Peptide, capable of regenerating bone tissue and binding with apatite

FIELD: medicine.

SUBSTANCE: group of inventions relates to field of biotechnology and transplantation medicine and represents peptide with amino acid succession SEQ ID NO: 40. Peptide has ability to regenerate bone tissue and specifically bind with surface of apatite mineral.

EFFECT: peptide is able to be stably immobilised on apatite surface for preservation of useful activity and rendering influence on bone regeneration for long time.

9 cl, 3 dwg, 3 tbl, 4 ex

 

Area of technology

The present invention relates to the peptide that has the ability to regenerate bone tissue and specifically binding to the surface of the mineral Apatite, and more specifically to the peptide having the ability to regenerate bone tissue and specifically binding to the surface of the mineral Apatite, is capable of being stably immobilized on the surface of Apatite, save useful activity and to have an impact on bone regeneration in a long time, thanks to sewn with each other amino acid sequence, having the ability to regenerate bone tissue, and amino acid sequence, having the ability to bind to Apatite, and thus the resulting peptide had the transplant effect, as well as the ability to contact with the surface of the mineral Apatite, and compositions for bone regeneration containing this peptide.

Prior art

Bones and teeth are called solid tissue of the body. Bone is made up of about 45% of inorganic substances, 35% organic matter and 20% water. As for the teeth, the enamel approximately 97% consists of inorganic substances, the dentin is 70% of inorganic substances, and the cement of the tooth is 50% of reorganizes�their substances. The content of inorganic substances differs depending on the animal species, body parts, age and the like.

Regarding the components of the bone substance, the majority of organic matter is a collagen involved in the formation of bones and the maintenance of the hardness and flexibility of bones, and is present as a matrix, causing selective adhesion of bone cells, thereby to Orient the bone inorganic particles. The mineral Apatite is the main component of bone in vivo, i.e., inorganic substance of the bones of vertebrates, and is known as hydroxyapatite (Ca10(PO4)6(OH)2), hydroxyapatite, carbomethoxyamino (the outside of the And-type: Ca10(PO4)6[(OH)2-2x(CO3)x], B-type: CA10-x[(PO4)6-2x(CO3)2](OH)2). It is known that instead of CA2+contains very small amounts of Mg2+, Na+or K+and instead of HE-contains very small amounts of Cl-or F-. Therefore, studies were conducted in dental materials and bone graft materials intended for healing bone defects, is made of the mineral Apatite, or surfaces coated with Apatite.

The purpose of the transplantation of a bone graft �is the restoration of morphological and physiological functions of bones in order to maintain biomechanical function. Therefore, as used herein, Apatite-containing bone graft material must meet the basic conditions, such as immediate use, lack of immune response, promoting rapid bone formation and revascularization, the support bone tissue and integrity, etc. However, Apatite as such can serve as an intermediary, which has bone conduction, but does not possess the ability to induce primary bone morphogenesis, required to reduce the duration of treatment. To remedy this drawback, there have been studies in which a biologically active substance containing chemotaxin (chemotactin), such as extracellular matrix protein, tissue growth factor, or bone morphogenetic protein, is used together with Apatite, and have developed products such as INFUSE (containing BMP-2), GEM21S (containing PDGF) and the like. However, these proteins are not stably immobilized on the surface of Apatite and released from Apatite, are then released into the systemic circulation and thus the collapse, and therefore difficult to maintain the activity of these proteins on the surface of Apatite for bone regeneration. Therefore, in order to increase�th effect bone regeneration, the necessary material, stably immobilized on the surface of Apatite and thus preserving useful activity.

The present inventors have attempted to overcome the problems of prior art as described above. As a result, the inventors of the present invention have confirmed that the peptide that has both and transplant capacity and the ability to bind to Apatite, can be in steady state when binding to surface of Apatite, and thus to assist the movement, proliferation and differentiation of cells associated with regeneration, and ultimately to maximize the ability of bone tissue to regenerate and to demonstrate a strong therapeutic effect in bone regeneration, and thus undertook the present invention.

Disclosure of the invention

The purpose of the present invention is to provide a peptide that has the ability to restore bone tissue and contact Apatite, and this peptide is stable immobilized on the surface of the mineral Apatite and retains its activity.

Another objective of the present invention is to provide a bone graft material and a biomaterial for bone regeneration, in which the peptide is immobilized on the surface of Apatite.

