In silico design, expression and initial immunoreactivity of TL5,6 multi-epitope chimeric protein derived from Tilapia Lake Virus S5 and S6 segment proteins

Pakkakul  Sangsuriya; Benjamaporn  Plysup; Apisit Lueangyangyuen; Prit  Khrisanapant; Saengchan  Senapin; Triwit  Rattanarojpong; Wasusit  Somsoros; Pongsak  Khunrae

doi:10.34044/j.anres.2026.60.5.08

Authors

Pakkakul Sangsuriya Aquatic Molecular Genetics and Biotechnology Research Team, BIOTEC, NSTDA, Pathum Thani 12120, Thailand
Benjamaporn Plysup Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand
Apisit Lueangyangyuen Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand
Prit Khrisanapant Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand
Saengchan Senapin Fish Health Platform, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Triwit Rattanarojpong Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand
Wasusit Somsoros Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand
Pongsak Khunrae Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand

DOI:

https://doi.org/10.34044/j.anres.2026.60.5.08

Keywords:

Bioinformatic, Immunoreactivity, Multi-epitope chimeric protein, Nile tilapia, Tilapia Lake Virus

Abstract

Importance of the work: The application of multi-epitope antigen design for TiLV remains limited, prompting investigation of its potential as an alternative antigen strategy for TiLV vaccine development. Objectives: To develop the TL5,6 protein by fusing predicted epitopes from TiLV segments 5 and 6 and to perform an initial evaluation of its immunoreactivity. Materials and Methods: Predicted B-cell epitopes from TiLV segment proteins 5 and 6 were used to design the TL5,6 protein. Structural and physicochemical properties were analyzed bioinformatically. TL5,6 was expressed in Escherichia coli and then purified and formulated with Freund’s incomplete adjuvant for immunization in Nile tilapia. Serum immunoreactivity was assessed based on dot blot assay, while molecular docking and immune simulation were used as supportive computational analyses. Results: TL5,6 was developed using molecular design and immunoinformatic analysis, in which the B-cell epitopes of segments 5 and 6 were combined into a single molecule. A soluble form of the TL5,6 protein was expressed successfully and purified from E. coli BL21 (DE3). The dot blot analysis showed that the serum obtained from TL5,6- immunized fish reacted with both the S5196–272 and S6200–317 antigens and also recognized TiLV particles. Molecular docking analysis predicted an interaction between TL5,6 and TLR4, with the lowest binding energy of -1,170 kJ/mol and 29 hydrogen bonds observed at the interface region. Furthermore, immune simulation predicted increased Thcell and B-cell populations, elevated IgM and IgG levels and upregulation of IL-2 and IFN-γ related responses following TL5,6 immunization. Main finding: TL5,6 demonstrated preliminary immunoreactivity and may serve as a potential antigen for future TiLV vaccine development. However, further studies are needed to evaluate its protective efficacy, including quantitative immune analyses and challenge experiments