Methods and compositions for expressing negative-sense viral rna in canine cells

FIELD: chemistry.

SUBSTANCE: invention is an isolated nucleic acid comprising a canine RNA polymerase I regulatory sequence and containing (i) at least 250 or at least 350 or at least 450 adjoining nucleotides or an entire nucleotide sequence, which is in form of SEQ ID NO:26, (ii) a nucleotide which is at least 80% identical to said nucleotide sequence (i) or includes a complementary or reverse complementary (i) or (ii) sequence. The nucleotide sequence (i) or (ii) is operably linked to cDNA which encodes influenza virus vRNA, and when produced in MDCK cells, is capable of directing expression of said influenza virus vRNA. The present invention also describes expression vectors and cells containing such nucleic acids, as well as methods of using such nucleic acids to obtain influenza viruses, including infectious influenza viruses.

EFFECT: canine plasmid rescue system pol I enables to obtain recombinant influenza viruses in a canine cell culture with high titre.

25 cl, 16 dwg, 7 tbl, 12 ex

 

The text descriptions are given in facsimile form.

1. The selected nucleic acid comprising a regulatory sequence of RNA-p is limarzi I dogs and containing (i) at least 250, or at least 350, or at least 450 contiguous nucleotides or the entire nucleotide sequence represented as sequences of SEQ ID NO:26, (ii) polynucleotide that at least 80% identical to the above nucleotide sequence (i), and the specified nucleotide sequence (i) or (ii)being functionally related to the cDNA, which encodes wrnc influenza virus, and being introduced into MDCK cells, capable of directing the expression of the specified wrnc influenza virus or includes complementary or complementary back (i) or (ii) sequence.

2. Nucleic acid according to claim 1, in which the regulatory sequence is a promoter.

3. Nucleic acid according to claim 1, in which the regulatory sequence is an RNA polymerase I is a nucleotide 1-469 sequence SEQ ID NO:26, complementary sequence or her back she complementary sequence.

4. Nucleic acid according to claims 1, 2 or 3, in which the regulatory sequence functionally linked to cDNA encoding the viral genomic RNA with negative thread or appropriate crnc.

5. Nucleic acid according to claim 4, which further includes the sequence termination of transcription.

6. Nucleic acid according to claim,5, in which the viral genomic PH is from minus-strand is the genomic RNA of the influenza virus.

7. The expression vector comprising the sequence of claim 1, 2 or 3.

8. The expression vector according to claim 7, which includes a sequence represented in SEQ ID NO:29.

9. The expression vector comprising the nucleic acid of claim 6.

10. The method of obtaining the genomic RNA of influenza virus, including the transcription of the nucleic acid according to claim 6 in the cell, which is produced by the genomic RNA of the influenza virus.

11. A method of obtaining a recombinant influenza virus, comprising culturing cells of a dog, comprising the expression vector according to claim 9 and one or more expression vectors expressing mRNA encoding one or more polypeptides of influenza virus selected from the group comprising polypeptides: RW, RW, RA, NA, NP, NA, M1, M2, NS1 and NS2, and selection of recombinant influenza virus.

12. A method of obtaining a recombinant influenza virus, including
a) the introduction into a population of cells of dogs expression vectors that can Express in these cells the genomic segments wrnc to obtain the complete genomic segments wrnc given virus in which these expression vectors include i) at least 250, or at least 350, or at least 450 contiguous nucleotides or the entire nucleotide sequence represented as sequences of SEQ ID NO:26, includes (ii) polynuclear the ID, which at least 80% identical to the above nucleotide sequence (i), and the nucleic acid sequence being operatively linked to cDNA, which encodes wrnc influenza virus, and being introduced into MDCK cells, capable of directing the expression of the specified wrnc influenza virus or includes complementary or complementary back (i) or (ii) the sequence
(b) the introduction into cells of expression vectors capable of expression of mRNA that encodes one or more polypeptides of the given virus, and
(C) culturing these cells, which are formed viral particles.

