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

Ttazir, Yasmina

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

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: [phone] Fax: -5599890
[e-mail]
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.
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1. Auflage 2009
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st1 Edition 2009
© 2009 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
édition scientifique
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: [phone] Fax: [phone]
email: [e-mail]
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

1
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 Chara