V. S. Samoylenko
North Caucasus Federal University, Stavropol, Russia
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Volume 26 No. 6
Date of paper submission: 25.02.2026, date of review: 25.03.2026, date of acceptance: 30.04.2026.
Published: 06/30/2026
Abstract. The purpose of the study was to develop an effective method for engineering genetically modified probiotic microorganisms with therapeutic potential for the treatment of inflammatory bowel diseases and autoimmune disorders in young cattle. Genetic modification of Lactobacillus spp. and Bifidobacterium spp. strains was considered a tool for producing immunoregulatory cytokines IL-10 and IL-22. Traditional cloning methods and plasmid systems were characterized by structural instability, reduced expression, and limited capacity for coordinated expression of multiple genes. Methods. The study was conducted in 2024–2025 at the Genome Center of the North-Caucasus Federal University and the laboratory of the Stavropol Agrarian University. The experimental phase aimed to construct bicistronic plasmids for the simultaneous expression of IL-10 and IL-22 in Lactobacillus spp. A modified pLacDual vector was used. Bioinformatic sequence design ensured high hybridization specificity. Amplification was performed using the high-fidelity Q5 DNA polymerase with DMSO. Assembly of genetic cassettes was carried out using the HiFi DNA Assembly method. Correctness of the constructs was verified by capillary electrophoresis and diagnostic PCR; results were compared with ligation and Golden Gate Assembly. Results. The constructed bicistronic plasmids confirmed correct assembly: the 6421 bp fragment matched the expected length, and the 723 bp amplicon indicated proper gene integration. The HiFi method demonstrated higher accuracy, reproducibility, and efficiency compared to alternative techniques. Scientific novelty. For the first time, the possibility of stable co-expression of two therapeutically relevant cytokines in probiotic strains using a bicistronic construct with HiFi DNA Assembly was substantiated and experimentally confirmed, expanding methodological approaches for creating multigene probiotic systems. Conclusion. Achieving the stated aim confirmed the potential of the proposed strategy for developing bioengineered probiotics, relevant to medicine, synthetic biology, and food biotechnology.
Keywords: bicistronic constructs, recombinant plasmids, high-efficiency cloning, probiotic strains, gene therapeutic approaches, cell cultures
For citation: Samoylenko V. S. Highly efficient cloning of bicistronic constructs for cytokine co-expression in probiotic strains. Agrarian Bulletin of the Urals. 2026; 26 (06): 1105‒1111. https://doi.org/10.32417/1997-4868-2026-26-06-1105-1111 (In Russ.)
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