RU2664192C1 - Recombinant gene encoding hbd-epo protein, recombinant plasmid dna pl610, method for producing a recombinant hbd-epo protein, a recombinant hbd-epo protein, a composition for specifically inducing bone regeneration, method of specific induction of bone tissue regeneration - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, genetic engineering, biochemistry, medicine, veterinary medicine. Synthetic DNA sequence corresponding to gene encoding the protein sequence of the heparin-binding domain (HBD) from Danio rerio fused with human erythropoietin (Epo) sequence (HBD-Epo protein) that nucleotide composition of the codons is optimized for heterologous expression in the non-pathogenic laboratory strain of E. coli. This synthetic HBD-Epo gene is flanked by 5'-end of the NcoI site, and 3'-end site of Kpn2I and inserted into plasmid pQE6 at the NcoI and Kpn2I sites. Based on this, a recombinant plasmid pL610 is obtained containing an artificial bacterial operon of the recombinant HBD-Epo protein comprising the promoter region of the early promoter of bacteriophage T5, the recombinant HBD-Epo protein gene, the transcription terminator; bacterial operon beta-lactamase; a bacterial region of the initiation of replication of the ColE1 type encoding the recombinant HBD-Epo protein and providing its synthesis in E. coli strains. Invention also includes a method for purifying the HBD-Epo protein by ion exchange chromatography. Invention relates to HBD-Epo recombinant protein itself, the expression vector pL610 and the composition (demineralized bone matrix (DCM) containing the HBD-Epo protein, as well as a DCM containing the protein HBD-Epo and BMP-2), aimed at stimulating osteogenesis. Invention allows to obtain a stable purified active protein HBD-Epo, as well as HBD-Epo, immobilized on DCM, including DCM with BMP-2 bone morphogenetic protein.

EFFECT: invention further includes a method for specifically inducing bone regeneration, comprising filling the defects of bone tissue with a composition consisting of HBD-Epo immobilized on DCM, including on the DCM with bone morphogenetic protein BMP-2.

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Description

The invention relates to the fields of: biotechnology, genetic engineering, biochemistry, medicine, veterinary medicine.

The invention includes the preparation of a recombinant plasmid pL610 based on the pQE6 vector containing a synthetic nucleotide sequence of a gene encoding the protein sequence of a heparin binding domain (HBD) from Danio rerio, and a nucleotide sequence of a gene encoding a human erythropoietin (EPO), the sequence being optimized for heterologous expression of hybrid HBD-Epo recombinant protein in non-pathogenic laboratory strains of Escherichia coli. The invention also includes a method for purifying HBD-Epo protein by cation exchange and affinity chromatography on heparin sepharose. The invention relates to the most recombinant HBD-Epo fusion protein, pL610 expression plasmid, a method for using a protein in demineralized bone matrix (DCM) containing HBD-Epo, and a method for using a protein in DCM containing, in addition to HBD-Epo, a recombinant bone morphogenetic protein 2 (BMP-2) for the regeneration of bone defects. The invention allows to obtain a stable biologically active purified protein HBD-Epo, as well as its form, immobilized on DCM, including DCM with recombinant BMP-2.

Erythropoietin is a hormone protein, or cytokine, under the influence of which red blood cell precursors in the bone marrow differentiate. EPO is the main regulator of erythropoiesis in the human body, participating in the formation of red blood cells and hormonal regulation. By binding to specific receptors, EPO stimulates the division and differentiation of competent progenitors of red blood cells in the bone marrow [Krantz S.B. Erythropoietin. Blood 1991. Vol. 77 (3), P. 419-434.]. In addition to influencing erythropoiesis, EPO is involved in the healing process of wounds, in the restoration of damaged nerves, and also performs other biological functions.

EPO is a glycoprotein, the molecular weight of the EPO polypeptide without sugar residues is 18.236 Da. The natural intact molecule EPO has approximately 40 mol%. the masses are carbohydrate residues, EPO glycosylation occurs at specific glycosylation sites [Sasaki N., Bothner B., Dell A., Fukuda M. Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J. Biol. Chem. 1987. Vol. 262 (25), P. 12059-12076.].

EPO is used in clinical practice in the form of a solution for parenteral use [Martindale, the extra pharmacopoeia. Martindale, William, 1840-1902. Royal Pharmaceutical Society of Great Britain. Department of Pharmaceutical Sciences Edition 31st ed. / Ed. James E.F. Reynolds London: Royal Pharmaceutical Society, 1996. Martindale, 1996 WHO Drug Information, Vol. 10 (3), P. 146 1-165-erythropoietin (human clone λHEPOFL13 protein moiety), glycoform ω.].