To achieve the above �firs the present invention provides a peptide having the ability to regenerate bone tissue, and binding to Apatite, wherein the peptide has the ability to regenerate bone, and the peptide having the ability to bind to Apatite communicate with each other.

The present invention also provides a bone graft material and a biomaterial for bone regeneration, in which the surface of Apatite immobilized peptide.

Brief description of the drawings

Fig.1 is obtained using a confocal microscope image of tissue taken 4 weeks after transplantation FITC-labeled bone mineral in the defect region at the base of the skull of the rabbit (Fig.1A - bone mineral without a linked peptide, and Fig.1B - bone mineral with an associated peptide having the sequence of SEQ ID NO:40, labeled with FITC).

Fig.2 shows the results of the study blood samples taken one day before and 1 day, 3 days, 7 days, 14 days, 21 days and 28 days after transplantation FITC-labeled bone mineral in the defect region at the base of the skull of a rabbit, and then released into the blood peptides were measured using fluorimetry (RFI - relative fluorescence index, the blue line is a negative control showing the results of measurement in the plasma without fluorescent substances).

Fig.3 - image showing the extent of bone regeneration after 2weeks after transplantation of bone mineral with an associated peptide in the defect region at the base of the skull of the rabbit (Fig.3A - the material for a bone graft with an associated peptide SEQ ID NO:36, Fig.3b - material for bone graft with an associated peptide SEQ ID NO:35, and Fig.3C - material for bone graft with an associated peptide SEQ ID NO:40).

Detailed description of preferred embodiments

Unless otherwise noted, all used in the description of the technical and scientific terms have the same values, which in most cases is understandable to experts in the field of technology to which belongs the present invention. Generally used in the description of the item is well known and typically used in the art.

To identify the peptide according to the present invention obtained amino acid sequence of the active sites of bone morphogenetic protein and extracellular matrix protein and then subjected to chemical modification so as to maintain their active structure.

One aspect of the present invention relates to the peptide that has the ability to regenerate bone tissue, and binding to Apatite, in which at least one peptide selected from the group consisting of amino acid sequence SEQ ID NO:1 to SEQ ID NO:35 and at least one peptide selected from the group consisting of amino acid sequence SEQ ID NO:36 �of SEQ ID NO:39, communicate with each other.

In the present invention, the peptide that binds to the mineral Apatite, may be selected from the group consisting of the amino acid sequences of SEQ ID NO:36 (STLPIPHEFSRE), SEQ ID NO:37 (VTKHLNQISQSY), SEQ ID NO:38 (SVSVGMKPSPRP) and SEQ ID NO:39 (NRVFEVLRCVFD). The peptide is chemically attached to the N-Terminus of the peptide that has the ability to regenerate bone tissue, to increase ability to bind to the Apatite, which is a part of the bone, and thus is stably associated with the bone graft material or the implant covered with Apatite.

In the present invention, the peptide having the ability to regenerate bone tissue, can be selected from the group consisting of amino acid sequence SEQ ID NO:1 to SEQ ID NO:35.

In particular, a peptide that has the ability to regenerate bone tissue, can be at least one peptide selected from the group consisting of:

(a) amino acid sequence at positions 2-18 each of the bone morphogenetic protein (BMP)-2, 4 and 6 [SEQ ID NO:1 to BMP-2, SEQ ID NO:2 to BMP-4, and SEQ ID NO:3 for BMP-6]; amino acid sequence in positions 16-34 (SEQ ID NO:4), amino acid sequence in positions 47-71 (SEQ ID NO:5), amino acid sequence in positions 73-92 (SEQ ID NO:6), the amino acid sequence in positions 88-105 (SEQ ID NO:7), amino�slot sequence in the provisions 83-302 (SEQ ID NO:8), the amino acid sequence in positions 335-353 (SEQ ID NO:9) and amino acid sequence in positions 370-396 (SEQ ID NO:10) BMP-2; the amino acid sequence at positions 74-93 (SEQ ID NO:11), the amino acid sequence in positions 293-313 (SEQ ID NO:12), the amino acid sequence in positions 360-379 (SEQ ID NO:13) and amino acid sequence in positions 382-402 (SEQ ID NO:14) BMP-4; the amino acid sequence in positions 91-110 (SEQ ID NO:15), the amino acid sequence in positions 407-418 (SEQ ID NO:16), the amino acid sequence in positions 472-490 (SEQ ID NO:17) and amino acid sequence in positions 487-510 (SEQ ID NO:18) BMP-6; and the amino acid sequence at positions 98-117 (SEQ ID NO:19), the amino acid sequence in positions 320-340 (SEQ ID NO:20), the amino acid sequence in positions 400-409 (SEQ ID NO:21) and amino acid sequence in positions 405-423 (SEQ ID NO:22) BMP-7;