13. The method according to claim 11, in which the titer of particles of influenza virus obtained by culturing these cells for 48-72 h, is at least 1.0×104plaque-forming units (PFU)/ml

14. The method according to claim 11, in which the titer of particles of influenza virus obtained by culturing these cells for 48-72 h, is at least 1.0×105PFU/ml

15. The method according to claim 11, in which the obtained particles of influenza virus are infectious.

16. The method according to item 12, in which the resulting particles of influenza virus are infectious.

17. Cell, ensuring the expression of the RNA of influenza virus and comprising the expression vector of claim 9.

18. Cell is on 17, which is the cage of the dog.

19. Cell dogs on p, which are the cells of the kidney.

20. Cells of the kidney of the dog in claim 19, which belong to the cell line MDCK.

21. A method of obtaining a recombinant influenza virus, including
a) the introduction into a population of cells of dogs expression vectors capable of expression in said cells genomic segments wrnc to ensure complete genomic segments wrnc given virus, and one or more of these expression vectors include (i) at least 250, or at least 350, or at least 450 contiguous nucleotides or the entire nucleotide sequence represented above as a sequence of SEQ ID NO:26, includes (ii) polynucleotide that at least 80% identical to the above nucleotide sequence (i), and the specified sequence of nucleic acid being functionally related to the cDNA, which encodes wrnc influenza virus, and being introduced into MDCK cells, capable of directing the expression of wrnc influenza virus or includes complementary or complementary back (i) or (ii) the sequence
b) is also capable of expression in these cells mRNA that encodes one or more polypeptides of influenza virus selected from the group consisting of polypeptides: RW, RW, RA, NA, NP, NA, M1, 2, NS1 and NS2, and
C) culturing these cells, and, as a result, obtaining particles of influenza virus.

22. The method according to item 21, in which the titer of particles of influenza virus produced by culturing these cells for 48-72 h, is at least 1.0×104PFU/ml

23. The method according to item 21, in which the titer of particles of influenza virus produced by culturing these cells for 48-72 h, is at least 1.0×105PFU/ml

24. The method according to item 12 or 21, in which is used a virus helper.

25. The method of obtaining the genomic RNA of influenza virus, including the introduction of the expression vector of claim 9 in cells, and thus obtaining the genomic RNA of the virus.



 

Same patents:

FIELD: biology, medicine, nanotechnology.

SUBSTANCE: group of inventions is referred to the area of biology, medicine, nanotechnology and involves processing of new platform carriers for formation of complexes with biologically active compounds. The proposed platform carriers are of spherical shape and are made by the way of thermal reconfiguration of structure of tobacco mosaic virus (TMV) or another tobamovirus or another plant virus with spiral structure or their fragments. The platform carriers are also produced by the way of thermal reconfiguration of coat protein of TMV group virus (tobamoviruses) or another phytovirus with spiral structure or their fragments. There were also proposed the compositions containing the platform-carrier of said spherical carrier connected with the alien protein or another biologically active compound. The advantages of new platform carriers include the simplicity and quickness of production, possibility to get spherical particles of regulated sizes, long term storage, absence of aggregation and preservation of spherical shape during its storage, centrifugation and resedimentation, the convenience of spherical particles shape for formation of complexes with target compound.

EFFECT: important feature of a new carrier platform is the capability to adsorb on the surface and to form compositions with different structurally and functionally alien proteins/antigens.

3 cl, 6 ex, 10 dwg

FIELD: medicine.

SUBSTANCE: what is described is a composition containing an ordered antigen pattern where antigen represents IL-1, mutein IL-1 or fragment IL-1. There is also offered a based vaccine. The compositions offered in the invention can be applied for producing vaccines for inflammatory diseases and chronic autoimmune diseases, transmittable diseases and cardiovascular diseases.

EFFECT: compositions effectively induce immune responses, particularly humoral immune responses; compositions are the most suitable for effective induction of autogenic immune responses.

46 cl, 2 dwg, 3 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: disclosed is a conditionally defective particle of influenza virus with the absence of a nucleic acid segment of influenza virus selected from a group of segments, mainly coding acid polymerase (PA), basic polymerase 1 (PB1) and basic polymerase 2 (PB2). The conditionally defective particle of influenza virus is used for induction of influenza virus defence. Also, a method of producing such particles, a pharmaceutical composition containing such particles, its application and a method of induction of influenza virus defence are described.

EFFECT: higher efficacy.

