Development of a recombinant fusion protein subunit vaccine comprising two truncated proteins effectively protects largemouth bass against infectious spleen and kidney necrosis virus infection.

The infectious spleen and kidney necrosis virus (ISKNV) is a major pathogen causing infectious spleen and kidney necrosis disease in largemouth bass. As its major capsid protein (MCP) is considered a candidate subunit vaccine, its protective efficacy in practical applications remains suboptimal, failing to meet clinical prevention and control demands. This study employed bioinformatics techniques to analyze the sequences and predict the functions of MCP and candidate protein 051 L of ISKNV. Two truncated proteins (designated tMCP and t051L) with rich antigenic epitope were screened, which were linked via a rigid linker peptide to construct the fusion truncated protein t051L-tMCP. tMCP, t051L, and t051L-tMCP were induced and purified by using the prokaryotic expression system. The immunogenicity and protective efficacy of the different proteins were evaluated by intraperitoneal injection. Serum ELISA results showed that serum antibody titers in all immunized groups gradually increased with time within 28 days post-immunization. Simultaneously, optimal protection was achieved at an immunization dose of 1 mg/mL: The survival percentages of fish in the tMCP group and in the t051L group were 85.01 % and 79.01 %, while the t051L-tMCP group demonstrated a significantly survival percentages of 95.50 %, superior to the tMCP and t051L groups. Furthermore, viral load detection 7 days post-challenge revealed significantly reduced viral replication in the head kidney liver, and spleen tissues of vaccinated fish compared to the PBS group. Above all, a clear dose-dependent relationship was also observed in the immunoassay results. Consequently, immunological studies demonstrate that the fusion protein t051L-tMCP exhibits excellent immunogenicity. Furthermore, t051L-tMCP significantly enhances the immune protective capacity compared to tMCP and t051L. Our study provides an efficient and viable recombinant fusion truncated protein candidate vaccine for the prevention of infectious spleen and kidney necrosis virus disease.