The three­dimensional shape of a DNA molecule is a key property influencing its functional specificity and the nature of its molecular interactions. The characteristic shape into which a DNA molecule folds under certain conditions is a manifestation of its micromechanical and structural features, which are sequence­dependent. DNA shape­related properties can there fore be determined in a predictable manner. A number of models have been designed to describe intrinsic DNA curvature, incorporating a set of helical parameters which can be applied to operative three­dimensional reconstruction of the DNA structures. Alternatively, desired base pair parameters can be computed based on publicly available information about atomic DNA structures. Further, taking the base pairs as rigid bodies, their relative location in space can be estimated based on these parameters. Matrices are a common method to implement any rigid body transformations and are widely used in the modeling of DNA structures. Quaternions are the more straightforward and robust alternative for matrices. Unit quaternions can represent only a rotation, whereas dual quaternions combine rotation and translation into a single state. In the present guide, the algebra of unit and dual quaternions is applied for the first time to modeling of the DNA helical path, based on conformational parameters of the base pair steps. Although dual quaternions are preferable for  modeling of DNA structure in detail, the use of unit quaternions is sufficient to predict the DNA trajectory and all calculations of DNA shape features. In order to analyze DNA shape and chain sta ­ tistics, and predict the micromechanical properties of DNA molecules based on coordinates of the helical path, the widely used as well as original algorithms for computing DNA curvature, radius of gyration, persistence length and phasing of DNA bends are described. Taken together, these algorithms will be useful both in the in silico analysis of relatively short DNA fragments as well as in topological mapping of whole genomes.

The interactions between protein and DNA in essence underlie all processes in a living cell. Understanding the principles of specific recognition of DNA sites will open the way to understand how these processes are controlled and to interfere in their operation. In the paper we studied contacts between the protein and DNA at the atomic level in the structures of all the 3 518 protein­DNA complexes available in PDB by the Voronoi–Delaunay tessellation method. The method unambiguously defines contacts between atoms without any parameters, and characterizes each contact by the distance between atoms and the contact area, which is determined by the corresponding face of the Voronoi polyhedron. It was shown that most contacts are formed between the protein atoms and the sugarphosphate backbone of the DNA (72.9 %). The contact with the atoms of the nucleic bases emerging into the grooves of DNA is 17.0 % for a major groove and 10.1 % for all atomic contacts for a minor groove. Totally, the interaction between protein atoms and nucleic base atoms accounts for 27.1 % of all contacts. Analysis of the accessible surface area of atoms in the major and the minor grooves showed a correlation with the number of contacts (coefficient of linear correlation 0.94 and 0.93, respectively), however, nucleic acid atoms forming hydrogen bonds make contacts more often than may be expected from statistical considerations. It was shown that conformationally stable peptides occur sometimes in the binding regions with DNA. Analysis of the residues in a predefined conformation in 3 518 protein­DNA complexes revealed 159 amino acid residues in a predefined β­bend type I conformation, 15 residues in the conformation of β­bend type I’, and 6 residues in the conformation of β­bend type II. No residues in the conformation of β­bend type II’ were found. Analysis of contacts showed that such residues virtually do not form contacts with DNA. Contacts with nucleic base atoms are found only in the two homologous structures 3qea and 3qe9, where threonine atoms form contacts with atoms of nucleotide bases of the AT­pair.

