| Popis: |
Plants, or cell suspension cultures derived from them, are a promising platform for the production of biologics and pharmaceuticals. In this work transient expression utilising the pEAQ vector system was deployed for the expression of virus-like particles (VLPs) in Nicotiana benthamiana or N. tabacum BY-2 cell suspension cultures. The results presented in this thesis demonstrate the potential of plant systems for the production of VLP-based vaccines. VLPs of the fish virus, Nervous necrosis virus (NNV), were successfully produced in plants by transient expression of the coat protein. The protein self-assembled into T = 3 particles, which appeared to be morphologically identical to the wild-type NNV when analysed by high resolution microscopy but were devoid of nucleic acid. In addition, transgenic BY-2 cell suspension lines were generated expressing correctly assembled NNV VLPs. Poliovirus (PV)-like particles from all three PV serotypes, containing either the wt coat protein or coat proteins with stabilising mutations, were successfully expressed in plants. These were generated by co-expression of the structural polyprotein P1 and the proteinase 3CD. Sufficient quantities of purified particles could be obtained for structural and immunological analysis. Mice carrying the gene for the human PV receptor were protected from wild-type PV when immunised with the plant-made stabilised PV VLPs. Structural analysis of the stabilised mutant of PV3 at 3.6 Å resolution revealed a structure almost indistinguishable from wild-type PV3, with the stabilising mutations having no effective on the antigenic surface of the particle. To make the product more attractive to the vaccine industry, tobacco BY-2 cells have been successfully tested for the transient expression of the above-mentioned PV mutant VLPs using the cell-pack method. |
