Construction and analysis of a secreting expression vector for fish cells

Area of Science:

• Aquaculture Biotechnology
• Molecular Biology
• Gene Expression Systems

Background:

• Efficient protein production and secretion in fish cells are crucial for aquaculture applications.
• Existing expression vectors often lack optimized systems for high-level protein secretion in fish.
• Development of specialized vectors is needed to enhance recombinant protein yield in fish.

Purpose of the Study:

• To design and evaluate a novel expression plasmid (pcDNA3-LP) for enhanced protein secretion in fish cells.
• To compare the secreting efficiency of the new vector against a non-secreting control.
• To assess the in vivo efficacy of the vector for protein expression in rainbow trout.

Main Methods:

• Constructing secreting (pcDNA3-LP-LUC) and non-secreting (pcDNA3-LUC) plasmids using a luciferase reporter gene.
• Transient transfection experiments in salmonid (RTG-2) and cyprinid (EPC) cell lines.
• Isolation and analysis of stable cell clones for secretion efficiency.
• Intramuscular injection into rainbow trout to determine in vivo serum luciferase activity.

Main Results:

• The pcDNA3-LP vector demonstrated significantly higher luciferase secretion in both RTG-2 (7-fold) and EPC (85-fold) cell lines compared to the control.
• Stable EPC clones transfected with pcDNA3-LP-LUC secreted approximately 90% of total luciferase activity, versus 5% for pcDNA3-LUC clones.
• Intramuscular injection in rainbow trout showed 2.7-fold higher serum luciferase activity with pcDNA3-LP-LUC compared to pcDNA3-LUC.

Conclusions:

• The pcDNA3-LP plasmid effectively enhances protein secretion in fish cells, validated by reporter gene assays.
• The vector proves efficient in both in vitro cell culture and in vivo applications in rainbow trout.
• This novel vector presents significant opportunities for advancing fish DNA vaccinology and recombinant protein production.