FUSION PROTEIN, POLYNUCLEOTIDE, GENETIC CONSTRUCT, PRODUCER, PREPARATION FOR REGENERATION OF CARTILAGE (VARIANTS)

Abstract

The invention relates to molecular biology, biotechnology, medicine, veterinary science. A group of inventions is proposed: a fusion protein comprising a ligand to MATN1 protein, and a growth factor of EGF, TGF, FGF, IGF, the construct components are connected via a flexible link, characterized by the amino acid sequence of FIG. 1; such fusion protein further comprising the Fc-fragment of an antibody or a polypeptide binding with FcRn and/or transferrin or a fragment thereof, the construct components connected via a flexible link, characterized by the amino acid sequence of FIG. 1; encoding polynucleotide, codon-optimized for expression in the producer cells or a target organism, a genetic construct for the synthesis of the fusion protein in producer cells, or cells of a target organism, the fusion protein producer, a producer of a genetic construct based on a bacterial cell, a preparation for the regeneration of cartilage containing at least 1 fusion protein or genetic construct as the active agent, in an effective amount, and a physiologically acceptable carrier or buffer solution, for parenteral or, in the case of the preparation based on at least 1 fusion protein containing the transport domain, oral administration, in the latter case the preparation is enclosed in an enteric coating, all variants

Claims

1. A fusion protein comprising a ligand to MATN1 protein, characterized by the amino acid sequence of FIG. 1A, and a growth factor of EGF, TGF, FGF, IGF, characterized by the amino acid sequence of FIG. 1B, either C or D, or J, respectively; construct components are connected via a flexible link characterized by the amino acid sequence of FIG. 1E.

2. A fusion protein comprising a ligand to MATN1 protein, characterized by the amino acid sequence of FIG. 1A, the growth factor of EGF, TGF, FGF, IGF, characterized by the amino acid sequence of FIG. 1B, either C or D, or J, respectively, and the Fc-fragment of an antibody, characterized by the amino acid sequence of FIG. 1F or a polypeptide binding with FcRn, characterized by the amino acid sequence of FIG. 1G, and/or transferrin, characterized by the amino acid sequence of FIG. 1H or its fragment characterized by the amino acid sequence of FIG. 1I; construct components are connected via a flexible link characterized by the amino acid sequence of FIG. 1E.

3. A polynucleotide that encodes the fusion protein according to claim 1, codon-optimized for expression in the producer cells, or a target organism.

4. A genetic construct for the synthesis in cells of the producer or a target organism of the fusion protein according to claim 1, including the polynucleotide according to claim 3 and additional elements, allowing to realize the specified function.

5. A producer of the fusion protein according to claim 1.

6. A producer of the genetic construct according to claim 4 on the basis of a bacterial cell.

7. A preparation for the regeneration of cartilage, containing at least one fusion protein according to claim 1 as an active agent in an effective amount and a physiologically acceptable carrier or buffer solution, for parenteral administration.

8. A preparation for the regeneration of cartilage, containing at least one genetic construct for the synthesis in the cells of the target organism according to claim 4 of one type as an active agent, in an effective amount, and a physiologically acceptable carrier or buffer solution, for parenteral administration.

9. A preparation for the regeneration of cartilage, containing at least one fusion protein according to claim 2 as an active agent, in an effective amount, and a physiologically acceptable carrier or buffer solution, for oral administration, the drug is enclosed in an enteric coating.

10. A polynucleotide that encodes a fusion protein according to claim 2, codon-optimized for expression in the producer cells, or a target organism.

11. A genetic construct for the synthesis in cells of the producer or a target organism of the fusion protein according to claim 2, including the polynucleotide according to claim 3 and additional elements, allowing to realize the specified function.

12. A producer of the fusion protein according to claim 2.

13. A preparation for the regeneration of cartilage, containing at least one fusion protein according to claim 2 as an active agent in an effective amount and a physiologically acceptable carrier or buffer solution, for parenteral administration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1. Components of the fusion proteins of the invention: Athe ligand to MATN1 protein, BEGF, CTGF1, DFGF2, Ea flexible link, FFc-fragment of an antibody, Gpolypeptide binding with FcRn, Htransferrin, Ifragment of transferrin, JIGF-1. CEAconnection example of components in a fusion protein.

EXAMPLE 1

Modeling of Fusion Proteins

[0082] For the modeling of proteins the following steps were carried out:

[0083] 1. Search of Components of the Fusion Protein

[0084] 2. Build of a Model of the Whole Protein to Determine the Orientation of Domains

[0085] 3. Build of Models for Each Domain (Using Samples of the 3D Structures and Ab Initio)

[0086] 4. Docking of Models Using Models of the Whole Protein.

[0087] To obtain the most realistic results in an automated regime the algorithm I-Tasser was used for modeling the proteins.

[0088] Simulated fusion proteins represented by a combination of components (FIG. 1) A, B/C/D/J, as well as those additionally containing component(s) F/G and/or H/I, components in all proteins connected via a E flexible link, were analyzed using the ProtParam program (http://au.expasy.org/tools/protparam.html), on amino acid sequences of these proteins. The following data were obtained.

[0089] Simulated fusion proteins are represented by a combination of components (FIG. 1) A, B, linked via E, consist of 77 aa, A, C linked via E136 aa, A, D, linked via E147 aa, A, J, linked via E, consist of 177 aa, and have a molecular weight 8.4 kDa, pI 5.1, 15 kDa, pI 8.74, 16.3 kDa, pI 9.81, 19.2 kDa, pI 9.46, respectively.

