Аңдатпа
A protein B18R is encoded by Western Reserve (WR) strain of Vaccinia virus. B18R works as a decoy receptor for the type I interferons (IFNs), attaches to cell surfaces, binds IFNs without prominent species-specificity and prevents an establishment of the antiviral state. At a same time B18R has no effect on cell viability itself. Thereunder B18R has become an essential aid in cell technologies which use RNA-mediated gene delivery. Despite an established high demand for this protein in cell technologies and also attractive prospects for use in mammalian expression systems to maintain RNA-vectors, little information is available on obtaining of B18R. In majority of works utilizing it, this protein has not been produced in a pure form. Rather, conditioned media (CM) are collected from cell cultures infected with Vaccinia virus or transfected with a B18R mRNA. The CM containing secretory-expressed B18R are used without an isolation of B18R. As the production in cell cultures is expensive, a development of a bacterial expression system is warranted. Here is described the bacterial expression and a method for refolding of recombinant B18R. Multi-milligram quantities of recombinant B18R are easily produced and the product is biologically active despite bacterially-expressed B18R is not glycosylated. It has been successfully utilized to support persistence of a RNA vector derived from a genome of a RNA-virus in cell cultures capable of the antiviral state. A synthetic gene B18R which encodes the aminoacid residues His20-Glu351 from the Genbank entry D01019 is used in this work. The B18R gene sequence is codon-optimized for the optimal expression in E.coli. A sequence of an expression plasmid pET28-B18R(HisTag) is deposited to the Genbank MG356786. The expression product has a hexahistidine tag at the N-terminus which is used for a metal-affinity purification at one step of the protocol.