Expression of the human respiratory syncytial virus (RSV) fusion protein (F) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter was analyzed by enzyme-linked immunosorbent assay (ELISA) in polyethylene glycol-transfected apple leaf protoplasts. In particular, we examined whether RSV-F gene expression could be enhanced by addition of a viral leader and a plant enhancer to the chimeric gene construct. Insertion of the 5'-untranslated leader from alfalfa mosaic virus (AMV) RNA 4 between the CaMV 35S promoter and the RSV-F gene increased viral expression by 5.5-fold compared to the construct without the leader. The addition of a transcriptional enhancer from the pea plastocyanin gene (PetE) upstream of the CaMV 35S promoter to a construct containing the AMV leader further increased RSV-F gene expression by 1.4-fold. Immunoblot assays showed that the RSV-F expressed in transfected apple protoplasts reacted with RSV-F monoclonal antibodies and was of the expected molecular mass of 68 kDa. These results demonstrated that the RSV-F recombinant protein was expressed in an antigenic form in plant cells. Furthermore, protein expression was enhanced by modifying the transfection vector using both a leader and an enhancer linked to a promoter.
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Acknowledgements We thank Nancy McCoppin of the United States Department of Agriculture/Agriculture Research Service for help with ELISA and immunoblotting, and Alexandre do Amaral of the Department of Natural Resources and Environmental Sciences for providing proliferating apple shoot cultures. This work was supported by grants from the Illinois Council on Food and Agricultural Research (C-FAR) and the State of Illinois Value-Added Program.
- Apple protoplasts
- RSV-F expression