[0090] Simulated fusion proteins are represented by a combination of components (FIG. 1) A, B, F, linked via E, consist of 316 aa, A, C, F, linked via E,375 aa, A, D, F, linked via E386 aa, A, J, F, linked via E, consist of 416 aa, and have a molecular weight 34.7 kDa, pI 6.34, 41.3 kDa, pI 8.4, 42.7 kDa, pI 9.1, 45.5 kDa, pI 8.94, respectively. When H component is added with the use of E, proteins increase by 709 aa, the molecular massby 77.7 kDa. When I component is added with the use of E instead of using the H component with the use of E, proteins increase by 27 aa, molecular massby 2.4 kDa.

[0091] Simulated fusion proteins are represented by a combination of components (FIG. 1) A, B, G, linked via E, consist of 126 aa, A, C, G, linked via E185 aa, A, D, G, linked via E196 aa, A, J, G, linked via E, consist of 226 aa, and have a molecular weight 13.3 kDa, pI 5.3; 19.9 kDa, pI 8.7, 21.2 kDa, pI 9.65, 24.1 kDa, pI 9.35, respectively. When H component is added with the use of E, proteins increase by 709 aa, the molecular massby 77.7 kDa. When I component is added with the use of E instead of using the H component with the use of E, proteins increase by 27 aa, molecular massby 2.4 kDa.

[0092] Simulated fusion proteins are represented by a combination of components (FIG. 1) A, B, H, linked via E, consist of 786 aa, A, C, H, linked via E845 aa, A, D, H, linked via E856 aa, A, J, H, linked via E, consist of 886 aa, and have a molecular weight 86,1 kDa, p1 6.52, 92.6 kDa, pI 7.70, 94 kDa, pI 8.3, 96.9 kDa, pI 8.25, respectively.

[0093] Simulated fusion proteins are represented by a combination of components (FIG. 1) A, B, 1, linked via E, consist of 104 aa, A, C, I, linked via E163 aa, A, D, I, linked via E174 aa, A, J, I, linked via E, consist of 204 aa, and have a molecular weight 10.8 kDa, pI 5.83, 17.4 kDa, pI 8.88, 18.7 kDa, pI 9.89, 21.6 kDa, pI 9.55, respectively.

[0094] Based on the calculations of the above-mentioned programme, in all types of cells an optimal half-life of any of presented in this example proteins is supported in the presence of methionine at the N-terminus of this protein. When expression of a fusion polynucleotide (variants) in any cell, a protein with methionine at the N-terminus is produced, because the translation always starts with the start codon. Further, the methionine may be cleaved, either naturally, for example, if the protein is a secreted one, in the composition of the secretory peptide, or it can be cleaved during the protein purification, as, for example, in the case of some proteins produced in cells of Escherichia coli and other bacteria. Thus, a fusion protein according to the invention may be with methionine at the N-terminus, or without it.

EXAMPLE 2

Obtaining of Highly Purified Fusion Proteins According to the Invention with the Use of a Prokaryotic Organism

[0095] Amino acid sequences of designed fusion proteins were transferred into the nucleotide ones, at the same time a codon optimization was performed for expression in E. coli cells using the program http://molbiol.ru/scripts/01_19.html and adding restriction sites flanking a gene. The designed genes were chemically synthesized.

[0096] The obtained genes are cloned in bacterial expression vector pET28a(+) at the restriction sites flanking the target genes, on the instruction to the vector.

[0097] For the creation of a producer strain cells of E. coli of strain BL21 Star (DE3) (Invitrogen, USA) were used, with genotype FompT hsdSB (rB-mB-) gal dcm rne131(DE3), containing in the genome De3 lysogen and the rne131 mutation. The mutated me gene (rne131) encodes a truncated form of RNase E, which reduces the intracellular destruction of mRNA, resulting in an increase in its enzymatic stability, Ion and ompT-mutations in the genes of proteases allow to obtain non-proteolyzed recombinant proteins in large quantities.

[0098] Cells of E. coli strain BL21 with genotype FompT hsdSB (rB-mB-) gal dcm rne131 (DE3) were prepared as follows. Cells were incubated at +37 C. overnight in 5 ml of L-broth containing 1% tripton, 1% yeast extract and 1% sodium chloride. The culture was bred with a fresh L-broth 50-100 times and grown on a shaker at +37 C. to an optical density of 0.2-0.3 at a wavelength of 590 nm. Upon reaching an optical density of 0.3, the culture was diluted with a fresh L-broth to an optical density of 0.1 and grown for 30 min. 100 ml of culture were transferred in a sterile centrifuge tube, and cells were precipitated at +4 C. at 5000 g for 10 min. The supernatant was discarded, the cells were resuspended in deionized water in the original volume followed by centrifugation. The procedure of washing was repeated three times. After washing the precipitate of cells was resuspended in a small volume of deionized water and centrifuged for 30 seconds at 5000 rpm on microcentrifuge.

[0099] Transformation of competent cells was performed by the method of electroporation. For this 1 l of tenfold diluted ligase mixture was added to 12 l of the competent cells, mixed, and electroporation was performed on electroporator Eporator (Eppendorf, Germany) in sterile cuvettes for electroporation (Eppendorf, Germany), of a volume of 100 l, gap 1 mm, the electric tension impulse of 1.7 kV with duration of 5 msec.

