Abstract
The ApxII toxin and outer membrane lipoprotein (Oml) ofActinobacillus 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 inEscherichia 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. pleuropneumoniaeinfection. 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 .
Keywords : bicistronic T7 expression system, PCP subunit vaccine
protein, hyper-production, Escherichia coli , recombinant
expression