Gene networks are molecular genetic systems that ensure the formation of phenotypic characteristics of organisms (molecular, biochemical, structural, morphological, behavioral, etc.) based on  information encoded in their genomes. Reconstruction of gene networks provides a methodological basis for modern systems biology. In this regard, the information on the structural and functional organization of gene networks accumulated in modern databases is extremely valuable. This review characterizes a number of Internetaccessible information resources oriented to humans and animals and containing data on gene networks and their functional modules. Without pretending to fully cover all information resources containing data related to humans and animals on the subject, the current review was created to report the current status of the problem and to present the criteria according to which we propose to evaluate the utility of webresources for specific research tasks. On this basis, we compiled and characterized a collection of databases containing information on metabolic and signaling pathways, as well as pathways of regulation of biological processes at the cellular and organismal levels. In addition, we observed the characteristics of several well­known databases containing data on interactions between biomolecules of various types. The following characteristics of databases were considered: (1) the types of information accumulated in the databases; (2) methods of data presentation; (3) methods of data collection; (4) data sources; (5) special search tools and options for data analysis. A comparison of the above characteristics showed that the databases are very heterogeneous according to their scopes, sources and types of data, interfaces, as well as according to their search options and data analysis tools. It was concluded that at the first step of the gene network reconstruction it is important to form a full set of information resources from which the data can be obtained. The web portals accumulating information about the databases that may be useful for the reconstruction and analysis of gene networks are specified.

The paper presents the results of a study of the tissue­specificity of the circadian phase characteristics of biological processes in the mouse liver and kidneys. We performed a comparative analysis of the translatomes in these two organs based on experimental data on the daily dynamics of the level of translation of mouse genes from the GEO database (GSE67305 and GSE81283) obtained by ribosome profiling. Genes with a pronounced daily dynamics of translation were revealed (3 358 genes in the liver and 2 938 in the kidneys). Further, for each of the 12 time points (ZT0–ZT22), for each tissue (liver, kidneys), groups of genes that were in a phase with an increased level of translation were identified. It was assumed that the gene is in a phase with an increased level of translation if at a given time point its ribosome profiling rate for both replicas exceeded the daily average value for this gene. The greatest number of rhythmic genes in the liver has an increased level of translation at the beginning of the dark phase of the day corresponding to increased animal activity. In the kidneys, the differences in the distribution of the number of genes in the phase of an elevated translation level by the time of day were less pronounced, and the maximum number of such genes was observed from the middle of the light phase of the day to the middle of the dark one. A statistical analysis of enrichment of Gene Ontology terms in these twelve gene groups in the liver and kidneys was perform ed.  Analyzing the processes, the rhythmicity of which is typical of both liver and kidneys, we have identified the processes, the circadian phase characteristics of which in these tissues coincide and the processes having essentially different temporal phase patterns for these tissues. Processes with strict tissue­specific rhythmic translation have also been identified. The approach used in our work allows us to analyze the organo/tissue­specificity of the phase characteristics of biological processes, and the results emphasize the need to take into account the phase circadian characteristics when comparing the features of the course of biological processes in various organs.

Aggressive behavior in animals plays an important role in protecting the territory, offspring, establishing social hierarchical relations, etc. Increased aggression is observed in a number of diseases ( schizophrenia, bipolar disorder, brain degenerative disorders). Neuronal apoptosis is crucial in the maintenance of developmental processes during neurogenesis. Alterations in neuronal apoptosis are observed in aging and neuropathologies accompanied by changes in psycho­emo­ tional state (epilepsy, Alzheimer’s disease, neurotrauma). The expression of key neuronal apoptosis genes (Casp3, Bax and Bcl-xl) in the brain of highly aggressive rats is significantly altered. The aim of this work was to analyze associative networks that describe genetic interactions between genes/proteins involved in neuronal apoptosis, differentially expressed genes and genes with polymorphisms in grey rats with aggressive behavior. Analysis revealed 819 differentially expressed genes in the hypothalamus, ventral tegmental region and periaqueductus Sylvii grey matter in grey rats with aggressive and tame behavior. The Stx1a, Mbp and Th genes have the highest index of betweenness centrality in the associative network of differentially expressed genes. Genome analysis revealed 137 polymorphic genes. Three of them (Lig4, Parp1 and Pigt) were involved in neuronal apoptosis. It was shown that polymorphic and differentially expressed genes were statistically significantly overrepresented among ge nes interacting with neuronal apoptosis genes (p value < 0.01). Three molecular­genetic chains describing connections between polymorphic and neuronal apoptosis genes mediated by differentially expressed genes were reconstructed. Chains included the polymorphic genes Tsc1, Adamts4 and Lgals3, differentially expressed genes Ezr, Acan, Th and 19 neuronal apoptosis genes. It was shown that neuronal apoptosis is closely related to aggressive behavior in animals.