For clinical use, EPO is obtained by biosynthesis in eukaryotic cells using recombinant DNA technology [Egrie J.C., Strickland TW, Lane J., Aoki K., Cohen AM, Smalling R., Trail G., Lin FK, Browne JK, Hines Dk Characterization and biological effects of recombinant human erythropoietin. Immunobiology. 1986. Vol. 172 (3-5), P. 213-224.]. Such EPO is a product of expression of the human EPO gene cloned in a culture of Chinese hamster ovary cells [RF Patents No. 2125093, No. 2070931, No. 2070931].

Recombinant human EPOs are commercially available in the form of molecular forms of Epoetin alfa and Epoetin beta. They are used to treat anemia in chronic renal failure (CRF). These forms of EPO have the same sequence at 165 aa, but differ in the glycosylation pattern and pharmacokinetic profile. Epoetin alfa is slowly and not completely absorbed by subcutaneous administration, its bioavailability is approx. 10-50% of bioavailability with intravenous administration. Epoetin beta is also absorbed slowly, its bioavailability is approx. 40%

Other forms of EPO have been developed, for example, Epoetin delta, gamma and omega, as well as Darbepoetin alfa, a hyperglycosylated analogue of the recombinant human EPO alpha, the half-life of which is about three times higher when administered intravenously than the recombinant human EPO alpha and native hormone [Egrie J. C, Dwyer E., Browne JK, Hitz A., Lykos MA Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin. Exp Hematol. 2003. Vol. 31 (4), P. 290-299.].

Since human EPO is necessary for the formation of red blood cells, the hormone has been successfully used to treat diseases that lead to a decrease in the formation of red blood cells, for example, for the treatment of anemia in chronic renal failure and in patients with immunodeficiency [Eschbach JW, Egrie J.C., Downing MR, Browne JK Adamson JW NEJM. 1987. Vol. 316, P. 73-78; Eschbach J.W., Abdulhadi, M.H., Browne J.K., Delano B.G., Downing M.R., Egrie J.C., Evans R.W., Friedman E.A., Graber S.E., Haley N.R.,. Ann. Intern. Med. 1989. Vol. 111, p. 992; Egrie J.C., Eschbach J.W., McGuire T., Adamson J.W. Kidney Intl. 1988. Vol. 33, p. 262; Lim V.S., Degowin R.L., Zavala D., Kirchner P.T., Abels R., Perry P., Fangman J. Ann. Intern. Med. 1989. Vol. 110, P. 108-114 .; Danna R.P., Rudnick S.A., Abels R.I. In: Garnick M. B., ed. Erythropoietin in Clinical Applications - An International Perspective. New York, N.Y .: Marcel Dekker; 1990: P. 301-324.].

To treat these diseases, the protein is administered parenterally (including intravenously), and when it enters the bloodstream, it is destroyed by the protease system for a short time, which leads to a decrease in the effectiveness of therapy.

Attempts have been made to improve the pharmacokinetic properties of EPO, namely, lengthening the half-life to reduce the frequency of injections, which was achieved by obtaining derivatives of EPO.

Several approaches were used along this path: (1) conjugation with (poly) peptides, i.e. obtaining a hybrid EPO protein with highly glycosylated peptides and other EPO protein conjugates; (2) chemical modifications of EPO (for example, addition of the residue of polyethylene glycol - PEG, polysaccharides); (3) obtaining a highly sialylated protein in a recombinant eukaryotic system; (4) Encapsulation of EPO.

The first approach is implemented in patents [20160176940; 20170114110], in which they obtained recombinant hybrid proteins - EPO conjugates with a highly glycosylated peptide or part of an immunoglobulin polypeptide, which increased the in vivo half-life and biological activity compared to natural or recombinant human EPO.

PEGylation, addition of polysaccharide and other residues to EPO was achieved by obtaining EPO muteins (using the cell-free synthesis technique) followed by their chemical derivatization (covalent modification) [EP 1219636]. Basically, PEG was attached to the free sulfhydryl groups of EPO muteins.

Examples of this approach are the product developed by Roche and known as CERA (Constant Erythropoiesis Receptor Activator), as well as Hematide, a pegylated synthetic peptide for the treatment of anemia in chronic renal failure and cancer. It is described by Fan et al. [Fan Q., Leuther K.K., Holmes C.P., Fong K.L., Zhang J., Velkovska S., Chen M.J., Mortensen R.B., Leu K., Green J.M., Schatz P.J., Woodburn K.W. Preclinical evaluation of Hematide, a novel erythropoiesis stimulating agent, for the treatment of anemia. Exp. Hematol. 2006. Vol. 34 (10), P. 1303-1311.], Its analogue is the drug MIRZERA (produced according to the patent [WO No. 2002/049673]). Methods of obtaining EPO conjugated with PEG are described in patents [RU 2433134 C1; U.S. Pat. No. 7,128,913; 20170008941; US 2004/0082765; 20160317674]. Covalent conjugation of EPO with non-antigenic hydrophilic polymers (polyalkenoxides, polyamides, carbohydrates - anionic polysaccharide, polysialic acids) covalently bound to it also increases the half-life of the resulting conjugate [U.S. Pat. No. 7,074,755; U.S. Pat. No. 5,846,951; WO-A-0187922; 20170119893].

