Generation and characterization of single chain antibody fragments binding to the coat protein of grapevine leafroll-associated virus 2 and 3 [Elektronische Ressource] / vorgelegt von Martin Orecchia

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“Generation and characterization of single chain antibody fragments binding to the coat protein of grapevine leafroll-associated virus 2 and 3” Von der Fakultät für Mathematik, Informatik and Naturwissenschaften der Rheinisch- Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von M. Sc. Martin Orecchia aus Rosario, Argentinien Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer, RWTH Aachen Universitätsprofessor Dr. rer. nat. Dirk Prüfer, University of Münster Tag der mündlichen Prüfung: 18 Januar 2007. Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Publication from this work: Rainer Fischer, Greta Nölke, Martin Orecchia, Richard Twyman, Stefan Schillberg (2004) Improvement of grapevine using current biotechnology. Acta Horticulturae 652: 383-390. Table of Contents ____________________________________________________________________________________________________ I Introduction………………………………………...………………….1 I.1 Economic importance of grapevine…………….……………….…………………1 I.2 Grapevine leafroll associated virus 2………………………………….…………...2 I.3 leafroll associated virus 3……………………………….……………...4 I.4 Resistance mechanism to plant viruses…………………………….……………....5 I.4.
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01 janvier 2007

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“Generation and characterization of single chain antibody
fragments binding to the coat protein of grapevine leafroll-
associated virus 2 and 3”





Von der Fakultät für Mathematik, Informatik and Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften genehmigte Dissertation







vorgelegt von


M. Sc. Martin Orecchia
aus Rosario, Argentinien









Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer, RWTH Aachen
Universitätsprofessor Dr. rer. nat. Dirk Prüfer, University of Münster


Tag der mündlichen Prüfung: 18 Januar 2007.
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.







































Publication from this work:

Rainer Fischer, Greta Nölke, Martin Orecchia, Richard Twyman, Stefan Schillberg (2004)
Improvement of grapevine using current biotechnology. Acta Horticulturae 652: 383-390. Table of Contents
____________________________________________________________________________________________________

I Introduction………………………………………...………………….1
I.1 Economic importance of grapevine…………….……………….…………………1
I.2 Grapevine leafroll associated virus 2………………………………….…………...2
I.3 leafroll associated virus 3……………………………….……………...4
I.4 Resistance mechanism to plant viruses…………………………….……………....5
I.4.1 Natural resistance to plant viruses…………………………………………...5
I.4.2 Pathogen-derived resistance………………………………………………....6
I.4.3 Antibody-based resistance against viruses…………………………………..6
I.5 Recombinant antibody engineering………………………………………………..7
I.6 Phage display………………………………………………………………………9
I.6.1 Antibody phage display libraries…………………...………….…...……… 10
I.7 Antibody production in plants……………………………………………............ 12
I.8 Aim of this thesis………………………………………………………………… 14
I.8.1 Generation of GLRaV-2 coat protein specific scFv fragments……………. 14
I.8.2 of GLRaV-3 coat e14

II Materials and Methods………………………………......................... 17
II.1 Materials………………………………………………………….…………...…. 17
II.1.1 Chemicals and consumables…………………………………………...…. 17
II.1.2 Buffer, media and solutions……………………………………………....... 17
II.1.3 Enzymes and reactions kits……………………………………………..…. 17
II.1.4 Primary antibodies, secondary antibodies and substrates………………….. 18
II.1.5 Vectors……………………………………………………………………... 18
II.1.5.1 Bacterial expression vectors……………………………………….. 18
II.1.5.2 Plant expression vectors……………………………………………. 19
II.1.6 Bacterial material…………………………………………………………... 20
II.1.6.1 Bacterial strains…………………………………………………….. 20
II.1.6.2 Plants……………………………………………………………….. 20
II.1.6.3 Animals…………………………………………………………….. 20
II.1.6.4 Hybridoma mAb R19………………………………………………. 20
II.1.6.5 Phages……………………………………………………………… 21
II.1.6.6 Viruses……………………………………………………………... 21
II.1.7 Oligonucleotides…………………………………………………………… 21
ITable of Contents
____________________________________________________________________________________________________

