Strongyloides ratti [Elektronische Ressource] : identification, isolation and characterisation of heat shock protein 10 and heat shock protein 60 / eingereicht von Yasmina Tazir
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Strongyloides ratti [Elektronische Ressource] : identification, isolation and characterisation of heat shock protein 10 and heat shock protein 60 / eingereicht von Yasmina Tazir

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129 Pages
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YASMINA TAZIRSTRONGYLOIDES RATTI:IDENTIFICATION, ISOLATION AND CHARACTERISATION OF HEAT SHOCK PROTEIN 10 AND HEAT SHOCK PROTEIN 60INAUGURAL-DISSERTATIONzur Erlangung des Grades eines Dr. med. vet. beim Fachbereich Veterinärmedizinder Justus-Liebig-Universität Gießen édition scientifiqueVVB LAUFERSWEILER VERLAGISBN 3-8359-5471-7VVB LAUFERSWEILER VERLAGSTAUFENBERGRING 15D-35396 GIESSENTel: 0641-5599888 Fax: -5599890redaktion@doktorverlag.dewww.doktorverlag.de 9 7 8 3 8 3 5 9 5 4 7 1 7édition scientifiqueVVB LAUFERSWEILER VERLAGVVBYASMINA TAZIR STRONGYLOIDES RATTI HSP10/HSP60. Das Werk ist in allen seinen Teilen urheberrechtlich geschützt. Jede Verwertung ist ohne schriftliche Zustimmung des Autors oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme.1. Auflage 2009All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Author or the Publishers.st1 Edition 2009© 2009 by VVB LAUFERSWEILER VERLAG, GiessenPrinted in Germany édition scientifiqueVVB LAUFERSWEILER VERLAGSTAUFENBERGRING 15, D-35396 GIESSENTel: 0641-5599888 Fax: 0641-5599890 email: redaktion@doktorverlag.dewww.doktorverlag.

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YASMINA TAZIR
STRONGYLOIDES RATTI:
IDENTIFICATION, ISOLATION AND
CHARACTERISATION OF HEAT SHOCK PROTEIN 10
AND HEAT SHOCK PROTEIN 60
INAUGURAL-DISSERTATION
zur Erlangung des Grades eines
Dr. med. vet.
beim Fachbereich Veterinärmedizin
der Justus-Liebig-Universität Gießen
édition scientifique
VVB LAUFERSWEILER VERLAG
ISBN 3-8359-5471-7VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15
D-35396 GIESSEN
Tel: 0641-5599888 Fax: -5599890
redaktion@doktorverlag.de
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 4 7 1 7
édition scientifique
VVB LAUFERSWEILER VERLAGVVB
YASMINA TAZIR STRONGYLOIDES RATTI HSP10/HSP60. Das Werk ist in allen seinen Teilen urheberrechtlich geschützt.
Jede Verwertung ist ohne schriftliche Zustimmung des Autors
oder des Verlages unzulässig. Das gilt insbesondere für
Vervielfältigungen, Übersetzungen, Mikroverfilmungen
und die Einspeicherung in und Verarbeitung durch
elektronische Systeme.
1. Auflage 2009
All rights reserved. No part of this publication may be
reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical,
photocopying, recording, or otherwise, without the prior
written permission of the Author or the Publishers.
st1 Edition 2009
© 2009 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
édition scientifique
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: 0641-5599888 Fax: 0641-5599890
email: redaktion@doktorverlag.de
www.doktorverlag.deAus dem Institut für Parasitologie
der Justus-Liebig-Universität Giessen
Betreuer: Prof. Dr. C. Grevelding

und

dem Bernhard-Nocht-Institut für Tropenmedizin
Hamburg
Betreuer: PD Dr. K. Erttmann



Strongyloides ratti:
Identification, isolation and characterisation of
Heat Shock Protein 10 and Heat Shock Protein 60





INAUGURAL-DISSERTATION
zur Erlangung des Grades eines
Dr. med. vet.
beim Fachbereich Veterinärmedizin
der Justus-Liebig-Universität Giessen



eingereicht von

Yasmina Tazir
Tierärztin aus Wiesbaden


Giessen 2009

Mit Genehmigung des Fachbereichs Veterinärmedizin der
Justus-Liebig-Universität Giessen





















