DEVELOPMENT OF AUTONOMOUSLY REPLICATING VIRAL RNA TO EXPRESS THE RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR

Makhabbat Kaliyeva, Alexander Shustov

Abstract


Recombinant human granulocyte colony-stimulating factor (G-CSF) produced in cultured mammalian cells undergoes proper posttranslational modifications and, thereby, possesses better pharmacological properties in comparison with the homologous protein expressed in bacteria. Biopharmaceuticals derived from cell culture tend to be expensive because of lower yields compared to bacterially expressed competitors and numerous issues with the scalability of production. Particular limitation of scalability pertains to delivery of expression vectors to the cell culture. Natural and efficient way to deliver foreign DNA or RNA to cell is a viral infection. We intended to develop the viral genome capable of G-CSF expression.

Objectives: To develop autonomously replicating viral RNA capable of heterologous expression of the G-CSF in cultured mammalian cells.

Methods: Genetic engineering, cell culture, and virology.

Results: Viable genome of the Venezuelan equine encephalomyelitis virus (VEE) was constructed. The G-CSF gene was synthesized de novo. Gene cassette GFP-2A-G-CSF was constructed for simultaneous expression of the fluorescent marker of viral replication (GFP) and the product of interest. Recombinant viral genome VEE/GFP-2A-G-CSF was assembled; its viability was confirmed upon transfection to the BHK-21 cells.

Conclusion: Genetic engineering of the cDNA copies of the genomes of RNA viruses provides excessive opportunities to develop mammalian expression systems. We achieved production of recombinant proteins in cultured mammalian cells transfected with the VEE viral RNA carrying the inserted genes of marker protein and G-CSF.


Keywords


RNA virus, autonomously replicating RNA, replicon, Venezuelan equine, encephalomyelitis virus, granulocyte colony-stimulating factor, eukaryotic expression

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References


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DOI: http://dx.doi.org/10.12955/cbup.v3.629

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