Construction and analysis of a secreting expression vector for fish cells

B Collet, C J Secombes

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 languageEnglish
Pages (from-to)1534-1539
Number of pages6
JournalVaccine
Volume23
DOIs
Publication statusPublished - 2005

Keywords

  • fish
  • vector
  • secretion
  • TROUT ONCORHYNCHUS-MYKISS
  • RAINBOW-TROUT
  • MX1 PROMOTER
  • DNA
  • PROTEIN
  • ADJUVANT
  • CLONING
  • GENE
  • LINE

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