Dekan: Prof. Dr. Dr. habil. G. Baljer


Gutachter: Prof. Dr. C. G. Grevelding
PD Dr. K. D. Erttmann





Tag der Disputation: 23.07.2009
Table of Contents
I. Table of Contents
I. Table of Contents ____________________________________________________ 1
II. List of Figures _____________________________________________________ 6
III. List of Tables______________________________________________________ 8
1 Introduction ________________________________________________________ 9
1.1 Intestinal parasites____________________________________________________ 9
1.2 Strongyloides ratti 13
1.3 Heat shock proteins __________________________________________________ 15
1.3.1 Heat shock protein 60 _____________________________________________ 16
1.3.2 Heat shock protein 10 17
1.4 Objective of this thesis________________________________________________ 18
2 Materials and Methods _______________________________________________ 19
2.1 Reagents, disposals, instruments _______________________________________ 19
2.1.1 Solutions and buffers______________________________________________ 19
2.1.2 Media and additives 22
2.1.3 Enzymes _______________________________________________________ 23
2.1.4 Molecular weight standards_________________________________________ 24
2.1.5 Primer _________________________________________________________ 24
2.1.6 Plasmids 27
2.1.7 Bacteria and yeast strains __________________________________________ 35
2.1.8 Antibodies ______________________________________________________ 35
2.1.9 Computer-based sequence analysis___________________________________ 36
2.2 Strongyloides ratti life cycle and culturing________________________________ 37
2.2.1 Animals ________________________________________________________ 37
2.2.2 Host infection ___________________________________________________ 37
2.2.3 Host immunisation _______________________________________________ 37
2.2.4 Baermann technique ______________________________________________ 37
2.3 Molecular biological methods__________________________________________ 38
2.3.1 Production of competent bacteria (Nishimura et al 1990) _________________ 38
2.3.2 Transformation of DNA into bacteria _________________________________ 39
2.3.3 Purification of plasmid DNA _______________________________________ 39

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Table of Contents
2.3.3.1 Plasmid purification from bacterial cultures __________________________ 39
2.3.3.1.1 Mini-scale plasmid isolation___________________________________ 39
2.3.3.1.2 Alternative mini-scale plasmid isolation 39
2.3.3.1.3 Plasmid-DNA isolation from 100 ml cultures (Midi preps) ___________ 40
2.3.3.1.4 DNA-fragment purification 40
2.3.3.1.5 DNA-fragment extraction from agarose gels ______________________ 40
2.3.4 Total RNA isolation from S. ratti infectious larvae or parasitic females ______ 40
2.3.4.1 Total RNA isolation ____________________________________________ 40
2.3.4.2 Phenol/chloroform extraction _____________________________________ 41
2.3.4.3 Precipitation of RNA 41
2.3.5 gDNA isolation from S. ratti infectious larvae and precipitation ____________ 42
2.3.6 Polymerase chain reaction (PCR) ____________________________________ 42
2.3.7 5’and 3’ cDNAs amplification ______________________________________ 42
2.3.8 DNA agarose gel electrophoresis 43
2.3.9 Sequencing of DNA ______________________________________________ 43
2.3.10 Enzymatic manipulation of DNA 43
2.3.10.1 Restriction analysis ___________________________________________ 43
2.3.10.2 Plasmid DNA fragment________________________________________ 44
2.3.10.3 Enzymatic manipulation of vector DNA prior to ligation _____________ 44
2.3.10.4 Ligation of plasmid vector and insert DNA ________________________ 44
2.3.11 Photometric quantification of nucleic acids ____________________________ 45
2.3.12 Southern blot analysis _____________________________________________ 45
2.3.12.1 Random prime RNA labelling __________________________________ 46
2.3.13 Whole mount in situ hybridisation ___________________________________ 46
2.3.13.1 Tissue preparation____________________________________________ 46
2.3.13.1.1 Primary fixation of iL3 ______________________________________ 46
2.3.13.1.2 Fixation of iL3 onto slides 47
2.3.13.1.3 Kryo-block preparation 47
2.3.13.2 Dig-labelling of RNA probes 48
2.3.13.3 Prehybridisation, hybridisation and posthybridisation procedures_______ 48
2.3.13.4 Detection procedure __________________________________________ 49
2.4 Cell cultures ________________________________________________________ 49
2.4.1 Yeast cell culture_________________________________________________ 49
2.5 Protein biochemical methods 50
2.5.1 Mammalian two-hybrid____________________________________________ 50

