Enhanced expression of the human respiratory syncytial virus-F gene in apple leaf protoplasts

Expression of the human respiratory syncytial virus (RSV) fusion protein (F ) gene under the control of the cauliflower mosaic virus (CaMV) 35S promote r was analyzed by enzyme-linked immunosorbent assay ( ELISA) in polyethylen e glycol-transfected apple leaf protoplasts. In particular, we examined whe ther 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'-un translated leader from alfalfa mosaic virus (AMV) RNA 4 between the CaMV 35 S promoter and the RSV-F gene increased viral expression by 5.5-fold compar ed to the construct without the leader. The addition of a transcriptional e nhancer from the pea plastocyanin gene (PetE) upstream of the CaMV 35S prom oter 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 i n transfected apple protoplasts reacted with RSV-F monoclonal antibodies an d 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 pl ant cells. Furthermore. protein expression was enhanced by modifying the tr ansfection vector using both a leader and an enhancer linked to a promoter.