A genetic analysis to elucidate the function of the Plasmodium falciparum parasitophorous vacuole protein, PfPV1 [Elektronische Ressource] / corgelegt von Trang T. T. Chu

Trang

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

DECKBLATT















































A genetic analysis to elucidate the function of the Plasmodium

falciparum parasitophorous vacuole protein, PfPV1.






DISSERTATION
zur Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)



dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von



Trang T.T. Chu
aus Bac Ninh, Viet Nam



Marburg/ Lahn 2009































Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation am
angenommen.

Erstgutachter: Prof. Dr. Klaus Lingelbach
Zweitgutachter: Prof. Dr. Uwe G. Maier



Tag der mündlichen Prüfung am:




































To my Parents












TABLE OF CONTENTS

List of Figures...............................................................................................................v
List of Tables ...............................................................................................................vi
Abbreviations .............................................................................................................vii
1. Introduction..................................................................................................1
1.1. The life cycle of Plasmodium falciparum....................................................1
1.2. The parasite compartments ........................................................................6
1.3. The parasite induces alterations of the human erythrocyte.....................8
1.3.1. Structural alterations ..............................................................................8
1.3.1.1. Parasitophorous vacuole ................................................................8
1.3.1.2. The Maurer’s Clefts .......................................................................9
1.3.1.3. The tubulovesicular network..........................................................9
1.3.1.4. Knobs .............................................................................................9
1.3.2. Biochemical/ physiological alterations ................................................10
1.4. The parasitophorous vacuole- form and function...................................10
1.4.1. Invasion of erythrocytes and the PV formation ...................................10
1.4.2. The PV – a transit compartment...........................................................12
1.4.3. The PV – nutrition acquisition and regulation of the ionic environment.
..............................................................................................................13
1.4.4. The PV – preparation of merozoite egress...........................................14
1.5. Genetic manipulation of P. falciparum.....................................................16
1.5.1. Difficulties with P. falciparum transfection.........................................17
1.5.2. Functional analysis by integrative transfection....................................18
1.5.2.1. Gene targeting by single-crossover..............................................18
1.5.2.2. Gene targeting by double-crossover homologous recombination
using negative selection marker...................................................19
1.5.3. Other gene technique advances in P. falciparum.................................23
1.6. PfPV1 – a novel parasitophorous vacuole protein..................................24
1.7. Objective .....................................................................................................25
2. Materials and Methods..............................................................................28
2.1. Materials .....................................................................................................28
2.1.1. Equipment ............................................................................................28
2.1.2. Chemicals.............................................................................................29
i 2.1.3. Antibodies and working concentration ................................................31
2.1.4. Enzymes ...............................................................................................31
2.1.5. Molecular biological kits and reagents ................................................32
2.1.6. Cell culture materials ...........................................................................32
2.1.7. Cells and organisms .............................................................................32
2.1.8. Media and solutions .............................................................................33
2.1.8.1. Solutions for protein-based experiments .....................................33
2.1.8.2. Solutions for DNA-based experiments ........................................35
2.1.8.3. Bacteriological media ..................................................................38
2.1.8.4. Media and solutions for parasite culture and transfection ...........39
2.1.9. Plasmids ...............................................................................................41
2.1.10. Synthetic oligonucleotides ...................................................................42
2.2. Methods.......................................................................................................44
2.2.1 Bioinformatics methods .......................................................................44
2.2.2 Transfection of plasmid constructs ......................................................44
2.2.2.1 pHTK- PV1 ................................................................................44
2.2.2.2 pARL-DHFR-PV1g .....................................................................45
2.2.2.3 pARL-BSD-PV1g........................................................................45
2.2.2.4 pARL- PV1g...............................................................................46
2.2.2.5 pARL-mutPV1.............................................................................46
2.2.3 Parasite .................................................................................................50
2.2.3.1 Parasite culture.............................................................................50
2.2.3.2 Parasite transfection .....................................................................50
2.2.3.3 Drug selection of integrated plasmid containing parasites ..........51
2.2.3.4 Parasite cloning by limiting dilution............................................52
2.2.4 Monitoring transfectants: genetic analysis...........................................52
2.2.4.1 PCR analysis ................................................................................52
2.2.4.2 Southern blot analysis ..................................................................53
2.2.4.3 Pulsed field gel analysis (PFGE) .................................................54
2.2.5 Preparation of nucleic acid materials ...................................................54
2.2.5.1 Preparation of transfection plasmids............................................54
2.2.5.2 Preparation of P. falciparum genomic DNA ...............................54
2.2.5.3 Preparation of P. falciparum RNA ..............................................55
ii
DD2.2.5.4 Preparation of P. falciparum chromosome blocks.......................55
2.2.6 Methods on parasite proteins ...............................................................56
2.2.6.1 Fractionation of infected erythrocytes by SLO............................56
352.2.6.2 Labelling the newly synthesised parasite proteins with [ S]L-
methionine ...................................................................................56
2.2.6.3 Fluorescence microscopy.............................................................56
2.2.7 Immunoblotting analysis......................................................................57
2.2.8 Expression and purification of recombinant proteins ..........................57
2.2.8.1 Constructing the expression vector..............................................57
2.2.8.2 Over-expression and solubility test of recombinant proteins in E.
coli ...............................................................................................58
2.2.8.3 Purification of GST fusion protein from E. coli ..........................58
2.2.9 GST pull-down assay ...........................................................................59
3. Results.............................