(b) the amino acid sequence in positions 62-69 (SEQ ID NO:23), the amino acid sequence in positions 140-148 (SEQ ID NO:24), the amino acid sequence in positions 259-277 (SEQ ID NO:25), the amino acid sequence in positions 199-204 (SEQ ID NO:26), the amino acid sequence at positions 151 to 158 (SEQ ID NO:27), the amino acid sequence in positions 275-291 (SEQ ID NO:28), the amino acid sequence in position�deposits 20-28 (SEQ ID NO:29), the amino acid sequence in positions 65-90 (SEQ ID NO:30), the amino acid sequence in positions 150-170 (SEQ ID NO:31) and amino acid sequence in positions 280-290 (SEQ ID NO:32) bone sialoprotein II (BSP II); and

(c) the amino acid sequence in positions 149-169, YGLRSKS (SEQ ID NO:33), KKFRRPDIQYPDAT (SEQ ID NO:34), and YGLRSKSKKFRRPDIQYPDAT (SEQ ID NO:35) bone sialoprotein I (BSP I, osteopontin).

Another aspect of the present invention is related to the bone graft material and biomaterial for bone regeneration, in which the peptide having the ability to regenerate bone tissue immobilized on the surface of Apatite.

In the present invention, the biomaterial for bone regeneration may differ in that it is chosen from the group consisting of metals, natural polymers and synthetic polymers.

In the present invention to provide the possibility of binding of the peptide that has the ability to regenerate bone tissue, and binding to Apatite, where the surface of the Apatite material bone graft or biomaterial for bone regeneration, bone graft material composed of Apatite or biomaterial, such as a metal, a natural polymer or a synthetic polymer having a surface coated with Apatite, can be immersed in the peptide solution, and in this case �imicheskij perekrestivshis agent for binding is not required.

The peptide that has the ability to regenerate bone tissue, and binding to Apatite, according to the present invention stably immobilized on the surface of the Apatite, thereby to increase its stability and to retain its activity for a long time. Thus, when transferring the peptide in the body, the peptide is stably retained in the local area of transplantation, and thereby retained its influence on bone regeneration that meets the requirements to methods of treatment intended for bone regeneration and periodontal disease.

The peptide that has the ability to regenerate bone tissue, and binding to Apatite, in accordance with the present invention can contact the Apatite is selected from the group consisting of derived from the body's bone mineral hydroxyapatite, synthetic hydroxyapatite, carbonate-Apatite, tricalcium phosphate and monocalcium phosphate.

In the present invention, the content of the peptide that has the ability to regenerate bone tissue, and binding to Apatite, preferably is 1~100 mg per unit weight (1 g) bone graft material, and more preferably is 20~80 mg per unit weight of bone graft material.

In one embodiment of the present the image�t, peptide SEQ ID NO:36 having the ability to bind to the Apatite, and the peptide SEQ ID NO:35 having the ability to regenerate bone tissue, were related, in order to obtain the peptide of SEQ ID NO:40, having the ability to regenerate bone tissue, and binding to Apatite. Then, it was checked whether the stable binding of the peptide obtained with bone graft material. In addition, the ability to regenerate bone tissue was tested by transplantation of bone graft material, in which the peptide was stably immobilized on the surface of Apatite, in the region of a defect in the skull base.

Examples

Hereinafter the present invention will be described in detail using examples. These examples are only to illustrate the present invention, and specialists in the art it is clear that the scope of the present invention is not limited to these examples.

Example 1. Cooking smart peptide that has the ability to regenerate bone tissue and specifically binding to the surface of the mineral Apatite

Method F-moc solid-phase chemical synthesis using a peptide synthesizer was sequentially from N-Terminus of the synthesized peptide containing the sequence YGLRSKSKKFRRPDIQYPDAT (SEQ ID NO:35), with the ability to regenerate bone tissue�ü, derived from osteopontin, and peptide STLPIPHEFSRE (SEQ ID NO:36), having the ability to bind to Apatite. In other words, the synthesis was carried out using Rink resin (0,075 mmol/g, 100~200 mesh, stitching 1% DVB) with Fmoc-(9-fertilitycare) binds as a blocking group. 50 mg Rink resin was placed in a synth, and then subjected to swelling with DMF. After that, 20% solution of piperidine/DMF was used to remove the Fmoc-group. A 0.5 M solution of amino acid (solvent: DMF), A 1.0 m solution of DIPEA (solvent: DMF&NMP) and 0.5 m solution of HBTU (solvent: DMF) were successively introduced in 5, 10, and 5 equivalent amounts, respectively, with C-end of the peptide, and then the reaction was carried out for 1~2 hours in a nitrogen atmosphere. When the removal of the protective groups and the binding was completed, twice washed with DMF and NMP. Even when the final amino acid was carried out the removal of the protective groups for the removal of Fmoc-groups.