CIMP+ (CpG­Island Methylator Phenotype) tumors are characterized by dense methylation of promoter CpG islands of many genes at once and represent a separate group of malignant neoplasms of the colon. Despite the fact that the diagnostics of CIMP+ tumors has a significant prognostic value, an effective set of markers has not been developed yet. For the identification of CpG sites, the methylation level of which could be used to detect CIMP+ tumors, an analysis of expression and methylation profiles of 297 primary colon tumors and 38 histologically normal tissues paired to them, which are presented in the TCGA (The Cancer Genome Atlas) project database, was performed by us using the CrossHub tool created previously. We developed the scoring, which takes into account the methylation level of CpG sites, their location, and the expression level of the corresponding genes. It was revealed that the methylation status of CpG sites of the AMOTL1, ZNF43, ZNF134, and CHFR genes is a promising marker of CIMP+ tumors. Moreover, specific regions of promoters of these genes, the methylation level of which was associated with the examined phenotype, were identified. To verify the obtained data in independent sampling, first, the quantitative PCR was used to assess the relative mRNA level of the AMOTL1, ZNF43, ZNF134, and CHFR genes in 30 paired (tumor/histologically normal tissue) colon samples. For all the genes, a frequent (50–60 % of cases) and significant (2–30­fold) expression decrease was revealed. Then, the bisulfite conversion of DNA followed by cloning and sequencing was applied to examine the methylation status of CpG sites that were selected as the result of bioinformatics analysis. We observed a high methylation level (β­value = 0.3–0.9) of the CpG sites in the samples with simultaneous downregulation of all 4 genes and a low methylation level (β­value = 0.0–0.2) in the samples with the unchanged expression level of 4 genes and in histologically normal tissues. Thus, the methylation status of the CpG sites of promoter regions of the AMOTL1, ZNF43, ZNF134, and CHFR genes is a promising potential marker of CIMP+ colon tumors.

One of the most important branch of modern molecular genetics and biomedicine is the search for predictive markers that help choose the most effective way of treatment, drug and also determine its individual dosage. Among the markers, those that can provide the possibility of using a non­invasive, so­called “liquid biopsy” are considered particularly promising. This method allows the condition of the tumor to be assessed by analyzing the body’s natural fluids, such as blood, urine or saliva. Such studies are most convenient in those cases when it is necessary to monitor the effectiveness of therapy in order to record the time of the onset of resistance of tumor cells, the onset of relapse and to move on to the next line of therapy. In the treatment of aggressive and rapidly became metastatic malignant tumors, such as melanoma, the presence of reliable markers that allow quick and accurate determination of treatment tactics is especially important. Nowadays, there is an increasing number of studies devoted to the search for predictive markers of the effectiveness of immunotherapy. Melanoma is one of the most immunogenic tumors and, as a result, has become a model object for research into and introduction of new approaches to immunotherapy. In this study, we compared two groups of patients with metastatic skin melanoma, with different responses to immunotherapy with blockers of immune control points, to identify new predictive expression biomarkers among microRNAs and mRNAs, and to identify the genes responsible for the occurrence of an objective response to therapy. As a result, the study detected several microRNAs with a significant change in expression level within the tumor tissue of patients responding differently to immunotherapy. Differences in the level of expression of their target genes have also been found, that will allow a more detailed analysis of the molecular mechanisms that determine the sensitivity or resistance of malignant melanoma cells to the immunotherapy. Based on the obtained data, we have proposed expression markers (mRNAs and microRNAs) that can be used as predictors of malignant melanoma tumors to immunotherapy.