[0100] After transformation, the cells were incubated in SOC-medium (2% bacto-tripton, 0.5% of yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) for 40 min. at +37 C. 10-100 l of the cell suspension were plated on a selective LB-medium (Gibko BRL, USA) containing kanamycin (100 g/ml), for selection of clones containing plasmids (producer strains).

[0101] The grown colonies of E. coli were examined for the presence of plasmids with the insert of the target gene. A clone of cells containing a plasmid DNA was considered a producer strain of the fusion protein. Thus the producer strains of fusion proteins according to the invention were obtained.

[0102] For the cultivation of the obtained producer strains a standard agar-agar LB-medium was used containing kanamycin at a concentration of 100 g/ml and glucose at a concentration of 1% to block a non-specific expression.

[0103] Induction of expression was carried out when the cell culture reached optical density of 0.6-0.8 od units at a wavelength 600 nm. 0.2% lactose was used as the inductor (Studier, 2005).

[0104] For autoinduction expression by the method of Studier (Studier, 2005) PYP-5052 medium was used comprising 1% of peptone (Gibco, USA), 0.5% yeast extract (Gibco, USA), 50 mM Na.sub.2HPO.sub.4, 50 mM K2HPO4, 25 mM (NH.sub.4).sub.2SO.sub.4, 2 mM MgSO.sub.4, 0.5% glycerol, 0.05% glucose and 0.2% lactose.

[0105] A single colony of the producer strain was inoculated in PYP-5052 medium containing kanamycin at a concentration of 100 g/ml. Fermentation was carried out at +37 C. in a thermostated rotary shaker at 250 rpm min for 20 hours until no significant changes of OD.sub.600 for 1 hour. An aliquot of cells was taken for analysis of expression of the gene encoding the fusion protein by electrophoresis in PAGE, and the remaining biomass was precipitated by centrifugation at 9000 g.

[0106] The precipitated cells were lysed with 3 cycles of sonication for 30 sec with a break of 2 min on ice. Then the destruction of the inclusion bodies was performed by incubation with a lysing buffer containing 500 mM sodium phosphate buffer, pH 8.0, 6M guanidine hydrochloride, 500 mM sodium chloride, within an hour. 8 ml of lysing buffer were added to the cells collected by centrifugation from 50 ml of culture.

[0107] A column containing Ni-NTU sepharose, pre-equilibrated with a loading buffer (500 mM sodium-phosphate buffer, pH 8.0, 8 M urea, 500 mM sodium chloride, 10 mM imidazole). The destroyed inclusion bodies were placed on the column. Further the column was washed with two volumes of loading buffer (500 mM sodium-phosphate buffer, pH 8.0, 8 M urea, 500 mM sodium chloride, 10 mM imidazole). Then the column was washed with three volumes of washing buffer (500 mM sodium-phosphate buffer, pH 8.0, 8 M urea, 500 mM sodium chloride, 30 mM imidazole). The protein was eluted with 5 ml of elution buffer (500 mM sodium-phosphate buffer, pH 8.0, 8M urea, 500 mM sodium chloride, 200 mM imidazole). Fractions of 1 ml were collected and analyzed by electrophoresis in 12% PAGE-DDS-Na, the fractions with a target protein were combined, the concentration of protein was determined according to Bradford.

[0108] Preparations of proteins were received with purity of about 97-98%, according to SDS-PAGE, the concentration of the fusion protein in each preparation was 1-2 mg/ml.

EXAMPLE 3

Obtaining of Highly Purified Fusion Proteins According to the Invention with the Use of an Eukaryotic Organism

[0109] 3.1. Obtaining of Highly Purified Fusion Proteins Using the Yeast Cells.

[0110] Amino acid sequences of designed fusion proteins were transferred into the nucleotide ones, at the same time a codon optimization was performed for expression in Pichia pastoris yeast cells using the program http://molbiolsu/scripts/01_19.html and adding regions to obtain a secreted protein flanking a gene, according to the instruction to the vector. The designed genes were chemically synthesized.

[0111] The obtained genes are cloned in eukaryotic expression vector pHIL-SI on the instruction to the vector.

[0112] Yeast cells were prepared for transformation. Culturing and freezing of cells of Pichia pastoris of strain SMD1163 deficient in multiple proteases of yeast, resulting in a stability of a secreted protein, were conducted. Cells were seeded in sterile conditions on agar in the YPD medium (1%yeast extract, 2% peptone, 2% glucose, 1 mM dithiotreitol), cultivated at 30 C., then passaged into suspension and cultured for 16 h. A part of the cells was resuspended in YPD medium with addition of 15% glycerol and frozen at 86 C. To obtain competent cells colonies of cells have been pre-grown in an agar plate in YPD medium at 30 C. for two days. Then, the content of a single colony was grown in 10 ml YPD medium at 30 C. for 16 h. The suspension was diluted in YPD to 0.2 OD.sub.600 and the final volume of 10 ml, and the culture was grown to OD.sub.600 0-8 for 4 hours. The cell suspension was centrifuged for 5 min at 500 g, the supernatant was poured, resuspension was performed in 10 ml of solution I from the kit for transformation EasyComp Transformation Kit, re-centrifuged, and the precipitate was resuspended in solution I. Aliquots of competent cells 50-200 l, were poured in sterile tubes with a volume of 1.5 ml, which were stored at a temperature of 90 C. before use.