To confirm the synthesis used the ninhydrin reaction. Tested and fully synthesized resin was dried over THF or DCM. Then injected TFA mixture for splitting at a ratio of 20 ml per 1 g of the resin, and then stirred for 3 hours, followed by filtration to separate the resin and the mixture with dissolved peptide. The filtered solution was removed using a rotary evaporator, after which he entered here Hal�a national broadcast or large quantities of cold air were injected directly into TFA mixture for crystallization of the peptide to the solid phase, which was then isolated by centrifugation. At this point the mixture is completely TFA was removed with several washes with ether and centrifugation. The thus obtained peptide was dissolved in distilled water, and then dried by sublimation.

NH2-STLPIPHEFSRE-YGLRSKSKKFRRPDIQYPDAT-COONH2□ (SEQ ID NO:40)

The peptide was synthesized tsalala from the resin, washed, and dried by sublimation, and then was isolated and purified by liquid chromatography. The purified peptide was subjected to MALDI-analysis to confirm its molecular mass.

To test stability in vivo, carried out the binding 10 equivalent amounts fluoresceinisothiocyanate (FITC) with N-end with the use of triethylamine (1 ml per 1 g resin) during preparation of the peptide of SEQ ID NO:40. The synthesis of the peptide was confirmed by determination of its molecular weight using MALDI-TOF.

Example 2. Confirmation of in vitro stability of smart peptide that has the ability to regenerate bone tissue and specifically binding to the surface of the mineral Apatite

1200 mg of a peptide prepared in example 1 was dissolved in 1 ml tricotillomania water, and then added 4 g of bovine bone material (OCS-B, NIBEC), left for sedimentation for 24 hours and then dried by sublimation.

4 g of bone graft material with the bound peptide was placed in20 ml of PBS, after that test was performed on the release of peptide at 37°C. After 7 days, 20 ml of solvent for elution (which was originally entered) is completely removed, and then re-added 20 ml of a new solvent for elution to continue elution at 37°C. In the above procedure, the test elution of the peptide was performed for 14 days, 28 days, 56 days, 84 days and 100 days. After elution, the bone graft material was collected and measured the concentration of the peptide.

Method for obtaining test fluid

Accurately weighed 3 g of bone graft material with the bound peptide and then crushed into powder. Accurately weighed about 2 grams of powder, corresponding to 160 mg of peptide was placed in 40 ml of solution A (mobile phase), and then treated with ultrasound for 1 hour. Thereafter, while stirring the extraction was carried out at 37±2°C for 24 hours, and then the upper layer is extracted solvent was separated by centrifugation at 3000 rpm for 10 minutes and then filtered through millionby 0.22 μm filter. From there was taken and 1 ml was mixed with 3 ml of solution A, which is then filtered through millionby 0.22 μm filter, then the filtered solution was used as the test fluid.

A method of obtaining a standard liquid

Standard peptide product Vassilev a drying oven (silica gel) for 5 hours, then accurately weighed 10 mg and dissolved by adding a solution As the mobile phase, and exactly 10 ml was used as the standard liquid.

Test method

To obtain peak areas of the sample liquid and the standard liquid, ATand AS10 ml of each of the liquids was investigated using liquid chromatography under the following conditions.

