Objective: To construct the recombinant Lactobacillus casei and Lactobacillus fermentum for the expression of codonoptimized M2e: HBc fusion gene.
Material and Method: The ectodomain of the Matrix 2 (M2e), a conserved gene of influenza viruses, was fused with the gene
encoding hepatitis core protein (HBc) to generate M2e: HBc fusion gene. The M2e: HBc fusion gene was changed to obtain
the optimized codon usage in L. casei. The codon-optimized M2e: HBc fusion gene was cloned into an E. coli/L. casei shuttle
vector under the control of nisA-inducible promoter for the expression in L. casei EM116, and under the control of ldhL strong constitutive promoter for expression in two probiotic strains L. casei RCEID02 and L. fermentum RCEID01. The expression of M2e: HBc fusion gene in lactobacilli was determined by western blotting using a specific antibody against M2e.
Results: Codon optimization of the M2e: HBc fusion gene in L. casei was performed on 14 rare codons, consisting of 12 AGA
and 2 AGG. The optimized M2e: HBc fusion gene with the size of 638 bp was successfully generated. Two recombinant expression plasmids based on nisA inducible- and ldhL constitutive-promoter were successfully constructed and electro transformed into lactobacilli. Western blotting analysis revealed that the codon-optimized M2e: HBc fusion gene under the control by those two bacterial promoters was successfully expressed in Lactobacillus species.
Conclusion: In this study, the codon optimization strategy can be used for the expression of M2e: HBc fusion gene in two
lactobacilli prototypes, L. casei and L. fermentum. The expression of the M2e: HBc fusion gene in both lactobacilli would
provide the opportunity to apply these two bacteria as an alternative broad-spectrum vaccine candidate for influenza A virus.

Keywords: Lactic acid bacteria, Lactobacillus casei, Lactobacillus fermentum, Ectodomain Matrix 2 protein, Influenza virus, Heterologous protein expression