Molecular and immunological characterisation of Acanthocheilonema viteae chitinase [Elektronische Ressource] / von Babila Julius Tachu
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Molecular and immunological characterisation of Acanthocheilonema viteae chitinase [Elektronische Ressource] / von Babila Julius Tachu

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155 pages
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Molecular and immunological characterisation of Acanthocheilonema viteae chitinase Dissertation zur Erlangerung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von Babila Julius Tachu (M.Sc. Biochemistry) geboren am 14.02.74 in Limbe (Victoria), Kamerun Präsident in Vertretung der Humboldt-Universität zu Berlin Prof. Dr. Hans Jürgen Prömel Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Thomas Buckhout, PhD Gutachter/innen: 1. Prof. Dr. Richard Lucius 2. Prof. Dr. Wolfgang Höhne 3. Prof. Dr. Rainer Borriss Tag der Einreichung: 29.11.2005 Tag der mündlichen Prüfung: 04.05.2006 Eidesstattliche Erklärung Hiermit erkläre ich an Eides statt, die vorliegende Dissertation selbständig und ohne unerlaubte Hilfe aufgefertigt zu haben. Bei einer Abfassung der Dissertation wurden keine anderen als die im Text aufgeführten Hilfsmittel benutzt. Berlin den 23.11.2005 Babila Julius Tachu Table of Contents 1 Introduction 1 1.1 Structure and function of chitinases 1 1.2 Filarial chitinases are potential targets for interventions 4 1.3 A. viteae as a model to investigate parasitological parameters of filarial diseases 6 1.4 Aims and objectives of the study 7 2 Results 9 2.1 Characterisation of A. viteae chitinase genes 9 2.1.

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Publié par
Publié le 01 janvier 2006
Nombre de lectures 30
Langue Deutsch
Poids de l'ouvrage 7 Mo

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Molecular and immunological
characterisation of
Acanthocheilonema viteae chitinase

Dissertation
zur Erlangerung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
Babila Julius Tachu (M.Sc. Biochemistry)
geboren am 14.02.74 in Limbe (Victoria), Kamerun

Präsident in Vertretung der Humboldt-Universität zu Berlin
Prof. Dr. Hans Jürgen Prömel
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Thomas Buckhout, PhD

Gutachter/innen: 1. Prof. Dr. Richard Lucius
2. Prof. Dr. Wolfgang Höhne
3. Prof. Dr. Rainer Borriss
Tag der Einreichung: 29.11.2005
Tag der mündlichen Prüfung: 04.05.2006





















Eidesstattliche Erklärung

Hiermit erkläre ich an Eides statt, die vorliegende Dissertation selbständig und ohne
unerlaubte Hilfe aufgefertigt zu haben.

