In planta expression of anti-viral coat protein single-chain Fvs

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.