The amount of peptide (mg) = amount of standard peptide product (mg) X

Mode

Measuring instrument: analytical HPLC (Shimadzu, Japan)

Column: filled with C18-bound silica gel with a size of 5 μm (length: 250 nm, inner diameter: 4.6 mm)

Mobile phase: a 0.1% trifluoroacetic acid/DDW (solution A), 0,098% trifluoroacetic acid /acetonitrile (solution B)

Detector: UV absorption spectrometer (measurement wavelength: 230 nm)

Flow rate: 1 ml/min

Column temperature: constant at approximately 40°C

100
Table 1
The conditions of the gradient:
Time (min)The composition of the solution (%)
15
35100
45
505
605

Testing elution was performed in 100 days to measure the peak of the peptide in the elution liquid. As a result (Fig.2), peptide peak was observed in Lots No. 1, 2 and 3 with the retention time of the 14.196 min After elution were measured amounts of peptide for Lots No. 1, 2 and 3, which were 9.2 mg; 9,18 mg and 9,78 mg, respectively (Fig.3). This suggests that the peptide is associated with the bone graft material cannot be released in the elution liquid, and therefore, the peptide was stably immobilized on the bone graft material.

Table 2
The results of the release of the peptide SEQ ID NO:40 from bone mineral
Lot No..dayNameRetention timeAreaHeightmg
17RT14.1960.0000 00
14RT14.1960.000000
28RT14.1960.000000
56RT14.1960.000000
84RT14.1960.000000
100RT14.1960.000000
27RT14.1960.000000
14RT14.1960.0000 00
28RT14.1960.000000
56RT14.1960.000000
84RT14.1960.000000
100RT14.1960.000000
37RT14.1960.000000
14RT14.1960.000000
28RT14.1960.00000 0
56RT14.1960.000000
84RT14.1960.000000
100RT14.1960.000000

Table 3
The content of peptide SEQ ID NO:40 remaining in the bone mineral
Lot No.NameRetention timeAreaheightmg
1RT14.19614.2441176751511329.2
2RT14.19614.23511744995111 9.19
3RT14.19614.229124963560189.78

Example 3. The confirmation of the stability in vivo of smart peptide that has the ability to regenerate bone tissue and specifically binding to the surface of the mineral Apatite

Peptide SEQ ID NO:40, labeled with FITC, was besieged in bovine bone mineral (OCS-B, NIBEC). Obtained bone mineral transplanted rabbit in the region of the defect in the skull, and then he scarificial 4 weeks. After that, bone mineral, transplanted into the defect area in the skull, examined using a confocal microscope (Olympus, Japan). Bone mineral with an associated peptide SEQ ID NO:40, labeled with FITC, in the amount of 10 mg and 20 mg transplanted into the defect area in the skull. Blood samples were collected before transplantation and after 1 day, 3 days, 7 days, 14 days, 21 days and 28 days after transplantation, and then by means of fluorimetry determined, the released peptide in the bloodstream.

As a result, it was confirmed that the peptide was completely immobilized on the surface of the material bone graft transplanted into the defect area in the skull and does not spread into the surrounding tissue (Fig.1B). The RFI values of imediatley compared to the values before transplantation, confirming that the peptide contained in the bone mineral, is not released into the blood, and this fact proved that the peptide was indeed immobilized on the surface of bone mineral (Fig.2).

Example 4. Confirmation of ability to regenerate bone tissue in vivo "smart" peptide that has the ability to regenerate bone tissue and specifically binding to surface of Apatite

Each of the peptides SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:40, obtained in the example provided the opportunity to contact the bone graft material in the same manner as in example 2, it is then transplanted into the defect area in the skull of the rabbit. The ability of peptides to regenerate bone tissue was confirmed. In the area of the cranial vault rabbit under anesthesia was established round the bone defect with a diameter of 8 mm (white new Zealand rabbit, species name: cuniculus). The bone graft material and the peptide-containing bone graft material were transplanted into the area of the bone defect (50 mg for each defect), then you have stitches and on the periosteum and the skin. The animal was sacrificed 2 weeks after transplantation, and taken the sample was fixed in formalin solution. The fabric was subjected to casting to prepare a sample with a thickness of 20 μm. The smear was stained with basic fuchsin and toluidine�Vym blue to obtain non-demineralized sample. Thus produced sample was studied using optical microscope and photographed.

Fig.3 shows the result of bone regeneration using bone graft material that contains "smart" peptide that possesses the ability to regenerate bone tissue and specifically binds to surface of Apatite. The lowest result of bone regeneration was observed in the application of bone graft material with the bound peptide SEQ ID NO:36 (a, a peptide that is associated with Apatite). It was observed that the bone graft material with the bound peptide SEQ ID NO:35 (b, a peptide having the effect on bone regeneration) had an increased effect on bone regeneration compared to a), and the bone graft material with the bound peptide SEQ ID NO:40 (c, "smart" is a peptide that has the ability to regenerate bone and specifically binding to the surface of the mineral Apatite) had a significantly higher effect on bone regeneration in comparison with (a) and (b). From the above it follows that as such a peptide, associated with Apatite, was not effective for bone regeneration, and there is reason to believe that this peptide is not stably associated with the bone graft material, and therefore has less impact on bone regeneration in comparison with "smart" peptide tha�th have both these abilities. Thus, the expected large effect on bone regeneration with the use of smart peptide that has the ability to regenerate bone and specifically binding to surface of Apatite in the bone graft material made of Apatite, or implant, is covered with Apatite.