[0113] For transformation the kit EasyComp Transformation Kit was used, being a part of the Pichia Easy Select Kit (Invitrogen), the reaction was performed according to the instruction to the kit. The resulting suspension of cells was seeded in sterile petri dish on an agar gel, prepared on the YPD medium with the addition of 1M sorbitol and ampicillin antibiotic at a final concentration of 100 g/ml. After 3 days several dozens of colonies per cup have been obtained. Cells from grown colonies have been transferred to a cup with the MMD (minimal medium-dextrose)-agar and cultured for 2 days at 30 C.

[0114] Cells of colonies grown on the selective medium have been transferred to flasks and cultured in 5 ml of MGY medium on the shaker (250 rpm min) for 1 day until OD.sub.600 5. Then the cells have been precipitated by centrifugation at 3000 g for 10 min. Control of expression of the target gene was performed by the method of SDS-PAGE.

[0115] After precipitation of cells the culture medium was filtered (pore size 45 m), then Tris-HCl pH 6.0 was added to a final concentration of 20 mM. The cultivation medium containing the fusion protein was concentrated in 5-10 times using concentrators for proteins with a molecular weight over 10 kDa of a company Millipore.

[0116] After concentrating, the preparation of the fusion protein has been heated on a water bath to boiling (t=100 C.) and boiled for 2 minutes, then centrifuged at 4 C., 15000g for 15 minutes.

[0117] Ion-exchanging chromatography was performed on KM-sepharose. Column with KM-sepharose was equilibrated with a buffer containing 20 mM Tris-HCl pH 6.0. The preparation of the fusion protein was applied at a speed of 60 ml/hour. The column was washed with 20 mM Tris-HCl pH 6.0; 20 mM Tris-HCl pH 6.0, 200 mM NaCl. Elution was conducted with 20 mM Tris-HCl pH 6.0, 1 M NaCl, and fractions of 1 ml were collected.

[0118] The preparation of the received fusion protein was diluted 2 times, the phosphate pH 8.0 was added to a concentration of 50 mM, and was placed on the column. After washing the column with loading buffer ballast proteins were removed by washing with a solution of 20 mM imidazole in the same buffer. Protein was eluted by a solution containing 200 mM imidazole.

[0119] As a result, preparations of the fusion protein with a purity of over 95% were obtained. The presence of bands corresponding to molecular weight of target proteins was revealed on electrophoregram. For the resulting strains a high level of expression of target proteins is characteristic.

[0120] 3.2. Obtaining of Highly Purified Fusion Proteins Using Mammalian Cells.

[0121] At the N-terminus of the amino acid sequence of each fusion protein TPA (tissue-type plasminogen activator isoform 1 preproprotein [Homo sapiens], NCBI Reference Sequence: NP_000921.1) signal sequence was placed. Amino acid sequences of designed fusion proteins were transferred into the nucleotide ones, at the same time codon optimization was performed for expression in mammalian cells (CHO) in a manual mode, and adding restriction sites and the Kozak sequence flanking the gene. The calculated genes were chemically synthesized.

[0122] The synthesized gene was cloned in vector pcDNA3.1(+) due to instruction to the vector. A producer strain of this plasmid DNA was created based on E. coli DH10 B/R cells, as in protocol described in paragraph 4.1.1.

[0123] The transfection of mammalian cells by the created plasmids was carried out by calcium phosphate deposition.

[0124] For transformation of mammalian cells (CHO) by plasmid DNA the cells were cultured in 12-well plate (Costar, USA) with a seeding density of 510.sup.4 cells/cm.sup.2. The next day, for synchronization of cell divisions, the culture medium was replaced. Three hours later, plasmid DNA precipitated with calcium phosphate was added to the cells. To prepare the precipitate, 250 l of solution containing 50 g of DNA in 250 mM CaCl.sub.2 was slowly mixed with 250 l of solution (1,64% NaCl, 1.13% HEPES pH of 7.12 and 0.04% Na.sub.2HPO.sub.4). After 24 hours incubation at 37 C. in an atmosphere of 5% CO.sub.2, the medium has been replaced with the same containing 100 g/ml neomycin for selection of clones containing plasmid with insert of the target gene and, therefore, expressing fusion proteins, the selection has been carried out for 20 days, in the wells containing live cells, the medium has been changed (thus the previous culture medium has not been poured, but used to determine the amount of the secreted proteins by ELISA), and a day later the cells have been removed from the substrate and analyzed for the expression of the transformed genes. Analysis of the efficiency of transfection was performed on a flow cytometer Beckman Coulter EPICS XL (Beckman Coulter, USA).

[0125] The level of fusion proteins in the culture medium of the obtained stable transfectomas of line CHO was assessed using a standard solid-phase ELISA.

[0126] As a result of the clonings, stable CHO transfectomas had been obtained that have been accumulated for cryopreservation and production of an experimental batch of the fusion proteins. Productivity of created CHO transfectomas expressing fusion proteins was 420-540 g/10.sup.7 cells/day.

[0127] Cultivation of producer cells was carried out using the bioreactor BIOSTAT Bplus and autoclaved medium IMDM with the addition of 45 g DFBS (0.5%) and 25.8 g (100 mM) of heptahydrate zinc sulphate (ZnSO.sub.47H.sub.2O) per 9 l of the medium. An operating mode was set: temperature 37 C., pH 6,9-7,2, oxygen concentration 50% of saturation of the air. After reaching the predetermined mode, seeding of the bioreactor was performed, for which a seed material was inoculated in aseptic conditions. Time of cultivation was 3 days.

