Selection of novel antigens from Leishmania spp. and design of live recombinant salmonella vaccines against experimental visceral leishmaniasis [Elektronische Ressource] / Juliane Schroeder. Gutachter: R. Lucius ; Tamás Laskay ; Maurice Gallagher

Juliane Schroeder

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• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 40
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Selection of novel antigens from Leishmania spp.
and design of live recombinant salmonella vaccines
against experimental visceral leishmaniasis



D i s s e r t a t i o n

zur Erlangung des akademischen Grades
d o c t o r r e r u m n a t u r a l i u m
( Dr. rer. nat.)


im Fach Biologie

eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Diplom-Ingenieurin (FH) Juliane Schroeder


Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Lutz-Helmut Schön


Gutachter: 1. Prof. Dr. R. Lucius
2. Prof. Dr. Tamás Laskay
3. Dr. Maurice Gallagher


Tag der mündlichen Prüfung: 29.09.2010

Acknowledgement

First, I would like to thank Dr. Toni Aebischer for giving me the great opportunity to
do my PhD in his lab at the University of Edinburgh.

Furthermore I would like to thank the members of the Leishmania lab, but especially
Dr. Athina Paterou and Dr. Toni Aebischer, without whom I would still be sitting at
the bench homogenizing foot pads and who both read and corrected my thesis and my
English. I also would like to thank Anna Lehle for excellent lab work.

It has been a pleasure to work at the 3IR at Edinburgh University and I would like to
thank everyone for providing such a friendly working atmosphere. Special thanks are
going to Dr. Graeme Cowan for showing me the magical world of protein
purification, Dr. Martin Waterfall for showing me how to work the LSR, Dr. Dianne
Murray for work at the confocal microscope, the collaborators at the University of
York, Prof. Paul Kaye and Dr. Najmeeyah Brown and finally the guys from the
animal house. Bette Boyle, who as lab fairy did provide me not only with clean lab
ware, tip boxes and solutions, but also with an open ear for the everyday lab sorrows,
a friendly word or the every so often chit-chat between experiments.

Mein besonderer Dank gilt meinen Freunden und meiner Familie, speziell meiner
Mutter, die mir das Studium in erster Linie ermöglicht hat, aber auch für die seelische
und moralische Unterstützung während der Doktorarbeit, auch wenn es bedeutet auf
mich des Öfteren verzichten zu müssen.

And last but certainly not least I would like to thank Andrew for his patience and
tolerance towards a very busy and stressed PhD student and also for providing me
with an excellent late night and weekend taxi service to and from Edinburgh
University.





II Table of contents
ACKNOWLEDGEMENT ..................................................................................................... II
ABBREVIATIONS ...............................................................................................................VI
ZUSAMMENFASSUNG.......................................................................................................IX
SUMMARY..............................................................................................................................X
1. INTRODUCTION................................................................................................... 1
1.1 LEISHMANIA LIFE CYCLE ........................................................................................ 1
1.2 LEISHMANIA RELATED DISEASES............................................................................ 2
1.2.1 CUTANEOUS LEISHMANIASIS..................................................................................... 3
1.2.2 VISCERAL LEISHMANIASIS 4
1.3 TREATMENT OF VISCERAL LEISHMANIASIS ........................................................... 5
1.4 IMMUNOLOGY OF LEISHMANIASES......................................................................... 6
1.5 LEISHMANIASES – A GROWING PROBLEM?............................................................ 8
1.6 ANTI-LEISHMANIAL VACCINES ............................................................................... 9
1.6.1 REQUIREMENTS......................................................................................................... 9
1.6.2 LEISHMANIZATION .................................................................................................. 10
1.6.3 ANTIGENS................................................................................................................ 11
1.6.4 ADJUVANT AND DELIVERY SYSTEMS ...................................................................... 11
1.7 ATTENUATED LIVE SALMONELLA AS VACCINE CARRIERS.................................. 12
1.8 LOCALISATION OF ANTIGEN IN THE SALMONELLA CARRIER ............................. 14
1.9 OBJECTIVES ........................................................................................................... 16
2. MATERIALS AND METHODS ......................................................................... 17
2.1 ELECTRICAL LABORATORY EQUIPMENT ............................................................. 17
2.2 CHEMICALS AND REAGENTS ................................................................................. 18
2.3 MEDIA..................................................................................................................... 19
2.4 BUFFERS AND SOLUTIONS...................................................................................... 21
2.5 BIOLOGICALS......................................................................................................... 26
2.6 MOLECULAR BIOLOGICAL METHODS................................................................... 30
2.6.1 SITE-DIRECTED MUTAGENESIS AND PRIMER DESIGN .............................................. 30
2.6.2 PREPARATION OF DNA FOR LIGATION 30
2.6.3 PCR PURIFICATION.................................................................................................. 31
2.6.4 DNA SEQUENCING 31
2.6.5 LIGATION................................................................................................................. 31
2.6.6 PREPARATION OF CHEMICALLY COMPETENT CELLS ............................................... 31
2.6.7 TRANSFORMATION 32
2.6.8 AGAROSE GEL ELECTROPHORESIS........................................................................... 32
2.6.9 DNA EXTRACTION FROM GELS ............................................................................... 32
2.6.10 COLONY PCR .......................................................................................................... 33
2.7 PROTEIN TECHNIQUES........................................................................................... 33
2.7.1 AMIDOBLACK PROTEIN QUANTIFICATION............................................................... 33
2.7.2 SDS-PAGE.............................................................................................................. 34
2.7.3 WESTERN BLOT ANALYSIS ...................................................................................... 35
2.8 PROTEIN PURIFICATION ........................................................................................ 36
2.8.1 INDUCTION OF RECOMBINANT PROTEIN.................................................................. 36
2.8.2 PURIFICATION OF SOLUBLE PROTEINS..................................................................... 36
2.8.3 PN OF PROTEINS FROM INCLUSION BODIES ........................................... 37
III Table of contents
2.9 PARASITE CULTURES ............................................................................................. 38
2.9.1 MAINTENANCE OF LEISHMANIA PROMASTIGOTE CULTURES .................................. 38
2.9.2 FREEZE/THAWING OF LEISHMANIA PARASITES ....................................................... 38
2.10 ANIMALS, IMMUNISATION AND PROTECTION EXPERIMENTS.............................. 39
2.10.1 MICE........................................................................................................................ 39
2.10.2 PREPARATIONS OF FROZEN SALMONELLA STOCKS FOR IMMUNISATION................. 39
2.10.3 PURIFICATION OF OUTER MEMBRANE VESICLES ..................................................... 40
2.10.4 DETERMINATION OF BACTERIAL FITNESS BY COLONISATION ASSAY ..................... 40
2.10.5 INFECTION OF MICE WITH L. MAJOR AND FOOT PAD MEASUREMENTS 41
2.10.6 D L. MAJOR BURDEN IN MURINE ORGANS .................................. 41
2.10.7 DETERMINATION OF HEPATOSPLENOMEGALLY AND L. DONOVANI BURDEN IN
IMPRESSION SMEARS ............................................................................................... 42
2.10.8 GIEMSA STAINING ................................................................................................... 42
2.10.9 BLOOD COLLECTION AND SERUM PREPARATION .................................................... 42
2.11 IMMUNOLOGICAL METHODS................................................................................. 43
2.11.1 ELISA ....................................................................................................