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

Citation
Js. Sandhu et al., Enhanced expression of the human respiratory syncytial virus-F gene in apple leaf protoplasts, PL CELL REP, 18(5), 1999, pp. 394-397
Citations number
20
Categorie Soggetti
Plant Sciences","Animal & Plant Sciences
Journal title
PLANT CELL REPORTS
ISSN journal
07217714 → ACNP
Volume
18
Issue
5
Year of publication
1999
Pages
394 - 397
Database
ISI
SICI code
0721-7714(199901)18:5<394:EEOTHR>2.0.ZU;2-T
Abstract
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.