Composition for amplification of transgene expression in eucariotic cells, and method for increasing generation of target protein coded with transgene

FIELD: biotechnologies.

SUBSTANCE: invention relates to compositions for intensive generation of a target protein in eucariotic cells, which includes a DNA vector with an insert of target protein gene and an agonist of cell receptors. Besides, the invention relates to methods for increasing generation of a target protein coded with a transgene in eucariotic cells by using the above compositions.

EFFECT: invention allows effective increase of generation of a target protein in eucariotic cells.

28 cl, 4 dwg, 7 tbl, 10 ex

 

The scope to which the invention relates

The invention relates to the field of biotechnology and medicine, in particular to the production of DNA vector with an insert of the gene of the target protein, the production of recombinant proteins in eukaryotic cell cultures, generation of modified cells for cell therapy, cell and gene therapy in humans and animals.

Description of the prior art

The DNA vectors used in various fields of biology to deliver into cells and expression of foreign genetic material. They are used as molecular biological tools such as in vitro studies, for example, to study the functions of any gene, and in vivo to transfer genetic information into cells for the purpose of therapy or vaccination. A separate area for the use of DNA vectors is a modification with the help of their cells, which are then used as producers target protein or tools for gene therapy.

In all the above cases, the use of DNA vectors their important characteristic is the level of expression of a target gene. Therefore, a particular challenge in developing systems based on DNA-vectors is the increased expression level delivered into the cells of the transgene. Depends on the level of production of the transgene in the genetic therapy youroven immune response in genetic immunization. Furthermore, the increase of the level of expression of the transgene may allow to reduce the dose of vector administered in vitro or in vivo.

To increase the level of expression of a target gene using two main approaches. The first approach is the inclusion in the composition of the expression cassette DNA vectors various regulatory elements: promoters, polyadenylation signals, introns, exons, 5' and 3'-noncoding elements, such as PARS, IRES, WPRE [Dorokhov Y. L., Skulachev M. V., Ivanov P. A., Zvereva S. D., Tjulkina L. G., Merits, A., Y. Y. Gleba, Hohn T., Atabekov J. G. Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry // Proc. Natl. Acad. Sci. USA, 2002, V. 99, p.5301-5306. Lee Y. B., Glover C. P., A. S. Cosgrave, Bienemann, A., Uney J. B. Optimizing regulatable gene expression using adenoviral vectors. // Exp Physiol., 2005 Jan; 90(1):33-37. Li Z. L., Tian X. P., W. J. Xue, J. Wu Co-expression of sCD40Llg and CTLA4lg also been other ideas where by adenovirus prolonged mouse skin allograft survival. J Zhejiang Univ Sci B 2006 Jun; 7(6):436-44. US 20080241883 A1 Recombinant expression vector elements (rEVEs) for enhancing expression of recombinant proteins in host cells. WO 2008000445 Expression vector(s) for enhanced expression of a protein of interest in eukaryotic or procariotic host cells].

The second approach is the impact on the cells in which the introduced DNA vector, the outer molecular agents, such as sodium butyrate and trichostatin A [Siavoshian S., J-P Segain, M. Kornprobst, C. Bonnet, C. Cherbut, J-P. Galmiche, H. M. Blottierea. Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression. // Gut, 2000; 46:507-514].

Replicative defective DNA vectors, e.g., recombinant pseudovirus particles �e able to multiply [US 6019978 Replication-defective adenovirus human type 5 recombinant as a vaccine carrier], therefore, the efficiency of expression of the target transgene is dependent on the ability of viral particles to transducible cells and the effectiveness of further developments of the target protein.

Improve the efficiency of in vivo achieved by increasing the dose of administered virus [Tutykhina I. L., Sedova E. S., Gribova I. Y., Ivanova T. I., Vasilev L. A., Rutovskaya M. V., Lysenko A. A., M. M. Shmarov, Logunov D. Y., B. S. Naroditsky, Tillib S. V., Gintsburg A. L. Passive immunization with a recombinant adenovirus expressing an HA (H5)-specific single-domain antibody protects mice from lethal influenza infection. // Antiviral Res., 2013 Mar; 97(3):318-28] or improving the quality of injected viral particles [RU 2443779 Method for producing recombinant adenovirus, characterized by a reduced ratio of physical and infectious viral particles, and gene therapeutic drug obtained in this way].

You know the invention [US 20080311095 Methods and compositions for increased transgene expression], in which the method for increasing the expression of the transgene in T cells by pre-activated in vitro with antibodies to CD3/CD28. This invention was chosen as a prototype. Obvious following significant disadvantages of the prototype: (1) the present invention is suitable only for activation of T cells and cannot be used to activate other types of cells because the CD3 receptor is synthesized exclusively by T-cells; (2) proposed in the prior art type of activation T-cell�OK can only be performed in vitro; (3) proposed in the prior art method of activation using antibodies to CD3/CD28 all T cells actively proliferate (polyclonal activation). Such polyclonal activated in vitro T cells cannot be inserted into the body of animals or humans, because it will lead to serious autoimmune, systemic inflammatory, lymphoproliferative processes and total disruption of the normal functions of the immune system with very high risk of death.

The objective of this invention was to develop compositions for enhancing the expression of transgenes in eukaryotic cells and method of increasing production of target protein encoded by the transgene, as well as providing the possibility of applying the composition and method as in cell culture in vitro, and in vivo in a living organism without causing hazards to his health and life.

The problem is solved due to the fact that the composition for intensive production of the target proteins in eukaryotic cells comprising a DNA vector with an insert of the gene of the target protein and the cell receptor agonist class of receptors PRR - pattern recognition receptors, chosen from the following agonists: agonists of the receptors TLR2, or agonists of TLR4 receptors, or receptor agonists TLR5, or receptor agonists TLR7, or agonists of TLR8 receptors, or receptor agonists TLR9, or NOD1 agonists- �of eception, or NOD2 agonists receptors, taken in optimal ratios. At the same time as the agonist of the receptor TLR2 use lipoteichoic acid, either as an agonist of the receptor TLR2 use lipopeptide. As an agonist of TLR4 receptor using bacterial lipopolysaccharide, either as an agonist of TLR4 receptor use acid peptidoglycan with a molecular weight of 1200-40000 CD. As a receptor agonist TLR5 flagellin use. As an agonist of the receptor TLR7 use imiquimod, either as an agonist of the receptor TLR7 using the drug CL097, a derivative imidazoquinolines. As an agonist of the receptor TLR8 use imiquimod, either as an agonist of the receptor TLR8 using the drug CL097, a derivative imidazoquinolines. As an agonist of the receptor TLR9 use of oligonucleotide CpG ODN 1826, or as an agonist of the receptor TLR9 use of oligonucleotide CpG ODN 2006. As a NOD1 agonist-receptor use C12-iE-DAP - Lauroyl-g-D-Glu-D-mDAP and Lauroyl-g-D-Glu-L-mDAP - synthetic fragments of bacterial peptidoglycan. As an agonist of NOD2 receptors-use L18-MDP - derived muramyl dipeptide, namely the fragment of bacterial peptidoglycan. As the PRR receptor agonist can be used in the pharmaceutical preparation in an effective dose. As an agonist of receptors PR use pharmaceutical drug "Immunomax" - R N001919/02. As the PRR receptor agonist can also be used pharmaceutical drug Pirogenal" - R N003478/0. As the PRR receptor agonist can also be used pharmaceutical drug licopid" - LS-001438. As the DNA vector used nonreplicating recombinant adenoviral nanoparticles based on human adenovirus serotype 5. The DNA vector used to insert the target gene encoding the secretory protein, or use of a DNA vector with an insert of the target gene encoding a cytoplasmic protein. Or use of a DNA vector with an insert of the target gene encoding a membrane protein.

