臺灣博碩士論文加值系統

中文摘要
創傷弧菌為一嗜鹽性革蘭氏陰性桿菌， 對人類造成嚴重的傷口感染和致命性敗血症。在創傷弧菌的基因體被完整解出及註解後，我們在其基因體中找到一段包含多白氨酸重複序列基因。多白氨酸重複序列是一種常見的蛋白質反應區域，在某些微生物中也找到包含此重複序列的基因，例如Shigella flexneri中的IpaH家族，Yersinia specie中的YopM ，以及Listeria monocytogenes中的internalin家族，以上基因皆和細菌的致病性有關。為了探討多白氨酸重複序列基因在創傷弧菌中可能扮演的功能，我選殖此段基因並在大腸桿菌中重組以生產重組蛋白及製造抗體。我發現多白氨酸重複基因是一個外膜蛋白並且在生物一型和生物二型的創傷弧菌中皆會產生。此外，鐵對創傷弧菌而言是一非常重要的毒力調控因子，我發現多白氨酸重複蛋白在缺乏鐵的環境下會大量產生。利用同源染色體互換的技術，我製造了缺乏白氨酸重複蛋白的突變株，但在以腹腔注射為感染途徑的小鼠毒力測試中，突變株並不會降低對小鼠的毒力。在細胞感染實驗方面，我發現當野生株感染細胞株時，細胞會出現分散的型態，而當突變株感染細胞株時，細胞分散的型態已不明顯。我認為創傷弧菌中多白氨酸重複蛋白可能影響細胞株之間的連結，以幫助創傷弧菌具有侵略性的感染。

Abstract
Vibrio vulnificus is a halophilic, Gram-negative, curved rod-shaped bacterium. This bacterium has the ability to cause serious wound infections and often-fatal septicemia in human. After the genome of V. vulnificus has been completely sequenced and annotated, we have identified a gene named lrv that contains Leucine-rich repeats (LRRs) domain, a common property involving in protein-protein interaction. Several bacterial genes encoding an LRR protein have been reported, which include the IpaH family in Shigella fexneri, YopM in Yersinia species, and internalin family in Listeria monocytogenes, and are normally associated with pathogenesis. In order to explore the possible function of lrv in V. vulnificus, I cloned and expressed the gene, and produced recombinant Lrv and an anti-Lrv antibody. I found that Lrv is an outer membrane protein and expressed in both biotypes 1 and 2 of V. vulnificus. Lrv expression is up regulated in iron-deficient condition. I have constructed a lrv deletion mutant by the allelic exchange technique, and found that the LD 50 of the mutant did not change in animal intraperitoneal (i.p.) model of infection. The mutant strain appeared to be less able to cause cell dispersion compared to the parental strain. In conclusion, our data suggest that Lrv may disturb the cell-cell junction and facilitate the invasive infection of V. vulnificus.

CONTENTS
ABSTRACT (CHINESE)……………………………………………………… i
ABSTRACT……………………………………………………………………… ii
ACKNOWLEDGMENT…………………………………………………………iii
CONTENTS……………………………………………………………………….iv
TABLE CONTENTS…………………………………………………………….vi
FIGURE CONTENTS…………………………………………………………..vii
INTRODUCTION
1.The organism…………………………………………………………………. 1
2. Biotypes……………………………………………………………………… 1
3. Host susceptibility…………………………………………………………… 2
4. Clinical symptoms and treatment……………………………………………. 2
5. Virulence of V. vulnificus…………………………………………………………… 3
5.1. Polysaccharide capsule and LPS……………………………………….. 3
5.2. Pili…………………………………………………………………………3
5.3. Hemolysins…………………………………………………………………4
5.4. Metalloprotease…………………………………………………………….4
5.5 Enterotoxin………………………………………………………………….5
5.6 Other Extracellular Proteins……………………………………………….. 5
5.7 Iron………………………………………………………………………….5
6. This study……………………………………………………………………….6
MATERIAL AND METHODS
Bioinformatic analysis……………………………………………………………8
Bacterial strains, mammalian cell lines and plasmids …………………………...8
Media, cultivation and storage of bacteria and cell lines……………………….. 8
RT-PCR……………………………………………………………………… ….9
Genomic DNA preparation and PCR……………………………………………9
Construction of expression plasmids……………………………………………10
Protein expression, purification and refolding…………………………………..10
Polyclonal antibody production…………………………………………………10
Sub-cellular fractionation……………………………………………………….11
Western blot analysis……………………………………………………………11
Cell line assay with treatment of recombinant Lrv……………………………..12
Construction of lrv deletion mutant in V. vulnificus YJ016……………………12
Southern hybridization ………………………………………………………13
Determination of bacterial density ……………………………………………13
Cell line infection assay ………………………………………………………14
Virulence assay…………………………………………………………………14
RESULTS……………………………………………………………………….. 15
Features of lrv……………………………………………………………………………15
lrv is expressed in V.vulnificus…………………………………………………….15
Production of Lrv protein in E. coli……………………………………………………16
Generation of a Lrv-specific polyclonal antibody……………………………….16
lrv expression level increased as the bacterium grows…………………………...16
Both biotypes 1 and 2 of V. vulnificus express lrv……………………………….17
lrv expression is associated with iron deficiency………………………………...17
Lrv is an outer membrane protein …………………………………………….17
Recombinant Lrv alters MDCK cell morphology………………………………..18
Isolation of V. vulnificus lrv -deletion mutant……………………………………18
The ability to cause cell dispersion is reduced in the V. vulnificus lrv deletion
mutant……………………………………………………………………………19
The virulence of V. vulnificus lrv deletion mutant is not reduced……………….19
DISCUSSION…………………………………………………………………. 20
REFERENCES………………………………………………………………… 23
TABLES AND FIGURES ………………………………………………………28

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