Bei einer Abfassung der Dissertation wurden keine anderen als die im Text
aufgeführten Hilfsmittel benutzt.
Berlin den 23.11.2005
Babila Julius Tachu
Table of Contents
1 Introduction 1
1.1 Structure and function of chitinases 1
1.2 Filarial chitinases are potential targets for interventions 4
1.3 A. viteae as a model to investigate parasitological parameters of filarial
diseases 6
1.4 Aims and objectives of the study 7
2 Results 9
2.1 Characterisation of A. viteae chitinase genes 9
2.1.1 Determination of the gene structure of A. viteae chitinase genes 9
2.2 Comparative analysis of A. viteae genomic chitinase sequences with
genomic data bases of nematodes 18
2.3 A. viteae chitinase transcripts 21
2.3.1 Screening of A. viteae L3 and adult female cDNA libraries for chitinase
transcripts 21
2.3.2 Identification of A. viteae chitinase transcripts 23
2.4 Identification of homologous chitinase sequences from nematode
databases 25
2.5 Cloning, expression and purification of recombinant A. viteae chitinase 32
2.5.1 Cloning, periplasmatic/ cytoplasmatic expression and purification of N-
terminal portion of A. viteae chitinase in the expression vector pET 22 b (+) in E. coli 32
2.5.2 Activity of N-terminal chitinases 34
2.6 Immunisation studies with A. viteae chitinase in Meriones unguiculatus 35
2.6.1 Immunisation studies with A. viteae N-terminal chitinase fragment 36
2.6.2 Immunisation studies with synthetic peptides from the active site of A.
viteae chitinase 38
3 Discussion 42
3.1 Analysis of A. viteae chitinase genes and transcripts 42
3.2 Expression, purification and activity of N-terminal chitinase 47
3.3 Immunological control 49
3.4 Outlook 52
4 METHODS 53 4.1 Identification of recombinant cDNA and genomic clones from phage
libraries by plaque hybridisation 53
4.1.1 Preparation of plating bacteria 53
4.1.2 Plating of lambda phage 54
4.1.3 Titration of phage libraries 54
4.1.4 Amplification and cryopreservation of phage stocks 54
4.2 Screening of libraries 54
4.2.1 Screening of libraries with a DIG-labelled A. viteae chitinase probe 54
4.3 Purification and manipulation of DNA 56
4.3.1 Isolation of lambda DNA and sub-cloning of genomic inserts 56
4.3.2 Small scale Plasmid isolation from E. coli 57
4.3.3 Electrophoresis and detection of DNA on agarose gels 58
4.3.4 Isolation of DNA from agarose gels 58
4.3.5 Isolation and concentration of DNA from aqueous solutions 58
4.3.6 Isolation of high molecular weight genomic DNA from A. viteae 59
4.3.7 Determination of DNA Concentration 59
4.3.8 Restriction digestion of DNA 60
4.3.9 5' Dephosphorylation of digested DNA 60
4.3.10 Ligation of DNA fragments 61
4.3.11 mRNA isolation, reverse transcription and RT-PCR 62
4.3.12 Polymerase chain reaction (PCR) 62
4.4 Southern blotting 64
4.4.1 Digestion, electrophoresis, and blotting of genomic DNA 64
4.4.2 Radioactive labeling of A. viteae chitinase probe 65
4.4.3 Hybridisation and detection 65
4.5 Microbiological methods 66
4.5.1 Preparation of competent E. coli 66
4.5.2 Transformation of competent E. coli 66
4.5.3 Screening of bacterial colonies for plasmids/ recombinant plasmids 66
4.5.4 Bacteria cultures and long-term storage of bacterial stocks 67
4.5.5 Expression and purification of recombinant proteins from E. coli 67
4.6 Protein analytical methods 70 4.6.1 Determination of protein concentration 70
4.6.2 Sodium dodecly sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) 70
4.6.3 Chitinase activity assays 70
4.7 Immunochemical and immunological methods 71
4.7.1 Western blot 71
4.7.2 Coupling of peptides to KLH 71
4.7.3 Immunisation studies 72
4.8 Parasitological methods 72
4.8.1 Maintenance of the life cycle of A. viteae 72
4.8.2 Quantification of microfilarial load in blood of jirds 72
4.8.3 Isolation of filariae 72
4.9 Computer analysis and statistical methods 73
4.9.1 Analysis of DNA sequences 73
4.9.2 Statistical analysis 74
5 Materials 75
5.1 Commercial Kits and Enzymes 75
5.2 Laboratory Equipment and consumables 75
5.3 Synthetic oligonuceotides 76
5.3.1 PCR Primers 76
5.3.2 Sequencing primers 77
5.3.3 Primers used for sequencing A. viteae chitinase genomic clone 1 78
5.3.4 Primers used for sequencing A. viteae chitinase genomic clone 9 79
5.3.5 Primers used for sequencing A. viteae chitinase genomic clone 12 80
5.4 Plasmids 81
5.4.1 Cloning plasmids 81
5.4.2 Plasmids harbouring genomic and cDNA inserts: λ Dash II and λ Zap,
respectively 81
5.5 Buffers and solutions 83
5.5.1 Molecular biology 83
5.6 Media and buffers for E. coli 85
5.7 Protein and immuno- chemistry 86 5.7.1 SDS-PAGE 86
5.7.2 Western blot 87
2+
5.7.3 Buffers for native purification of N-terminal chitinase with Ni -NTA 88
2+
5.7.4 Buffers for denaturing purification of N-terminal chitinase with Ni -NTA 88
5.7.5 Synthetic peptides 88
5.7.6 Buffers and solutions for chitinase enzyme activity assay 89
5.7.7 Stock solutions 89
5.8 Antibiotic stock solutions 89
5.9 E. coli host strains and plasmids 90
5.10 Databanks, softwares and online services 90
5.11 Softwares 90
6 Abbreviations 91
7 References 93
8 Appendix 101
9 Publications and conference abstracts 144
Dedication I
Dedication

This work is dedicated to:

Elizabeth Abimwoe and the one born after she transisted
Feh Susanna
Jeremiah Tchinda and Tanyi-Mbombo
Acknowledgements II
Acknowledgements

I wish to extend my sincere gratitude to all those who contributed to the success of
this work and my sojourn in the Chair of Molecular Parasitology, Humboldt-University
of Berlin.
I wish to thank Professor Dr. Richard Lucius for his supervision, moral support and for
offering me the possibility to do my thesis at the Chair of Molecular Parastology.

I wish to thank Prof. Dr. Vincent P.K. Titanji and the members of his research team at
the Biotechnology Unit, University of Buea for their support and cooperation.

My particular thanks go to Prof. Dr. W. Höhne and Dr. Rudolf Volkmer-Engert of
Charité Medical faculty, Humboldt-University of Berlin for designing and synthesizing
the synthetic peptides, respectively.
My thanks also go to Dr. Jörg Hirzmann (University of Gießen) for providing the A.
viteae genomic library used in this study.

My sincere gratitude goes to Dr. Thomas Pogonka for the close supervision and for
regular and helpful discussions on the progress of this work.

I thank the staff and students at the Chair of Molecular Parasitology Berlin, for their
support and discussions. Karin Biermann, Grit Meusel and Bettina Sonenburg are
particularly thanked for their help in animal experiments. Many thanks go to Smitha
Pillai and Michal Sereda for their moral support and collaboration. My thanks go to
Chris, Jörg, Florian, Oliver and Katja for their interest in the work.

I also wish to thank my friends and family. Cécile Barra, Aysun Kara and Sabrina
Werl are thanked for reviving my down-trodden spirit.

My special gratitude goes to the German Academic Exchange Services (DAAD) for
financing my stay and study, as well as Dr. Bernd Kalinna and Dr. Susanna Hartmann
for employing me within the framework of their projects. Summary III
Summary
Chitinases are enzymes that break down chitin, a homopolymer of N-
acetylglucosamine (GlcNAc) to its monomers. They are important molecules in the
life cycle of parasitic nematodes representing important drug and vaccine targets. In
this thesis, three genomic chitinase sequences (I, II and III) were obtained by
characterising nine clones from a genomic library of Acanthocheilonema viteae.
Southern blots independently confirmed the existence of three chitinase genes in A.
viteae. The organisation of all three genomic sequences is similar, with the greatest
difference occurring in exons encoding

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