Abstract
A new expression plasmid (pcDNA3-LP) was designed to produce and secrete proteins in fish cells by fusion with the rainbow trout TGF-beta leader peptide. The luciferase reporter gene was used to test the secreting ability of this vector. Secreting (pcDNA3-LP-LUC) and non-secreting (pcDNA3-LUC) constructs were made and compared in transient transfection experiments in salmonid (RTG-2) and cyprinid (EPC) cell lines. The amount of luciferase secreted into the supernatants of RTG-2 or EPC cells transiently transfected with pcDNA3-LP-LUC relative to cells transfected with pcDNA3-LUC was 7- and 85-fold, respectively. Two stable clones of EPC transfected with pcDNA3-LUC and four clones transfected with pcDNA3-LP-LUC were isolated. Approximately 90% of the total luciferase activity produced was secreted by stable EPC clones containing pcDNA3-LP-LUC whereas only 5% of total activity was secreted by clones containing pcDNA3-LUC. The two constructs were injected intra-muscularly into rainbow trout and the luciferase activity present in the serum of fish determined. The luciferase activity in serum from fish injected with pcDNA3-LP-LUC was 2.7-fold higher (P < 0.05) than that fish injected with pcDNA3-LUC. This new vector opens up opportunities in fish DNA vaccinology and in the production of fish recombinant proteins. (c) 2004 Elsevier Ltd. All rights reserved.
Original language | English |
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Pages (from-to) | 1534-1539 |
Number of pages | 6 |
Journal | Vaccine |
Volume | 23 |
DOIs | |
Publication status | Published - 2005 |
Keywords
- fish
- vector
- secretion
- TROUT ONCORHYNCHUS-MYKISS
- RAINBOW-TROUT
- MX1 PROMOTER
- DNA
- PROTEIN
- ADJUVANT
- CLONING
- GENE
- LINE