequence analysis•••••••• 23
3.4 Expression and purification of recombinant IgA1 protease•••••• 26
3.5 Purification of human IgG••••••••••••••••••• 27
3.6 IgA1 protease activity assay•••••••••••••••••• 28
3.7 Adherence assay••••••••••••••••••••••• 28
Chapter 4 Discussion•••••••••••••••••••••• 32
4.1 DNA sequencing of iga gene and sequence analysis•••••••• 32
4.2 The ability of IgA1 protease on the enhanced-adherence of H. influenzae••••••••••••••••••••••••••• 33
4.3 The effect of human IgA1 or IgA1 protease on the adherence of H. influenzae••••••••••••••••••••••••••• 34
4.4 The effect of serum IgG or IgA1 protease on the adherence of H. influenzae••••••••••••••••••••••••••• 34
References•••••••••••••••••••••••••• 36











List of Tables
Table 1. Bacterial strains, and plasmids••••••••••••••• 12
Table 2. Design of PCR primers for amplification of iga gene•••••• 14

Figures and legends
Figure 1 Amino acid sequence of the hinge region of IgA1 and IgA2 differ mainly in the hinge region. ••••••••••••••••••••40
Figure 2 Peptide bonds in the human IgA heavy chain cleaved by known microbial IgA proteases. •••••••••••••••••••••41
Figure 3 Mechanism of processing and secretion of IgA1 protease. •••• 42
Figure 4 Polymerase chain reaction amplification of non-typable H. influenzae and N. meningitidis430 iga gene•••••••••••••••••• 43
Figure 5 Cloning of iga gene to the intermediate vector, pGEM-T easy Vector•••••••••••••••••••••••••••• 44
Figure 6 Sub- cloning of iga gene to the expression vector pTrcHisA••• 45
Figure 7 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified recombinant IgA1 protease•••••••••••••••••••• 46
Figure 8 IgA1 protease activity examined by 12% SDS-polyacrylamide gel electrophoresis••••••••••••••••••••••••• 47
Figure 9 IgA1 protease activity examined by 12% SDS-polyacrylamide gel electrophoresis••••••••••••••••••••••••• 48
Figure 10 Comparision of the deduced amino acid sequences of the NTHi500 IgA1 protease domain with other published H. influenzae IgA1 protease from strain Rd KW20 and HK61•••••••••••••••••••• 49
Figure 11 Comparison of NTHi500 iga beta domain with other H. influenzae type 1 IgA1 protease••••••••••••••••••••••• 50
Figure 12 The deduced amino acid sequences of the NTHi465 protease domain••••••••••••••••••••••••••••51
Figure 13 Comparison of NTHi465 IgA1 protease beta domain with other H. influenzae type 1 IgA1 protease•••••••••••••••••• 52
Figure 14 Neither NTHi465 nor NTHi500 IgA1 protease contain the α-protein••••••••••••••••••••••••••• 53
Figure 15 Effect of the recombinant IgA1 protease from NTHi500 on the relative adherence (ratio of the percentage of adherence with and without IgA1 protease) of four strains of H. influenzae••••••••••••••• 54
Figure 16 Effect of recombinant IgA1 protease or human antibody on the relative adherence (percentage of adherence compared with inoculum) of NTHi500••••••••••••••••••••••••••• 55
Figure 17 Effect of recombinant IgA1 protease or human antibody on the relative adherence (percentage of adherence compared with inoculum) of NTHi558••••••••••••••••••••••••••• 56
Figure 18 Effect of recombinant IgA1 protease or human antibody on the relative adherence (percentage of adherence compared with inoculum) of THi46679••••••••••••••••••••••••••• 57
Figure 19 Effect of recombinant IgA1 protease or human antibody on the relative adherence (percentage of adherence compared with inoculum) of THi 46644•••••••••••••••••••••••••••• 58

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