Introduction: Antibiotic resistance is recognized as a major public health threat. Antimicrobial peptides provide an alternative to antibiotics. Human β-defensin 1 (hBD1), produced by epithelial cells, demonstrates broad antimicrobial activity and retains stability after proteolysis. However, its clinical application is constrained by high synthesis costs. This study aimed to generate a eukaryotic cell platform for hBD1 expression and secretion.

Methods: Plasmids pHBD1-Out and pHBD1RFP-Out were designed using the pcDNA3.1(+) vector with the CMV promoter. Cloning was performed in E. coli XL1-Blue, followed by plasmid purification and restriction analysis. HEK-293T cells were transfected via lipofection. Transfection efficiency was evaluated after 48 h by fluorescence microscopy. hBD1 production was confirmed by PAGE and HPLC. Antimicrobial activity was assessed against E. coli using agar diffusion assay.

Results: The pHBD1-Out plasmid carried the hBD1 sequence and the SRP9 signal peptide for extracellular expression, while pHBD1RFP-Out additionally contained A2P peptide and RFP marker. Transfection was confirmed by the presence of fluorescent cells, although the efficiency was relatively low. hBD1 production was detected in both cell lysates and supernatants, showing similar expression patterns for pHBD1-Out and pHBD1RFP-Out. Lysates demonstrated pronounced antimicrobial activity comparable to ampicillin. Supernatants also inhibited E. coli growth, though less effectively.

Conclusions: The constructed plasmids enabled efficient hBD1 expression in HEK-293T cells. The recombinant peptide exhibited antimicrobial activity comparable to conventional antibiotics, highlighting the potential of cell-based systems for AMP production and the development of novel antimicrobial therapeutics. This work was supported by the Ministry of Science and Higher Education of the Russian Federation in 2025 (No. 0088-2025-0004).