Colorectal cancer (CRC) is one of the most common malignant neoplasms in the world, and is characterized by a high mortality rate. The study of the key aspects of colorectal cancer formation and progression is necessary to develop new approaches to its therapy, as well as to search for new diagnostic, prognostic and predictive biomarkers of CRC. In many types of tumors, one of the key changes in metabolism is the  activation of glycolysis, which is associated with alterations in the expression of the main glycolytic enzymes and regulatory molecules. There is often an increase in hexokinase 2 (HK2) exogenous expression in tumor cells, which makes it a promising target for anticancer therapy. Quantitative expression analysis of 15 genes (GAPDH, ADPGK, ALDOA, ENO3, PFKL, PGK1, PGAM1, PKM2, ENO1, PDK1, PDK3, PFKP, ENO2, GPI, and BPGM), encoding the key glycolysis enzymes, as well as HIF1A gene was carried out in a modified RKO cell line, which constantly expresses the short hairpin RNA (shRNA) for the inhibition of hexokinase 2. A significant decrease in the expression of PFKP, BPGM, and GPI genes both at the mRNA (5­, 86­, and 93­fold, respectively) and protein (2.5­, 3.5­, and 19­fold, respectively) levels was revealed. Probably, the downregulation of GPI and PFKP is associated with a decrease in the amount of their substrates, glucose­6­phosphate and fructose6­phosphate, under the inhibition of hexokinase 2. Nevertheless, the cause of a decreased mRNA level of these three enzymes, while the expression level of other glycolytic participants is constant, requires further investigation.

The use of some assisted reproductive technologies, in particular, embryo transfer, may cause various physiological and behavioral changes in the offspring. The purpose of our study was to study the effects of surgery (which is used for embryo transfer) done with pregnant dams on the weight, blood pressure and behavior in the open field and elevated plus­maze tests in adult offspring. Thus, long­term effects on the offspring after maternal exposure to surgical stress given to dams at the 4th day of pregnancy were studied in OXYS rats. OXYS females were mated in estrus with fertile males of the same strain. 96 hours after spermatozoa were found in vaginal smears the surgery (sham operation, imitating embryo transfer) was performed. Body weight (BW), systolic (SAP) and diastolic (DAP) arterial pressure as well as behavior in open field (OF) and elevated plus maze (EPM) tests were studied in the offspring of females exposed to surgical treatment during pregnancy (OXYS­PS) at the age of 3 mo. Untreated offspring of OXYS rats were used as controls. BW in naturally born OXYS rats did not differ from those of the OXYS­PS group. OXYS and OXYS­PS rats exhibited higher SAP (more than 150 mm Hg) and DAP; it is noteworthy that both SAP and DAP were higher in the OXYS­PS group than in the control group. The time spent in the center of arena, the area studied, the time and number of rearing were decreased in OXYS­PS rats in the OF test as compared to the OXYS controls. Moreover, OXYS­PS rats were characterized by the absence of grooming in the OF test. As was demonstrated by the EPM test, the duration and numbers of peeking out from closed arms were decreased in the OXYS­PS rats as compared to the OXYS controls. Thus, OXYS dams’ exposure to surgical stress at their early pregnancy led to such effects in the offspring as elevated SAP and DAP, decreased overall activity and increased anxiety.

The populations of honeybees and bumblebees have been decreasing around the world in the recent decades. A variety of pathogens and parasites, including  bacteria, fungi, protozoa, nematodes, mites and insects play signi ficant role in honeybee and bumblebee colonies loss. Pa rasites of the genus Nosema (Microsporidia: Nosematidae) and the genera Crithidia and Lotmaria (Kinetoplastida: Trypanosomatidae) have a significant negative impact on honeybee and bumblebee colonies. Recent  studies of nuclear DNA markers of these parasites allowed to describe new species and genetic variants. The aim of this study was to investigate the Microsporidia (Nosema spp.) and Trypanosomatidae (Crithidia spp. and Lotmaria  passim) prevalence and genetic diversity in honeybee and bumble bee populations of Indian territories that haven’t been studied before. In total 119 specimens of 4 honeybee and 5 bumblebee species were analyzed in this study. The prevalence of parasites in honeybee and bumblebee po pulations of the two Indian states (Jammu and Kashmir, Karnataka) were identified using PCR with primers specific for the ribosomal RNA genes cluster of Nosema, Crithidia and Lotmaria species. Co­infection by microsporidian and trypanosomatid parasites was detected in several honeybee and bumblebee specimens from Jammu and Kash mir state. Comparative analysis of ribosomal RNA genes sequences showed that honeybee samples from India studied were infected by N. bombi, N. ceranae and L. passim.  Bumblebee populations were infected by Nosema D, Crithidia bombi and Crithidia expoeki. No honeybee’s specimen with trypanosomatid infection was found in Karnataka state. For the first time N. bombi infection was detected in the honeybee population. The studies of distribution of microsporidia and trypanosomatid parasites among the honeybee and bumblebee populations all over the World were summarized and supplemented.