II.1.7.1 Oligonucleotides used for PCR amplification of recombinant DNA
(E. coli and A. tumefaciens)…………………………………………... 21
II.1.7.2 used for synthesis of cDNA from total RNA
(in 5´-3´ orientation)………………………………………………….. 22
II.1.7.3 Sequences of murine V domain specific forward primers H
(in 5′-3 ′ orientation)…………………………………………………... 22
II.1.7.4 Sequences of murine V domaers L
(in 5′-3 ′23
II.1.7.5 Sequences of murine V -domain specific backward primers H
(in 5′-3 ′23
II.1.7.6 Sequences of murine κ V -domain specific backward primers L
(in 5′-3 ′24
II.1.7.7 Oligonucleotides used for sequencing DNA
(in 5′-3 ′ orientation)…………………………………………………... 24
II.1.7.8 Oligonucleotides used for the generation of the truncated versions
of the MBP-GLRaV-3 coat protein (in 5 ′-3 ′ orientation)……..……… 24
II.1.8 Matrices and membranes……………………………………………... 24
II.1.9 BIAcore reagents……………………………………………………. 25
II.1.10 Equipment and applications…………………………………….......... 25
II.2 Methods.…………………………………………………………………………. 27
II.2.1 Recombinant DNA technology…………………………………………….. 27
II.2.1.1 Preparation of electrocompetent E. coli cells…………………...…… 27
II.2.1.2 Transformation of E. coli by electroporation………………………… 27
II.2.1.3 PreparatiAgrobacterium cells………………. 27
II.2.1.4 Transformation of Agrobacterium by electroporation……………….. 28
II.2.1.5 ation efficiency …………………………………………... 28
II.2.1.6 Culturing of E. coli and glycerol stock preparation………………….. 29
II.2.1.7 Growth of recombinant A. tumefaciens and preparation of glycerol
stocks……………………………………………………………….. 29
II.2.1.8 Isolation of plasmid DNA from E. coli………………………………. 29
II.2.1.9 Agarose gel electrophoresis………………………………………….. 29
II.2.1.10 Preparative agarose gel electrophoresis……………………………… 30
II.2.1.11 Quantification of nucleic acids………………………………………. 30
II.2.1.12 PCR amplification……………………………………………………. 30
IITable of Contents
____________________________________________________________________________________________________

II.2.1.13 DNA sequencing……………………………………………………... 31
II.2.1.14 Sequence analysis……………………………………………………. 31
II.2.2 Generation and characterisation of transgenic plants……………………… 32
II.2.2.1 Preparation of recombinant A. tumefaciens………………………….. 32
II.2.2.2 Transient assay in tobacco leaves by vacuum infiltration………...…. 32
II.2.2.3 Vacuum infiltration of intact leaves………………………………….. 32
II.2.2.4 Agrobacterium-mediated stable transformation of Nicotiana
benthamiana………………………………………………………….. 33
II.2.2.5 Growth of N. benthamiana…………………………………………... 34
II.2.2.6 Preparation of total soluble proteins from plant leaves……………… 34
II.2.3 Expression and purification of recombinant proteins……………………… 35
II.2.3.1 Bacterial expression and purification of full length
MBP-GLRaV-3 CP and truncated MBP-GLRaV-3 CP versions......... 35
II.2.3.2 Bacterial expression and purification of full length GLRaV-3
coat protein...………………………………………………………….36
II.2.3.3 Large scale production and purification of soluble scFv by
IMAC………………………………………………………………… 37
II.2.4 Protein analysis…………………………………………………………….. 38
II.2.4.1 Quantification of proteins……………………………………………. 38
II.2.4.2 SDS-PAA gel electrophoresis and Coomassie brilliant blue
staining………………………………………….……………………. 38
II.2.4.3 Immunoblot analysis…………………………………………………. 38
II.2.4.4 Horseradish peroxidase coupling to scFvLR3cp-1…………………... 39
II.2.5 Immunization of mice……………………………………………………… 39
II.2.5.1 Determination of antisera titers by ELISA…………………………... 40
II.2.6 Construction of phage displayed scFv libraries and scFvLR2cp-35………. 40
II.2.6.1 Isolation of total RNA from spleen cells…………………………….. 40
II.2.6.2 of total RNA from cells hybridoma line mAb R19………… 40
II.2.6.3 First strand cDNA synthesis…………………………………………. 41
II.2.6.4 Construction of scFv libraries from immunized mice……………….. 41
II.2.6.5 of scFvLR2cp-35 from the hybridoma cell line R19….. 41
II.2.6.6 BstNI fingerprinting………………………………………………….. 42
II.2.7 Phage displayed antibody selection………………………………………... 42
II.2.8 Characterisation of scFv derived from immunized mice…………………... 43
IIITable of Contents
____________________________________________________________________________________________________

II.2.8.1 Soluble expressions of scFv fragments and direct ELISA…………… 43
II.2.8.2 Direct ELISA of selected scFv fragments…………………………… 43
II.2.8.3 Capture ELISA analysis using soluble scFv fragments……………… 43
II.2.8.4 Electrophoretic mobility shift assay (EMSA)………………………... 44
II.2.8.5 Characterization of scFv fragments by surface plasmon resonance…. 44
II.2.8.6 Epitope mapping……………………………………………………... 44
II.2.8.6.1 PEPSCAN analysis…………...……………………………… 44
II.2.8.7 Electron microscopy…………………………………………………. 45

III Results……………….……………………………………………..... 46
III.1 Generation and characterization of a GLRaV-2 specific scFv antibody………… 46
III.1.1 Total RNA isolation from hybridoma cells………………………………... 46
III.1.1.1 First strand cDNA synthesis and PCR amplification of variable
antibody domains…………………………………………………..… 47
III.1.1.2 Cloning of the scFvLR2cp-35 and sequence analysis……………….. 48
III.1.2 Generation and characterization of bacterial produced scFvLR2cp-35……. 51
III.1.2.1 of the bacterial expression construct and bacterial
expression…………………………………………………………….. 51
III.1.2.2 Reactivity of affinity purified scFvLR2cp-35 to virus particle………. 52

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