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United States Department of Agriculture

Agricultural Research Service

Title: ENHANCED EXPRESSION OF THE HUMAN RESPIRATORY SYNCYTIAL VIRUS-F GENE IN APPLE PROTOPLASTS

Authors
item Sandhu, Jagdeep - UNIV OF ILLINOIS
item Osadjan, Mark - UNIV OF ILLINIS
item Krasnyanski, Sergei - UNIV OF ILLINOIS
item DOMIER, LESLIE
item Korban, Schuyler - UNIV OF ILLINOIS
item Buetow, Dennis - UNIV OF ILLINOIS

Submitted to: Plant Cell Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 28, 1998
Publication Date: N/A

Interpretive Summary: Through molecular biological techniques, it is possible to introduce new genes directly into plants cells. For many of these new genes to produce their desired effects, a relatively high amount of protein accumulation is required. However, the amount of protein expressed from these genes, especially genes that do not come from plants cells, is often very low. In nthis study, we examined methods to enhance the production of protein from a gene that was isolated from a human virus. Even though plants can not be infected with this virus, we found that by manipulating the environment in which the gene was placed we could significantly increase the amount of protein that plant cells produced from the non-plant gene. The results of these studies illustrate one mechanism by which the activities of genes introduced into plants for insect control or disease resistance can be enhanced, which could make plants produced by these technologies more attractive alternatives to traditional plant varieties and chemical control measures. These findings should be useful to scientists researching improved means of managing diseases and insects in plants.

Technical Abstract: 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 the 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 the 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 the RSV-F gene expression by 1.4-fold. In-ununoblot 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.

Last Modified: 8/27/2014
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