doi: 10.15389/agrobiology.2016.6.837eng

UDC 636.4:619:616.636:578:[577.2.08+51-76

Acknowledgements:We thank Prof. V.M. Balyshev, Dr S.P. Zhivoderov and Dr I.A. Titov for assistance in carrying out the work.
Supported by Russian Science Foundation (the research project «Design of African swine fever virus candidate vaccine based on chimeric viruses», contract No 16-16-00090)



A.R. Imatdinov, A.D. Sereda, I.R. Imatdinov, A.S. Kazakova,
O.A. Dubrovskaya, D.V. Kolbasov

All-Russian Institute of Veterinary Virology and Microbiology, Federal Agency of Scientific Organizations, 1, ul. Akademika Bakuleva, pos. Vol’ginskii, Petushinskii Region, Vladimir Province, 601125 Russia, e-mail

Received August 30, 2016


Control of African swine fever (ASF) is complicated by the lack of specific prevention medications. The attempts to obtain live attenuated vaccines by conventional methods were not promising, and the inactivated or subunit vaccines have not been developed so far (N.J. Petiska, 1965; D.V. Kolbasov et al., 2014; V. Makarov et al., 2016). The investigation of protective immune response against ASF virus (ASFV) enabled determination of a critical role of cellular defense mechanisms and the most important viral proteins p30, p54 and CD2v (or gp 110-140) involved (P. Gomez-Puertas еt al., 1998; J.M. Argilaguet et al., 2012; A.D. Sereda et al., 2015). In view to develop a DNA vaccine against ASFV seroimmunotype 3 we have constructed a set of hybrid plasmids containing fragments of ASFV genes CP204L, E183L and EP402R from attenuated strain MK-200 (pCI-neo/ASFV/p30, pCI-neo/ASFV/p54 and pCI-neo/ASFV/CD2v). To study expression of the antigenically active polypeptide products for recombinant proteins rp30, rp54 and rCD2v in the eukaryotic cells, we transfected human embryonic kidney cells HEK293T, which stably express the SV40 large T antigen, with recombinant plasmids pCI-neo/ASFV/p30, pCI-neo/ASFV/p54 and pCI-neo/ASFV/CD2v. By immunoblotting, the polypeptides of the expressed recombinant proteins were identified in the HEK293T cell lysates and characterized for their molecular weights. Regarding size, some antigenically active recombinant polypeptides were as calculated, whereas the other ones apparently resulted from post translational modification. We identified a 21.6 kDa polypeptide after pCI-neo/ASFV/p30 transfection, a major (20.9 kDa) and a minor (36.3 kDa) polypeptides after pCI-neo/ASFV/p54 transfection, and, finally, major polypeptides of 39.8 kDa and 63.1 kDa, together with minor polypeptides of 28.8 kDa and 104.7 kDa when pCI-neo/ASFV/CD2v transfected. These genetic constructions will be helpful to investigate antigenic, immunogenic and protective properties of ASFV recombinant proteins rp30, rp54 and rCD2v.

Keywords: African swine fever, recombinant genes and proteins, transfection, antigenicity.


Full article (Rus)

Full text (Eng)