In the method of increasing production of target protein encoded by the transgene, in eukaryotic cells when they transducible DNA vectors used in the claimed composition. In this case, the amplification products of the target protein is achieved in a culture of eukaryotic cells in vitro. Also increased production of the target protein is achieved in the conditions of the organism in vivo. While eukaryotic cells are obtained from the body of the mouse, or eukaryotic cells obtained from the human body.

Can also be used composition for intensive production of the target proteins in eukaryotic cells comprising a DNA vector with an insert of the gene of the target protein and the agonist of cell recipe�the moat from the class of cytokine receptors, in an optimum ratio. In this case, as an agonist of receptors of cytokines used tumor necrosis factor. Used method of increasing production of target proteins in eukaryotic cells when they transducible DNA vectors using the above composition.

Thus, the technical result - improving the production of transgenic protein target was achieved by the use of PRR agonists receptors (from the English. pattern-recognition receptors) and receptors cytokines.

A distinctive feature of the invention is the use of PRR agonists receptors of the classes TLR - and NOD-like receptors and cytokine receptors (the receptor of tumor necrosis factor, TNF).

The present invention provides the following advantages: (1) the effect is achieved both in vitro and in vivo; (2) the effect is achieved in any types of cells, which can Express the DNA vector and which are functionally active PRR receptors; (3) our proposed method of enhancing the expression of the transgene leads to polyclonal proliferation, nor to any other pathological effects that are dangerous to animals and humans, and for enhancing the expression of the transgene by our proposed method can be used pharmaceutical drugs PRR agonists receptors.

Summary of the invention

This �turbine zobretenie provides a composition for enhancing the expression of transgenes in eukaryotic cells and a method of increasing production of target protein encoded by the transgene.

The problem solved by this invention consists in a significant increase in expression of the target transgene and production of target proteins in eukaryotic cells both in cell culture in vitro, and in conditions of humans and animals.

It is known that the transcription of genes and production of proteins in eukaryotic cells can vary considerably under the influence of external signals, which can be attributed to a few principal types:

a) contact eukaryotic cells with infectious agents or components of infectious agents, which in scientific literature is called PAMP (pathogen associated molecular patterns);

b) contact eukaryotic cells with damaged cells or their components, which in scientific literature is called DAMP (damage-associated molecular patterns);

b) cytokines and danger signals (danger signals), which is a chemical regulatory signals for communication between cells;

g) contact interaction of eukaryotic cells with each other;

d) the contact of cells with the extracellular matrix polymers.

In the present invention, the task of enhancing the expression of the transgene and production of the target protein is solved by activating translationindia cells with the AGENCY, DAMP, acting on cells via PRR receptors, or by using cytokines, days�existing cells through receptors of cytokines, or by simulating such effects using substances-agonists acting on eukaryotic cells via PRR receptors or receptors of cytokines.

In the present invention for enhancing the expression of the transgene and production of target protein in eukaryotic cells that transducers DNA vector with an insert of the transgene, the use of substances that activate cellular metabolism via PRR receptors or through cytokine receptors. The result proposed in the present invention, the combination of DNA vectors with specified types of activators of cellular metabolism is enhanced 2-10 times the production of the target proteins related to cytoplasmic, membrane or secretory proteins.

In this description of the invention it is proved that in eukaryotic cells, which transducer DNA vector with an insert of the transgene, the increase in expression of the transgene and production of target protein by using activators operating through the PRR receptors or receptors of cytokines that are equally effectively implemented in eukaryotic cells in vitro and in vivo, i.e. in terms of the body.

The amplification effect is observed both in animal cells (mouse) and humans. The gain observed in those cell types that are able to transducerless and to Express this DNA vector and are functionally active PRR-prescription�ry.

Proposed in the present invention, the amplifiers of expression of the transgene can be used in vivo, as demonstrated in laboratory animals have also shown that the PRR agonists receptors can be used pharmaceutical drugs.

List of figures

Fig.1A - Response cells HEK-Blue TLR4 products reporter SEAP protein, confirming that acid peptidoglycan activates NF-kB via receptors TLR-4.

The activity of NF-κb was measured by the expression of NF-kB-dependent reporter gene SEAP cells HEK-Blue without TLR receptors (null), and in cells expressing one of the following TLR receptors 2, 3, 4, 5, 7, 8, or 9. Cells were incubated for 18 hours in the presence of the CNG acid peptidoglycan (5 µg/ml, ■) or without (negative control, □). As a positive control for each type of cells was used as standard agonist (): TNF-α (10 ng/ml) for null-cells, lipopeptide (1 μg/ml) for TLR2 cells, poly I:C (10 μg/ml) for TLR3 cells, LPS (1 μg/ml) for TLR4 cells, flagellin (1 μg/ml) for TLR5 cells, imiquimod (1 μg/ml) for TLR7-cells, CL097 (1 mg/ml) for TLR8 cells and ODN 2007 (10 μg/ml) for TLR9 cells.

The results are given in the form of a multiplicity of increasing the production of NF-kB-dependent reporter SEAP protein in relation to control cells (without activator). Presents average values for�scientists in three independent experiments. Conclusion - acid peptidoglycan (CNG, patent RU No. 2195308) activates NF-kB via the receptor TLR4.

Fig.1B - dependence of the production of NF-kB-dependent reporter protein SEAP cells HEK-Blue TLR4 on the concentration of acid of peptidoglycan.

CNG induces the expression of NF-kB-dependent reporter gene (SEAP) in cells HEK-Blue TLR4. Cells were incubated in the presence of the indicated concentrations of CNG within 18 hours. Intact cells HEK-Blue TLR4, cells, HEK-Blue-TLR-null were used as a negative control. The results are given in the form of a multiplicity of increasing the production of NF-kB-dependent reporter SEAP protein in relation to control cells (without activator). Presents the average values obtained in three independent experiments.

Fig.2 (A, B, C, D, E, F, G, H) - identification of the cell receptor drug "Immunomax" (P N001919/02).

Cell collection NECK Blue TLR (InvivoGen) expressing one of the TLR receptors (TLR2, 3, 4, 5, 7, 8, or 9), or non-TLR receptors (null), have been used to identify receptor drug "Immunomax" (P N001919/02). Presents the results of the study:

(A) on the cells of TLR-null;

(B) cells with receptors TLR2 and coreceptors CD14;

(C) cells with receptors TLR3;

(D) cells with receptors TLR4 and coreceptor CD14, and MD2;

(E) cells with receptors TLR5;

(F) cells with Retz�Ptolemy TLR7;

(G) cells with receptors TLR8;

(H) cells with the receptor TLR9.

Cells of these lines were incubated in complete culture medium in vitro for 18 hours at 37°C and 5% CO2in the presence of the drug "Immunomax" (from 0.02 to 15 mg/ml) or without it.

As a positive control for each type of cells was used as standard agonist: TNF-α (10 ng/ml) for null-cells (2 A), lipopeptide (1 μg/ml) for TLR2 cells (2 B), poly I:C (10 μg/ml) for TLR-cells (2 C), LPS (1 μg/ml) for TLR4 cells (2 D), flagellin (1 μg/ml) for TLR5 cells (2 E), imiquimod (1 μg/ml) for TLR7-cells (2 F), CL097 (1 mg/ml) for TLR8-cells (2 G) and ODN 2007 (10 μg/ml) for TLR9-cells (2 H). The activation of TLR-receptors (TNF-receptors in the case of control cells TLR-null) was assessed by the activation products of reporter SEAP protein, a gene which is under NF-kB-dependent promoter used in all types of cells. The results are given in the form of a multiplicity of increasing the production of NF-kB-dependent reporter SEAP protein in relation to control cells (without activator). Presents the average values obtained in three independent experiments.

