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