Immunogenicity of Riemerella anatipestifer DnaK, OmpA and OmpA-DnaK proteins

Abstract:

[Background] Riemerella anatipestifer (RA) causes septicemia and infectious serositis in a variety of birds such as duck, leading to serious economic losses in the poultry industry. Protein vaccination is considered to be one of the most important strategies for preventing RA infection in ducklings. At present, there are few studies on the immunogenicity of RA recombinant proteins, and their application is also limited by the insufficient specific immune response induced by the single protein antigen. [Objective] The study aims to explore the immune response induced by the molecular chaperone DnaK, outer membrane protein A (OmpA) and OmpA-DnaK vaccines in ducklings, and to evaluate whether the recombinant fusion protein OmpA-DnaK has enhanced immunogenicity, which is conducive to the further development of RA vaccines. [Methods] We amplified the DnaK and OmpA genes and linked them to the pET-32a(+) vector, respectively. OmpA gene was ligated to the upstream region of DnaK gene through the restriction sites of Nco I and BamH I. DnaK, OmpA and OmpA-DnaK proteins were prepared by prokaryotic expression and purification. The DnaK, OmpA and OmpA-DnaK vaccines were respectively used to immunize ducklings twice via subcutaneous injection, and then the serum antibody titers, lymphocyte proliferation and cytokine (IL-2 and IL-4) levels were determined. The ducklings were then challenged by RA-GH5 strain through intramuscular injection, and the histopathological changes and immune protection rate were examined. [Results] The recombinant DnaK, OmpA and OmpA-DnaK proteins were expressed, with molecular weights of approximately 90, 60 and 130 kDa, respectively. The three recombinant protein vaccines induced the humoral and cellular immune responses of ducklings, and mitigated the histopathological damage caused by RA. Compared with DnaK or OmpA alone, OmpA-DnaK significantly increased serum antibody titer, lymphocyte proliferation index, and IL-2 and IL-4 levels after vaccination. The immune protection rate of OmpA-DnaK to ducklings was 50%, and those of both DnaK and OmpA were 30%. [Conclusion] The recombinant OmpA, DnaK and OmpA-DnaK protein vaccines all induced immune responses in ducklings. Moreover, the fusion of DnaK and OmpA caused stronger humoral and cellular immune responses.