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Information × Registration Number 0222U003722, 0120U104112 , R & D reports Title SARS-CoV-2 vaccine development based on the yeast expression of S protein fragments and studying immune response in the laboratory animals popup.stage_title Head Sibirny Andriy A., Доктор біологічних наук Registration Date 25-05-2022 Organization Institute of Cell Biology of the National Academy of Sciences of Ukraine popup.description2 Based on "humanized" strains of yeast K. phaffii and O. polymorpha, a collection of transformants was obtained containing in its genome the cassettes for expression of fragments of subunits S1 and S2 of the SARS-CoV-2 spike glycoprotein under the control of the AOX1 gene promoter induced by methanol. It was found that the fragments of the spike protein with N-terminal α-factor are not being secreted into the cultivation medium. Target proteins accumulated in the insoluble membrane fraction of cell-free extracts. A number of detergents were used to isolate the corresponding proteins, the most effective being guanidine hydrochloride. Because the process of purification of proteins from insoluble membrane fractions was found to be inefficient, additional strains of K. phaffii and O. polymorpha were constructed that produce RBD with flanking linker sites rich in the amino acids glycine and serine. This variant of RBD is secreted into the cultivation medium and can be easily purified without the use of detergents. Recombinant strains of K. phaffii containing NTD-RBD fragment of S protein, M- and E-proteins of SARS-CoV-2 and small S protein (dS, scaffolding protein) of duck hepatitis virus were also constructed to induce the production of SARS-CoV2 virus-like particles. Electron microscopy showed the presence of structures in yeast cells with a size of about 20 nm. Acetone powders and samples of cells inactivated with heating and subsequent lyophilization were obtained from such strains. Experiments were performed on mice to assess the tolerability and immunogenicity of the obtained samples, as well as on the Vero cell line infected with SARS-CoV-2 virus. The most effective were the sample of the protein FP-HR1-HR2 purified from cells of recombinant strain K. phaffii and acetone powders from cells of K. phaffii containing virus-like particles SARS-CoV-2.  Product Description popup.authors Vasylyshyn Roksolana V. Dmytruk Kostyantyn V. Zazulya Anastasiya Z. Semkiv Marta V. Fayura Lyubov R. Fedorovych Daria V. popup.nrat_date 2022-05-25 Close