[0128] After cultivation the culture fluid was filtered through sterile capsule Sartopure (Sartorius, Germany) with a pore diameter of 1.2 m, with a speed of 1 l/min, Then the clarified liquid was concentrated on the system of Viva Flow 200 (Sartorius, Germany) by using a filter. Concentration was performed until the total volume was 200 ml.

[0129] A chromatographic purification was carried out in two stages, using sterile solutions. On the first stage BioLogic DuoFlow Pathfinder (Bio-Rad) system was used, with automatic collector of fractions BioFract, and semi-preparative chromatographic column YMC TriArt, 2504,6 mm, sorbent C18. Before operating, the column was equilibrated with 200 ml of buffer (1 kg of water for injection and 1 g of trifluoroacetic acid) in manual mode through the pump of the chromatograph at the speed of 2 ml/min.

[0130] The prepared material in a volume of 200 ml was introduced into the chromatograph through the pump of the chromatograph at the speed of 0.5 ml/min. Elution was performed with buffer (2 kg of acetonitrile, 2 g of trifluoroacetic acid) at a rate of 0.5 ml per minute. A fraction was collected at the absorption maximum at 260 nm. The fraction volume was about 500 ml.

[0131] The second stage of purification was performed using gel chromatography column BioSil SEC 125-5, 3007,8 mm. The column was equilibrated preliminarily with 0.02 M of PBS-buffer. The resulting material was introduced into the chromatograph through the pump of chromatograph at the speed of 0.5 ml/min. Elution was performed by buffer (0.6 M solution of NaCl) with a concentration gradient from 0.1 to 0.6 M. A fraction was collected with absorbance at A280 nm of not less than 3.4 optical units. The fraction was collected into vials. The volume of the resulting solution of each preparation of protein was approximately 1 1 with a concentration of the fusion protein 2-2,7 mg per 1 ml.

[0132] The fusion proteins according to the invention may be obtained using other mammalian cells, e.g. HEK293, COS.

[0133] 3.3. Obtaining of Highly Purified Fusion Proteins Using Plants.

[0134] The amino acid sequences of the calculated fusion proteins were transferred into nucleotide ones, at the same time codon optimization was performed for expression in Nicotiana benthamiana cells using the program http://molbiolsu/scripts/01_19.html and adding regions flanking a gene, according to the instruction to the vector. The designed genes were chemically synthesized and cloned in eukaryotic expression vector pTRV1. It is possible to use a viral vector (for example, described in the article Komarova T. V., Skulachev M. V., Zvereva A. S., Schwartz M. A., Dorokhov Y. L., Atabekov I. G. (2006) A new virus-vector for efficient production of target proteins in plants. Biochemistry, 71(8), 1043-1049).

[0135] The resulting vector was introduced into Agrobaterium tumefaciens strain GV3101, which was used for infiltration of leaves of N. benthamiana. The resulting strain of Agrobaterium tumefaciens carrying the fusion gene, was cultured for 12 h at 30 C. in a shaker. Cells (1.5 ml) were precipitated by centrifugation (4000 g, 5 min), the precipitate was resuspended in buffer (1.5 ml: 10 mM MgCl.sub.2, 10 mM MES (pH 5.5)), OD.sub.600 was adjusted to 0.2. A suspension of Agrobacterium was syringed without a needle on the leaves of growing N. benthamiana plants. Maximum level of protein synthesis was observed 7-11 days after infiltration.

[0136] The expression of fusion proteins in cells of leaves of producer plants was analyzed using electrophoresis in SDS PAGE. A fragment of a leaf was triturated in buffer (10 mM KCl, 50 mM Tris pH 8.0, 5 mM MgCl.sub.2, 10 mM -mercaptoethanol, 0.4 M sucrose, 10% glycerol) on day 10 after infection. The resulting extract was subjected to centrifugation (14000 g, 10 min), the precipitate and the supernatant were analyzed using SDS-PAGE. On the electrophoretogram proteins were revealed corresponding to the molecular weight of the fusion proteins according to the invention, in the membrane fraction of cells. In control, in plants that have not undergone transformation, the corresponding proteins have not been identified. The yield of proteins was approximately 12-14% of the fraction of insoluble proteins.

[0137] Based on the obtained results, the inventive fusion proteins may be obtained using both prokaryotic and eukaryotic cell systems, highly purified preparation of each protein can be obtained using various types of protein purification. Given conditions of extraction and purification were selected experimentally and may vary in values known to the average expert in this field.

EXAMPLE 4

Obtaining of Highly Purified Genetic Constructs According to the Invention

[0138] The amino acid sequences of the calculated fusion proteins were transferred into nucleotide ones, at the same time codon optimization was performed for expression in mammalian cells using the program http://molbiol.ru/scripts/01_19.html and adding restriction sites and the Kozak sequence flanking a gene, according to the instruction to the vector. The designed genes were chemically synthesized.

[0139] 4.1. Obtaining of a Plasmid DNA Coding the Fusion Protein of the Invention (Options)

[0140] 4.1.1. Creation of a Producer Strain of the Plasmid DNA

[0141] The obtained genes were placed in an eukaryotic expression vector pVAX I (Invitrogen), or pcDNA3.1+ (Invitrogen) at restriction sites flanking the target genes, due to the instruction to the vector.