The present invention is described based on its individual characteristics, while specialists in the art it is obvious that these specific technologies are only preferred embodiments of the implementation, and thus the scope of the present invention is not limited to these options for implementation. Thus, the actual volume of the present invention defined by the accompanying claims and their equivalent. All modifications and changes of the present invention can easily be used by a person skilled in the art, and these modifications or changes are included in the scope of the present invention.

The peptide having the ability to contact the mineral Apatite and the ability to regenerate bone tissue, in accordance with the present invention binds to the surface of the Apatite, thereby is in a stable condition, and therefore can be used in substitute bone material, metal, natural polymers or si�thetic polymers, covered with Apatite, for use in dentistry or orthopedics; may assist the movement, proliferation and differentiation of cells associated with regeneration, and as a result, the maximum increase bone regeneration; can be present stably, while maintaining the activity of the peptide, being implanted in the body, and thus, is suitable for the development of therapeutic technologies of regeneration of bone tissue using a peptide.

1. The peptide that has the ability to regenerate bone tissue, and binding to Apatite, in which a peptide consisting of the amino acid sequence SEQ ID NO:35, and a peptide consisting of the amino acid sequence SEQ ID NO:36, linked.

2. The peptide that has the ability to regenerate bone tissue, and binding to Apatite, in which the peptide represented by the amino acid sequence of SEQ ID NO:40.

3. The peptide according to claim 1 or 2, having the ability to regenerate bone tissue, and binding to Apatite, where the peptide immobilized on the surface of Apatite.

4. The peptide according to claim 3, having the ability to regenerate bone tissue, and binding to Apatite, in which the Apatite is selected from the group consisting of derived from the body's bone mineral hydroxyapatite, synthetic hydroxyapatite, carbon�Apatite tricalcium phosphate and monocalcium phosphate.

5. The peptide according to claim 3, in which the peptide is 1-100 mg per unit weight (1 g) bone graft material.

6. The peptide according to claim 1 or 2, having the ability to regenerate bone tissue, and binding to Apatite, where the peptide immobilized on the surface of the biocompatible material, coated with Apatite.

7. The peptide according to claim 6, characterized in that the biocompatible material is selected from the group consisting of metals, natural polymers and synthetic polymers.

8. The peptide according to claim 6, having the ability to regenerate bone tissue, and binding to Apatite, in which the Apatite is selected from the group consisting of derived from the body's bone mineral hydroxyapatite, synthetic hydroxyapatite, carbonate-Apatite, tricalcium phosphate and monocalcium phosphate.

9. The peptide according to claim 6, in which the peptide is 1-100 mg per unit weight (1 g) bone graft material for bone regeneration.



 

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25 cl, 70 dwg, 56 tbl, 61 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a combined recombinant protein of the formula: S-L-R, including SR10, SR13, SR15, SdR10, SdR13 or SdR15, which specifically recognises melanoma cells, where S - streptavidin monomer, L - linker having amino-acid sequence Ser-Arg-Asp-Asp-Asp-Asp-Lys containing a restriction site with enteropeptidase and marked as "d", or amino-acid sequence Ser-Arg-Ala-Gly-Ala,R - melanoma-addressing oligopeptide representing R10 having amino-acid sequence Asp-Gly-Ala-Arg-Tyr-Cys-Arg-Gly-Asp-Cys-Phe-Asp-Gly, or R13 having amino-acid sequence Leu-Ser-Gly-Cys-Arg-Gly-Asp-Cys-Phe-Glu-Glu, or R15 having amino-acid sequence Asp-Gly-Phe-Pro-Gly-Cys-Arg-Gly-Asp-Cys-Ser-Gln-Glu. This invention also describes recombinant plasmid DNAs pSR and pSdR for expression of the specified combined proteins, bacterial strains Escherichia coli MG1655/pSR and MG1655/pSdR, producers of the specified combined proteins and a producing method of melanoma-addressing oligopeptide R from combined recombinant proteins SdR10, SdR13 or SdR15.