A known method of producing a recombinant cell line [20170129932], the cells of which synthesize recombinant glycoproteins, in particular EPO, containing a large number of sialic acid residues.

Examples of encapsulated forms of EPO are the nanocapsulated form of the recombinant human EPO [Patent No. 2518329] (in addition to lengthening the half-life, nanocapsules with EPO have high penetration), the liposomal form of EPO [RF No. 22118914], the lipid phase of which contains a charged lipid compound and cholesterol as well as microencapsulated (in the form of microspheres) form of EPO [CN 102233129], which includes EPO. as an active component, and dextran as a protective (stabilizing) agent for the active component.

The above methods for the production of recombinant EPO in eukaryotic cells have a common disadvantage - low protein yield and the associated high cost of the target product. The search for a way to overcome this problem led to the development of a system for heterologous expression of a gene encoding a recombinant protein in prokaryotic cells [Wang YJ, Liu YD, Chen J., Hao SJ, Hu T., Ma GH, Su ZG Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli. Int. J. Pharm. 2010. Vol. 386 (1-2), P. 156-164.]. From the point of view of obtaining EPO protein, the prototype and the closest analogue of the present invention is the specified method. In this way, the production of a non-glycosylated recombinant protein (molecular weight ~ 18.4 kDa) in the inclusion bodies, which are dissolved under denaturing reducing conditions, is achieved, then the protein is purified by cation exchange chromatography and gel filtration to a homogeneous state during electrophoresis.

Compared with said prototype [Wang Y.J., Liu Y.D., Chen J., Hao S.J., Hu T., Ma G.H., Su Z.G. Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli. Int J Pharm. 2010. Vol. 386 (1-2), P. 156-164.], The method of producing recombinant EPO proposed by the present invention is characterized in that the sequence of the recombinant protein contains, in addition to EPO, also a sequence of a heparin binding domain (HBD). The presence of a heparin-binding domain allows the immobilization of EPO on a heparin-containing sorbent. Due to the presence of HBD, the affinity purification of the target HBD-Epo protein on a heparin-containing sorbent (for example, heparin-sepharose) is also possible, and thus the HBD-Epo purification step by gel filtration is excluded. The introduction of EPO immobilized on a sorbent containing heparin, for example, demineralized bone matrix (DCM), into the body should lead to a gradual dosed release of the factor into the surrounding tissues and, thus, provide a prolonged action and a high local concentration of EPO, which may be useful for some options for using the factor, in particular, when it is used for the repair of bone defects.

and Linhardt, Heparin-binding domains in vascular biology. Arterioscler. Thromb. Vasc. Biol. 2004. Vol. 9, P. 1549-1557.]. Аффинность HBD к гепарину нашла практическое применение в биотехнологии и медицине. Например, она используется при очистке белков аффинной хроматографией на гепарин-сефарозе, а также для электростатического удержания VEGF на гепарин-содержащем носителе с целью дальнейшего ковалентного связывания этого фактора [

and Linhardt, Heparin-binding domains in vascular biology. Arterioscler. Thromb. Vasc. Biol. 2004. Vol. 9, P. 1549-1557; Ducheyne P., Healy K., Hutmacher D.E., Grainger D.W., Kirkpatrick C.J. Comprehensive Biomaterials. Elsevier 1-st Ed. 2011. Vol. 1, P. 269.]. Гепарин-связывающий домен фибрина (фибриногена), иммобилизованный на синтетическом заменителе (имитирующем фибрин и использующемся для заполнения ран), связывает ростовые факторы, активируя процесс заживления ран [Martinoa М.М., Briqueza P.S., Rangaa A., Lutolfa М.Р., and Hubbell J.A. Heparin-binding domain of fibrin(ogen) binds growth factors and promotes tissue repair when incorporated within a synthetic matrix. Proc. Nat. Acad. Sci. 2013. Vol. 110, P. 4563-4568.]. Наличие природного гепарин-связывающего домена в последовательности фактора дифференцировки - костного морфогенетического белка ВМР-2 [Gandhi N.S. and Mancera R.L. Prediction of heparin binding sites in bone morphogenetic proteins (BMPs). Biochim. Biophys. Acta. 2012. Vol. 1824(12), P. 1374-1381.], позволяет производить аффинную очистку этого белка на гепарин-сефарозе и иммобилизировать ВМР-2 на ДКМ [Карягина А.С., Бокша И.С., Грунина Т.М., Демиденко А.В., Попонова М.С., Сергиенко О.В., Лящук A.M., Галушкина З.М., Соболева Л.А., Осидак Е.О., Семихин А.С., Громов А.В., Лунин B.Г. Оптимизация получения активной формы rhBMP-2 в гетерологичной системе экспрессии с помощью микробиологических и молекулярно-генетических подходов. Молекулярная генетика, микробиология и вирусология. 2016. Т. 4, С. 132-137.].Heparin-binding domains - natural amino acid sequences that form a complex with heparin due to ionic interactions, are present in many proteins [

