ABSTRACT
Introduction. Hyperhomocysteinemia is a dangerous metabolic disorder that leads to a number of diseases. Problem Statement. Urgent task is to develop pharmaceutical product for lowering the homocysteine levels without causing side effects. Purpose. To develop the dietary supplement for reducing high homocysteine levels, which has a minimum content of components that may cause side effects;to test the supplement effect on the cognitive abilities of animals and to commercialize the product. Materials and Methods. The developed dietary supplement Alfacognitin contains vitamins B6, B9, B12, C, and choline. For modelling experimental hyperhomocysteinemia in rats, the animals are kept on a diet rich in L-methionine. Blood homocysteine concentrations are determined by the ion exchange liquid column chromatography method with the use of an automatic amino acid analyzer. The behavioral responses and cognitive abilities of the rats have been studied with the use of behavioral tests (open field test, fear conditioning test, and social interaction test). The production of Alfacognitin dietary supplement has been launched with Nutrimed Ltd. (Kyiv). Results. Alfacognitin has been shown to reduce homocysteine levels, to improve cognitive abilities, social interaction and communication skills, and to compensate functional memory and learning disorders in animals with hyperhomocysteinemia. Specifications for the dietary supplement have been approved, a pilot technology for obtaining the capsule form of the drug has been developed, and an experimental batch has been manufactured. Conclusions. Alfacognitin may reduce the homocysteine levels. Therefore, it may be used to normalize the functional state of the cardiovascular and nervous systems in patients with hyperhomocysteinemia, as well as to improve the cognitive functions, in particular in patients after COVID-19.
ABSTRACT
The quickly emerged global COVID-19 pandemic raised a desperate need in the development of pro-tecting vaccines targeting this disease. Therefore, a generation of effective producers of recombinant SARS-CoV-2 proteins became an urgent task. Its resolving contributes to the study of functional SarS-CoV-2 properties, as well as will allow developing the domestic COVID-19 vaccine in Ukraine, thus playing an important strategic role in tackling the pandemics. The aim of the study was to generate prokaryotic and eukaryotic producers of recombinant SarS-CoV-2 proteins and to isolate nucleocapsid (N) protein, receptor-binding do-main (RBD) of spike (S) protein, as well as RBD fused to the carrier – diphtheria toxoid CRM197. For this purpose, appropriate genetic constructs, in particular, replication deficient recombinant AdvC5-based adenoviral vectors expressing the SarS-CoV-2 proteins and CRM197-fused conjugate were created through methods of molecular biology and genetic engineering. Restriction analysis and/or dna sequencing confirmed that we created the correct constructs. Immobilized metal affinity chromatography was used to purify the recombinant proteins. Compliance of their properties was confirmed by the results from polyacrylamide gel electrophoresis, Western blotting, immunoenzymatic assay and MALDI-TOF mass spectrometry. As a result, we generated E. coli Rosetta (de3) bacterial strain and HEK293 cell line producing recombinant SARS-CoV-2 proteins and CRM197-based fusion. In addition, pure N protein, RBD of S protein and RBD-crm197 fusion protein were isolated. The obtained recombinant SarS-CoV-2 proteins can be used to study immunogenic and antigenic properties of the SarS-CoV-2 proteins. Cells producing recombinant SarS-CoV-2 proteins and RBD-crm197 fusion protein are able to provide cheap and safe synthesis of the antigenic substances for domestic development and production of immunodiagnostics for COVID-19 and COVID-19 vaccines in Ukraine. © 2021 Krynina O. I. et al.