Fig.3 - Analysis methods, confocal microscopy and flow cytometry enhancing the expression of the transgene Ad-GFP in peritoneal macrophages under the influence of PRR agonists receptors.

A - peritoneal macrophages were transducible Ad-GFP (2×107The FIGHT/ml)in the absence (left) or in the presence of a TLR4 agonist (CPG, 10 μg/ml, right photo).

B and C are successive stages cytometric selection of populations of living macrophages by stray light and DAPI staining of specific DNA dye.

D and E - analysis of the intensity of GFP-fluorescence of peritoneal macrophages, translationindia Ad-GFP (2×107The FIGHT/ml) in the absence (D) or in the presence (E) of the agonist of TLR7/8 receptors CL097 (2.5 ág/ml).

Cell cultures were incubated at 37°C and 5% CO2during the 4 days. After incubation the cells were washed with cold solution Version and using a flow cytometer FACS Aria II determined the percentage and absolute number of cells with green fluorescence, and the fluorescence intensity of cells. The absolute content of cells was normalized by calibration beads of known concentration (CountBright Invitrogen).

Fig.4 - increased expression of the target cytoplasmic GFP protein in peritoneal macrophages in vitro using pharmaceutical PRR agonists receptors.

Peritoneal mouse macrophages were transducible Ad-GFP (2×105The BOUT) without any additional activation (control) or when you activate one of the cells pharmaceutical PRR agonists receptors, in particular medicines "Immunomax" (Reg. No. R N001919/02-171011) or "Pirogenal" (P N003478/0), or "licopid" (HP-001438). Cells were incubated in complete culture medium at 37°C and 5% CO 2in for 2 days. After incubation the cells were washed with cold solution Version and using a flow cytometer FACS Aria II determined the number of macrophages with green fluorescence (A), and average fluorescence intensity (B). Integrated fluorescence of macrophages (C) were calculated as the product of the number of fluorescent cells on the average fluorescence intensity of cells. Data are normalized to values in control (Ad-GFP without additional activation of cells).

Disclosure of the invention

The problem solved by this invention consists in a significant increase in expression of the target transgene and production of target proteins in eukaryotic cells both in cell culture in vitro, and in conditions of humans and animals.

As DNA vectors of the present invention used nonreplicating recombinant adenoviral nanoparticles (NREN) that encode cytoplasmic, membrane or excreted proteins. DNA-vector expressible in cultures of the following eukaryotic cells: primary cells of the spleen, bone marrow, peritoneal cavity of mice, bone marrow cells of mice differentiated in vitro culture in the presence of granulocyte-macrophage colony stimulating factor GM-CSF, the fraction of mononuclear cells from human blood. Analysis Express�and the transgene depended on the vector used. When using NRAN with an insertion of the gene of green fluorescent protein (Ad-GFP) expression analysis was performed using fluorescence and confocal microscopy and flow citofluorimetry. When using NRAN with insert hemagglutinin genes of influenza viruses H1N1 (Ad-HA1), H3N2 (Ad-3) and (Ad-HA-B) the production of the target protein was analyzed by the method of citofluorimetry after staining of cells with monoclonal antibody-specific binding to the protein ON. The expression of NRAN with insert gene secretory embryonic alkaline phosphatase (Ad-SEAP) was determined by the secretion of the target protein SEAP, the concentration of which was measured by the colorimetric method on the enzymatic activity of SEAP.

Enhancing the expression achieved with PRR agonists receptors. Modern conceptions of PRR-receptors are reduced to the presence in the cells of humans and animals of several families the AGENCY - and DAMP-recognizing receptors that allow cells not only detect and react on contact with almost any microorganism or its components, but also to learn about the damage to their own body tissues and to respond adequately to these injuries.

To PRR-receptors are TLR (toll-like receptors), NLR (NOD-like receptors), RLR (RIG-like receptors) and some other families of receptors [her R. Thompson, John J. Kaminski, Evelyn A. Kurt-Jones, and Katherine A. Fitzgerald. Pattern Recognition Receptors and the Innate Immune Response to Viral Infection. // Viruses, June 2011; 3(6): 920-940. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. // Cell, Mar 2010 19; 140(6):805-20]. Detailed intracellular signaling pathway beginning with the activation of PRR receptors and culminates in the activation of NF-kB, AP-1, IRF and other transcription factors that control the expression of specific groups of genes and the subsequent production of proteins encoded by those genes.

In the following examples 4, 5, 6, 7, 8, 9 and 10 demonstrated that increased expression of the target transgene is achieved when exposed to a wide range of PRR receptors from class TLR receptors selected from the list: TLR2, TLR4, TLR5, TLR7, TLR8 and TLR9, as well as from the class NLR, in particular, NOD1 - and NOD2-receptors.

Enhancing the expression of the target protein GFP was achieved using the following natural PRR agonists receptors or their synthetic analogues:

ligands of TLR2 - lipoteichoic acid (LTC) and lipopeptide (Lipopeptide, Lot A11, EMC microcollections GMBH);

ligands of TLR4 lipopolysaccharide (LPS) from E. coli serotype 055: B5 (Sigma L-2880) and acid peptidoglycan with a molecular weight of 1200-40000 CD (CNG, patent RF №2195308);

TLR5 ligand - flagellin (Invivogen);

ligands of TLR7 and TLR8 - imiquimod and CL097 (derived imidazoquinolines, Invivogen);

ligands of TLR9 - CpG-oligonucleotides ODN 1826 and ODN 2006 (Invivogen).

In example 4 demonstrated that increased expression of the target transgene is achieved when exposed to h�cut class NOD-receptors using:

NOD1 ligand-receptor C12-iE-DAP (Lauroyl-g-D-Glu-D-mDAP and Lauroyl-g-D-Glu-L-mDAP, a synthetic fragments of bacterial peptidoglycan, Invivogen), and

the NOD2 ligand-receptor L18-MDP (a derivative of muramyldipeptide, a synthetic fragment of bacterial peptidoglycan, Invivogen).

A distinctive feature of this invention is the use of the effect of amplification products of the target transgene in vivo, and that is especially valuable when using pharmaceutical PRR agonists receptors.

In the example 10 demonstrated an increase in expression of the target protein using pharmaceutical receptor agonists:

TLR4, in particular, the medicinal product "Immunomax" (Reg. No. R N001919/02-171011, Immupharma, Russia) and the drug, "Pirogenal" (P N003478/0, Megamall, branch of the Institute. N. F. Gamaleya RAMS, Russia), as well as an agonist of NOD2 receptors drug "licopid" (HP-001438, Ltd PEPTECH, Russia).

Having a positive effect on the enhancement of the expression of the target protein not only in the use of PRR agonists receptors, but also in the use of cytokines has been demonstrated on the example of TNF-α (example 6).

In examples 6, 7, 8 demonstrated increased expression of the secretory protein, for which we used a DNA vector with an insert of the gene SEAP.

In example 9 trademonster�Vano increased expression of the target membrane protein, why used DNA vector with an insert of hemagglutinin gene of influenza viruses NM, H3N1, or B.

The implementation of the invention was demonstrated on cells of the mouse (examples 4, 5, 6, 7, 8 and 10) and humans (example 9). In example 8 demonstrated an increase in expression of the transgene and production of target protein in the conditions of a living organism, in particular in laboratory mice, which does not restrict the dissemination of the invention in other animals and humans.

The following are examples that reveal the most preferred embodiment of this invention for a better explanation of its essence.

The examples of the invention

Example 1. The creation of plasmid constructs encoding cytoplasmic, secreted or membrane protein.

As the DNA vector used NRAN based on the human adenovirus 5-th serotype.