27 cl, 4 dwg, 4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention is intended for inhibiting reproduction of influenza A viruses in infected eukaryotic cells by means of virus-specific catalytically active oligodeoxyribonucleotide (deoxyribozyme). Method is realised by obtaining deoxyribozyme, consisting of nucleotide sequence: 5'-GAAATAAGAGGCTAGCTACAACGACCTTCATTA, intended for inhibiting reproduction of influenza A viruses.

EFFECT: extension of spectrum of anti-viral action of deoxyribozymes and increase of their anti-viral activity.

3 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to new compounds which are N-substituted 1,4-diazabicyclo[2.2.2.]octane derivatives. The offered compounds exhibit antiviral activity and can find application in medicine as active components for the development of dosage forms used for treating viral diseases.

EFFECT: higher antiviral activity of the compound.

7 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of veterinary and deals with mutant virus of bull diarrhea. Essence of invention includes virus of bull diarrhea, containing at least one amino acid mutation of helicase domaine, where mutation in domain NS3 results in loss of recognition by monoclonal antibody, generated against wild type of NS3, but were virus RNA replication and generation of infectious virus are preserved. Second version includes virus of bull diarrhea containing at least one mutation in helicase domain, where mutation is in helicase domain in IGR loop and/or in KHR loop and/or in SES loop.

EFFECT: advantage of invention lies in obtaining mutant virus of bull diarrhea, where virus RNA replication and generation of infectious virus are preserved.

21 cl, 10 ex, 7 tbl, 5 dwg

FIELD: medicine.

SUBSTANCE: recombinant virus of cowpox contains virus of cowpox, which includes modified gene of thymidine-kinase (TK), modified gene HA and modified gene F3 or broken locus F3. At the same time each of TK, HA and F3 gene or locus of cowpox virus is inactivated, and F3 gene or locus is located in fragment HindIII-F of cowpox virus between open reading frames F14L and F15L.

EFFECT: improved compositions and methods for treatment of cancer and inhibition of immune-privileged cells or tissues in animals.

34 cl, 3 dwg, 2 tbl, 13 ex

FIELD: medicine.

SUBSTANCE: invention can be applied for blocking of chronic integration diseases caused by human immunodeficiency virus (HIV), bovine leucosis virus (BLV), bovine immunodeficiency virus (BIV), mouse leucosis virus (MuLV), equine infection anemia virus (EIA) and hepatitis B virus. Effect is achieved by application of live cells of Mycoplasma arginini microbe non-pathogenic to humans, used for superinfection of T lymphocyte infected with HIV.

EFFECT: enhanced specificity and efficiency of stepwise removal of integrated DNA provirus from cell genome preserving cell vitality, despite of any virus mutations.

5 dwg, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: method provides target modification of virulent properties of influenza virus by genetic modification of caspase and granzyme viral proteolysis sites. There is also described application of such genetically modified influenza virus as vaccines and therapeutic agents, translation and expression vectors of biologically active genetic elements, polypeptides and fragments thereof in an organism-recipient for metabolism targeting in bodies and tissues and as agents for selective elimination of damaged and tumour cells in an organism. The invention can be used in medicine.

EFFECT: well-directed attenuation or enhancement of viral virulence.

11 cl, 4 dwg, 2 tbl, 7 ex

FIELD: medicine, virology.

SUBSTANCE: invention relates to method for virus controlling using substance based on 2,8-dithioxo-1H-pyrano[2,3-d;6,5-d']dipyrimidine and 10-aza- analogs thereof. Claimed method includes sharing said substances together with integrase inhibitor, reverse transcriptase inhibitor, ore protease inhibitor in various ratio (variants).

EFFECT: method for virus inhibiting of increased effectiveness.

7 cl, 8 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention describes Mus museums hybrid cultivated cell strains of Russian National Collection of Industrial Microorganisms H-117, Russian National Collection of Industrial Microorganisms H-116, Russian National Collection of Industrial Microorganisms H-115, Russian National Collection of Industrial Microorganisms H-113 and Russian National Collection of Industrial Microorganisms H-114 - producers of the monoclonal antibodies showing various specificity to human recombinant erythropoietin (EPO) and enabling distinguishing between a glycolised form of EPO and accompanying hypo- and deglycolised impurities. Estimating the strain efficacy and the produced antibody specificity is ensured by immune-enzyme assay with using a number of specificity markers: EPO recombinant protein; hypoglycolised EPO (o-glycolised) produced by natural antigen periodate oxidation; deglycolised EPO (de-EPO) produced by enzymatic treatment of recombinant EPO (PNGasa).

