PRODUCTION AND PURIFICATION OF RECOMBINANT AFRICAN SWINE FEVER VIRUS ATTACHMENT PROTEIN P12

The conditions for cultivation of Spodoptera frugiperda (Sf9) insect c ells for production of recombinant baculoviruses have been studied, to scale-up and improve the efficiency of the process for production of the African swine fever virus attachment protein p12 in the baculoviru s expression system. It was shown that the total virus and recombinant protein production in insect cells infected with the Acp12 recombinan t baculovirus were slightly dependent on cell density, but largely dep endent on the serum concentration, in the case of suspended cells, but not in static monolayer cultures. The yield of recombinant protein p1 2 exceeded 50 mg per 1 of 2x10(9) cells, representing more than 10% of total cell proteins, a level > 20-fold higher than that observed with other eukaryotic expression systems. The presence of p12 in the cytop lasmic fraction of infected cells has allowed the purification of the protein by a simple two-step procedure of aqueous phase partition and octyl-glucoside solubilization. The recombinant protein p12 was able t o inhibit, in a dose-dependent manner, the African swine fever virus p roduction in swine macrophages infected with a number of different vir us isolates, including attenuated, virulent, highly passaged on tissue culture, and non-haemadsorbing strains, indicating ii fundamental rol e for p12 in the early interaction of the virus with the natural targe t cell receptors. However, pigs immunized with purified recombinant p1 2 did not develop protective immunity against African swine fever.