To obtain QUAN initially establish plasmid constructs carrying expressing cassette containing the nucleotide sequences that encode cytoplasmic protein GFP, SEAP secreted protein, membrane proteins HA1, HA3, or HA-B. Thus, plasmid construction pShuttle-CMV-GFP, pShuttle-CMV-SEAP, pShuttle-CMV-HAI, pShuttle-CMV-HA3, pShuttle-CMV-HA-B.

To obtain plasmid constructions pShuttle-CMV-GFP, pShuttle-CMV-SEAP, pShuttle-CMV-HAI, pShuttle-CMV-3, pShuttle-CMV-HA-In as the vector used plasmid con�tructio pShuttle-CMV with portions of the genome of a human adenovirus 5-th serotype to obtain rereplacenocase recombinant adenoviral nanoparticles included in the set) system AdEasy Adenoviral vector system" ("Stratagene" Cat. No. 240009). Plasmid construction pShuttle-CMV hydrolyze site for the restriction enzyme EcoRV, and then carry out the insertion of nucleotide sequences that encode protein GFP, SEAP, HA1, HA3, or-B, respectively, get the design of pShuttle-CMV-GFP, pShuttle-CMV-SEAP, pShuttle-CMV-HAI, pShuttle-CMV-HA3 or pShuttle-CMV-HA-B. Nucleotide sequence of the cytoplasmic GFP protein secreted SEAP protein, membrane proteins HA1, HA3 and HA-B to insert into pShuttle-CMV prepared by hydrolysis of the corresponding plasmid constructs pGREEN (USA, Carolina Biological Supply Company), pAL-SEAP, pAL-HA1, pAL 3 and pAL-HA-B (chemical synthesis, Russia, Evrogen) the sites for the restriction enzyme Ase I (pGREEN), EcoRV (pAL-SEAP, pAL-HA1, pAL 3 and pAL-HA-B). The presence of protein gene GFP, SEAP, HA1, HA3,-B within the respective plasmid constructs pShuttle-CMV-GFP, pShuttle-CMV-SEAP, pShuttle-CMV-HAI, pShuttle-CMV-3, pShuttle-CMV-HA-B confirmed by restriction analysis using the restriction enzyme EcoRI, NotI and EcoRV and PCR.

Thus, plasmid construction pShuttle-CMV-GFP, pShuttle-CMV-SEAP, pShuttle-CMV-HAI, pShuttle-CMV-3, pShuttle-CMV-HA-B, bearing expressing cassette containing the nucleotide sequences that encode cytoplasmic protein GFP, SEAP secreted protein, membrane proteins HA1, HA3, or-B, which are further used for receiving NRAN.

Example 2. Accessed� and testing nonreplicating recombinant adenoviral nanoparticles with inserts genes of target proteins GFP, SEAP, HA1, HA3, or-B.

Getting nonreplicating recombinant adenoviral nanoparticles Ad-GFP, Ad-SEAP, Ad-HA1, Ad-3 and Ad-HA-In carrying expressing cassette containing the nucleotide sequences that encode cytoplasmic protein GFP, SEAP secreted protein, membrane proteins HA1, HA3, or-B, respectively, is carried out according to the methodology "AdEasy Adenoviral vector system" (Stratagene, Cat. No. 240009), which is based on homologous recombination sites of the adenovirus genome in E. coli cells. The presence of genes GFP, SEAP, HA1, HA3 and-B in the composition of the relevant QUAN confirmed by PCR. Next, determine the titers of NRAN Ad-GFP, Ad-SEAP, Ad-HA1, Ad-3 and Ad-HA-B by the method of Blasco education on the cell culture NEC (cells embryonic kidney) [Graham F. L., Prevec L. Manipulation of adenovirus vectors. // Methods in Mol. Biol., 1991, v.7, p.109-127].

The expression of the DNA vectors and products of target proteins was tested in cell culture lines A. The expression of cytoplasmic target protein GFP was detected using an inverted fluorescence microscope (ICM 405, Leitz) and using the method of running citofluorimetry on the instrument FACS Aria II (BD Biosceinces).

The secretory expression of the target protein SEAP was determined by the method of Berger (Berger, J., Hauber, J., Hauber, R., Geiger, R., Cullen B. R. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. // Gene, 1988 Jun 15; 66 (1):1-10) with minor modification�I. An aliquot of test fluid was clarified by centrifugation 14000 g for 2 min, heated at 65°C for 5 min to inhibit endogenous phosphatases, made of 20 μl in the wells of 96-hole tablet in 130 µl of reaction buffer (0.5 M NaHCO3, 0.5 mm MgCl2pH of 9.8) and incubated at 37°C for 10 minutes was Then added 50 μl of a 60 μm para-nitrophenyl phosphate in the reaction buffer and fixed intervals of time measured optical density at a wavelength of 405 nm. The SEAP activity was expressed in IU/ml on the basis that 1 IU/ml corresponds to an increase of the optical density of 0.04 units in 1 minute.

The expression of membrane-targeted proteins HA1, HA3 and-B were determined by staining of cells with monoclonal antibodies to HA1, HA3 and-B, respectively, with subsequent analysis by flow of citofluorimetry on the instrument FACS Aria II (BD Biosceinces).

Thus were obtained DNA-vectors - NRAN (Ad-GFP, Ad-SEAP, Ad-HA1, Ad-3 and Ad-HA-B) bearing expressing cassette containing the nucleotide sequences that encode cytoplasmic protein GFP, SEAP secreted protein, membrane proteins HA1, HA3, or-B, respectively, and confirmed the expression of relevant proteins.

Example 3. Acid peptidoglycan with a molecular weight of 1200-40000 CD (RF patent No. 2195308) and pharmaceutical drug "Immunomax" (P N001919/02) are agonists of TLR4.

The presented data prove that CNG (patent RF №2195308) and pharmaceutical drug "Immunomax" (P N001919/02) are agonists of TLR4.

Example 4. Increased expression of the target cytoplasmic GFP protein in peritoneal mouse macrophages in vitro with PRR agonists receptors of the classes TLR - and NOD-receptors.

Peritoneal macrophages of the mouse (BALB/c females, kennel "Pillar") was washed using�se of saline, besieged by centrifugation 1200 rpm for 10 min, suspendirovanie at a concentration of 1 million cells in 1 ml complete culture medium (PS, consists of MOM-1640 plus 10% fetal calf serum, 2 mM L-glutamine, 50 µm β-mercaptoethanol and 10 μg/ml gentamicin), incubated overnight in Petri dishes with a diameter of 90 mm at 37°C and 5% CO2. The next day, not adherent cells were washed with warm phosphate buffered saline (PBS, pH 7,4), adherent macrophages were filled with a solution of Versene, withstand 60 minutes in the refrigerator at 4°C and then washed off by a jet of solution Version of a 5 ml pipette. Macrophages were precipitated by centrifugation 1200 rpm for 10 min, suspendirovanie in PS and incubated in the wells of 96-well plates at a concentration of 0.1 million/ml in a volume of 200 μl per well in triplets with the addition of 2×107The FIGHT/ml of Ad-GFP (obtained as in example 2) in combination with agonists of TLR and NOD receptors or without (control). The plates with cells were incubated for 4 days at 37°C and 5% CO2. Table 1 presents the used agonists of the receptors (TLR) and NOD to their final concentration in vitro.

