title: Construction and analysis of a secreting expression vector for fish cells.
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.
authors.affiliation: Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen, Scotland, UK. colletb@marlab.ac.uk
authors.forename: Bertrand
authors.initials: B
authors.lastname: Collet
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Secombes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics*; D002460:Cell Line; D005822:Genetic Vectors / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000235:genetics*; D014162:Transfection / Q000379:methods*