Hyper-production of porcine contagious pleuropneumonia subunit vaccine proteins in Escherichia coli by developing a bicistronic T7 expression system

The ApxII toxin and outer membrane lipoprotein (Oml) of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) are vital vaccine antigens against porcine contagious pleuropneumonia (PCP), a prevalent infectious disease in the swine industry worldwide. Previous studies have reported the recombinant expression of ApxII and Oml in Escherichia coli (E. coli). However, their yields were not satisfactory. Here, we aimed to enhance the production of ApxII and Oml in E. coli by constructing a bicistronic expression system based on the widely used T7 promoter. To create efficient T7 bicistronic expression cassettes, 16 different fore-cistron sequences were introduced downstream of the T7 promoter. The four most potent expression vectors were screened, and the expression of three vaccine antigens Oml1, Oml7, and ApxII in these four bicistronic vectors were enhanced compared to the monocistronic control. Further optimization of the fermentation conditions in micro-well plates led to improved production of Oml1, Oml7, and ApxII. Finally, the production yields reached unprecedented levels of 2.43 g/L, 2.59 g/L, and 1.21 g/L, respectively, in a 5 L bioreactor. These three antigens also demonstrated well-protective immunity against A. pleuropneumoniae infection. In conclusion, this study established a highly efficient bicistronic T7 expression system and achieved the hyper-production of PCP vaccine proteins. This bicistronic T7 expression system could be a valuable tool for the improved production of other proteins, especially recombinant vaccines, in E. coli.