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.
This study aimed to develop a method for producing human recombinant insulin-like growth factor (IGF-1) based on a prokaryotic expression system and to characterize the highly purified protein. To achieve the study’s goal, the following methods were conducted: we performed automated chemical synthesis of DNA, constructed the expression plasmid, obtained Escherichia coli cell-producers of human recombinant IGF-1, cultivated the obtained producer cells with the induction of recombinant protein synthesis by isopropyl-β-D-1-thiogalactopyranoside and lactose, and purified human recombinant IGF-1 with affinity and cation exchange chromatography. The recombinant protein IGF-1 forms inclusion bodies during synthesis in Escherichia coli BL21 cells that contain plasmid pET28-IGF-1. Purified recombinant protein was obtained with a purity of 98% using affinity and cation exchange chromatography methods. The protein yield was 6 mg of human recombinant IGF-1 from 1 g of raw biomass. The resulting protein has the ability to protect Neuro 2a neuroblastoma cells from death caused by the deprivation of serum in the culture medium and can stimulate the differentiation of cells into neurons. Thus, a highly purified human recombinant IGF-1 was obtained. This protein has biological activity and is suitable for preclinical studies.
BACKGROUND: Rotaviruses are the main cause of acute gastroenteritis in children in both developed and developing countries. Vaccination is the only way to prevent severe and fatal course of this disease. Live attenuated viruses-based vaccines currently available can have a number of side effects. A candidate rotavirus vaccine reported is based on a hybrid recombinant protein FliCVP6VP8, which includes a VP6 protein fragment, a rotavirus A VP8 protein fragment, and S. typhimurium FliC flagellin components. AIM: The aim was to evaluate the immunogenicity and safety of а preparation Rotavirus vaccine, recombinant in preclinical studies. MATERIALS AND METHODS: The immunogenicity of vaccine (blood antibody titers, antigen-specific proliferative response of spleen cells) was evaluated in BALB/c mice. The acute and subchronic toxicity, the possible irritating effect, pyrogenicity and the anaphylactic effect and delayed type hypersensitivity were evaluated in laboratory mice, rats, Guinea pigs, and rabbits. RESULTS: Double immunization of mice with the candidate vaccine demonstrated a significant increase in antibody titers in mouse sera compared to that in control mice. Evaluation of antigen-specific proliferative response after double immunization with a candidate vaccine demonstrated a significant increase in the values of stimulated proliferation. Evaluation of safety through acute and chronic toxicity studies demonstrated no toxicity. The immunostimulatory effect of vaccine was demonstrated when evaluating the number of antibody-producing cells with sheep red blood cells as antigens. The number of white blood cells was demonstrated to increase after the prolonged vaccine administration. CONCLUSIONS: The preclinical studies have demonstrated safety of the candidate rotavirus vaccine and its capability to produce the immune response.
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