Staphylococci are capable of penetrating many human tissues and organs, causing superficial and deep purulent infections, respiratory and urinary tract infections, food poisoning and intoxication. Last years, coagulase­negative staphylococci were the cause of infection in many cases. Infectious agents, namely Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis, were detected more often as nosocomial infections. A particular danger of these infections is a high virulence and  pathogenicity of bacterial strains and their resistance to various anti ­ biotics. Methicillin­resistant staphylococci are especially difficult to treat. The correct identification of staphylococci and their sensitivity to antibiotics are important for clinical diagnosis and appointment of adequate drug therapy. Rapid and accurate identification of Staphylococcus species and detection of their sensitivity to antibiotics is quite important. The aim of this study was to study staphylococci isolated in Novosibirsk from human, animal and environmental samples. A collection of 100 staphylococcus strains was analyzed. Staphylococcus species were identified by sequencing the 16S rRNA gene. Eleven staphylococcus species were identified. Among the strains obtained from hospitalized patients, Staphylococcus aure us dominated (79.1 %), Staphylococcus  epidermidis amounted to about 12.5 %. However, S. aureus and S. epi dermidis strains were isolated in an approximately equal proportion from community­associated samples. Identification of coagulase positive strains was performed using a standard biochemical method and by real­time PCR of the coa gene. 100 % coincidence between the presence of the gene and coagulase activity for S. aureus strains was recorded, which suggests that detection of the coa gene can be used as a correct method for S. aure us identification. A high coincidence rate (99 %) was reveal ed between the phenotypic resistance to oxacillin and the presence of the staphylococcal mecA gene. The study of staphylococci for the presence of the mecA gene can be considered as an alternative to the phenotypical method for identification of methicillin­resistant strains of staphylococci.

Hydrolytic bacteria (in particular, proteolytics) are the primary destructors in hot springs. The proteolytic bacteria are able to secrete enzymes that are active in wide ranges of pH and temperature. The aim of this work was to study the taxonomic composition, the structure of the bacterial microbial mat, and to study the distribution of peptidases in the thermophilic microbial Garga community. For the study, we sampled the microbial mat at a water temperature of 54.2 °C and a pH of 8.3. Hydrochemical analysis of water showed a high content of sulfates, 390 mg/dm3. The microelement composition of water showed that the Garga water had increased concentrations of B, Rb, Li, Ba, Sr. We analyzed the taxonomic diversity of the microbial community in the hot spring Garga at a temperature zone of 54 °C. The structure of the microbial mat is represented by various phylogenetic groups of mesophilic and thermophilic bacteria, with various metabolic and ecological functions. The dominant group in this community was the phylum Firmicutes (64 %). The analysis of the collected metagenomic sequences of the microbial community allowed the detected peptidases in the microbial community in the hot spring Garga to be for the first time systematized and characterized. Comparisons of metagenomic sequences of representative data showed a dominance of serine peptidase class enzymes. Natural peptidases in the investigated microbial community ensure the hydrolysis of biopolymers at the first stages of the destruction of organic matter and may have biotechnological relevance.

