臺灣博碩士論文加值系統

胃幽門螺旋桿菌養在含有血的培養基上時，可以發現到有溶血現象的發生。溶血蛋白是其中一個可能造成此現象的因子。然而，對於胃幽門螺旋桿菌的溶血蛋白，在臨床疾病及感染機制上所扮演的角色還有待進一步的研究。在本研究中，我們比較了來自胃癌、胃潰瘍、十二指腸潰瘍和非潰瘍性腸炎這四種疾病病人菌株的溶血性高低，藉以找出胃幽門螺旋桿菌溶血蛋白與疾病的相關性。其次，從具有最高及最低溶血性的菌株中複製出溶血蛋白的基因，並做進一步的特性分析。接著，製造溶血蛋白基因有缺陷的突變株，來評估突變株體外環境下的黏附力大小有無變化。之後，我們表現及純化溶血蛋白用來製作多株抗體。
在所有98個菌株中，我們並沒有發現到此蛋白質與疾病的發生有特別相關性。比較我們及國外兩個菌株(26695, J99)的溶血蛋白基因（tlyA），發現彼此之胺基酸序列有大於９0％的相同度，這暗示著TlyA有相當高的保守性，非常穩定的存在胃幽門螺旋桿菌中。溶血蛋白突變的突變株其溶血力與黏附力都變的比原生菌株低，這或許暗示著此蛋白質與細菌的黏附力有關連，會影響其感染能力高低。這些實驗數據暗示著，胃幽門螺旋桿菌溶血蛋白在導致疾病過程中所扮演的角色，溶血性與感染力有一定的關係性。

Helicobacter pylori shows hemolytic activity when cultured on blood agar plates. Hemolysin is one of potential factors in such an activity. However, the role of H. pylori hemolysin in the clinical pathogenesis and infection mechanism remains to be elucidated. In this study, we compared the hemolytic activities of strains from gastric cancer (GC), gastric ulcers (GU), duodenal ulcers (DU), and non-ulcer dyspepsia (Dys) patients to investigate the relationship among hemolysin and four different diseases. Secondly, we cloned and the tlyA gene that was considered as a key hemolysin in H. pylori from isolates with the highest and lowest hemolytic activity. Thirdly, we constructed the isogenic mutants for assessment of in vitro adherence and hemolytic activity. We also expressed and purified recombinant TlyA in E. coli for future biochemical studies.
Among these 98 cases, we found that there were no significant differences of the hemolytic activity among four groups. An alignment and comparison of tlyA between Taiwanese isolates and H. pylori 26695 and J99 showed > 90 % amino acid sequence identity, suggesting that tlyA is a relatively conserved gene in H. pylori. The hemolytic activity of the tlyA knockout mutant was reduced and so did the adherent activity, implicating that TlyA might be related with adherence and infection. These data collectively suggest a positive role of TlyA in H. pylori infection.

Contents
I. 摘要 1
II. ABSTRACT 2
III. 致謝 (ACKNOWLEDGEMENTS) 3
IV. CONTENTS 4
V. INTRODUCTION 6
DISCOVERY OF HELICOBACTER PYLORI (H. PYLORI) 6
EPIDEMIOLOGY OF H. PYLORI 6
DIAGNOSIS AND THERAPY OF H. PYLORI INFECTION 7
VIRULENCE OF H. PYLORI 8
HEMOLYSIN 9
THE AIM OF MY STUDY 11
VI. MATERIALS AND METHODS 13
BACTERIA STRAINS, PLASMIDS AND GROWTH CONDITION 13
DETERMINATION OF HEMOLYTIC ACTIVITY BY MICROPLATE ASSA 13
PREPARATION OF GENOMIC DNA OF H. PYLORI 14
ISOLATION OF PLASMID DNA (VIOGENE) 14
POLYMERASE CHAIN REACTION (PCR) 15
PURIFICATION OF PCR PRODUCT FROM AGAROSE GEL (VIOGENE)16
DNA SEQUENCING OF TLYA GENE BY CLONING WITH PGEM-T VECTOR SYSTEM (PROMEGA) 16
CONSTRUCTION OF H. PYLORI TLYA MUTANT STRAINS BY NATURAL TRANSFORMATION 17
DETERMINATION OF HEMOLYTIC ACTIVITY OF HEMOLYTIC MUTANT STRAINS BY LIQUID HEMOLYSIS ASSAY 18
EXPRESSION AND PURIFICATION OF RECOMBINANT H. PYLORI TLYA 19
PREPARATION OF POLYCLONAL ANTIBODY 20
WESTERN BLOTTING 20
CELL CULTURE 21
ADHERENCE TO AGS CELLS 21
COMPETITION ASSAY OF ADHERENCE TO AGS CELLS 22
STATISTICAL ANALYSIS 22
VII. RESULTS 24
RELATIONSHIP BETWEEN HEMOLYTIC ACTIVITY AND DISEASES 24
SEQUENCE ANALYSIS OF THE TLYA GENE 24
HEMOLYTIC ACTIVITY IN DIFFERENT H. PYLORI FRACTIONS 25
CONSTRUCTION OF AN ISOGENIC H. PYLORI TLYA MUTANT 26
REDUCED HEMOLYTIC ACTIVITY OF TLYA MUTANT 26
REDUCED ADHERENCE TO AGS CELLS OF TLYA MUTANT STRAINS 27
H. PYLORI TLYA CONFERS THE HEMOLYTIC ACTIVITY TO THE NONHEMOLYTIC E. COLI STRAIN JM109 27
EXPRESSION & PURIFICATION OF TLYA 28
VIII. DISCUSSION 31
IX. REFERENCE 35
X. FIGURES AND TABLES 41

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