EFFECT: hybridoma produced monoclonal antibodies with said specificity are required for recovery of glycolised forms of EPO, and the quantitative and qualitative analysis of EPO mixtures.

5 cl, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: there is constructed a recombinant plasmid DNA pAP271 containing a rhFVII protein gene, a MAR matrix attachment region of an avian lysozyme gene, CMV virus transcription amplifier, a promoter of translational factor of human EF-1α elongation, an internal site of translational initiation of encephalomyocarditis IRES virus, a mouse DHFR gene, a SV40 virus polyadenylation signal, a cartridge comprising all elements required for aminoglycoside-3'-phosphotransferase (APH) gene expression, a cartridge for expression in bacterial cells of β-lactamase gene, as well as unique recognition sites of the following restriction endonucleases: Agel (850 base pairs), BbvCI (1657 base pairs), BmgBI (4202 base pairs), BssHII (6672 base pairs), Eco47III (11269 base pairs), EcoRI (10929 base pairs), EcoRV (11863 base pairs), Fsel (1455 base pairs), NotI (4812 base pairs), RsrII (6790 base pairs), Sbfl (2330 base pairs), Sfil (6027 base pairs), Tthllll (6390 base pairs), Xcml (2404 base pairs).

EFFECT: presented invention provides producing stably high-yield recombinant human blood coagulability factor VII.

2 cl, 4 dwg, 2 ex

Fused partner cell // 2431667

FIELD: medicine.

SUBSTANCE: invention describes fused partner cells making it possible to produce heterohybridomas of cells of biological species different from a mouse. Heterohybridomas are produced by fused myeloma cells produced from an animal of a first biological species with leukemia cells produced from an animal of a second biological species which have an additional S-phase in a cell cycle and exhibit a diploidisation property. The fusion partner cell SPYMEG is deposited, No. FERM BP-10761 and can be used for hybridoma production. What is described is hybridoma producing antibodies produced by fusion of fusion partner cell and a third cell with antibody-producing ability. The invention describes methods for producing a fusion partner cell, a hybridoma and an antibody-producing cell, and also methods for producing antibodies. Use of the invention provides stable and simple production of the substances with the use of hybridomas in a wide range of species of animals.

EFFECT: hybridomas stably keep a phenotype throughout the whole process of cloning.

20 cl, 10 dwg, 2 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: described is strain of hybrid cultivated cells of animals Mus. Musculus RIM9 - producer of monoclonal antibodies, specific to peptide of human milk - lactaptin and its recombinant analogues, which possess apoptotic activity with respect to human cancer cells. Invention allows to obtain monoclonal antibodies, recognising general linear antigen determinant of recombinant and natural lactaptin.

EFFECT: possibility to elaborate sensitive and specific test-systems for detecting lactaptin, antibodies to it and for isolation of lactaptin from human milk by method of affinity chromatography.

2 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to anti-M-CSF-specific antibodies based on RX1 or originating from RX1, and which more than 785% compete with monoclonal antibodies RX1, MC1 and/or MC3 for bonding with M-CSF (macrophagal colony-stimulating factor). The non-mouse antibody is two-stranded, contains a certain amino acid sequence (given in the formula of invention and list of sequences) and retains high affinity towards M-CSF. The invention discloses an isolated nucleic acid which codes the said antibody, an expression vector, a host cell and a method of producing the anti-M-CSF-antibody using a host cell or hybridome, particularly ATCC PTA-6263 or ATCC PTA-6264 hybridome. The invention describes a pharmaceutical composition containing said antibodies, sets containing pharmaceutical compositions and methods of preventing and treating osteoporosis in a person suffering from an osteolytic disease.

EFFECT: disclosed antibodies can inhibit osteoclast differentiation, which facilitates their use as highly effective preparations for treating osteolysis, cancer with metastases and osteoporosis associated with cancer metastases.

