Stable Recombinant-Gene Expression from a Ligilactobacillus Live Bacterial Vector via Chromosomal Integration

Area of Science:

• Microbiology
• Immunology
• Veterinary Science

Background:

• Antibiotic use in animal production raises concerns about resistance.
• Live vaccination offers a promising alternative for mucosal immunity.
• Ligilactobacillus agilis La3 is a potential candidate for a live vaccine vector.

Purpose of the Study:

• To develop and characterize a stable expression system in L. agilis La3 for delivering therapeutic proteins.
• To demonstrate the feasibility of using L. agilis La3 as a live vaccine vector in chickens.

Main Methods:

• Genome sequencing and transcriptome analysis of L. agilis La3.
• Construction of recombinant expression cassettes and shuttle plasmids.
• Integration into the L. agilis La3 chromosome via homologous recombination.
• Stable expression of a Clostridium perfringens antigen (rNetB) was assessed over 100 generations.

Main Results:

• A modular and stable expression system was successfully developed in L. agilis La3.
• Recombinant protein expression levels correlated with promoter strength.
• Stable chromosomal integration and expression of rNetB were achieved without selection over 100 generations.
• Two recombinant L. agilis La3 strains were constructed and characterized for potential vaccine applications.

Conclusions:

• L. agilis La3 can be effectively engineered as a live delivery vehicle for recombinant proteins.
• The developed system provides stable, selectable integration for therapeutic protein expression.
• This approach holds potential for developing novel live vaccines against poultry diseases.