2
Table of Contents
2.5.1.1 Transfection___________________________________________________ 52
2.5.1.2 Renilla luciferase assay__________________________________________ 53
2.5.2 Yeast two-hybrid _________________________________________________ 53
2.5.2.1 Filter and fluid ß-Galactosidase assays ______________________________ 55
2.5.3 Western blot analyses _____________________________________________ 55
2.5.3.1 Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) __ 55
2.5.3.2 Electropheric transfer (semi dry blot) and immunological detection of proteins
56
2.5.3.3 Coomassie staining of polyacrylamide gels __________________________ 56
2.5.3.4 Silver staining of polyacrylamide gels ______________________________ 56
2.5.3.5 Ponceau-S staining _____________________________________________ 56
2.5.3.6 Drying of polyacrylamide gels ____________________________________ 57
2.5.4 Protein expression ________________________________________________ 57
2.5.4.1 Expression of recombinant protein in E. coli _________________________ 57
2.5.4.2 Transfection and expression of recombinant protein in the Baculovirus system
57
2.5.5 Enzyme-linked immunosorbent assay (ELISA) 59
2.5.6 Mass spectrometry (Liquid Chromatography Electrospray Ionisation Tandem MS-
LC ESI MS/MS) _________________________________________________________ 59
2.5.6.1 Supernatant extraction___________________________________________ 59
3 Results____________________________________________________________ 61
3.1 Identification and cloning of the putative S. ratti HSP10 sequence ___________ 61
3.2 Characterisation of the S. ratti HSP10 transcript__________________________ 62
3.3 Identification and cloning of the putative S. ratti HSP60 transcript___________ 65
3.4 Characterisation of S. ratti HSP60 iL3 transcript _________________________ 66
3.5 Characterisation of putative S. ratti HSP10 and HSP60 transcripts of
parasitic females_____________________________________________________ 69
3.6 Localisation of putative HSP10 and HSP60 transcripts in S. ratti iL3 by
in situ hybridisation__________________________________________________ 70
3.7 Identification of HSP10 and HSP60 proteins in supernatants of S. ratti iL3 and
parasitic females 72
3.8 Characterisation of the putative S. ratti HSP10 and HSP60 genes ____________ 73
3.8.1 S. ratti HSP10 and HPS60 Southern blot analyses _______________________ 73
3.8.2 S. ratti HSP10 and HSP60 gene analyses ______________________________ 74
3.8.3 Analyses of the intergenic region of S. ratti ____________________________ 75

3
Table of Contents
3.9 Characterisation of the S. ratti HSP10 and HSP60 as putative binding partners
using two-hybrid approaches __________________________________________ 78
3.9.1 Mammalian two-hybrid analysis _____________________________________ 78
3.9.2 Yeast two-hybrid analysis 81
3.10 DNA immunisation with recombinantly expressed S. ratti HSP10 ____________ 85
3.11 Putative S. ratti HSP60 expression in E. coli ______________________________ 87
3.12 Putative S. ratti HSP10 expression in the baculovirus system ________________ 88
4 Discussion _________________________________________________________ 89
4.1 Identification of SrHSP10 and SrHSP60 _________________________________ 89
4.2 Characterisation of SrHSP10 and SrHSP60 ______________________________ 90
4.3 Characterisation of SrHSP10 and SrHSP60 genes _________________________ 92
4.4 Characterisation of SrHSP10 and SrHSP60 as binding partners _Fehler! Textmarke
nicht definiert.
4.5 The immunological role of SrHSP10 ____________________________________ 95
4.6 Outlook ____________________________________________________________ 97
5 Summary__________________________________________________________ 98
6 Appendix 99
6.1 Mammalian and yeast two-hybrid SrHSP60 bait-constructs ________________ 99
6.1.1 SrHSP60 cDNA sequences of the mammalian two-hybrid bait-constructs ____ 99
6.1.2 SrHSP10 cDNA sequences of the mammalian two-hybrid prey-constructs___ 100
6.1.3 SrHSP60 cDNA sequences of the yeast two-hybrid bait-constructs_________ 101
6.1.4 SrHSP10 cDNA sequences of the yeast two-hybrid prey-constructs ________ 101
6.2 SrHSP10 and SrHSP60 Southern blot probes____________________________ 102
6.2.1 SrHSP60 Southern blot probe cDNA sequences________________________ 102
6.2.2 SrHSP10 Southern blot probe cDNA sequence 103
6.3 SrHSP60 subfragment cDNA sequence for E. coli expression excluding the
mitochondrial leading sequence _______________________________________ 103
6.4 SrHSP60 cDNA and amino acid sequences including the 5’ and 3’ UTR _____ 104
6.5 SrHSP10 cDNA and amino acid sequences including the 5’ and 3’ UTR _____ 105
6.6 SrHSP60 and SrHSP10 parasitic female cDNA sequences _________________ 106
6.6.1 SrHSP60 parasitic female cDNA sequence____________________________ 106
6.6.2 SrHSP10 parasitic female cDNA sequence 108
6.7 SrHSP60 and SrHSP10 gDNA sequences _______________________________ 109

4
Table of Contents
7 References________________________________________________________ 110
8 Abbreviations and Units _____________________________________________ 119
8.1 Abbreviations______________________________________________________ 119
8.2 Units _____________________________________________________________ 120
8.3 Amino acids _______________________________________________________ 121

5