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Studies on cloned antibody genes for virus resistance in plants

Dwyer, Geoffrey Ian (1998) Studies on cloned antibody genes for virus resistance in plants. PhD thesis, Murdoch University.

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Viruses are important pathogens of plants. The consequence of virus infection is of most concern in the large monocultural crops produced in the agricultural and horticultural industries. Infection usually results in a significant reduction in crop yield and is responsible for considerable economic loss. Inherent resistance to plant viruses is not always available in the germplasm used in breeding programs. Therefore, synthetic resistance strategies need to be investigated so those economically important crops can be provided protection against plant viruses. The initial aim of this study was to construct a single-chain variable fragment antibody (scFv) that recognises virions of clover yellow vein virus (C1YVV), and to assess its potential for conferring resistance to C1YVV in plants. The recombinant antibodies were constructed from mRNA purified from a mouse monoclonal hybridoma culture (MablO) that reacts specifically with coat protein virions of C1YVV and other members of the bean yellow mosaic potyvirus group. The light chain variable domain (VL), the heavy chain variable domain (Vh), the light chain Fab domain (CK), and the heavy chain Fab domain (CH) were amplified from MablO antibody mRNA by reverse transcriptase-polymerase chain reaction (RTPCR). The four antibody domains were cloned individually into an intermediary cloning vector pGEM-T. The cDNA sequences for all four inserts was determined to ensure that the amplified fragments were in-frame and that a full-length protein was produced. The two variable domains were amplified by PCR from the pGEM-T plasmid, purified and assembled into a scFv using PCR by overlap extension. The scFv PCR product was subcloned into pGEM-T, the cDNA sequence obtained to ensure that nucleotide misincorporation had not occurred during PCR and that the insert remained in-frame. The insert was digested out of pGEM-T and transferred into the E. coli phagemid expression vector pHEN. The CK and CH Fab domains were amplified by PCR from the pGEM-T plasmid, purified and cloned into the E. coli phagemid expression vector pFAB60. The pHEN/MablO scFv and pFAB60/Mab 10 Fab phagemid vectors were transformed into two E. coli strains and analysed for protein expression by western blot and antigen binding activity by enzyme-linked immunosorbent assay (ELISA). Preliminary investigations showed that the Fab fragments were produced in E. coli but there was no evidence for the expression of the scFv. However, the results of ELISA analysis indicated that both recombinant antibodies bind antigen and may therefore retain the antigen binding capacity of the parent antibody. For expression in plants, the scFv gene was amplified by PCR from the pHEN/MablO scFv plasmid using three sets of primers that incorporated a range of signal sequences. The signal sequences were designed so that the scFv would be targeted to, and expressed in either the cytoplasm. the endoplasmic reticulum or the apoplast. The three targeted scFv sequences were cloned into pGEM-T and sequenced to ensure that they remained in-frame. All three scFv inserts were then cloned into the plant binary vectors pART27 and pPZBIOlexp for transfonnation into tobacco and lupins, respectively. The binary vectors were transformed into Agrobacterium tumefaciens and are awaiting transformation into An additional experiment plants. was undertaken to develop a recombinant bean yellow mosaic virus (BYMV) protein to use for producing a scFv that was specific to BYMV. The RNA-dependent RNA polymerase gene (Nib) was chosen because of its importance in virus replication. The BYMV Nib gene sequence was amplified from RT-PCR from genomic BYMV-MI RNA. The PCR product was cloned into pGEM-T, digested out and transferred to the prokaryotic protein expression vector pQE-30. The entire Nib gene (1554 bp) was sequenced to ensure that it was translatable and that it was in-frame with the translational start codon of pQE-30. The recombinant plasmid was transformed into E. coli M15(pREP4), a strain used for protein production, and induced for expression. Following induction, a crude E. coli lysate preparation was analysed by SDSPAGE for expression of recombinant Nib protein. There was no evidence for expression.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Western Australian State Agricultural Biotechnology Centre
Division of Science
School of Biological Sciences and Biotechnology
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Jones, Michael
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