METHOD FOR PRODUCING BACTERIOPHAGE HPV AND METHOD FOR PRODUCING MODIFIED BACTERIOPHAGE HPV Russian patent published in 2023 - IPC C12N7/00 

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. A method for reassembly (disassembly and assembly) of a bacteriophage capsid, including bacteriophage MS2, including recombinant ones, is presented. The method includes two stages: disassembly of the capsid of a natural or recombinant bacteriophage, and then assembly with various toxic ions and their compounds, for example, thallium and other elements, including radioactive isotopes and without them. Also presented is a method for obtaining bacteriophage HPV, including: the bacteriophage is concentrated to the required volume; to one volume of bacteriophage concentrated in step A), two volumes of pre-chilled 100% acetic acid are added; the mixture obtained in step B) is kept in a refrigerator at plus 4°C for 30 minutes; after 30 minutes, the mixture of bacteriophage and acetic acid is precipitated by centrifugation at 14000 g for 10 minutes in a centrifuge cooled to plus 4°C; the supernatant after precipitation is applied to a previously prepared column for desalting and centrifugation is performed for 2 min at 1000 g; collecting a slip containing the disassembled bacteriophage; disassembled bacteriophage diluted 3 times with 50 mm Tris pH 7; the mixture obtained in step G) is adjusted to pH 7.3 by adding 10 M NaOH; the mixture obtained in step G) is concentrated 7.5 times using a centrifugal concentrator at 3600 rpm in a centrifuge cooled to plus 4°C; the resulting concentrate of disassembled bacteriophage is divided into two parts; one part of the disassembled bacteriophage concentrate is incubated for 24 hours at plus 4°C in the presence of 25 mM TMAO per 50 mM Tris pH 7.10 mM yeast tRNA at a concentration of 1 mg/ml per 50 mM Tris pH 7; the second part of the disassembled bacteriophage concentrate is kept for 24 hours at plus 4°C in the presence of 25 mM TMAO, 10 mM yeast tRNA at a concentration of 1 mg/ml, and a thallium solution at a concentration of 0.16 mg/ml; after 24 hours, samples with collected bacteriophage are desalted, thus obtaining bacteriophage HPV. At the same time, bacteriophage HPV can have a capsid modified with a peptide, in particular, an IRGD peptide or a YGRKKRRQRRR peptide, and can also be obtained from recombinant capsid proteins with further modification - the addition of a peptide by conjugation. HPV can also be obtained from recombinant peptide-linked capsid proteins, and thallium is a radioactive isotope.

EFFECT: invention makes it possible to use bacteriophages as an effective means of delivering, in particular, thallium salts or peptides to the recipient's body; this method also does not allow sufficient control of the packaging of thallium, to reduce nonspecific immunogenicity; patented method makes it possible to control the packaging of thallium, to reduce the nonspecific immunogenicity of the drug by ensuring the absence of bacteriophage RNA, thus, the effect of the claimed invention is to purify the preparation of a bacteriophage virus-like particle (VLP) from bacteriophage RNA in order to reduce nonspecific immunogenicity; also, the effect consists in controlling the packaging of thallium, the method allows to overcome the barrier of the minimum concentration of thallium when obtaining the drug substance, the concentration is 0.16 mg/ml, which is important when using radioactive isotopes in virus-like particles; the claimed method allows to obtain recombinant bacteriophage HPV.

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