The article examines the data of comparative bioinformatic analysis of the V and C regions of monoclonal antibody (MA) mRNA and substantiation of the potential auto- and polyreactivity of MA in cancer immunotherapy. It was shown that the limitations in coding are strictly followed in MA mRNAs, which is manifested in the linear neighborhood of codons with predominantly close values of complementarity indices. The features of the syntax of mRNA V region, manifested in their nucleotide composition and, accordingly, in the translational code, are associated with the mechanism of the occurrence of somatic hypermutations in them, caused in germinal centers by induced activation by cytidine deaminase, and they can be considered as limitations in mRNA recoding. They argue for the need to revise the ideas about the size of the Ab repertoire in the organism in the direction of its significant reduction, taking into account the subtle molecular mechanisms of their diversification. The autopolyreactivity of MA is justified by the existence of a peptide continuum of protein affinity. A violation of the global continuum of the organism regulation by MA is considered as a cause of the frequent occurrence of systemic complications in the cancer immunotherapy with MA.
In eukaryotic cells, distinct genome regions are associated with nuclear matrix which is the protein backbone of the nucleus. Recently, we identified genomic sites attached to nuclear pores through nucleoporin Elys in late Drosophila embryos. Many of these regions contained A/T-rich DNA motifs. Since according to published data, matrix attachment regions also frequently contain A/T-rich DNA sequences, we hypothesized that genomic regions attached to nuclear pores may be bound to nuclear matrix. The goal of this study was to check an association with nuclear matrix and the presence of DNase I hypersensitive sites within a nuclear pore attachment region from Drosophila embryonic cells. To this end, we have isolated nuclear matrix from Drosophila S2 cells using lithium 3,5-diiodosalicylate. The enrichment in the nuclear matrix fraction of a DNA fragment attached to nuclear pores was estimated by semi-quantitative PCR. Identification of DNase I hypersensitive sites in the same DNA fragment in late Drosophila embryos was carried out by Southern hybridization. As a result, we have shown that a genomic region attached to nuclear pores through nucleoporin Elys in Drosophila embryos is indeed associated with nuclear matrix and contains DNase I hypersensitive sites. These findings confirm an association of the analyzed genomic region with the nuclear envelope via nuclear pore complexes and deepen our understanding of mechanisms that maintain the localization of heterochromatin at the nuclear periphery.
The aim of this study was to analyze the CRISPR-Cas system of V. cholerae serogroup O1 strains of the classical biovar and nontoxigenic isolates of the El Tor biovar and determine their resistance to cholera phages. Materials and methods. Fifty-six strains of V. cholerae serogroup O1 were analyzed. Phage assays were performed using the two-layer method proposed by A. Gratia. Rostov 7 cholera phage was used as a model phage. Sequencing was performed on the MGI DNBSEQ-G50 platform (MGI, China). CRISPRCas typer ( https://github.com/Russel88/CRISPRCasTyper ) and CRISPRCas Finder ( https://github. com/dcouvin/CRISPRCasFinder ) were used to analyze the CRISPR-Cas system. Results and discussion. Classic biovar strains contain a class I CRISPR-cas system of type I-E, which is also prevalent in the studied nontoxigenic ctxAB–tcpA– strains of V. cholerae O1 El Tor. The latter strains also exhibit a canonical variant of the I-F system, as well as I-C, III-B, and a reduced I-Fmini type. The CRISPR array of classical vibrios contains spacers homologous to the protospacer sequences of cholera phages only, while El Tor vibrios possess spacers homologous to both cholera phages and phages of the Vibrio genus, unrelated bacteria, as well as plasmids and a transposon of V. cholerae. The obtained data on the presence of spacers to various cholera phages in the CRISPR array of a number of studied classical strains, as well as nontoxigenic isolates of V. cholerae O1 El Tor, demonstrate its activity. It was established that 82
The objective of the study was to develop an algorithm for constructing recombinant Yersinia pestis strains with regulated gene expression. Materials and methods. The suicide vector pCVD442 was used as a basis for constructing the plasmid pCVD442-URsurA-Lt0TT-PBAD-SDsurA, which provided the replacement of the native promoter of the Y. pestis surA gene with the arabinose-regulated araC PBAD promoter of Escherichia coli. The E. coli strain S17-1 served as a donor for plasmid transfer. Results and discussion. The plasmid pCVD442-URsurA-Lt0TT-PBAD-SDsurA was constructed based on the suicide vector pCVD442. A mutant strain 231(ΔPsurA::Lt0TT araC PBAD SDsurA) was obtained from the highly virulent Y. pestis strain 231. Using the creation of this strain as an example, an algorithm for obtaining attenuated mutants with arabinose-regulated gene expression from virulent strains was developed. The essence of the algorithm lies in the assembly and cloning of a cassette in the suicide vector pCVD442, which can be generally represented as URgeneX-Lt0TT-araC PBAD-SDgeneX, and finally obtaining a strain with the genotype ΔPgeneX::Lt0TT araC PBAD SDgeneX, where Lt0TT is the phage λ transcription terminator t0, araC PBAD is the promoter of the E. coli arabinose operon, and geneX is the target gene. Conclusions. A universal algorithm has been proposed that allows to obtain the araC PBAD mutants for any target genes of the plague microbe and other bacteria in which this system can function.
Adenovirus vector vaccines became an important class of vaccines following their approval for the prevention of Ebola virus fever and COVID-19. Quality data collected in the process of the production of adenovirus vectors include the determination of the number of viral particles and ratio of these particles to infectious ones. The determination of viral particle size is another quality parameter, which is of significant importance in characterizing the homogeneity of the preparation. Aim of the study. In this article, the dynamic light scattering (DLS) method for the particle size determination was developed; previously, it was used for studying adenovirus particles, but was not used for monitoring the quality characteristics of the preparations. Materials and Methods. Adenoviruses were grown in the adhesive culture of HEK 293 cells, concentrated, and purified by ultracentrifugation method in a cesium chloride density gradient. The number of viral particles in the preparation was determined spectrophotometrically. The measurement of viral particle size was performed using the dynamic light scattering method with the Malvern Nano ZS zetasizer (Malvern Instruments, United States). Results. Based on the results of the conducted work, two important parameters were determined for the analysis of adenovirus particle size using the DLS method. A concentration of adenovirus preparations from 1×1010 to 1 ×1011 virus particles/mL in the buffer with pH 7.5 allows for a correct determination of the particle size, which ranges from 100 to 127 nm regardless of the adenovirus species and type. Conclusions. The DLS method is a rapid and efficient analysis for assessing the sizes of adenovirus particles and determining the purity level of the obtained preparations. The minimal volume of required reagents, high rate of the analysis, and ease of execution make this method attractive for the routine use. The ability to detect foreign impurities is an important advantage of DLS, since their presence can reduce the effectiveness of the final preparation and negatively affect its properties.