  1. Sanchez-Vizcaíno J.M., Mur L., Gomez-Villamandos J.C., Carrasco L. An update on the epidemiology and pathology of African swine fever. J. Comp. Pathol., 2015, 152(1): 9-21 CrossRef
  2. Makarov V.V., Sukharev O.I., Tsvetnova I.V. Veterinarnaya praktika, 2013, 1(60): 6-16 (in Russ.).
  3. Kolbasov D.V., Sereda A.D. Veterinariya, 2013, 1: 19-23 (in Russ.).
  4. Rowlands R.J., Michaud V., Heath L., Hutchings G., Oura C., Volsoo W., Dwarka R., Onashvili T., Albina E., Dixon L.K. African swine fever virus isolate Georgia, 2007. Emerg. Infect. Dis., 2008, 14(12): 1870-1874 CrossRef
  5. Gogin A., Gerasimov V., Malogolovkin A., Kolbasov D. African swine fever in the North Caucasus region and the Russian Federation in years 2007-2012. Virus Res., 2013, 173(1): 198-203 CrossRef
  6. Smietanka K., Wozniakowski G., Kozak E., Niemczuk K., Fraczyk M., Bocian L., Kowalczyk A., Pejsak Z. African swine fever epidemic, Poland, 2014-2015. Emerg. Infect. Dis., 2016, 22(7): 1201-1207 CrossRef
  7. Petisca N.J. Quelques aspects morphologiques à la suite de la vaccination contre la peste porcine Africaine (Virose L) au Portugal. Bull. Off. Int. Epizoot., 1965, 63: 199-237.
  8. Vigario I.D., Terrinha A.M., Nunes J.F.M. Antigenic relationships among strains of African swine fever virus. Archiv für die gesamte Virusforschung, 1974, 45(3): 272-277 CrossRef
  9. Kolbasov D.V., Balyshev V.M., Sereda A.D. Veterinariya, 2014, 8: 3-8 (in Russ.).
  10. Makarov V., Nedosekov V., Sereda A., Matvienko N. Immunological conception of African swine fever. Zoology and Ecology, 2016, 26(3): 236-243 CrossRef
  11. Takamatsu H.H., Denyer M.S., Lacasta A., Stirling C.M.A., Argilaguet J.M., Netherton C.L., Oura C.A.L., Martins C., Rodríguez F. Cellular immunity in ASFV responses. Virus Res., 2013, 173(1): 110-121 CrossRef
  12. Oura C.A.L., Denyer M.S., Takamatsu H., Parkhouse R.M.E. In vivo depletion of CD8+ T lymphocytes abrogates protective immunity to African swine fever virus. J. Gen. Virol., 2005, 86(9): 2445-2450 CrossRef
  13. King K., Chapman D., Argilaguet J.M., Fishbourne E., Hutet E., Cariolet R., Hutchings G., Oura C.A.L., Netherton C.L., Moffat K., Taylor G., Le Potier M.F., Dixon L.K., Takamatsu H.H. Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunization. Vaccine, 2011, 29(28): 4593-4600 CrossRef
  14. Bachmann M.F., Kundig T.M., Freer G., Li Y., Kang C.Y., Bishop D.H., Hengartner H., Zinkerna R.M. Induction of protective cytotoxic T cells with viral proteins. Eur. J. Immunol., 1994, 24: 2228-2236 CrossRef
  15. Gomez-Puerta P., Rodriguez F., Oviedo J.M., Ramiro-Ibanez F., Ruiz-Gonzalvo F., Escribano J.M. Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization. Virology, 1996, 70(8): 5689-5694.
  16. Gómez-Puertas P., Rodriguez F., Oviedo J.M., Brun A., Alonso C., Escribano J.M. The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response. Virology, 1998, 243: 461-471 CrossRef
  17. Sereda A.D. Aktual'nye voprosy veterinarnoi biologii, 2013, 4(20): 31-35 (in Russ.).
  18. Argilaguet J.M., Perez-Martin E., Nofrarias M., Gallardo C., Accensi F., Lacasta A., Mora M., Ballester M., Galindo-Cardiel I., Lopez-Soria S., Escribano J.M., Reche P.A., Rodriguez F. DNA vaccination partially protects against African swine fever virus lethal challenge in the absence of antibodies. PLoS ONE, 2012, 7(9): e40942 CrossRef
  19. Sereda A.D., Kazakova A.S., Imatdinov A.R., Kolbasov D.V. Humoral and cell immune mechanisms under African swine fever (review). Agricultural Biology, 2015, 50(6): 709-718 CrossRef (in Engl.).
  20. Mima K.A., Burmakina G.S., Titov I.A., Malogolovkin A.S. African swine fever virus glycoproteins p54 and CD2v in the context of immune response modulation: bioinformatic analysis of genetic variability and heterogeneity. Agricultural Biology, 2015, 50(6): 785-793 CrossRef (in Engl.).
  21. Sereda A.D., Balyshev V.M. Voprosy virusologii, 2011, 4: 38-42 (in Russ.).
  22. Malogolovkin A., Burmakina G., Titov I., Sereda A., Gogin A., Baryshnikova E., Kolbasov D. Comparative analysis of African swine fever virus genotypes and serogroups. Emerg. Infect. Dis., 2015, 21(2): 312-315 CrossRef
  23. Balysheva V.I., Prudnikova E.Yu., Gal'nbek T.V., Balyshev V.M. Voprosy virusologii, 2015, 2: 43-47 (in Russ.).
  24. Green M.R., Sambrook J. Molecular sloning: a laboratory manual. Cold Spring Harbor Laboratory Press, NY, 2012.
  25. Maniatis T., Fritsch E. F., Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY, 1982.
  26. Graham F.L., van der Eb A.J. Transformation of rat cells by DNA of human adenovirus 5. Virology, 1973, 54(2): 536-539 CrossRef
  27. Laemmle U.K. Clevage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227: 680-685 CrossRef
  28. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods, 1984, 10(3/4): 203-209.
  29. Escribano J.M., Tabares E. Proteins specified by African swine fever virus. V. Identification of immediate early, early and late proteins. Arch. Virol., 1987, 92: 221-238. 
  30. Gibson D.G., Young L., Chuang R.Y., Venter J.C., Hutchison C.A. III, Smith H.O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 2009, 6: 343-345 CrossRef
  31. Rodriguez F., Alcaraz S., Yanez R.J., Rodriguez J.M., Alonso C., Rodriguez J.F., Escribano J.M. Characterization and molecular basis of heterogeneity of the African swine fever virus envelope protein p54. J. Virol., 1994, 68(11): 7244-7252.
  32. Goatley L.C., Dixon L.K. Processing and localization of the African swine fever virus CD2v transmembrane protein. J. Virol., 2011, 85(7): 3294-3305 CrossRef