METHOD FOR OBTAINING THERAPEUTIC AGENT FOR CURING OF TICK-BORNE ENCEPHALITIS

摘要

FIELD: biotechnologies.;SUBSTANCE: method involves production of aptamers that are capable of forming a complex with tertiary structure of surface virus protein. Fragment of surface protein E of tick-borne encephalitis virus, aminoacid from 50 to 250 from N-end of the protein, the gene of which is amplificated and cloned in a plasmid vector, is used. Cloned recombinant protein is expressed. The obtained protein is cleaned and immobilised on a plane table. From initial pool of degenerated oligonucleotides composed of 5'-CTCCTCTGACTGTAACCACG-(N40)-GGCTTCTGGCTACCTATGC-3', where N - any of nucleotides (G, A, T, C) taken in equivalent amount at synthesis of 40-link oligonucleotides containing fluorescent colourant FAM on 5'-end by means of 15 cycles of exponential benefication, SELEX, there performed is extraction of aptamers specifically bonded to virus protein. At each sorption stage there performed is denaturation of 25 pM single-stranded oligonucleotide. Denaturated pool of aptamers is applied onto a basin with immobilised protein, incubated and washed from non-bonded oligonucleotides. Aptamers bonded to protein are removed from surface with buffer with low ionic force and high pH. Pool of aptamers is amplificated after each sorption stage. Obtained amplicons of two-stranded library of aptamers are cleaned and a reaction of asymmetric polymerase chain reaction (PCR) is performed. The product is cleaned by means of sorption so that target product is obtained in the form of aptamers.;EFFECT: invention allows obtaining aptamers that are used for prevention of tick-borne encephalitis disease.;5 cl, 1 ex