Deficiency of some carbohydrates in the traditional diet of native populations of the Far North contributed to a high population prevalence of inactive variants of genes encoding, for example, amylase (AMY2A gene) and sucrase­isomaltase (SI gene). Trehalose, which is found in algae, higher fungi, lichens and some higher plants, is another of the disaccharides, poorly digested by native people of the Far North. Here, in native populations of Siberia, we studied the polymorphism of the TREH gene, which encodes trehalase, an enzyme that cleaves trehalose. The analysis of exome polymorphism showed the presence of seven haplotypes of the TREH gene in the population. Three of them are determined by the variant rs2276064­A, which is associated with the lowest activity of trehalase. The maximum frequency of this group of haplotypes is observed in the samples of native populations of Northeast Asia (about 60 %), in the remaining samples of the Siberian populations, its frequency is 30–40 %. Thus, the high frequency of variant rs2276064­A, associated with a low­activity trehalase, explains why Northern aboriginal peoples avoid food containing trehalose. It is assumed that the increase in the frequency of this variant in the populations of Northeast Asia can have been facilitated by gene drift acting in populations of a small effective size. However, it is possible that artificially induced trehalose deficiency in the traditional diet of the indigenous peoples of the Far North (due to the tradition of rejecting fungi) could also cause an increase in the frequency of low­activity trehalase, provided that this tradition has been existing among Northern aboriginal peoples for many generations.

Yeasts are a convenient model eukaryote used for genome studies and genome editing. Saccharomyces cerevisiae is the species most widely employed in biotechnology, since it is easily cultivated in bioreactors and is absolutely safe. The last decade saw a significant development of methods of yeast genetic engineering and the creation of novel instruments adapted from other fields, which allowed one to significantly accelerate the construction of new strains. The most prominent examples are the proteins used for directed DNA editing. For a long time, yeast genome engineering was based on the yeasts’ system of homologous recombination. It was sufficient for several decades before the development of high­throughput methods. Many high­throughput methods were developed in the second decade of the XXI century, including those used in genomics, transcriptomics, proteomics, metabolomics, interactomics, etc. Modern bioinformatic databases now allow one to rapidly process the increasing flow of information and model cellular processes. As a result, the rate of analysis and prediction of targets for genome editing is currently higher than the rate of genome editing, which led to the development of new methods of genetic engineering. This process was particularly pronounced for microorganisms. Modern tasks require tens, hundreds, sometimes even thousands of genome modifications, which made researchers to look for new techniques. As a result, the instruments used for more complex objects, such as animals, plants, and cell lines, were adapted for yeasts. Modern methods for yeast genome editing allow introducing several modifications into the genome in a single step. In this study, we review the methods of directed genome editing and their applications and perspectives for yeasts.

The market of pharmaceutically valuable proteins is the fastest growing segment of the economy. Most biopharmaceuticals have been obtained in mammalian and microorganism cells, but both systems have a number of disadvantages. Plant cells combine the advantages of the eukaryotic system of protein production and the simplicity and cheapness of the bacterial, and the use of plants for the production of recombinant proteins is an economically important and promising direction. The advantage of plant systems is the lower cost of cell cultivation. They are free from unwanted components, such as bacterial endotoxins, hyperglycosylated proteins produced by yeast, animal and human pathogens in cell cultures of transgenic animals. In addition, plants are higher eukaryotes, and therefore fullvalue folding and the formation of multimeric protein complexes occur in their cells, as well as a significant portion of post­translational modifications similar to those in mammalian cells. The currently developed plant expression systems for recombinant proteins are extremely diverse and number more than 100 different technologies based on different plant species, gene transfer methods, expression strategies, methods for the subsequent extraction of the target protein, etc. This is nuclear and plastid transformation, transient and stable expression during transformation using agrobacterial transport, bombardment or electroporation, cultivation of whole terrestrial or aquatic plants, plant tissues or suspension cell cultures as expression systems. The review examines the current state of research in the use of plant expression systems for the production of recombinant proteins for pharmaceuticals. The emphasis was placed on the advantages of plant cell cultures in comparison with other expression systems. Specific examples discuss promising plant systems for the production of recombinant proteins, such as transplastomic plants, moss and aquatic plant cultures, as well as suspension cultures of cells of higher plants. The current state of the market for recombinant proteins obtained using plant expression systems is considered. The prospects of creating plant (“edible”) vaccines based on genetically modified plants are discussed.

