IRES-incorporated lactococcal bicistronic vector for target gene expression in a eukaryotic system

Area of Science:

• Molecular Biology
• Genetic Engineering
• Biotechnology

Background:

• Lactic acid bacteria (LAB) plasmids, like Lactococcus lactis (L. lactis) pNZ8048, are valuable tools in genetic engineering.
• Developing versatile vectors for eukaryotic gene expression is crucial for various biotechnological applications.

Purpose of the Study:

• To engineer the lactococcal plasmid pNZ8048 for efficient multi-gene expression in eukaryotic systems.
• To construct and validate a novel bicistronic vector, pNZ:vig, incorporating an internal ribosome entry site (IRES).

Main Methods:

• Cloning an IRES cassette between the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) and the green fluorescent protein (GFP) reporter gene.
• Utilizing a modified pNZ8048 derivative (pNZ:CA) with cytomegalovirus promoter (Pcmv) and polyadenylation signal.
• Verifying vector functionality using in vitro transcription/translation, SDS-PAGE, and Western blot analysis.

Main Results:

• The constructed bicistronic vector, pNZ:vig, successfully co-expressed multiple genes in a eukaryotic system.
• Western blot analysis confirmed the expression of VP2 (49kDa) and GFP (29kDa) proteins.
• The IRES element facilitated the simultaneous translation of both genes from a single transcript.

Conclusions:

• The novel lactococcal bicistronic vector, pNZ:vig, demonstrates feasibility for co-expressing multiple genes in eukaryotic systems.
• This vector holds potential for applications in in vitro cell culture transfection and advanced vaccine delivery systems.