Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2016, V. 51, № 6, pp. 837-844.

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)

 

EXPRESSION OF RECOMBINANT GENES ENCODING FRAGMENTS OF THE PROTECTIVE IMPORTANT PROTEINS OF AFRICAN SWINE FEVER VIRUS IN EUCARYOTIC CELLS

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 almazlcf@yandex.ru

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)

 

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