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Title: In planta expression of anti-viral coat protein single-chain Fvs
Author: Hendy, Simon Michael
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1998
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This thesis describes the expression of two anti-potyvirus single-chain Fvs in planta. The work was done with a view to obtaining functional single-chain Fv protein which could be used in virus diagnostics, and also which could possibly mediate plant virus resistance. The project aimed to achieve expression using two different approaches. In the first a potato virus X expression vector was used. The inclusion of single-chain Fv sequence did not appear to greatly influence the potato virus X infection process. Western blot analysis of vector-infected tissue showed single-chain Fv protein which migrated at an apparent molecular weight of 30 kDa. An apoplast targeting signal sequence was shown to significantly increase single-chain Fv accumulation levels. A single-chain Fv diabody, with a shortened variable domain linker, was also expressed in this way. In antigen binding tests single-chain Fv protein, expressed by the potato virus X vector, bound virus antigen with the same specific as the parental monoclonal antibodies. An association between the single-chain Fvs and negative control cucumber mosaic virus was detected. Further analysis however, indicated that this was not an antibody-antigen interaction. The second approach to obtaining single-chain Fv expression was to use Agrobacterium tumefaciens mediated plant transformation. A number of the resulting transgenic lines were shown to accumulate functional single-chain Fv protein. This accumulation was higher in those lines which included apoplast or chloroplast targeting sequences. However, chloroplast targeted single-chain Fv protein was not functional. Virus resistance tests, performed using both manual and aphid inoculation, showed that the accumulation of single-chain Fv protein in the transgenic plants produced no detectable potyvirus resistance effect.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available