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Tytuł:
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Towards development of plasmacytoma cells-based expression systems utilizing alphavirus vectors: An NS0-VEE model.
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Autorzy:
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Keyer VV; National Center for Biotechnology, Korgalzhin hwy 13/5, 010000, Astana, Kazakhstan. Electronic address: .
Shevtsov AB; National Center for Biotechnology, Korgalzhin hwy 13/5, 010000, Astana, Kazakhstan. Electronic address: .
Zaripov MM; Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Puschino, 142290, Russia. Electronic address: .
Baltabekova AZ; National Center for Biotechnology, Korgalzhin hwy 13/5, 010000, Astana, Kazakhstan. Electronic address: .
Ramanculov EM; National Center for Biotechnology, Korgalzhin hwy 13/5, 010000, Astana, Kazakhstan. Electronic address: .
Shustov AV; National Center for Biotechnology, Korgalzhin hwy 13/5, 010000, Astana, Kazakhstan. Electronic address: .
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Źródło:
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Journal of virological methods [J Virol Methods] 2019 Dec; Vol. 274, pp. 113734. Date of Electronic Publication: 2019 Sep 13.
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Typ publikacji:
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Journal Article; Research Support, Non-U.S. Gov't
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Język:
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English
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Imprint Name(s):
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Original Publication: [Amsterdam] : Elsevier/North-Holland Biomedical Press, 1980-
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MeSH Terms:
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Cell Line, Tumor*
Gene Expression*
Genetic Vectors*
Biotechnology/*methods
Encephalitis Virus, Venezuelan Equine/*growth & development
Recombinant Proteins/*biosynthesis
Animals ; Cytopathogenic Effect, Viral ; DNA, Complementary/genetics ; Electroporation ; Encephalitis Virus, Venezuelan Equine/genetics ; Mice ; Plasmids ; Promoter Regions, Genetic ; Recombinant Proteins/genetics ; Transfection ; Virus Cultivation/methods
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Contributed Indexing:
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Keywords: Alphavirus; Apoptosis; Cytopathogenicity; Electroporation; Myeloma; Plasmacytoma; Rescue; VEE
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Substance Nomenclature:
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0 (DNA, Complementary)
0 (Recombinant Proteins)
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Entry Date(s):
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Date Created: 20190917 Date Completed: 20200525 Latest Revision: 20200525
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Update Code:
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20240105
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DOI:
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10.1016/j.jviromet.2019.113734
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PMID:
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31525396
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Plasmacytoma (myeloma) cells have a large protein expression capacity, although their industrial use is confined to stable expression systems. Vectors derived from genomes of viruses from the genus Alphavirus allow obtaining of high yields of target proteins but their use is limited to transient expression. Little information has been published to date on attempts to combine the myeloma cells as hosts with alphaviruses as expression vectors. A plasmid construct which allows rescue of a model alphavirus Venezuelan equine encephalitis virus (VEE) upon transfection of a cell culture was created. Mutations in the capsid and nsP2 genes allow for less cytopathogenic propagation of the virus. A cDNA-copy of the genome was placed in a plasmid under the control of the CMV promoter for virus rescue following DNA transfection. Parameters for the virus rescue by electroporating of the infectious clone in murine myeloma cells (NS0) were optimized. The highest FFU counts (1.2 × 10 5 FFU per 10 ug DNA) were produced with 2 pulses (voltage 250 V, capacitance 960 u F) and the best electroporation buffer was selected from eight buffers. Self-sustained VEE infection was established in NS0 cultures with high titers (8 × 10 8 FFU/ml) of the virus, despite a fraction of infected cells dying during 5-days observation. Further development of the NS0-VEE expression system may require addressing of apoptosis induced by VEE.
(Copyright © 2019 Elsevier B.V. All rights reserved.)
