ABSTRACT
ApxII is a vaccine antigen used to protect against porcine contagious pleuropneumonia, which is a significant threat to the pig industry. Here, we aimed to improve the proteolytic degradation stability of ApxII during its secretion by establishing a complete screening process of stable variants through bioinformatics and site-directed mutagenesis. We employed a combination of semi-rational and rational design strategies to create 34 single-point variants of ApxII. Among them, R114E and T115D variants exhibited better stability without compromising antigen activity. Furthermore, we constructed a multi-site variant, R114E/T115D, which demonstrated the best stability, activity, and yield. Protein stability and molecular dynamic analysis indicated that the greater solubility and lower structural expansion coefficient might explain the increased stability of R114E/T115D. Additionally, site T115 was identified as a key point of truncated ApxII stability. The R114E/T115D variant, with its proven stability and intact antigenic activity, holds promising prospects for industrial-scale applications in the prevention of porcine contagious pleuropneumonia.