Table 1
Used agonists of receptors PRR
ReceptorAG�NIST (ligand) The final concentration
TLR2Lipoteichoic acid (Invivogen)10 m g/ml
TLR2Lipopeptide (EMC microcollections GMBH)1 mg/ml
TLR4Lipopolysaccharide from E. coli (Invivogen)10 mg/ml
TLR4CNG (the Russian Federation No. 2195308)10 mg/ml
TLR5Flagellin (Invivogen)1 mg/ml
TLR7/8Imiquimod (Invivogen)10 mg/ml
TLR7/8CL097 (Invivogen)2.5 mg/ml
TLR9ODN 1826 (Invivogen)0.6 ág/ml
TLR9ODN 2006 (Invivogen)10 mg/ml
NOD1C12-iE-DAP (Invivogen)20 mg/ml
NOD2L18-MDP (Invivogen)20 m�g/ml

Fig.3A shows a photograph of macrophages, obtained by confocal microscopy of cells, traduzioni Ad-GFP with additional activation of TLR4 agonist (10 mg/ml CNG, the right photo), in comparison with control macrophages, translationindia Ad-GFP without adding CNG (left photo). Clearly shows the intensity of fluorescence in the cells, additionally stimulated CNG, which indicates an increase of the synthesis of the target protein GFP. For quantitative analysis of GFP production used the method of flow cytometry, which allows to determine the absolute number of cells containing fluorescent protein GFP and the fluorescent protein content in each cell in the fluorescence intensity of individual cells. The product of the number of fluorescent cells on the fluorescence intensity of each cell is proportional to the amount of synthesized GFP molecules.

As described above, the peritoneal macrophages were transducible Ad-GFP (2×107The FIGHT/ml) in the absence or in the presence of one of the PRR agonists receptors. Cell cultures were incubated at 37°C and 5% CO2during the 4 days. After incubation the cells were washed with cold solution Version and using a flow cytometer FACS Aria II determined the percentage and absolute number of cells, with green fluorescent�Yu, as well as the intensity of fluorescein cells. The absolute content of cells was normalized by calibration beads of known concentration (CountBright Invitrogen), live and dead cells were separated using a DNA-specific dye DAPI. Fig.3B and Fig.3C shows a stage cytometric selection of living cells and macrophages. Typical histograms of the fluorescence signal of GFP after transduction of macrophages with Ad-GFP without additional activation of the cells of Fig.3D or with additional activation of cells CL097, agonist receptors TLR7 and TLR8 - Fig.3E.

Quantification of enhancing the expression of the target protein GFP was performed according to the following formula:

K=MFI×N/MFIcontr×r-Ncontr,(1)

where:

K - multiplicity enhancing the expression of GFP;

MFI - mean fluorescence intensity translationindia Ad-GFP cells for their further activation by an agonist;

MFIcontr- the average fluorescence intensity of translationindia Ad-GFP �notches in control without additional activation;

N is the number of fluorescent cells after transduction with Ad-GFP with additional activation by agonist;

Ncontr- the number of fluorescent cells in the control after transduction with Ad-GFP without additional activation.

The data presented in table 2 indicate that the increase in expression of GFP in peritoneal macrophages, translationindia Ad-GFP, was influenced by various agonists PRR receptors, selected from the classes of TLR - and NOD-like receptors, in particular, TLR2 agonists (lipopeptide, lipoteichoic acid), TLR4 (CPG, lipopolysaccharide), TLR5 (flagellin), TLR7/8 (CL097, imiquimod), TLR9 (CpG 2006, ODN 1826), NOD-1 (C12-iE-DAP) nNOD-2(L18-MDP).

Table 2
Increased GFP expression in peritoneal macrophages of the mouse, translationindia Ad-GFP (2×107The FIGHT/ml) and activated the specified receptor agonist PRR
PRR receptorAgonistThe multiplicity of enhancing the expression of GFP, by the formula (1)Significant differences
The average valueStandard deviation
TLR2Lipopeptide, 10 m g/ml7.5 1.3<0.05
TLR2Lipopeptide, 1 m g/ml16.63.2<0.05
TLR2LTK, 10 m g/ml2.100.12<0.05
TLR4CNG, 1.5 mg/ml10.52.2<0.05
TLR4LPS, 10 mg/ml1.960.08<0.05
TLR5Flagellin, 1 mg/ml2.130.18<0.05
TLR7/8CL097, 2.5 ug/ml14.21.60.05
TLR7/8Imiquimod, 10 mcg/ml2.030.250.05
TLR9CpG 2006, 10 mg/ml3. 0.60.05
TLR9ODN 1826, 0.6 ág/ml1.430.190.05
NOD1C12-iE-DAP, 20 mg/ml3.11.30.05
NOD2L18-MDP, 2 mg/ml3.71.20.05

Example 5. Increased expression of the cytoplasmic target protein GFP in vitro in dendritic cells mouse bone marrow origin using the agonist of TLR4.

Bone marrow cells mouse (BALB/c females, kennel "Pillar") was leached out from the femur and tibia using a saline solution, the erythrocytes were literally using hypotonic shock (15 seconds in distilled water), osmotic pressure instantly restored, making the required number of 10-fold Hanks ' solution, the cells were besieged by centrifugation 1200 rpm for 10 min, suspendirovanie in PS at a concentration of 1 million/ml and incubated in Petri dishes with a diameter of 90 mm in the presence of 20 ng/ml of growth factor GM-CSF. After 3-4 days in culture medium was replaced with fresh PS, dopolnennoe� GM-CSF. After 7 days, washed away the non-adherent cells were suspensively them in PS and incubated in the wells of 96-well plates at a concentration of 0.1 million/ml in a volume of 200 μl per well in triplets with the addition of 2×107The FIGHT/ml of Ad-GFP, obtained as in example 2, in combination with the addition of 5 μg/ml CNG (RU # 2195308) or without it. 2 days after making Ad-GFP cells were washed with cold versene solution and determined the percentage of fluorescent cells using a flow cytometer FACS Aria II. The results are presented in table 3. The expression of the target protein GFP in bone marrow dendritic cells was increased 5-fold when activated CNG (RU # 2195308).

Ad-GFP 2×107The FIGHT/ml + CNG (RU # 2195308), 5 mg/ml
Table 3
The increase in expression of the transgene in bone marrow dendritic cells as a result of their activation by TLR4 agonist (CPG, patent RF №2195308)
TransductionThe percentage of dendritic cells expressing GFP
The average valueStandard deviationSignificant differences
Ad-GFP 2×107The FIGHT/ml30,5-
1510,05
Gain5--

Example 6. Increased expression of the targeted secretory protein embryonic alkaline phosphatase in peritoneal mouse macrophages in vitro using cytokine TNF-α.

Peritoneal macrophages mice were obtained and incubated as described in example 4. Cells were incubated in triplets with the addition of Ad-SEAP at a concentration of 2×107The BATTLE on hole together with TNF-α (10 ng/ml) or CNG (RU # 2195308, 10 μg/ml). In the control wells were added Ad-SEAP (2×107The BOUT) without the addition of activators. After 4 days in culture liquid was measured the activity of secreted alkaline phosphatase SEAP as described in example 2. In the presence of TNF-α secretory expression of the target protein SEAP increased 1.8 times (table 4).

Table 4
The increase in expression of the transgene secretory SEAP protein in peritoneal macrophages as a result of their activation by TLR4 agonist or cytokine TNF
Transduction Activity (IU/ml) secreted alkaline phosphatase SEAP in the culture fluid
The average valueStandard deviationSignificant differences
Ad-SEAP (2×107The BATTLE on the hole)0,340,06-
Ad-SEAP 2×107The BATTLE on the hole + TNF-α (10 ng/ml)0,620,12<0,05
Ad-SEAP (2×107The BATTLE in the hole) + CNG(RU # 2195308, 10 µg/ml)0,820,03<0,05
The gain for TNF-α1,8--
The gain for CNG (RU # 2195308)2,4--

Example 7. Increased expression of the secreted target protein SEAP in vitro in dendritic cells mouse bone marrow origin using the agonist of TLR4.

