Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2017, Vol. 52, № 6, p. 1251-1258
(how to cite this article)

doi: 10.15389/agrobiology.2017.6.1251eng

UDC 636.4:619:578:577.2.08:51-76

Acknowledgements:
Supported financially by Russian Science Foundation (grant № 16-16-00090)

 

OBTAINING A STABLE CELL LINE EXPRESSING RECOMBINANT I329L
PROTEIN OF AFRICAN SWINE FEVER VIRUS

S.A. Katorkin, E.I. Katorkina, K.A. Mima, I.A. Titov, A.S. Malogolovkin

Federal Research Center for Virology and Microbiology, Federal Agency of Scientific Organizations, 1, ul. Akademika Bakuleva, pos. Vol’ginskii, Petushinskii Region, Vladimir Province, 601125 Russia, e-mail e-mail mima89@ya.ru, Amalogolovkin@vniivvim.ru (corresponding author)


ORCID:
Katorkin S.A. orcid.org/0000-0001-7473-6692
Mima K.A. orcid.org/0000-0001-7184-6968
Malogolovkin A.S. orcid.org/0000-0003-1352-1780
Katorkina E.I. orcid.org/0000-0003-3329-0182
Titov I.A. orcid.org/0000-0002-5821-8980

Received July 8, 2017

 

The African swine fever virus (ASFV), a large DNA virus with icosahedral morphology, is the only representative of the family Asfarviridae. ASFV has a wide list of mechanisms for evading the host's immune system. This fact hinders the development the vaccines against ASF. One of the approaches used by the virus in immune evasion is the mimicry of Toll-like receptors (TLR) by immunomodulating proteins. ASFV immunomodulatory proteins are the most valuable tools for the understanding of the pathogenesis of the disease and to create a means of combating the disease. One such ASFV protein is pI329L, the antagonist and TLR3 signaling inhibitor, which reduces the interferon response of the body. Protein pI329L inhibits TLR3-mediated activation of NF-κB and induction of INF-b through the activation of TLR3 with its ligand — viral DNA, RNA and poly (I:C). Removing this protein from ASFV particles is a rational approach to developing a weakened virus vaccine. Therefore, I329L is characterized as a viral TLR3 antagonist, which negatively affects interferon antiviral response of the host. Purpose of the work was to obtain a CHO cell line stably expressing a TLR3 antagonist, the recombinant I329L protein of ASFV. Here, we designed the plasmid pBMN-I329-his, carrying the full-length I329L gene with 6xHis-tag at the C-terminus. By electroporation with plasmid pBMN-I329-his of the CHO cell line and further stabilization on a selective antibiotic (5 μg/ml puromycin), a stable CHO-I329L-His cell line was derived. The insertion of the I329L gene into the genome of the cell was confirmed by PCR using primers of the specific gene, followed by nucleotide sequencing, using as the template DNA isolated from the cells CHO-I329L-His. Western Blot confirmed the presence of I329L protein in the cell lysates of CHO-I329L-His. As a result of the analysis it was established that the size of the recombinant protein was 55 kDa compared to calculated 35 kDa. The sequential deglycosylation of endoglycosidases PNGase and Endo H of the target protein resulted in an increase in its electrophoretic mobility and detection of specific bands of ~ 37 and ~ 35 kDa. This fact confirms the high degree of glycosylation of the target molecule, which leads to a lower electrophoretic mobility. Additionally, the recombinant I329L protein was recognized by hyperimmune sera against the ASFV, which indicated its authenticity. The obtained stable cell line CHO-I329L-His is deposited in the cell culture museum of Federal Research Center for Virology and Microbiology and can be used to study the mechanisms of action of immunomodulating proteins, such as pI329L of the ASFV, and, therefore, to get deeper insight of the African swine fever virus biology.

Keywords: African swine fever, ASFV, TLR3 signalling, protein expression, recombinant pI329L, stable cell line.

 

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