[0142] For the creation of producer strain, cells of E. coli strain DH10B/R (F-mcrA, (mrr-hsdRMS-mcrBC), 80dlacZM 15, lacX74, deoR, recA1, endA1, araD139, A(ara,leu)769, galU, galK-, rpsL, nupG) were used which were transformed by the obtained plasmid DNA by the method of electroporation using electroporator MicroPulser (BioRad). This strain does not contain methylase, which minimizes the possibility of mutations arising in DNA, including in a cloned in the plasmid supported in this strain gene. To 12 l of the competent cells 1 l of a dialyzed ligase mixture was added, placed between the electrodes of a poration cell and treated with a current pulse.

[0143] After transformation, cells were placed in 1 ml of SOC-medium (2% bacto-tripton, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) and incubated for 40 min at +37 C.

[0144] The identification of clones of E. coli cells containing the plasmid DNA obtained was conducted on the selective medium containing LB-agar, 50 g/ml kanamycin or ampicillin, respectively.

[0145] From the grown clones the plasmid DNA was isolated. Isolation of the plasmid DNA was performed using Wizard Minipreps DNA Purification System (Promega, USA). The purified recombinant plasmid DNA was verified by sequencing.

[0146] Sequencing of the cloned fragments was performed by the method of Sanger using a set Applied Biosystems BigDye Terminator (BDT) v3.1 Cycle Sequencing Kit (Applied Biosystems, CIIIA) according to the accompanying instruction.

[0147] For the labeling of the reaction products fluorescent dye-labeled ddNTP were used, and each ddNTP corresponded to the dye. For sequencing unlabeled plasmid-specific primers were used. A PCR reaction was conducted, then the reaction mixture was purified from free labeled ddNTP due to the instruction in the kit BigDye X-Terminator Purification Kit (Applied Biosystems, USA), and the sequencing reaction products were separated using capillary sequencer Applied Biosystems 3500/3500L Genetic Analyzer (Applied Biosystems, USA) and reagent 3500/3500L Genetic Analyzer Polymer POP-6 (Applied Biosystems, USA).

[0148] The results of the separation of the reaction products of sequencing were recorded by scanning by the laser and the detection of four fluorescent dyes included in all types of ddNTP.

[0149] The computer analysis of DNA sequences was performed using the personal computer using the programs Chromas and BioEdit. The nucleotide sequences of the investigated DNA fragments were aligned to the designed, the identity of the synthesized fragments to the calculated ones was demonstrated. As a result E. coli cell clones were selected containing the full sequences of the target genes in the composition of plasmidsthe DNA sequences encoding the fusion proteins.

[0150] 4.1.2. The Accumulation of the Plasmid DNA, Coding for the Fusion Protein (Variants)

[0151] A separate colony of E. coli grown on LB-agar in the Petri dish with the addition of kanamycin or ampicillin was placed in 10 ml of selective media. Cells have been grown for 12 h at +37 C. under constant stirring (250 rpm). The resulting cells were collected by centrifugation at 4000 g. Further isolation and purification of plasmid DNA was performed using the kit EndoFree Plasmid Mega Kit (Qiagen), which allows to obtain non-pyrogenic DNA. The isolated plasmid DNA was analyzed by electrophoresis in a 0.8% agarose gel, its concentration was measured using fluorometry. The yield of the plasmid DNA made up from 3.1 mg to 4.7 mg from 1 1 of culture medium.

[0152] 4.2. Obtaining a Vector Based on AAV Virus Encoding the Fusion Protein of the Invention (Options)

[0153] The obtained genes were placed in a vector based on AAV pAAVK-EF1-MCS (System Biosciences (SBI)), on the basis of which a producer strain of the given vector was created using E. coli cells (RecA-), then the vector was isolated for use in mammals, all due to the instruction to the vector. The output of the vector was from 2 mg to 3.2 mg from 1 1 of culture medium.

[0154] 4.3. Obtaining of a Short Linear Design, Coding for a Fusion Protein of the Invention (Options)

[0155] For production of short linear construct, plasmid DNA obtained according to claim 4.1. was used. Using specific primers and PCR a fragment of plasmid DNA was amplified that contains signals for initiation of transcription, a promoter, signals of initiation of translation, the start codon, the fusion gene, 1 or 2 stop codons, transcription termination sequences, regulatory sequences.

[0156] Amplification of the mentioned sequence was performed in a total volume of 50 l, in thin-walled polypropylene tubes with a volume of 650 l containing 5 l 10 Taq buffer (700 mM Tris-HCl, pH 8.6/25 C., 166 mM (NH.sub.4).sub.2SO.sub.4), 5 l MgCl.sub.2 (1.25 mm), 1 l dNTP, and 31.5 l of water, 1 ml of forward and 1 l of reverse primer, 5 l of plasmid DNA and 0.5 l of Taq polymerase (Fermentas, Lithuania).

[0157] The reaction mixture was heated for 5 min at 95 C. for denaturation of DNA. To prevent evaporation, the reaction mixture of volume of 50 l was layered with 30 l of mineral oil Bayol F (Sigma, USA). Reaction of amplification was performed in thermal cycler S1000 Thermal Cycler (Bio-Rad, USA). 35 Cycles were carried out: 95 C.20 seconds, 50-62 C. (depending on primers)20 sec., 72 C. 1 min. For completing the formed DNA chains, an additional cycle was added: 5 min at 72 C.