Dendritic cells mouse bone marrow origin were prepared as described in example 5. A suspension of dendritic cells in t� were incubated in the wells of 96-well plates at a concentration of 0.1 million/ml in a volume of 200 μl per well in triplets with the addition of 2×10 7FIGHT Ad-SEAP, obtained as in example 2, in combination with CNG (RU # 2195308, 5 μg/ml) or without it. After 6 days in culture medium was measured the activity of secreted phosphatase SEAP as described in example 2. Upon activation of dendritic cells by TLR4 agonist (CPG, Patent RF №2195308) SEAP expression was increased 6-fold (table 5).

Table 5
The increase of expression of the transgene secretory SEAP protein in dendritic cells in vitro with an agonist of TLR4
TransductionThe SEAP activity in culture medium (IU/ml)
The average valueStandard deviationSignificant differences
Ad-SEAP (2×107The COMBAT)0,480,07-
Ad-SEAP (2×107The COMBAT) + CNG (patent RF №2195308, 5 µg/ml)3,00,3<0,05
Gain6- -

Example 8. The increase of expression of the transgene secretory protein SEAP in the body of laboratory mice in vivo using pharmaceutical TLR4 agonist ("Immunomax").

Ad-SEAP were prepared as in example 2 was administered at a dose of 108The BATTLE on the mouse in 200 μl of saline into the peritoneal cavity of mice BALB/c (female, 14-16 grams, kennel RAMS "Pillar"), together with 10 μg of the drug "Immunomax" (Immupharma, Russia). After 3 days individually from each mouse were taken blood from retro-orbital sinus, received the serum, which was determined by SEAP activity as described in example 2. The introduction of "Immunomax" increased expression of SEAP in 7 times (table 6). While not noted any adverse (toxic) effects in animals given the drug "Immunomax" in combination with Ad-SEAP. This result proves the possibility of enhancing the expression of the transgene in animals and humans through pharmaceutical agonist of TLR4 receptors drug "Immunomax".

Table 6
Increased expression of SEAP transgene in the body of laboratory mice with a single injection of pharmaceutical TLR4 agonist ("Immunomax")
Transduc�Oia in vivo The SEAP activity in the serum IU/ml
The average valueStandard deviationSignificant differences
Ad-SEAP (108The BATTLE on the mouse)4,354,69-
Ad-SEAP (108The BATTLE on the mouse) + Immunomax 10 µg per mouse30,4212,470,02
Gain7--

Example 9. The increase of expression of transgenes targeted membrane proteins HA1, HA3 and-B in cultured human cells in vitro with an agonist of TLR4.

The blood from the cubital vein of healthy donors were taken into tubes with KsEDTA BD Vacutainer and was isolated mononuclear fraction, the blood was diluted 2 times with physiological saline and layered on ficoll with a density of 1.077 g/l (Paneco, Russia), centrifuged 25 min at 400 g at 20°C. the Fraction containing mononuclear cells was collected in a 15 ml test tube, the cells were washed in PBS solution (with addition of 0.5% BSA, 1% glucose, 10 mm HEPES, pH of 7.4). Cells were suspensively at a concentration of 3 million in 1 ml of N�, poured 0.5 ml into the wells of 24-well plates Nunclon in doublets. Then the cultures were made by one of the vectors Ad-HA1, Ad-3 or Ad-HA-B (5×106FIGHT) together with 5 μg/ml CNG (RU # 2195308) or without it. The plate was incubated for 2 days at 37°C in atmosphere of 5% CO2. After 2 days the cells from the wells were collected with versene solution, was transferred to a 15 ml tube and precipitated by centrifugation. The cell sediment was stained with monoclonal antibodies against hemagglutinin HA1, HA3, or-B (SinoBiological) for 20 min, washed with PBS solution (with addition of 0.5% BSA, 0.01% of sodium azide, 0.35 mm EDTA, 10 mm HEPES, pH of 7.4). Bound to antibody was revealed using FITC-labeled Fab-fragments of antibodies to murine IgG. The cells were washed with PBS solution (with addition of 0.5% BSA, 0.01% of sodium azide, 0.35 mm EDTA, 10 mm HEPES, pH 7.4), monocytes were stained with antibodies to CD14 PE-Cy7 (BD Biosceinces). On a flow cytometer FACS Aria II determined the percentage of cells expressing ON that in a population of CD14-positive cells. When you activate the CNG cells (RU # 2195308) expression of membrane-targeted proteins HA1, HA3 and-B was increased 1.75-4.1 times (table 7).

Table 7
The increase in expression of HA1, HA3 and-B upon activation of TLR-agonist mononuclear cells of human blood, translationindia vectors Ad-HA1, d-3 or Ad-HA-B
TransductionThe rate of increase in percentage of monocytes expressing ON*
The average valueStandard deviationSignificant differences
Ad-HA1 (5×106The COMBAT) + CNG (RU # 2195308), 5 mg/ml1,750,260,05
Ad-HA3 (5×106The COMBAT) + CNG (RU # 2195308), 5 mg/ml3,20,120,05
Ad-HA-B (5×106The COMBAT) + CNG (RU # 2195308), 5 mg/ml4,11,040,05
* Note: data normalized to the percentage of cells expressing corresponding TO the control cell cultures without the addition of CNG.

Example 10. The increase of expression of the transgene target cytoplasmic GFP protein in peritoneal mouse macrophages in vitro using pharmaceutical PRR agonists of the classes TLR - and NOD-receptors.

Peritoneal macrophages mice were obtained and incubated as described in example 4. Cells were incubated in triplets, transducer�Ali vector Ad-GFP at a concentration of 5×10 6The FIGHT/ml For macrophage activation in cell culture have made pharmaceutical agonists of TLR4 receptors, in particular drug "Immunomax" (Reg. No. R N001919/02-171011, Immupharma, Russia) or drug "Pirogenal" (P N003478/0, Megamall, branch of the Institute. N. F. Gamaleya RAMS, Russia), or pharmaceutical agonist NOD-receptors - drug "licopid" (HP-001438, Ltd PEPTECH, Russia). Served as control culture translationindia macrophages without the addition of activators. After 2 days and evaluated the expression of a target protein GFP as described in example 4. Figure 4 presents the results of the experiments. They prove that all of us used drugs and pharmaceutical PRR agonists cause a significant increase in expression of the target transgene. The stimulation of cells by the pharmaceutical PRR agonists the production of the target protein was increased from 2 to 11 times. The amplification effect depended on the dose of agonist. This result means that the increase in expression of the transgene can be made to the person using medicinal (pharmaceutical) drugs that activate translationindia cells through receptors PRR.

All of these examples confirm that the stated technical problem, namely the development of compositions for enhancing the expression of the transgene in a eukaryotic cell�x and method of increasing production of target protein encoded by the transgene, as well as providing the possibility of applying the composition and method as in cell culture in vitro, and in vivo in a living organism is implemented in this invention.

List of abbreviations

PRR (from the English. pattern recognition receptors, is a cellular receptor for molecular recognition of images associated with microbes, viruses, cellular stress and damage.

TLR (from the English. Toll-like receptors) - Toll-like cell receptors relating to PRR

NOD-like receptors (from the English. nucleotide-binding oligomerization domain-containing receptors) - cell receptors specific to PRR.

