Резюме. Белок CRM197 является нетоксичным производным дифтерийного токсина и характеризуется одной мутацией, а именно, заменой глицина на глутаминовую кислоту в положении 52. Белок CRM197 является перспективным адъювантом нового поколения, который может быть успешно использован в вакцинных и терапевтических препаратах. Классическим способом получения дифтерийного токсина и его нетоксичных производных является продукция в клетках Corynebacterium diphtheriae. Преимущество использования E. coli в качестве продуцента состоит в том, что данный метод является более простым и дешевым, и позволяет получать рекомбинантный CRM197 в короткие сроки с использованием непатогенного микроорганизма. В данной работе использовался запатентованный высокопродуктивный штамм-продуцент рекомбинантного CRM197 на основе клеток E. coli. В ходе исследования подобран оптимальный протокол индукции экспрессии гена crm197, разработан метод получения высокоочищенного препарата рекомбинантного CRM197 путем последовательного применения ионообменной, гидрофобной и молекулярноситовой типов хроматографии.

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So far, there is no effective treatment for a number of socially significant bacterial and fungal diseases. β-(1→3) glucans are polysaccharides consisting of glucose units linked together with β-(1→3)glycoside bonds. They are principal components of the fungal cell wall including such dangerous nosocomial pathogens as Candida albicans, Aspergillus fumigatus and others. At the same time, β-(1→3) glucans are absent in humans and other mammals, thus making them to be promising components of carbohydrate-protein conjugate vaccines for prevention and treatment of fungal infections. The CRM197 protein is a non-toxic derivative of diphtheria toxin, which is widely used as a safe carrier for conjugate vaccines. The CRM197 extraction from lysogenic C. diphtheriae cultures is a classic way of its production. An alternative to the classic method is a transgene-driven CRM197 protein expression in E. coli, B. subtilis and Pseudomonas fluorescens. The advantage for using Escherichia coli in this case is that this method is more simple and inexpensive, and allows of producing recombinant CRM197 within short terms employing a non-pathogenic microorganism. The purpose of this study was to investigate antigenic activity in the samples of experimental conjugate vaccines based on synthetic oligosaccharide ligands and CRM197 carrier protein, by means of a competitive ELISA. A double immunization of laboratory Balb/c mice with experimental samples of conjugate vaccines based on oligosaccharide ligands and CRM197 protein caused induction of specific antibodies at high titers. All the samples of oligoglucoside-based conjugate vaccines with different contents of monomeric units (pentasaccharides, heptasaccharides, nanosaccharides, and undecasaccharides) caused the formation high antibody titer at the level 1: 51,200,following tandem injections. High avidity of antibodies to their oligosaccharide ligands was shown by competitive ELISA reaction. These data suggest a relevance of further pre-clinical trials of conjugate vaccines against Candida and Aspergillus, as well as selection of the most immunogenic and effective version of the conjugate vaccine.

The aim of research: to synthesize the gene, coding the non-toxic variant of diphtheria toxin CRM197, to explore its expression in Escherichia coli cells, to construct bacterial strain - producer of recombinant CRM197 and to develop the method for purification of this CRM197 protein from the biomass of bacteria. Materials and methods of research. the gene of CRM197 was synthesized by chemical and enzymatic methods. Expression vector pColdII-CRM197 construction and assembly were performed by standard genetic engineering methods. CRM197 production by Escherichia coli cells was explored by electrophoresis in polyacrylamide gel and immunoblotting. The recombinant CRM197 was purified with the use of metal-chelate chromatography. Results. The application of the pColdII expression vector made possible to produce recombinant protein after decrease of the cell cultivation temperature from 37° C to 16° C. The method for purification of CRM197 recombinant protein was developed and the protein preparation with the purity 97% was obtained, having molecular mass 60 kDa; this protein was coupled with polyclonal antibodies against diphtheria toxin in the immunoblot. Conclusion. Successful production of CRM197 recombinant protein was reached with the use of pColdII vector at the decreased cell cultivation temperature. The purified CRM197 demonstrated nuclease activity pointing its proper folding. Production of the purified recombinant CRM197 protein allows its use for development of new conjugate vaccines.