131 cl, 44 dwg, 12 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology. Hybridoma strain is prepared by immunisation of BALB/c mice with bovine HSP 70 within 78 days. On the third day, splenocyte hybridisation of immune mice (10 cells) with murine myeloma cells P3-X63 Ag/8-653 (107 cells) is carried out. As a fusion agent, polyethylene glycol of molecular weight 4000 (Merk, Germany) is applied. Hybridisation is followed with selection, screening, cloning and cryopreservation of hybridoma.

EFFECT: invention can be used for preparing monoclonal antibodies (MCA) to heat shock protein 70 (HSP 70).

4 dwg, 1 tbl, 6 ex

FIELD: veterinary.

SUBSTANCE: obtained is strain 8C12 of inter-species hybrid cells of mouse Mus musculus and sheep Ovis aries - producent of monoclonal antibodies of sheep to glycoproteidal antigen of virus of cattle (C) leucosis. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 72. Strain is permanent hybrid line of cells and possesses high level of production of monoclonal antibodies of sheep. Antibody titers in native culture liquid constitute 1:32-1:64 in immuno-enzymatic analysys (IEA). Monoclonal antibodies are specific to general antigen determinant of glycoproteids of C leucosis - external gp51 and transmembranous gp30. When used in IEA for detection of antibodies in blood serum and milk of C infected with leucosis virus, antibodies provide strong selective binding with solid-phase carrier and optimal space orientation of glycoproteidal antigen.

EFFECT: strain 8C12 can be used in production of immuno-enzymatic test-system for diagnostics of cattle leucosis, which will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms and, as a result, reduce incidence of leucosis in cattle.

1 tbl, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biochemistry, specifically to obtaining a hybrid, and can be used for obtaining a strain-producer of monoclonal antibodies to the MUCI human antigen. Using monoclonal antibody technology, a strain of hybrid cultured animal cells "ВКПМ Н-105" - producer of clinical rat monoclonal antibodies, specific for hypoglycosylated and deglycosylated isoforms of tumours associated with the human MUCI antigen can be obtained.

EFFECT: possibility of identifying clinical isoforms of MUCI antigen using antibodies, produced by an obtained strain, the antibodies of which can be used determining concentration of MUCI in blood plasma of a person previously diagnosed with tumours.

1 dwg, 3 ex

FIELD: medicine, peptides, biochemistry, pharmacy.

SUBSTANCE: invention relates to modification of glycosylation of proteins for preparing polypeptides with improved therapeutic indices including antibodies with enhanced antibody-dependent cellular cytotoxicity. For preparing indicated polypeptides cell-host is used that is modified with nucleic acid encoding enzyme β-1,4-N-acetylglucosaminyltransferase III (GnTIII). Prepared polypeptide represents, in particular, IgG or its fragment. Invention discloses a method for preparing polypeptide and antibodies or its fragment and a fusion protein prepared by indicated method. Invention describes a pharmaceutical composition used for increasing Fc-mediated cellular cytotoxicity and comprising antibody and carrier, and its using in cancer treatment, and a method for treatment of disease associated with elevated amount or production of B cells using indicated antibody, in particular, against CD20, and representing antibody IDEC-C2B8 in the preferable variant. Invention provides preparing polypeptide and antibody possessing the enhanced Fc-mediated cellular cytotoxicity that decrease the content of B cells in a patient body.

EFFECT: improved preparing method, valuable medicinal properties of polypeptide and antibodies.

38 cl, 21 dwg, 4 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: there are offered: recovered polynucleotides and polypeptides for binding human EGFR as a part of an antibody, a vector and a host cell for antibody expression, a method for producing an anti-EGFR antibody and an antibody fragment, the anti-EGFR antibody and the antibody fragment. There are discussed a composition containing the anti-EGFR antibody or its fragment, as well as applying the antibody and its fragment for treating EGFR-associated disorders. Besides, there are described versions of glycosylation of the anti-EGFR antibody or its fragment.

EFFECT: invention can find further application in therapy of various EGFR-mediated diseases.

30 cl, 6 ex, 32 dwg, 39 tbl

Up!