EFFECT: invention allows producing combined proteins that provide selective and effective binding to receptors on the surface of melanoma cells and can be used in diagnostics and therapy of cancer of a human being.

9 cl, 7 dwg, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to biotechnology, and concerns a DLK1-Fc fused protein and using it for the metastases inhibition, a polynucleotide coding such a protein, an expression vector containing the polynucleotide, a host cell producing the above fused protein, a method for producing the fused protein by culturing the above host cell, a composition containing the above fused protein, and a method for the metastases inhibition. The characterised fused protein contains a DLK1 extracellular soluble domain consisting of the amino acid sequence SEQ ID NO:4 and Fc domain of a human antibody.

EFFECT: group of inventions can be used for preparing a therapeutic agent for reduction of cancer cell migration and the metastases inhibition.

11 cl, 36 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and medicine, and concerns an vaccine against influenza caused by known viral strains of influenza A and B, as well as potential reassortants. The presented polyvalent influenza vaccine is based on a hybrid protein containing N1, N3 and N5 protein fragments of influenza A virus, a haemagglutinin fragment of influenza B virus and FliC1 and FliC2 flagellin components (SEQ ID NO:1) bridged flexibly. The protein-coding nucleotide sequence (SEQ ID NO:2) is optimised for the high expression in Escherichia coli cells.

EFFECT: using the characterised vaccine enables providing the general protection against influenza.

3 cl, 4 dwg, 1 tbl, 13 ex

FIELD: biotechnologies.

SUBSTANCE: invention offers method of printing biological ligands in the form of oligosaccharides and/or polysaccharides, and/or peptides, and/or glycopeptides, and/or biotin. A layer of hydrophobic non-volatile fluid not mixing with biological ligand solvent and having lower specific weight than biological ligand solution is applied in advance onto a substrate. Then biological ligands are printed by contact-free method. Vaseline, mineral oil or higher saturated hydrocarbons or mix thereof are used as hydrophobic non-volatile fluid. Preferable thickness of hydrophobic nonvolatile fluid layer is 50-200 mcm.

EFFECT: more level drying of biological ligand solution drops on substrate along with protection of biological ligands against early hydrolysis and improvement of spot morphology.

3 cl, 2 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of immobilising protein molecules on the surface of magnetically controlled iron nanoparticles coated with a carbon coating. The method involves reacting powder with 4-carboxybenzene diazonium tosylate dissolved in water to form a covalent bond between organic functional groups and the surface of the powder of iron nanoparticles coated with a carbon coating. Carbodiimide activation is then carried out using the following systems: dicyclohexylcarbodiimide with N-hydroxysuccinimide in dimethyl sulphoxide (DCC/NHS in DMSO) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with N-hydroxysuccinimide in water (EDC/NHS in H2O) or a phosphate buffer solution. Covalent linking of protein molecules with the activated COOH group is then carried out in an aqueous or buffer medium.

EFFECT: invention enables to immobilise biomolecules on the surface of magnetically controlled iron nanoparticles coated with a carbon coating.

3 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: described is method of cell analysis by means of biochip, containing immobilised molecules of substances, able of binding with molecules, which are on the surface of cells, includes incubation of biochip with suspension of cells. Then, washing of biochip from nonspecifically bound cells is carried out. After that, fixation and staining of cells, reading of results and estimation of quantity of cells, which bound in one or several parts of biochip of the given area, are performed. In conclusion, analysis of the image of bound cells is carried out. Before carrying out fixation from biochip surface excess of liquid is removed, without permitting it to dry.

EFFECT: improvement of technology.

4 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention discloses a device based on SSB technology for cleaning, separating, modifying and/or immobilising chemical objects or biological objects in fluid medium. The disclosed device has one or more supports made from microwire tied by their ends and having a multilayer structure consisting of a centre rod and at least one coating layer suitable for binding chemical or biological objects with a functional coating or ligands lying on the surface of the support made from microwire in the absence of the effect of a magnetic field created between the supports made from microwire and particles in the fluid medium. Described also is a method of cleaning, separating, modifying and/or immobilising chemical or biological objects in a fluid medium using said device.

EFFECT: invention enables to clean, separate, modify or immobilise objects in a fluid medium with high flow rate.

24 cl, 2 tbl, 14 ex

FIELD: biotechnology of enzyme preparations for purifying and fractionation of cellulolitic enzymes.

