PLASMID pVAX-RBD DNA MATRIX OF mRNA-RBD MOLECULES, OBTAINED USING THE pVAX - RBD DNA MATRIX, PROVIDING THE SYNTHESIS AND SECRETION OF THE RBD PROTEIN SARS-CoV 2 IN MAMMALIAN CELLS AND A COMPLEX IN THE FORM OF NANOPARTICLES CONTAINING mRNA-RBD MOLECULES THAT INDUCE SARS-CoV-SPECIFIC ANTIBODIES WITH VIRAL NEUTRALIZING ACTIVITY

摘要

FIELD: biotechnology. ;SUBSTANCE: invention relates to biotechnology and genetic engineering. The pVAX-RBD plasmid DNA matrix was created, having a nucleotide sequence SEQ ID NO: 1 with a size of 3710 bp, a molecular weight of 2.6×103 kDa, containing the target gene encoding the chimeric protein 176-RBD, preceded by the promoter of phage T7 RNA polymerase, which provides in vitro synthesis of mRNA-RBD using T7 RNA polymerase. The mRNA-RBD molecule having the nucleotide sequence SEQ ID NO: 2 was obtained using the pVAX-RBD DNA matrix, contains an open reading frame encoding the amino acid sequence SEQ ID NO: 3, which provides synthesis and secretion of the RBD SARS-CoV 2 immunogen protein in the mammalian body, and contains a hybrid signal sequence 176, the Kozak consensus sequence, a 7-methylguanosine cap at the 5'-end of the mRNA, a polyadenosine (poly A) tail at the 3'-end of the mRNA, 150-200 nucleotides long, and 5'- and 3'-untranslated regions (UTR) before and after the open reading frame. A complex in the form of nanoparticles inducing SARS-CoV-specific antibodies with viral neutralizing activity, consisting of polyglucinepermidine conjugate molecules and mRNA-RBD molecules taken in different charge ratios from 1:1 to 1:30, corresponding to a weight ratio from 1:3 to 1:90, having a size in the range of 50-900 nanometers, and each nanoparticle contains a core consisting of mRNA-RBD molecules and coated with a layer of polyglucinepermidine conjugate retained due to the ionic interaction between a negatively charged nucleus and a positively charged spermidine. ;EFFECT: creation of a more effective means of delivering mRNA-RBD to the cells of the body for the synthesis of the RBD SARS-Cov-2 protein in them and increasing the level of secretion of this protein from the cells to ensure the presentation of the RBD protein to the immune system of the body. ;4 cl, 7 dwg, 1 tbl, 5 ex