Abstract:

To construct the immunogen of the S1 protein of avian infectious bronchitis virus (IBV), the amino acid sequences of the published S1 neutralizing epitopes were compared with the S1 protein of ck/CH/LDL/091022 (LDL091022), current prevalent GI-19 representative strain in China. The amino acid sequences at positions 22-100 and 410-492 of the S1 protein were recognized as principal neutralizing epitope areas, named as B1 and B2, respectively. Plasmids encoding LDL091022 B1 and B2 epitope peptides and plasmids with chicken IgY Fc fragment fused at the C-terminus of B1 and B2 were constructed. The significant increase of protein expression caused by IgY Fc fragment was demonstrated by indirect immunofluorescence assay (IFA) and Western blot assay. The recombinant plasmids were transfected into Expi 293F cells, and the recombinant proteins B1-Fc and B2-Fc with IgY Fc fragments were purified. Western blot and ELISA were used to assess the reactivity of B1-Fc and B2-Fc with LDL091022 serum, the results showed that B2-Fc had significantly better reactivity than B1-Fc. SPF chickens were immunized with the plasmid pCAGGS-B2-Fc and the recombinant protein B2-Fc. The serum antibody levels were detected by ELISA and IFA, the results demonstrated that B2-Fc booster immunization effectively induced antibodies against the B2 epitope. And the IFA result revealed the serum could react with LDL091022-S1 protein and B2-Fc. Moreover, the neutralization test showed that the serum neutralization titer was 16. These results demonstrated that the IBV S1 protein neutralizing epitope-Fc fusion protein prepared in this study showed good expression level and immunogenicity. It could induce certain levels of neutralizing antibodies, providing insights for the development of novel IBV subunit vaccines.

Key words: avian infectious bronchitis virus, S1 subunit, neutralizing epitope, IgY Fc fragment, immunogenicity