NLR (from the English. NOD-like receptors) receptors, similar NOD, refer to PRR

RLR (from the English. RIG-like receptors) receptors, similar RIG, are PRR

The AGENCY (from the English. pathogen associated molecular patterns) - molecular images associated with germs

DAMP (from the English. damage-associated molecular patterns) - molecular images associated with damage

NRAN - nonreplicating recombinant adenoviral nanoparticles

GM-CSF - granulocyte macrophage-colony stimulating factor

GFP - green fluorescent protein

Ad - NRAN based on the adenovirus type 5

Ad-GFP - NRAN with an insertion of the GFP gene

HA1 - hemagglutinin influenza a virus H1N1

HA3 - hemagglutinin of influenza virus H3N2

ON-IN - the hemagglutinin of influenza virus B

Ad-HA1 - NRAN with insert HA1 gene

Ad-HA3-NRAN with insert gene� HA3

Ad-HA-B - NRAN insert the gene IN-B

SEAP - secretory embryonic alkaline phosphatase

Ad-SEAP - NRAN with insert SEAP gene

LTK - lipoteichoic acid, a component of bacterial wall

LPS - lipopolysaccharide, a component of bacterial wall

KPG - acid peptidoglycan

CL097 - derived imidazoquinolines

ODN - oligonucleotide

C12-iE-DAP - Lauroyl-g-D-Glu-D-mDAP and Lauroyl-g-D-Glu-L-mDAP, synthetic fragments of bacterial peptidoglycan

L18-MDP - derived muramyl dipeptide

PCR - polymerase chain reaction

PS - full media for cultivation of cells

PBS - phosphate buffer solution

The BATTLE - Blasco-forming units

TNF - a tumor necrosis factor

BSA - bovine serum albumin

FITC - fluorescein isothiocyanate

EDTA - Ethylenediamine tetraacetate

FACS flow cytometer

Literature

1. Dorokhov Y. L., Skulachev M. V., Ivanov P. A., Zvereva S. D., Tjulkina L. G., Merits, A., Y. Y. Gleba, Hohn T., Atabekov J. G. Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry // Proc. Natl. Acad. Sci. USA, 2002, V. 99, p.5301-5306.

2. Lee Y. B., Glover C. P., A. S. Cosgrave, Bienemann, A., Uney J. B. Optimizing regulatable gene expression using adenoviral vectors. // Exp Physiol., 2005 Jan; 90(1):33-37.

3. Li Z. L., Tian X. P., W. J. Xue, J. Wu Co-expression of sCD40Llg and CTLA4lg also been other ideas where by adenovirus prolonged mouse skin allograft survival. J Zhejiang Univ Sci B, Jun 2006; 7(6):436-44.

4. US 20080241883 A1 Recombinant expression vector elements (rEVEs) for enhancing expression of recombinant proteins in host cells.

5. WO 2008000445 Expression vector(s) for enhanced expression of a protein o interest in eukaryotic or procariotic host cells.

6. Siavoshian S., J-P. Segain, M. Kornprobst, C. Bonnet, C. Cherbut, J-P. Galmiche, H. M. Blottierea. Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression. // Gut, 2000; 46:507-514.

7. EN 2443779 Method for producing recombinant adenovirus, characterized by a reduced ratio of physical and infectious viral particles, and gene therapeutic drug obtained in this way.

8. US 20080311095 Methods and compositions for increased transgene expression (prototype).

9. Her R. Thompson, John J. Kaminski, Evelyn A. Kurt-Jones, and Katherine A. Fitzgerald. Pattern Recognition Receptors and the Innate Immune Response to Viral Infection. // Viruses, June 2011; 3(6):920-940.

10. Takeuchi O., Akira S. Pattern recognition receptors and inflammation. // Cell, Mar 2010 19; 140(6):805-20.

11. Graham F. L., Prevec L. Manipulation of adenovirus vectors. // Methods in Mol. Biol., 1991, v.7, p.109-127.

12. Berger, J., Hauber, J., Hauber, R., Geiger, R., Cullen B. R. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. // Gene, 1988 Jun 15; 66(1):1-10.

13. US 6019978 Replication-defective adenovirus human type 5 recombinant as a vaccine carrier.

1. Composition for intensive production of the target proteins in eukaryotic cells comprising a DNA vector, namely nonreplicating recombinant adenoviral nanoparticles based on the human adenovirus 5-th serotype (NPN) with an insertion in the target gene protein and the cell receptor agonist class of receptors PRR - pattern recognition receptors, chosen from the following agonists: agonists of the receptors TLR2, or receptor agonists LR4, or agonist of TLR5 receptors, or receptor agonists TLR7, or agonists of TLR8 receptors, or receptor agonists TLR9, or NOD1 agonist-receptor, or agonists of NOD2-receptors, taken in optimal ratios.

2. A composition according to claim 1, characterized in that as an agonist of the receptor TLR2 use lipoteichoic acid.

3. A composition according to claim 1, characterized in that as an agonist of the receptor TLR2 use lipopeptide.

4. A composition according to claim 1, characterized in that as an agonist of TLR4 receptor used by bacterial lipopolysaccharide.

5. A composition according to claim 1, characterized in that as an agonist of TLR4 receptor use acid peptidoglycan with a molecular weight of 1200-40000 CD.

6. A composition according to claim 1, characterized in that as an agonist of the receptor TLR5 flagellin use.

7. A composition according to claim 1, characterized in that as an agonist of the receptor TLR7 use imiquimod.

8. A composition according to claim 1, characterized in that as an agonist of the receptor TLR8 use imiquimod.

9. A composition according to claim 1, characterized in that as an agonist of the receptor TLR7 using the drug CL097, a derivative imidazoquinolines.

10. A composition according to claim 1, characterized in that as an agonist of the receptor TLR8 using the drug CL097, a derivative imidazoquinolines.

11. A composition according to �. 1, characterized in that as an agonist of the receptor TLR9 using oligonucleotide CpGODN 1826.

12. A composition according to claim 1, characterized in that as an agonist of the receptor TLR9 using oligonucleotide CpGODN 2006.

13. A composition according to claim 1, characterized in that the NOD1 agonist-receptor use C12-iE-DAP-Lauroyl-g-D-Glu-D-mDAP and Lauroyl-g-D-Glu-L-mDAP synthetic fragments of bacterial peptidoglycan.

14. A composition according to claim 1, characterized in that as an agonist of NOD2 receptors-use L18-MDP-derived muramyl dipeptide, namely the fragment of bacterial peptidoglycan.

15. A composition according to claim 1, characterized in that as the PRR receptor agonist used pharmaceutical drug in the effective dose.

16. A composition according to claim 15, characterized in that as the PRR receptor agonist used pharmaceutical agonist of TLR-4 receptor - Ρ N001919/02.

17. A composition according to claim 15, characterized in that as the PRR receptor agonist used pharmaceutical agonist of TLR-4 receptor - ΡN003478/0.

18. A composition according to claim 15, characterized in that as the PRR receptor agonist used pharmaceutical agonist NOD2 receptor - LS-001438.

19. A composition according to claim 1, characterized in that the DNA vector with an insert of the target gene encoding the secretory protein.

20. A composition according to claim 1, characterized in that use� DNA vector with an insert of a target gene, encodes a cytoplasmic protein.

21. A composition according to claim 1, characterized in that the DNA vector with an insert of the target gene encoding a membrane protein.

22. A method of increasing production of target protein encoded by the transgene, in eukaryotic cells when they transducible DNA vectors, namely nonreplicating recombinant adenoviral nanoparticles based on the human adenovirus 5-th serotype (NPAN) with the use of a composition according to claim 1.

23. A method according to claim 22, characterized in that the amplification products of the target protein is achieved in a culture of eukaryotic cells in vitro.

24. A method according to claim 22, characterized in that the amplification products of the target protein is achieved in the conditions of the organism in vivo.

25. A method according to claim 22, characterized in that the eukaryotic cell is obtained from the body of the mouse.

26. A method according to claim 22, characterized in that the eukaryotic cell is derived from the human body.

27. Composition for intensive production of the target proteins in eukaryotic cells comprising a DNA vector, namely nonreplicating recombinant adenoviral nanoparticles based on the human adenovirus 5-th serotype (NPN) with an insertion in the target gene protein and the cell receptor agonist class of receptors of cytokines, namely tumor necrosis factor in the optimal sootnoshenie�.

