Cell Culture Evaluation of the Semliki Forest Virus Expression System As a Novel Approach for Antigen Delivery and Expression in Fish |
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Authors: | KV Phenix B McKenna R Fitzpatrick L Vaughan G Atkins P Liljestrom D Todd |
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Institution: | (1) Department of Veterinary Sciences, The Queen's University of Belfast, Stoney Road, Belfast BT4 3SD, Northern Ireland, IE;(2) Moyne Institute, Trinity College, Dublin, Ireland, IE;(3) Microbiology and Tumour Biology Centre, Karolinska Institute, Stockholm, Sweden, SE |
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Abstract: | Heterologous gene expression by Semliki Forest virus (SFV) expression vectors was investigated in fish cell culture. Experiments
performed using an infectious strain of SFV, replication-defective SFV particles, and recombinant SFV RNA constructs encoding
the Escherchia coli LacZ or firefly luciferase reporter genes indicated that levels of SFV-mediated expression in fish cells were dependent on
cell type and temperature. Maximal expression levels were observed in the two salmonid-derived cell lines CHSE-214 and F95/9
at 25°C and 20°C. As the temperature was lowered to 15°C or below, levels of reporter gene expression were reduced up to 1000-fold,
indicating that the SFV replication complex functioned inefficiently at low temperatures. The ability of SFV expression systems
to function in fish cells was further investigated by analyzing the expression of the protective VP2 antigen of infectious
pancreatic necrosis virus (IPNV) from the various constructs, including a novel DNA-based SFV plasmid. The VP2 protein produced
in CHSE-214 and F95/9 cells transfected or infected with the recombinant SFV-IPNV VP2 constructs appeared to be synthesized
in an antigenically correct form, as evidenced by the ability to react with several conformation-dependent IPNV-specific monoclonal
antibodies. Whether the temperature-restricted replication and expression displayed by SFV-based constructs in fish cell culture
also occurs in vivo remains to be determined.
Received January 29; accepted June 29, 1999. |
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Keywords: | : IPNV VP2 expression recombinant DNA vaccine |
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