声明
Table of Contents
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
摘要
ABSTRACT
CHAPTER 1 INTRODUCTION AND LlTERATURE REVIEW
1．1 Part 1 Avian Pathogenic Escherichia coli(APEC)
1．1．2 Diseases caused by avian pathogenic Escherichia coli(APEC)
1．1．3 Epidemiology of APEC infection
1．1．4 Characterization of APEC
1．1．5 Virulence factors of APEC strains
1．2 Part 2 Introduction to outer membrane porin proteins
1．2．1 Structure of porins
1．2．2 Role of bacterial porins
Objectives of the study
Reference
CHAPTER 2 ROLE OF OUTER MEMBRANE PROTEIN T IN PATHOGENICITY OF AVAIN PATHOGENIC ESCHERICHIA COLI
2．1．Introduction
2．2．Materials and methods
2．2．1 Reagents and equipment
2．2．2 Baeterial strains
2．2．3 Cell manipulations
2．2．4 DNA manipulations
2．2．5 Polyclonal antibodies production
2．2．6 Western blot analysis
2．2．7 Construction of ompT isogenic mutant
2．2．8 Complementation of ompT mutants
2．2．9 Adhesion and invasion assays
2．2．10 Virulence testing
2．2．11 Animal infection studies
2．2．12 RNA extraction and qRT-PCR analysis
2．2．13 Statistical analysis
2．3．Results
2．3．2．Expression and purification of recombinant protein OmpT
2．3．3 Deletion of ompT does not affect growth kinetics
2．3．4 Analysis of ompT expression
2．3．5 The ompT deletion reduced virulence in vivo
2．3．6 Effect of ompT during systemic infection in vivo
2．3．7 Adhesion and invasion were reduced in the isogenic mutant of ompT
2．3．8 Expression profiling of virulence genes
2．4 Discussion
Reference
CHAPTER 3 FUNCTIONAL ROLE OF ompF PORIN IN PATHOENESIS OF AVIAN PATHOGENIC ESCHERICHIA COLI
3．1 Introduction
3．2 Materials and methods
3．2．2 DNA manipulations
3．2．3 Expression and purification of recombinant protein OmpF
3．2．4 Polyclonal antibodies production
3．2．5 Western blot analysis
3．2．6 Construction of ompF mutant
3．2．7 Complementation of ompF mutant
3．2．8 Adhesion and invasion assays
3．2．9 Virulence testing
3．2．10 Animal infection studies
3．2．11 RNA extraction and qRT-PCR analysis
3．2．12 Statistical analysis
3．3 Results
3．3．2 Expression and purification OmpF porin
3．3．3 Deletion of ompF does not affect growth kinetics
3．3．4 Analysis of OmpF expression
3．3．5 ompF deletion attenuate virulence in vivo
3．3．6 Effect of ompF during systemic infection in vivo
3．3．7 Adhesion and invasion were reduced in the isogenic mutant of ompF
3．3．8 Expression profiling of virulence genes
3．4 Discussion
3．5 Conclusions
Referenee
CHAPTER 4 INACTIVATION OF ompC CONTRIBUTE TO DECREASED PATHOGENICITY AND BIOFILM FORMATION IN AVAIN PATHOGENIC ESCHERICHIA COLI
4．1 Introduction
4．2 Materials and methods
4．2．1 Bacterial strains，media and growth conditions
4．2．2 DNA manipuIations
4．2．3 Expression and purification of recombinant protein OmpC
4．2．4 Polyclonal antibody production
4．2．5 Western blot analysis
4．2．6 Construction of ompC isogenic mutant
4．2．7 Complementation of ompC mutant
4．2．8 Biofilm formation assay
4．2．9 Adhesion and invasion assays
4．2．11 Animal infection studies
4．2．12 RNA extraction and qRT-PCR analysis
4．3．1 Prevalence of the ompC in APEC O1 strain TW-XM
4．3．2 Expression and purification of OmpC
4．3．3 Deletion of ompC does not affect growth kinetics
4．3．4 Analysis of OmpC expression
4．3．5 The expression of ompC contributes to biofilm formation
4．3．6 ompC deletion attenuate virulence in vivo
4．3．7 Effect of ompC during systemic infection in vivo
4．3．8 Adhesion and invasion were reduced in the isogenic mutant of ompC
4．3．9 Expression profiling of virulence genes
4．4 Discussion
4．5 Conclusions
Reference
CHAPTER 5 EVALUATION OF nmpC IN DETERMINING PATHOGENCITY AND BIOFILM FORMATION IN AVIAN PATHOGENCI ESCHERICHIA COLI
5．1 Introduction
5．2 Materials and methods
5．2．1 Bacterial strains，media and growth conditions
5．2．2 DNA manipulations and genetic techniques
5．2．3 Expression and purification of recombinant protein nmpC
5．2．4 Polyclonal antibody production
5．2．5 Western blot analysis
5．2．6 Construction of nmpC isogenic mutant
5．2．7 Complementation of nmpC mutant
5．2．8 Biofilm formation assay
5．2．9 Bacterial adherence and invasion assay
5．2．10 Determination of lethal dose of the bacteria
5．2．11 Bacterial survival in vivo
5．2．12 Histological analysis
5．2．13 RNA extraction and qRT-PCR analysis
5．2．14 Statistical analysis
5．3 Results
5．3．2 Expression and purification of recombinant protein NmpC
5．3．3 Analysis of NmpC expression
5．3．4 Deletion of nmpC does not affects growth kinetics
5．3．5 The expression of nmpC contributes to biofiim formation
5．3．6 The nmpC deletion significantly reduced virulence in vivo
5．3．7 Effect of nmpC during systemic infection in vivo
5．3．8 Histopathological analysis
5．3．9 Adhesion and invasion were reduced in mutant nmpC
5．3．10 Expression profiling of virulence genes
5．4 Discussion
5．5 Conclusions
Reference
CHAPTER 6 RECOMBINANT PROTEINS VACCINE AGAINST INFECTION OF AVIAN PATHOGENIC ESCHERICHIA COLI
6．1 Introduction
6．2 Material and methods
6．2．1 Bacterial strains media and growth conditions
6．2．2 PCR amplification，cloning and sequencing
6．2．3 Expression and purification of recombinant proteins
6．2．4 Polyclonal antibodies production
6．2．5 Western blot analysis
6．2．6 Mouse immunization and challenge
6．2．7 Bacterial load during immunization
6．2．8 ELISA
6．2．9 Adherence inhibition assays
6．2．10 Statistical analysis
6．3．2 Expression and purification of recombinant proteins
6．3．3 Protection of mice against APEC TW-XM
6．3．4 Vaccination reduced the bacterial load
6．3．5 Serological responses
6．3．6 Adherence inhibition assays
6．4 Discussion
Reference
GENERAL CONCLUSIONS
PUBLICATIONS
INNOVATIONS
ACKNOWLEDGEMENT
南京农业大学;
