To study the immunogenicity of the novel outer membrane protein 5’-nucleotidase(5’-NT) from Vibrio parahaemolyticus.

Bioinformatics methods were used to analyze the sequence conservation, structure and B-cell epitopes of 5’-NT; the gene was cloned by polymerase chain reaction (PCR) and the recombinant plasmid pET-28a(+)-nutA was constructed and transformed into Escherichia coli for induced expression. The soluble recombinant protein was obtained by nickel-chelated nitrilotriacetic acid agarose affinity chromatography purification and gradient dialysis refolding; mice immunization, enzyme-linked immunosorbent assay, Western blotting, complement-mediated serum bactericidal assay and immunofluorescence confocal microscopy were used to systematically evaluate the protein’s surface exposure, immunological titer, cross-reactivity and immunoprotective effect.

The results showed that 5’-NT had high sequence conservation in the genus Vibrio. The recombinant plasmid of 5’-NT was successfully constructed and the high-purity recombinant 5’-NT was obtained through induced expression and purification. The antibody titer of 5’-NT against the whole-cell immune serum of Vibrio parahaemolyticus was 1:81k, which confirmed the surface exposure of this protein. The titer of 5’-NT immune serum against 5’-NT protein was 1:81k, and 1:3k against Vibrio parahaemolyticus. It had weak cross-reactivity with most other Vibrio species and the immune serum exhibited weak complement-dependent bactericidal activity (1:4). Western blot analysis demonstrated that the immune serum against recombinant 5’-NT could generate specific bands with whole-cell proteins of Vibrio parahaemolyticus and other Vibrio species. Confocal immunofluorescence microscopy analysis further confirmed that the recombinant 5’-NT immune serum specifically bound to Vibrio parahaemolyticus which also indicated that this protein possesses surface-exposed characteristics.

Although 5’-NT of Vibrio parahaemolyticus exhibits Vibrio-conserved and surface-exposed characteristics, its relatively low immune serum titer indicates limited exposure, immunogenicity, or expression abundance. This limits its application as a standalone vaccine or detection antigen. In subsequent studies, combination strategies can be adopted to explore its value in the prevention and control of Vibrio parahaemolyticus.This study reveals the surface exposure, immunogenicity, and serum protectivity of the outer membrane protein 5’-NT from Vibrio parahaemolyticus, providing a reference for the development of diagnostic antigens and subunit vaccines against this pathogen.