Development of recombinant apxIA vaccine using peptide transduction domain

Actinobacillus pleuropneumoniae(APP) is an important pig pathogen, which is responsible for swine pleuropneumonia, a high contagious respiratory infection. The Apx toxins are species specific and all field strains produce these toxins. Apx toxin is consist of virulence domain A subunit and cell binding domain B subunit.

Protein transduction domains (PTD) are small peptides able to carry proteins, peptides, nucleic acid, and nanoparticles, including viral particles, across the cellular membranes into cells.

We have constructed an expression system capable of connecting the peptide transduction domain (PTD) which is known to pass through the cell and ApxIA B subunit gene. The aim of this study was to development nasal spray vaccine to more effectively induce mucosal immunity.

Bacterial strains and vectors. PTD-ApxIA B subunit cloned with pET30a vector. E. coli BL21 was used as host for transformation and expression of the recombinant protein.

Expression. Proteins expressed in E. coli were analyzed by SDS-PAGE and Western blot using mono-specific polyclonal antibody against Apx IA.

Immunization and sample collection. The group 1 of mice was inoculated by IM route. Another Groups 2-4 were inoculated by nasal route. Group 1(n=10) was inoculated with 20ug recombinant Apx1A vaccine. Group 2 (n=10), 3(n=10), 4(n=10) were inoculated with 5, 10, 20 ug recombinant Apx1A vaccine. Group 5 (n=10) injected with PBS as controls. Blood was taken on 0 and 14 days after vaccination.

Immune response analysis. Antibody titers (IgA and IgG) against recombinant ApxIA were measured by ELISA in order to analyze the immune response in the mice. The ELISA was perfomed using a mouse IgG ELISA kit and a mouse IgA ELISA kit (komabiotech) as directed by manufacturer.

The expressed PTD-Apx IA B subunit proteins were shown to be 60kDa, respectively, by Western blot.

Serum IgG titers against antigen were increased compared to those of control group until the end of the study (p < 0.05). All mice were challenged with the mixture of the challenge strains at 3 WPPI. Among groups 1 and 3 mice, 2 and 1 were dead within 14 days after challenge, respectively. The challenge strains were isolated from lung swab of 2 mice with pneumonic lung lesions in gross examination. All mice of groups 2,4,5, however, the lungs were normal in gross examination.

Suceecssfully confirmed cloning and expression of PTD-ApxI B subunit. Recombinant ApxIA genes could be a promising nasal spray vaccine candidate for the prevention of pleuropneumoniae acute infection in pigs and may provide optimal protection at target mucosal sites.

Disclosure of Interest: None Declared.