28. A method of increasing production of target proteins in eukaryotic cells when they transducible DNA vectors, namely nonreplicating recombinant adenoviral nanoparticles based on the human adenovirus 5-th serotype (NPAN) using compositions according to claim 27.



 

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7 dwg, 4 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to orthopaedics, and can be used for producing a transplant for long bone tissue repair. That is ensured by collecting bone marrow aspirate into lithium heparin test tubes, diluting with phosphate salt buffer, filtered through a filter with pore size 70 mcm and centrifuging for 10 min at 400 g. That is followed by inoculating flasks 150 cm2 with the produced mesenchymal stem cells (MSCs) at 1×106 cells/cm2, culturing at temperature 37°C and 5% CO2 in air, in the Dulbecco's Modified Eagle's medium containing glucose 1 g/l and less, with 10% embryo calve serum. The medium is changed every 3 days. Before preparing the transplant, the cells are removed from the substrate with 0.05% trypsin EDTA, washed and re-suspended. The cell suspension is applied on the matrix, incubated for 3 hours at temperature 37°C with rocking in a rotation shaker at 25 rpm. The matrix is filled with the DMEM medium with 10% bovine foetal serum, dexamethasone 10-7 M, β-glycerophosphate 10 mM, ascorbate-2-phosphate 0.05 mM, 1% streptomycin 100 mcg/ml or penicillin 100 units/ml. The MSCs are cultured for 28 days. What is also presented is a method for long bone tissue repair.

EFFECT: group of inventions provides the uniform bone tissue repair in replacing the bone tissue defect of critical size with the transplant.

3 cl, 2 tbl, 6 ex

FIELD: veterinary medicine.

SUBSTANCE: presented subline of cells A4C2/9k is highly sensitive to the ASF virus. The growth medium is used as medium Needle-MEM with 10% blood serum of swine. The inoculating concentration is 80-100 thousand cells/ml. The mitotic index to 2-3 days of cultivation is 25-35. The subline of cells is deposited at the Specialized collection of cell cultures of agricultural and game animals at the All-Russian research institute of experimental veterinary medicine under the number of 87.

EFFECT: invention enables to isolate the African swine fever virus without prior adaptation in reaction of hemadsorption and provides its accumulation in titre.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to methods for cell and tissue protection against a hypoxic injury and can be used for developing remedies for hypoxic and ischemic injuries. The developed method for protection is based on cell treatment by substances increasing a level of glutathionylation of Na,K-adenosine triphosphatase catalytic subunit that reduces to its activation. The obtained results can be actual for biological research institutions, medical facilities, particularly cardiologic clinics, as well as biotechnological companies for transplantology and tissue engineering.

EFFECT: presented method can be used for research activities aiming at creating the therapeutic agents for relieving tissue injuries, particularly myocardial tissue in hypoxia and hypoxia/reoxygenation.

6 dwg

FIELD: medicine.

SUBSTANCE: invention refers to a method for developing Alzheimer disease cell models for testing medical effectiveness of chemical substances to be further used in medicine, more specifically in treating individual's neurodegenerative diseases caused by neurotoxic effects of beta-amyloid aggregates. A molecular cytotoxic agent is a synthetic analogue of human beta-amyloid 1-42 isomerised in asparagic acid residue in position 7 ([isoD]) (amino acid sequence: DAEFRH[isoD]SGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA). The additional advantage of the present invention is applicability of primary cell cultures as Alzheimer disease models.

EFFECT: advantage of these Alzheimer disease cell models as compared to currently used exogenous-induced models, wherein the cytotoxic effects are caused by a non-modified form of beta-amyloid is a higher ability of the isomerised beta-amyloid to induce both cell necrosis and apoptosis.

2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and concerns a method for producing progenitor cells from mammalian myocardial samples. A presented method involves milling the samples, implanting them on culture dishes coated so that to provide adhesion; culturing the samples in the hypoxic environment so that to provide the sample adhesion to form a cell culture; recovering cells from the cell culture by enzymatic treatment; culturing the recovered cells until forming cell aggregates, and processing the cell aggregates with an enzyme solution to produce the progenitor cells of myocardium.

EFFECT: presented invention can be used in medicine and veterinary science for studying the myocardium regeneration mechanisms and stem cell biology, as well as for treating cardiac diseases.

10 cl, 2 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and medicine. What is presented is the mus musculus's hybrid culture cell strain α-producing monoclonal antibodies specific to human granulocyte colony-stimulating factor (GCSF) deposited in Special Cell Culture Collection of Institute of Cytology of the Russian Academy of Sciences, under No. PKKK ("П") 662 "Д".

EFFECT: invention enables extending the range of strains producing GCSF-specific monoclonal antibodies used for research and medical applications.

2 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to gastroenterology, and can be applied as a method of treating hormone-dependent/hormone-resistant ulcerative colitis complicated by opportunistic infections. That is ensured by measuring herpes simplex virus type 1 IgG, herpes virus type 6 IgG, cytomegalovirus IgG, mycoplasm IgG, Chlamydia IgG. If herpes simplex virus type 1 IgG is 185 units/ml and more, and IgM is 0.4 units/ml and more, herpes virus type 6 IgG is 1.5 units/ml and more, cytomegalovirus IgG k is 185 units/ml and more, IgM is 3.4 units/ml and more, mycoplasm IgG is 119 units/ml and more, IgM is 0.4 units/ml and more, Chlamydia IgG is 105 units/ml and more, IgM is 0.8 units/ml and more, a systemic transplantation of allogenic mesenchymal stem cells in a dose of 150-250 million cells.

EFFECT: using the given method enables providing the more effective therapeutic treatment of hormone-dependent/hormone-resistant ulcerative colitis, promotes CMV elimination with no anti-viral therapy required and overcoming of hormone-dependency/hormone-resistance.

3 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and medicine. What is presented is a method for differentiating pluripotent stem cell population recovered from recognised pluripotent stem cell lines into a cell population expressing markers specific for a pancreatic entoderm line co-expressing PDX1, NKX6.1, but not expressing CDX2 and NGN3.

EFFECT: method can be used for medical and biotechnological applications.

9 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to gene engineering, molecular biology and biotechnology. The method can be used for medical and biotechnological applications.

EFFECT: what is presented is the method for differentiating a population of pluripotential stem cells into a population of marker-positive cells specific for pancreatic entoderm line, which co-expresses PDX1, NKX6,1, but does not express CDX2 and NGN3B.

2 cl, 3 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, ophthalmology and biotechnology. Claimed is method of preparing cellular structures in form of spheroids for formation of front layers of artificial cornea, which includes application of parts of limbus.

EFFECT: invention can be applied for construction of front layers of artificial cornea in order to compensate damaged tissues of eye cornea.

1 ex

FIELD: chemistry.

SUBSTANCE: inventions relate to chimeric proteins, nucleic acid, coding such a protein, an expression cassette, providing the expression of nucleic acid, a vector, including the expression cassette, a method of diagnostics and a set for diagnostics. The characterised chimeric Borrelia protein includes at least one sequence of an extracellular domain of the VlsE Borrelia protein of the first type, corresponding to a certain strain, and at least one sequence of IR6 area of the VlsE Borrelia protein of the second type or Borrelia of the first type, but corresponding to a strain, different from the strain of the first type, with Borrelia being selected from Borrelia stricto-sensu, Borrelia afzelii and Borrelia garinii.

EFFECT: claimed inventions make it possible to carry out diagnostics of Lyme-borreliosis with an increased specificity and sensitivity.

15 cl, 8 tbl, 7 ex

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