SLURP-1 is a three-finger human protein targeting nicotinic acetylcholine receptors (nAChRs). The recombinant forms of SLURP-1 produced in E. coli differ in added fusion fragments and in activity. The closest in sequence to the naturally occurring SLURP-1 is the recombinant rSLURP-1, differing by only one additional N-terminal Met residue. sSLURP-1 can be prepared by peptide synthesis and its amino acid sequence is identical to that of the natural protein. In view of recent NMR analysis of the conformational mobility of rSLURP-1 and cryo-electron microscopy structures of complexes of α-bungarotoxin (a three-finger snake venom protein) with Torpedo californica and α7 nAChRs, we compared conformations of sSLURP-1 and rSLURP-1 by Raman spectroscopy and CD-controlled thermal denaturation, analyzed their competition with α-bungarotoxin for binding to the above-mentioned nAChRs, compared the respective receptor complexes with computer modeling and compared their inhibitory potency on the α9α10 nAChR. The CD revealed a higher thermostability of sSLURP-1; some differences between sSLURP-1 and rSLURP-1 were observed in the regions of disulfides and tyrosine residues by Raman spectroscopy, but in binding, computer modeling and electrophysiology, the proteins were similar. Thus, sSLURP-1 and rSLURP-1 with only one additional Met residue appear close in structure and functional characteristics, being appropriate for research on nAChRs.

Leishmania tarentolae (LEXSY) system is an inexpensive and effective expression approach for various research and medical purposes. The stated advantages of this system are the possibility of obtaining the soluble product in the cytoplasm, a high probability of correct protein folding with a full range of post-translational modifications (including uniform glycosylation), and the possibility of expressing multi-subunit proteins. In this paper, a LEXSY expression system has been employed for obtaining the receptor binding domain (RBD) of the spike-protein of the SARS-CoV-2 virus and the homopentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis. RBD is actively used to obtain antibodies against the virus and in various scientific studies on the molecular mechanisms of the interaction of the virus with host cell targets. AChBP represents an excellent structural model of the ligand-binding extracellular domain of all subtypes of nicotinic acetylcholine receptors (nAChRs). Both products were obtained in a soluble glycosylated form, and their structural and functional characteristics were compared with those previously described.

Introduction. Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier. Objective. The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method. Materials and methods. Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct. Results. Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase. Conclusion. Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.

Aim. Study of changes in the phenotypic characteristics of the virulent A/WSN/33 (H1N1) strain of influenza A virus under the influence of the inclusion of site-specific mutations in the PB1-gene of this strain.Materials and methods. Using a two-step polymerase reaction in the PB1 gene of A/ WSN/33 (H1N1) strain were included ts mutations taken from the genome of attenuated CA donors-strains: A/Ann Arbor/6/60 (H2N2), A/Leningrad 134/17/57 (H2N2) and A/ Krasnodar/101/35/59. Ts-phenotype, att-phenotype, immunogenicity, as well as weight loss in mice infected with these mutants were studied in the obtained site-specific mutants.Results. It was shown that the inclusion of ts mutations from the genome of CA donorsstrains of attenuation in the PB1 gene of the virulent A/WSN/33 (H1N1) strain leads to a change in the phenotypic characteristics of this strain to different degrees.Discussion. Analysis of the genome of CA strains- donors of attenuation of influenza virus indicates the crucial importance of the presence of functional defects in the PB1– protein for the formation of the attenuation phenotype of the virus.Conclusion. The technology of site-specific mutagenesis canbe successfully used to modify the PB1 gene of a virulent influenza A virus strain in order to construct a new generation of live influenza vaccines.

The aim of study was to carry out comparative investigation of biological properties of site-specific mutants of Influenza A virus and variant of live cold-adapted (CA) influenza reassortant vaccine. Materials and methods. The genetic stability of site-specific mutants (SSM) of the A/WSN/33 (H1N1) strain with ts (temperature sensitive)-mutations in polymerase genes was studied using a stress-test in MadinDarby Canine Kidney (MDCK) culture. A comparative study of immunogenicity of U2 and M26 mutants with the high genetic stability and the CA-reassortant with similar surface proteins was carried out. The increase in the antibody titer was investigated using enzyme-linked immunosorbent assay and the reaction of delayed hemagglutination. Ability of the studied viruses to induce type 1 interferon in A549 cells was determined using real-time polymerase chain reaction (real-time PCR). Results. It was shown that U2 and M26 mutants, which have 3 ts-mutations or more in polymerase genes have high genetic stability. It was found that U2 and M26 mutants induced a higher antibody titers than the CA reassortant in mice following the intranasal immunization. The ability of site-specific mutants and CA reassortant to induce type 1 interferon was also investigated. Mutants U2 and M26 increased the level of interferon to a greater extent than the CA-reassortant. Conclusion. The data obtained indicate that SSM U2 and M26 with 3 ts-mutations or more in the genome have a significant level of genetic stability. Mutants U2 and M26 have a higher immunogenicity and a higher ability to induce interferon in comparison with the CA reassortant. These facts allow us to conclude that SSM of the influenza virus with a set of mutations in polymerase genes can be considered as promising candidates for live influenza vaccines.