The present work describes the construction of the gene encoding the recombinant protein flagG­protE, its synthesis, purification and study. The recombinant flagG­protE protein is a promising molecule for developing a candidate recombinant vaccine against tickborne encephalitis by the ability to bind to monoclonal antibodies (MCA) against native protein E of tick­borne encephalitis virus. The antigenic determinants of two recombinant proteins were studied: protE and flagG­protE using a panel of 8 MCA. The recombinant protein protE comprises the tick­borne encephalitis virus envelope protein and the flagGprotE recombinant protein has an additional flagG domain encoding flagellin G of Salmonella typhi. It was found that the MCA tested revealed epitopes on the recombinant protein protE. This indicates that the investigated recombinant protein has an antigenic structure similar to the antigenic structure of the native tick­borne encephalitis virus protein E. In the study of the recombinant protein flagG­protE by the ability to bind a panel of 8 MCA, only five of them react with epitopes of the tested protein. MCA 4F6, 7F10, and 6B9 did not recognize the corresponding epitope in the recombinant flagG­protE protein, while in the recombinant protein protE, these epitopes were detected successfully. Our data indicate that the antigenic structure of recombinant protE­protein can be changed under the influence of the flagellin domain, which in turn can lead to the unavailability of some antigenic determinants. This fact must be taken into account when constructing recombinant molecules with antigenic properties. Nevertheless, the fundamentally important regions in the region of the fusion peptide and III domain are antigenically present on the surface of the recombinant protein. This should ensure the formation of neutralizing antibodies, and the presence of a complete amino acid sequence of protein E in the recombinant protein induces the formation of a T­cell immune response. The emergence of a new generation of vaccines against tick­borne encephalitis with a higher level of safety and immunogenicity will improve the vaccine prevention of the population from tick­borne encephalitis. 

Fully­human antibodies have a great therapeutic importance; however, the development of stable strains providing a high level of production of full­size antibodies is a challenging task, as antibody molecules contain two types of polypeptide chains. To develop the producing strain, random integration of the plasmid containing the gene encoding the target protein into the genome of the host cells is commonly used. The aim of this study was the development of an original expression system, using gene targeting to integrate the gene encoding the fully­human antibody into the transcriptionally active region of the genome of eukaryotic suspension cells CHO­S. To develop a stable strain, the cassette vector plasmid pCDNA5/FRTDHFR­CH­CL containing the site of homologous recombination and the genes encoding heavy and light chains of the fully human antibody of the IgG1/kappa class was constructed at the first step. Notably, DNA of the plasmid pCDNA5/FRT­DHFR­CH­CL was organized in such a way that the restriction sites for rapid cloning of DNA fragments encoding the variable domains of heavy and light chains were inserted upstream of the sequences encoding constant domains of the heavy and light chains of the antibody. Secondly, DNA fragments encoding the variable domains of the heavy and light chains of antibody against orthopoxvirus protein p35 were inserted into the pCDNA5/FRT­DHFRCH­CL cassette plasmid. Then, CHO­S/FRT cells, which contain the FRT­site for homologous recombination and are able to produce green fluorescence protein GFP, were transfected with the constructed plasmid. After the insertion of the target genes into the FRT­site, GFP production was supposed to stop. Using this selection system, a stable clone producing target antibody fh8E was selected with the level of production of about 100 μg/ml. The binding affinity of purified antibody fh8E with the targeted protein, measured by surface plasmon resonance, was 12 nM. In addition, antibody fh8E demonstrated anti­vaccinia virus activity in the plaque reduction neutralization test in vitro.