Generation and characterisation of plant produced recombinant antibodies specific to LHRH for treatment of sex hormone dependent diseases [Elektronische Ressource] / vorgelegt von Richa Nath

Generation and characterisation of
plant produced recombinant antibodies
specific to LHRH for treatment of
sex hormone dependent diseases






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



vorgelegt von

Master of Science
Richa Nath
aus
Lucknow, Indien







Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer
Universitätsprofessor Dr. rer. nat. Fritz Kreuzaler




Tag der mündlichen Prüfung: 29 July 2003

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

CONTENTS
I INTRODUCTION..................................................................................................................1
I.1 LHRH structure ..................................................................................................................2
I.2 Physiological function of LHRH........................................................................................3
I.3 Mammalian isoforms of LHRH.........................................................................................5
I.4 LHRH manipulation...........................................................................................................6
I.5 LHRH analogues....7
I.6 Applications of pituitary suppression by LHRH manipulation .....................................8
I.6.1 Sex-steroid dependent cancers.......................................................................................8
I.6.1.1 Prostate cancer .......................................................................................................8
I.6.1.2 Gynaecological cancers .........................................................................................9
I.6.2 Miscellaneous ................................................................................................................9
I.7 Immunoneutralisation of LHRH .....................................................................................10
I.7.1 Active immunization against LHRH ...........................................................................10
I.7.2 Passive immunization against LHRH..........................................................................11
I.8 Recombinant antibodies ...................................................................................................12
I.8.1 Humanised antibodies..................................................................................................13
I.8.2 Phage display...14
I.8.3 Recombinant antibody fragments................................................................................15
I.9 Recombinant antibodies in therapy17
I.10 Plants as antibody production systems .........................................................................18
I.11 Research objectives.........................................................................................................19
II MATERIALS AND METHODS.......................................................................................22
ii
II.1 Materials...........................................................................................................................22
II.1.1 Chemicals and consumables.......................................................................................22
II.1.2 Buffers, media and solutions......................................................................................22
II.1.3 Matrices and membranes............................................................................................22
II.1.4 Enzymes and reaction kits..........................................................................................23
II.1.5 Primary antibodies, secondary antibodies and substrates ..........................................23
II.1.6 Vectors .......................................................................................................................24
II.1.6.1 Bacterial expression24
II.1.6.2 Plant expression..................................................................................................24
II.1.7 Biological material .....................................................................................................25
II.1.7.1 Bacterial strains25
II.1.7.2 Hybridoma P 2 .................................................................................................26 7 78
II.1.7.3 Plants ..................................................................................................................26
II.1.7.4 Animals...............................................................................................................26
II.1.8 Oligonucleotides ........................................................................................................26
II.1.8.1 Oligonucleotides used for synthesis of cDNA from mRNA ..............................26
II.1.8.2 Oligonucleotides complementary to FW4 of the mouse V Ab domain............26 L
II.1.8.3 Oligonucleotides complement1 of the mouse Vain27 L
II.1.8.4 Oligonucleotides complementary to FWouse V Ab domain ...........27 H
II.1.8.5 Oligonucleotides complement4 of the mouse Vain28 H
II.1.8.6 Oligonucleotides used for sequencing DNA ......................................................28
II.1.8.7 Oligonucleotides used for construction of the chimeric rAb P 2 ....................28 7 78
II.1.8.8 Oligonucleotide used for the construction of the diabody..................................28
II.1.8.9 Oligonucleotides for PCR based analysis of recombinant bacterial clones .......29
II.1.9 BIAcore reagents29
II.1.10 Equipment ................................................................................................................29
II.2 Methods ............................................................................................................................31
II.2.1 Hybridoma culture and purification of mouse mAb P 2 .........................................31 7 78
II.2.1.1 Subcloning of the hybridoma P 2 by limiting dilution ....................................31 7 78
II.2.1.2 Purification of mouse mAb P 2 from the hybridoma supernatant...................31 7 78
II.2.2 Recombinant DNA techniques...................................................................................32
II.2.2.1 Isolation of plasmid DNA from E. coli ..............................................................32
II.2.2.2 Analytical agarose gel electrophoresis ...............................................................32
II.2.2.3 Preparative gel electrophoresis...........................................................................32
II.2.2.4 Quantification of nucleic acids33
II.2.2.5 Growth and maintenance of bacterial strains .....................................................33
II.2.2.6 Bacterial transformation .....................................................................................33
II.2.2.6.1 Preparation of competent E. coli cells for heat-shock transformation........33
II.2.2.6.2 Transformation of E. coli by heat-shock.....................................................34
II.2.2.6.3 Preparation of electrocompetent E. coli cells .............................................34
II.2.2.6.4 Transformation of E. coli by electroporation..............................................34
II.2.2.6.5 Preparation of electrocompetent Agrobacterium cells................................34
II.2.2.6.6 Transformation of Agrobacterium by electroporation35
II.2.2.6.7 Determination of transformation efficiency of competent cells .................35
II.2.2.7 RNA Isolation and synthesis of first strand cDNA ............................................35
II.2.2.7.1 Total RNA extraction and mRNA preparation ...........................................35
II.2.2.7.2 cDNA synthesis ..........................................................................................36
II.2.2.8 PCR amplification ..............................................................................................36
II.2.2.8.1 PCR amplification of DNA fragments........................................................36
II.2.2.8.2 PCR based analysis of recombinant bacterial clones..................................37
iii
II.2.2.8.3 PCR amplification of variable domains of mouse mAb P 2 ....................37 7 78
II.2.2.8.4 Splice overlap extension PCR.....................................................................38
II.2.2.9 DNA sequencing and sequence analysis ............................................................38
II.2.2.10 Construction of anti-LHRH antibody fragments..............................................39
II.2.2.10.1 Construction of scFv P 2 ........................................................................39 7 78
II.2.2.10.2 Construction of diabody P 2 ..................................................................39 7 78
II.2.2.10.3 Construction of chimeric rAb P 2 ..........................................................39 7 78
II.2.3 Methods for protein analysis.............................................................................