[0158] The result of the PCR was analyzed by electrophoresis in agarose gel. If a result was positive, preparative electrophoresis was carried out.

[0159] The amplified DNA fragments were concentrated and purified by preparative electrophoresis in a 1.2% agarose gel (Gibko BRL, USA). A sample of the mixture after PCR was mixed with sixfold buffer (0.25% Bromphenol blue, 30% glycerol) (ThermoScientific, USA) and was applied to a gel, 18 l per a well. Electrophoresis was performed in a horizontal apparatus in TAE buffer (40 mM Tris-acetate, 2 mM EDTA pH 8.0, 0.5 g/ml Ethidium bromide) at a voltage of 5-10 V/cm. The result of the separation of DNA was recorded in a transmitted UV light (302 nm) of transilluminator Macrovue (LKB, Sweden). The length of the amplified fragment was determined by a logarithmic dependence of DNA mobility on the length of the fragments in the marker. As markers a proprietary mix of DNA fragments 1000 bp GeneRuler DNA Ladder(Fermentas, Lithuania) was used. The plot of agarose containing the DNA band of desired size was excised, and the DNA fragment was purified using a kit DNA&Gel Band Purification Kit (GE Healthcare, UK) in accordance with the instruction. An isolated short linear design was used in mammals.

EXAMPLE 5

Identifying the Regenerative Effect of the Fusion Proteins According to the Invention

[0160] Collagen-induced arthritis in rats is a model of human rheumatoid arthritis. This type of arthritis in rats was induced by introduction of native heterologous type II chicken collagen, with incomplete Freund's adjuvant. The target of this autoimmune attack is type II collagen. In collagen-induced arthritis leading role in the implementation of the effector reactions belongs to autoantibodies to collagen (a detailed description of the models: Kleinau S., 1991, Gromyko, Gritsuk 2012).

[0161] Fusion proteins according to the invention were analyzed, when one protein is introduced and when a mixture of proteins is introduced, and also when administered orally and when administered parenterally. The results were evaluated by analyzing histological sections of joints, microscopy.

[0162] 5.1. The Study of the Regenerative Potential of the Drug on the Basis of One Fusion Protein (Variants) for Parenteral Administration

[0163] 5.1.1. Introduction in the Periarticular Tissue

[0164] After the formation of arthritis the rats were injected with a fusion protein (variants), based on the components A, B/C/D/J, and in some embodiments, additionally F/G and/or H/I, components in the variants of the protein were presented in a different order and connected via E component, all the components are shown on FIG. 1, or a mixture based on them, in the amount of 20 g protein locally in periarticular tissues, mostly in muscle, which corresponds to the dose for humans 0.1 mg. The introduction was carried out daily for seven days. Starting from the 6th day, the morphology of joints has been analyzed, within 21 days. As a negative control saline was used. A reliable picture of a cartilage repair was obtained using all of the investigated fusion proteins, compared to control, while the best performance among the groups with the introduction of a single fusion protein was observed in the group AEJ, groups with the introduction of two fusion proteinsin the group AEJ+JEA, with a small gapin the group AES+AEB+DEA+AEJ, the components are shown on FIG. 1. The fusion proteins additionally containing component(s) of F/G and/or H/I, mainly, had an effect comparable to those of proteins of similar structure that do not contain that domains, so caused regeneration of a damaged cartilage, more poorly expressed in the case of the content of two additional domains.

[0165] 5.1.2. A Systemic Introduction

[0166] After the formation of arthritis the rats were injected with a fusion protein (variants), based on the components A, B/C/D/J, and in some embodiments, optionally F/G and/or H/I, components in the variants of the protein are presented in different order and connected via E component, all the components are shown on FIG. 1, or a mixture based on them, in the amount of 20-30 g of a protein systemicallyin the tail vein, which corresponds to a dose for humans of 0.2-0.3 mg. The introduction was carried out daily for seven days. Starting from the 6th day, the morphology of joints was analyzed, within 21 days. As a negative control saline was used. A reliable picture of the cartilage repair was received using all of the investigated fusion proteins, compared to control, while the best performance among the groups with the introduction of a single fusion protein was observed in the group of JEA, groups with the introduction of two fusion proteinsin the CEA group from methionine at the N-terminus of the protein (no formyl)+JEA, with a small gapin the group CEA+BEA+JEA, the components are shown on FIG. 1. When using fusion proteins containing the additional component(s) of F/G and/or H/I, a little less expressed regeneration of cartilage was observed in the experiment, however, after the experiment a longer circulation of these proteins in the body was observed that may have led to the continuation of their effect on regenerative processes in the cartilage.

[0167] A less expressed regeneration of cartilage as a result of this experience was observed, compared to that in the experiment described in paragraph 5.1.1.

