Background and Aim: African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process. Materials and Methods: SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA). Results: The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001). Conclusion: This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well. Keywords: African swine fever, diagnosis, indirect enzyme-linked immunosorbent assay, p30, protein expression, purification.

Foot-and-mouth disease (FMD) is one of the most infectious viral diseases of cloven-hoofed animals. The crucial strategy for FMD control is the combination of animal quarantine and a rapid onsite diagnostic assay capable of differentiation of infected from vaccinated animals (DIVA). Herein, we have developed a lateral flow immunoassay to detect specific antibodies against FMDV non-structural protein (NSP), based on recombinant mu3ABC produced by E. coli as an antibody detector, the so-called mu3ABC strip test. The performance of this test was examined using 366 field serum samples from cattle, pigs, and goats and compared with the commercial ELISA kit, resulting in a diagnostic sensitivity (DSn) and specificity (DSp) of 98.05% and 93.16%, respectively. Additionally, efficacies of this test and commercial ELISA were determined using 200 reference bovine serum samples (100 positives, 100 negatives) classified by the Regional Reference Laboratory for Foot-and-Mouth Disease in South East Asia (RRL). The result reveals that the DSn and DSp of the test were 78% and 91%, respectively, while those for the commercial ELISA kit were 69% and 100%, respectively. The developed mu3ABC strip tests are effective and can be used to differentiate the infected animals in parallel with the sero-surveillance ELISA test.

Foot-and-mouth disease (FMD) is one of the most infectious viral diseases of coven-hoofed animals. The crucial strategy for FMD control is the combination of animal quarantine and rapid onsite diagnostic assay capable of differential infected from vaccinated animals (DIVA). Herein, we have developed a lateral flow immunoassay to detect specific antibodies against FMDV non-structural protein (NSP), based on recombinant mu3ABC produced by E. coli as an antibody detector, so called mu3ABC strip test. Performance of mu3ABC strip test was examined using 566 field serum samples from cattle, pigs, and goats and compared with the commercial ELISA kit, resulting in the diagnostic sensitivity (DSn) and specificity (DSp) of 88.18% and 92.56%, respectively. Additionally, efficacies of the mu3ABC strip test and commercial ELISA were determined using 200 reference bovine sera (100 positives, 100 negatives) classified by the Regional Reference Laboratory for Foot and Mouth Disease in South East Asia (RRL). The result reveals that DSn and DSp of mu3ABC strip test were 78% and of 91%, respectively while the commercial ELISA kit had DSn of 69%and DSp of 100%. The developed mu3ABC strip tests are effective and can be used to differentiate the infected animals in parallel with the sero-surveillance ELISA test.