SUBSTANCE: invention relates to method for production of affinity adsorbent. Claimed method includes chemical attachment of cellulase substrates, such as carboxymethyl cellulose sodium salt to hydroxyapatite surface.

EFFECT: affinity adsorbent of high adsorption selectivity.

2 cl, 4 ex, 1 tbl

The invention relates to biotechnology, in particular to a method for affinity sorbent for purification of collagenases, and can be used for isolation of highly purified and highly active enzyme preparations that are used in medicine, biochemistry, food industry, enzymatic synthesis of peptides, as well as for research purposes

FIELD: medicine.

SUBSTANCE: biodegradable material for bone tissue replacement contains biodegradable hydroxyapatite 50-52 wt %, magnesium phosphate 30-32 wt %, calcium hydrophosphate 8-10 wt %, calcium hydroxide 4.8-5.3 wt %, the rest - sodium alginate. As a binding agent, the material contains casein in the form of an ammonia solution in the relation of a powder mass of the biodegradable material to the ammonia solution of casein of 1:1÷1.5.

EFFECT: extended range of the biodegradable materials for bone tissue replacement.

FIELD: medicine.

SUBSTANCE: invention refers to medicine. What is described is a bioresorbable hydrogel polymer composition for cardiovascular surgery in the form of a film prepared by a reaction of natural polymers, biologically active substances, a solvent and a softening agent wherein the polymers are presented by cross-linked bioresorbable polymers - gelatin, chitosan or a mixture of chitosan and gelatin, chitosan and polyhydroxybutyrate; the biologically active substance or mixtures thereof are presented by the antioxidant L-carnosine, the anticoagulant heparin, the antiaggregant dipyridamole, acetylsalicylic acid, the non-steroid anti-inflammatory preparation acetylsalicylic acid, the antimicrobial preparations - ciprofloxacin, metronidazole; mechanical strength of the film is not less than 1.2 MPa, the relative elongation is no more than 160%, and the elasticity modulus is 0.4-5 MPa.

EFFECT: there are used hydrogel polymer compositions with the control bioresorption period, prolonged length of biologically active substance release, having biocompatible and thrombus-resistant properties and improved mechanical characteristics - higher softness and elasticity.

7 cl, 12 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine. The invention aims at creating biodegraded implants of the regenerated silk fibroin Bombyx mori. A method for preparing the biodegraded composite matrix of the regenerated silk fibroin Bombyx mori involves the stages: - dissolving the regenerated fibroin in a buffer containing CaCl2, C2H5OH, H2O in molar ratio 1:2:8. That is followed by dialysis of the prepared solution to the fibroin concentration min. 20 mg/ml. The fibroin solution of the concentration min. 20 mg/ml is mixed with dimethyl sulphoxide (DMSO). Gelatin or polylysine is added to the prepared mixture. The prepared mixture is frozen and thawed with an organic solvent added; what is also presented is the use of the specified biodegraded composite matrix as a base for the biodegraded implant.

EFFECT: group of inventions enables improving the biocompatibility, increasing the adhesive properties of matrix and cell proliferation in a mammalian body.

2 cl, 3 dwg, 3 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely transplantology, traumatology, general surgery, dentistry, combustiology, plastic surgery, cosmetology. The organ-specific regenerant GI contains a fibrin matrix with thrombin, cryoprecipitate prepared of quarantine fresh-frozen donor plasma. The fibrin matrix is nanostructured and contains thrombolysate enriched by growth factors and cytokines. Cryoprecipitate, thrombolysate and human thrombin in the regenerate are found in equal proportions in the following quantitative relation of the ingredients, mg: cryoprecipitate - 1; thrombolysate - 1; human thrombin -1 0.9%; normal saline - 2 10%; calcium chloride brine -1.

EFFECT: use of the declared method enables higher effectiveness of the recovery of surface and deep wound tissues of a human body.

1 ex, 8 dwg

FIELD: medicine.

SUBSTANCE: method of obtaining wound bandage material is claimed. Method involves obtaining protein polymer by protein reaction with polyfunctional spacer or its activated derivative. Preferred polyfunctional spacer is polycarboxylic acid, especially dicarboxylic acid. Protein polymers obtained with such spacers can be applied in a wide range of therapeutic purposes, including wound bandage materials, therapeutic agent delivery to organism, and bioadhesive and sealing substances.

EFFECT: obtaining material taking exact shape of wound filling wound hollow completely without irritation of exposable tissues.

27 cl, 2 dwg, 13 tbl, 12 ex

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