[0168] 5.2. The Study of the Regenerative Potential of the Drug on the Basis of One Genetic Construct (Options) for Parenteral Administration

[0169] 5.2.1. Introduction in the Periarticular Tissue

[0170] After the formation of arthritis the rats were injected with at least 1 created genetic construct based on plasmid DNA or AAV or a linear fragment, from which in the cells of animals fusion protein was synthesized based on the components A, B/C/D/J, and in some embodiments, optionally F/G and/or H/I, components in the variants of the protein are presented in different order and connected via E component, all the components shown on FIG. 1, in the amount of 50 g of DNA locally in periarticular tissues, mostly in muscle, which corresponds to a dose for humans of 1 mg. The introduction was carried out once every seven days, in total five times. After the third injection the morphology of joints has been analyzed, within 21 days. As a negative control the same genetic construct was used, not containing the fusion gene. A reliable picture of cartilage repair was received using all of the investigated genetic structures (DNA), compared to control, while the best performance among the groups with the introduction of the same genetic structure was observed in the groups based on the pVAX plasmid DNA, coding fusion protein AEJ, and also based on the linear construct coding fusion protein AEDEF, with a small gap in the group based on the AAV encoding a protein FEBEA, groups with the introduction of two genetic structures in the group on the basis of the plasmid DNA pcDNA3.1, encoding fusion proteins AEJ+JEA(gene with a secretion signal at the N-terminus), with a small gap in the group based on the pVAX plasmid DNA, coding fusion proteins AES+AEB+DEA+JEA, the components shown on FIG. 1. Genetic constructs encoding the fusion proteins additionally containing the above-mentioned fourth component, mainly, effected comparable to proteins of similar structure not containing those domains, so caused regeneration of the damaged cartilage, but more weakly expressed. The study also showed that the synthesized fusion proteins longer were detected in the serum of animals after the experiment end with the introduction of genetic constructs that encode one transport domain of F/G/H/I. In the result of the introduction of the linearized vector slightly lower rates of regeneration were observed.

[0171] 5.2.2. Systemic Introduction

[0172] After the formation of the arthritis the rats were administered with the created genetic constructs based on plasmid DNA or AAV or a linear fragment, from which in the cells of an animal a fusion protein was synthesized based on the components A, B/C/D/J, and in some embodiments, optionally F/G and/or H/I components in the variants of the protein are presented in different order and connected via E component, all the components are shown on FIG. 1, or a mixture of such genetic structures, in the amount of 50 g of a genetic construct systemicallyin a tail vein, which corresponds to the dose for humans 1 mg. The introduction was carried out once every seven days, a total of five times. After the third injection the morphology of joints has been analyzed, within 21 days. As a negative control the same genetic construct not containing the fusion gene was used. A reliable picture of cartilage repair was received using all of the investigated genetic structures, compared to control, while the best performance among the groups with the introduction of the same genetic structure was observed in the groups based on the AAV encoding a protein JEA, groups with the introduction of two genetic constructsin the group on the basis of plasmid DNA encoding proteins CEA+JEA, with a small gapin the group based on linear fragments encoding proteins CEA+BEA(with a signal sequence at N-end)+JEA, the components are shown on FIG. 1. A little less expressed regeneration of cartilage was observed as a result of this experience, compared to that in the experiment described in paragraph 5.2.1.

[0173] Genetic constructs encoding the fusion proteins additionally containing component(s) F/G and/or H/I, effected comparable to proteins of similar structure that do not contain those domains, so induced regeneration of the damaged cartilage, but the synthesized fusion proteins were longer detected in the serum of animals after the end of experiment.

[0174] 5.3. The Study of the Regenerative Potential of the Drug on the Basis of One Fusion Protein (Variants) when Administered Orally

[0175] A fusion protein (variants), presented by the components A, B/C/D/J, F/G and/or H/I, in different sequences, connected via E component, the components are shown on FIG. 1, or a mixture of such fusion proteins, was placed in enteric coating and given to rats after the formation of arthritis, in the amount of 2-3 mg of protein orally, which corresponds to the dose for a man 20-30 mg. The introduction was carried out daily for 7-30 days. Starting from the 10th day, the morphology of joints has been analyzed, within 21 days. As a negative control water was used. As a positive control a preparation of chondroitin sulfate was used, 4 mg were administered, which corresponds to a dose for humans 750 mg, 2 times a day, for 3 weeks.

[0176] A reliable picture of cartilage repair was received using all of the investigated fusion proteins, compared to control, while the best performance among the groups with the introduction of a single fusion protein was observed in groups GEAEB/C/D/J, and GEB/C/D/JEA, groups with the introduction of two fusion proteinsin the group IEJEA+AEBEH, the components are shown on FIG. 1. It is revealed that when in conjunction with a three-component or a four-component (including transport domain) a two-component fusion protein (not including transport domain) is used, the difference in the results is insignificant in comparison with the introduction of a protein from three or four components.

[0177] Regeneration of cartilage was observed as a result of this experience, comparable to that observed in the experiment 5.1.1. In addition, a prolonged circulation of fusion proteins containing the transport domain was observed, in the blood. The use of a particular enteric coating does not affect the results of the study.

[0178] Also a similar study was conducted on rabbits, to whom a surgical tear of the meniscus was introduced. The results of that study correlate with the results obtained in the study on the model of collagen-induced arthritis in rats.

[0179] So, the possibility is proved of obtaining fusion proteins, polynucleotides, genetic constructs, producers, drugs for the regeneration of cartilage (all versions). It is also shown that the developed fusion proteins have a significant regenerative effect on the damaged cartilage, both directly imposed and with the introduction of genetic constructs encoding them. In all studies an increase in the number of chondrocytes in previously damaged joints was observed, which probably caused the regeneration of cartilage. This allows to conclude that in other types of cartilage defects the proposed invention (embodiments) will allow for their regeneration.