Novel Soluble apxIVA-Truncated Protein and Its Application to Rapid Detection and Distinction of Actinobacillus pleuropneumoniae Wild-Strain-Infected Samples from Those Vaccinated with apxIV-Partially Deleted Vaccine

Abstract

Actinobacillus pleuropneumoniae (APP) is a bacterial pathogen causing porcine pleuropneumonia, causing great economic loss to the global pig industry. Although natural apxIV contributes to the prevention and control of porcine pleuropneumonia, its isolation poses a great challenge, and recombinant soluble apxIV proteins tend to carry large molecular weight tags. The traditional serologic methods tend not to accurately detect the apxIV-partially deleted vaccine (GDV). In this study, we screened the soluble protein apxIVA N2 (756 bp) from six apxIV-truncated proteins and applied it to the enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatographic strip for detecting the samples vaccinated with APP GDV. The results indicate that N2 was close to the natural apxIV protein in terms of structure and function as it only contained a single His (0.86 kDa) tag and a single S (2 kDa) tag. Among the six candidate proteins, N2 exhibited the best performance in distinguishing APP-infected samples from those vaccinated with the APP GDV. Both ELISA and colloidal gold immunochromatographic strips based on this protein exhibited an excellent performance in detecting and distinguishing wild-strain-infected samples from those vaccinated with the subunit vaccine or the GDV. In addition, three monoclonal antibodies against different antigenic epitopes were identified using these truncated proteins. Our studies are of great significance for further research on APP, the differential diagnosis of wild strains and vaccine strains, and pig control breeding, exhibiting a broad application prospect in the on-site diagnosis of APP, particularly in remote areas lacking detection instruments and professionals.

Keywords: Actinobacillus pleuropneumoniae; ELISA; apxIV; colloidal gold immunochromatographic strip; gene-deleted vaccine; rapid detection; wild strain.

Figure 1

Schematic of the novel truncated protein and its application in distinguishing wild-type strain infection from vaccine immunization.

Figure 2

Truncation and expression of apxIVA. (a) Summary of the six truncated proteins’ information. (b) Hydrophilicity and hydrophobicity analysis of apxIV protein (ProtScale). (c) Transmembrane domain analysis of apxIV protein (TMHMM). (d) Three-dimensional structural prediction of the antigenic epitope of apxIV protein (SWISS-MODEL). (e) Amplification of target genes. (f) Purification of N1, N2, N3, and N2c2 proteins. (g) Western blot to verify the correct expression of N1, N2, N3, and N2c2 proteins. (h) Amplification of target genes.

Figure 3

Preparation of colloidal gold immunochromatographic strip. (a) Screening of the optimal colloidal-gold-coupled proteins. Ring a, the colloidal-gold-coupled proteins on the conjugate pad; Ring b, the proteins on the T line; Ring c, concentration of the proteins on the T line; Ring d, P represents positive serum, N represents negative serum; Ring e, “+” indicates a positive result, and “−” indicates a negative result; Ring f, naming of the combinations. (b) Schematic of the strip structure.

Figure 4

Preparation of monoclonal antibodies against apxIV protein. (a) Titer detection of monoclonal antibodies 11C3, 2G2, and 3D12. (b) Chromosome number (CN) analysis of hybridoma cells. (c) Western blot verification of monoclonal antibody specificity. Lane M: marker; Lane 1, 9: recombinant proteins; Lane 2: pET-30a vector bacterial lysate; Lane 3: APP 4074 strain bacterial lysate (in vitro culture); Lane 4–8: sequentially, Bacillus lysate, E. coli lysate, E. faecalis lysate, S. aureus lysate, and Salmonella lysate. (d) ELISA validation of monoclonal antibody specificity. Serovar 1-15: APP serovar 1-15 strain lysates.

Figure 5

Evaluation of colloidal gold immunochromatographic strip. (a) Detection results of positive and negative sera. (b) Detection results of sera from pigs vaccinated with APP GDV. (c) Evaluation of sensitivity. (d) Evaluation of specificity. (e) Evaluation of reproducibility. (f) Evaluation of shelf-life. Lane 0: the strips stored at 4 °C for 0 months; Lane 2: the strips stored at 4 °C for 2 months; Lane 4: the strips stored at 4 °C for 4 months; Lane 6: the strips stored at 4 °C for 6 months. Each test was performed in duplicate. (g) Clinical sample test results (partial results shown).