臺灣博碩士論文加值系統

黏質沙雷氏菌與尿道感染和菌血症等院內感染疾病有很大的關連性。由我們實驗室先前的實驗數據指出，黏質沙雷氏菌在MDCK細胞模式下，尿道感染菌株比血流感染菌株有更高的穿透能力和細胞毒性。由實驗數據指出，能穿透上皮細胞屏障或導致細胞毒性似乎是黏質沙雷氏菌能產生侵入性感染的重要毒性因子。跟穿透能力和細胞毒性能力相關的基因尚未被研究出來；因此這項研究目的是在尋找與黏質沙雷氏菌的穿透能力和細胞毒性相關的特定基因。我們建立了黏質沙雷氏菌cosmid基因資料庫來尋找跟穿透能力相關的基因，但在此cosmid基因資料庫內利用MDCK細胞模式並未找到跟穿透能力或細胞毒性相關的
基因。而由黏質沙雷氏菌跳躍子突變株的序列分析中指出，破壞纖毛組合蛋白基因papD有可能導致突變株的細胞毒性下降。希望我們的研究結果能有助於進一步了解這種重要病原菌的致病機制。

Serratia marcescens is associated with a large proportion of nosocomial infections, including urinary tract infections and bacteremia. Previous data from our laboratory indicated that the blood isolates of S. marcescens showed a significantly greater penetrative ability and cytotoxicity than the urinary isolates of S. marcescens by using a Madin-Darby Canine kidney cell model. It suggests that the ability to penetrate through epithelial barriers or to cause cytotoxicity seems to be important virulence factors in
causing invasive infections by S. marcescens. Genes associated with the expression of penetration and cytotoxicity have not yet been studied. The objective of this study was to identify specific genes that are associated with the expression of penetration and cytotoxicity in S. marcescens. To study the penetration-associated genes, a cosmid library was constructed but failed to identify any cosmid clone that demonstrated penetration/cytotoxicity in MDCK cells. Sequence analysis from a transpositioned mutant of S. marcescens indicated that the interruption of a pilus assambly protein gene, papD, may be responsible for the absence of the cytotoxicity in the mutant. It is hoped that our findings will provide a better understanding of the pathogenesis mechanisms regarding this important pathogen.

CONTENTS
Abstract …………………………………………………………… 1
中文摘要 ……………………………………………………………2
Chapter 1 Introduction …………………………………………… 3
1.1 Introduction of Serratia marcescens ……………… 4
1.2 Genomic Library ………………………………… 5
1.2.1 The lambda vector ………………………… 5
1.2.2 The cosmid vector ………………………… 6
1.3 Transposon ……………………………………… 7
1.4 Special Aims …………………………………… 9
Chapter 2 Materials ……………………………………………… 10
2.1 Bacteria Strains …………………………………… 11
2.2 Vectors and Transposon ………………………………… 11
2.3 Primers ……………………………………… 12
2.4 Chemicals and Reagents …………………………… 13
2.5 Nutrition Media …………………………………… 16
2.6 Solutions ………………………………………… 17
Chapter 3 Methods ………………………………………………… 20
3.1 Genomic Library Construction ……………………… 21
3.1.1 Bacteria and vectors ……………………… 21
3.1.2 Genomic DNA extraction ……………. 21
3.1.3 Preparation of DNA fragments ………… 22
3.1.4 Vector preparation and ligation ………… 23
3.1.5 Transduction ………………………………. 23
3.1.6 Coverage calculation ………………………… 24
3.1.7 Cell culture ………………………………… 25
3.1.8 Penetration assay …………………………… 25
3.1.9 Cytotoxicity assay ………………………… 26
3.2 Analysis of Transposon Mutagenesis …………… 27
3.2.1 Bacteria strains …………………………… 27
3.2.2 DNA purification ………………………… 27
3.2.3 Enzyme digestion and self ligation ……… 28
3.2.4 Transformation and rescue cloning ……… 29
3.2.5 Rescued plasmid DNA purification and
sequencing …………………………………… 29
Chapter 4 Results ………………………………………………… 31
4.1 Genomic Library Construction and Clone
Selection …………………………………………… 32
4.2 Analysis of Transposon Mutagenesis ……………… 33
Chapter 5 Discussion ……………………………………………… 35
5.1 Genomic Library Analysis ……………………… 36
5.2 Rescued Plasimd Analysis ……………………… 38
References ………………………………………………………… 40
Figures …………………………………………………………… 47

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