Expression of immunogenic fraction of Apx toxins of Actinobacillus pleuropneumoniae for the design of a quantitative assay

Abstract

Porcine pleuropneumonia is a highly contagious disease for pigs worldwide, and its presence represents a large economic loss. Actinobacillus pleuropneumoniae (App) is the etiological agent of porcine pleuropneumonia. Its virulence is correlated with the presence of four exotoxins, known as Apx toxins (Apx I, Apx II, Apx III, and Apx IV). 19 serotypes of App are recognized until today, and commercially available vaccines cannot ensure total protection against all, thus the relevance of new subunit vaccines to be developed and introduced into the market. To test vaccine efficiency, validation with quantitative immunoassays is required. Enzyme-linked immunosorbent assays (ELISAs) are the preferred technique to validate efficiency, but the available test only helps identify the serotype present, not specific antigen and immune reactions. Thus, an Apx antigen-specific ELISA should be developed to measure immune responses to each specific Apx toxin independently, and contribute to developing new treatments for App. Here, we generated recombinant fragments of Apx toxins IA, IIA, and IIIA, using Escherichia coli BL21 DE3 as expression host. This fragments were selected after an homology analysis to define the specific immunogenic regions for each toxin. The recombinant fragments were purified by affinity chromatography in an ÄKTA Avant purification system. Dot blot, SDS-PAGE, and Western blot techniques were used to validate the purification process from the obtained recombinant fragments. Lastly, a growth kinetics characterization of five serotypes was carried on in three different culture media. The results obtained confirm that the over-expression of the recombinant proteins of interest was possible using the pET-28b(+) plasmid with the insert to express in E. coli BL21 DE3. Further, affinity chromatography was a good purification process, allowing purification percentages around 90%. Lastly, that the five selected serotypes of App were able to grow in Brain Heart Infusion media supplemented with 5% (w/v) yeast extract, Tryptic Soy Broth supplemented with 5% (w/v) yeast extract and an Animal-free component media. Our results contribute for the development of analytical methods much needed in the veterinary industry.