title: Production and purification of recombinant African swine fever virus attachment protein p12.
abstract: The conditions for cultivation of Spodoptera frugiperda (Sf9) insect cells 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 baculovirus expression system. It was shown that the total virus and recombinant protein production in insect cells infected with the Acp12 recombinant baculovirus were slightly dependent on cell density, but largely dependent on the serum concentration, in the case of suspended cells, but not in static monolayer cultures. The yield of recombinant protein p12 exceeded 50 mg per 1 of 2 x 10(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 cytoplasmic 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 to inhibit, in a dose-dependent manner, the African swine fever virus production in swine macrophages infected with a number of different virus isolates, including attenuated, virulent, highly passaged on tissue culture, and non-haemadsorbing strains, indicating a fundamental role for p12 in the early interaction of the virus with the natural target cell receptors. However, pigs immunized with purified recombinant p12 did not develop protective immunity against African swine fever.
authors.affiliation: Centro de Biología Molecular (CSIC-UAM), Facultad de Ciencias, Universidad Autónoma de Madrid (Cantoblanco), Madrid, Spain.
authors.forename: A L
authors.initials: AL
authors.lastname: Carrascosa
authors.forename: I
authors.initials: I
authors.lastname: Sastre
authors.forename: E
authors.initials: E
authors.lastname: Viñuela
mesh_terms: D000358:African Swine Fever Virus / Q000737:chemistry* / Q000254:growth & development; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D002478:Cells, Cultured; D003470:Culture Media; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D016676:Macrophages, Alveolar / Q000821:virology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D018411:Spodoptera; D013552:Swine; D013556:Swine, Miniature; D014162:Transfection / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology; D014776:Virus Cultivation
chemical_list: D003470:Culture Media; D003902:Detergents; D011994:Recombinant Proteins; D014764:Viral Proteins; C074550:protein p12, African swine fever virus