and Linhardt, Heparin-binding domains in vascular biology. Arterioscler. Thromb. Vasc. Biol. 2004. Vol. 9, P. 1549-1557.]. The affinity of HBD for heparin has found practical application in biotechnology and medicine. For example, it is used in protein purification by heparin-sepharose affinity chromatography, as well as for electrostatic retention of VEGF on a heparin-containing carrier in order to further covalently bind this factor [

and Linhardt, Heparin-binding domains in vascular biology. Arterioscler. Thromb. Vasc. Biol. 2004. Vol. 9, P. 1549-1557; Ducheyne P., Healy K., Hutmacher DE, Grainger DW, Kirkpatrick CJ Comprehensive Biomaterials. Elsevier 1-st Ed. 2011. Vol. 1, P. 269.]. Heparin-binding domain of fibrin (fibrinogen), immobilized on a synthetic substitute (simulating fibrin and used to fill wounds), binds growth factors, activating the healing process [Martinoa M.M., Briqueza PS, Rangaa A., Lutolfa M.R. , and Hubbell JA Heparin-binding domain of fibrin (ogen) binds growth factors and promotes tissue repair when incorporated within a synthetic matrix. Proc. Nat. Acad. Sci. 2013. Vol. 110, P. 4563-4568.]. The presence of a natural heparin binding domain in the sequence of the differentiation factor, BMP-2 bone morphogenetic protein [Gandhi NS and Mancera RL Prediction of heparin binding sites in bone morphogenetic proteins (BMPs). Biochim. Biophys. Acta. 2012. Vol. 1824 (12), P. 1374-1381.], Allows affinity purification of this protein on heparin-sepharose and immobilize BMP-2 on DCM [Karyagina AS, Boksha IS, Grunina TM, Demidenko A.V., Poponova M.S., Sergienko O.V., Lyashchuk AM, Galushkina Z.M., Soboleva L.A., Osidak E.O., Semikhin A.S., Gromov A.V., Lunin B.G. Optimization of obtaining the active form of rhBMP-2 in a heterologous expression system using microbiological and molecular genetic approaches. Molecular genetics, microbiology and virology. 2016. V. 4, S. 132-137.].

There are various methods of introducing EPO into the body. In most cases, EPO is introduced into the human body through the parenteral route (injection) [Nikolaev A.Yu., Klepikov PV, Lashutin SV, Kukhtevich AV The effectiveness of the combination of Ero-calcitriol in patients with chronic renal failure undergoing hemodialysis. Therapeutic Archive. 1995. T. 67, S. 27-31; USSR patent No. 1801118].

The therapeutic effect of recombinant human EPO upon parenteral administration is obvious and described in many works, but the same works also indicate its undesirable side effects, leading to various complications. The introduction of parenteral dosage forms containing recombinant human EPO in large doses can lead to disruption of homeostasis and cause additional risks for the patient [Sinyukhin V.N. Stetsyuk E.A., Lovchinsky E.V. Pharmacokinetics of recombinant human erythropoietin. Therapeutic Archive. 1994. T. 66, S. 60-62.].

To solve this problem, a Reporon oral dosage form was developed [Application: 97108814/14, 05/22/1997 RU], which was obtained by mixing recombinant EPO with stabilizing additives in certain proportions.

Also known is the inhalation route of administration of EPO into the human lungs [N 94/17784, 1994.].

All previously described methods of introduction into the body involve the systemic intake of EPO through the blood.

However, given the numerous effects in various organs — the spinal cord and brain, peripheral nerves, retina, heart, and kidneys — the effects of EPO (including anti-apoptotic), realized through cascades of protein phosphorylation and involving PI3K, Akt, NF-κB and, thus, activating transcription factors [Sharples EJ, Thiemermann C., Yaqoob M.M. Novel applications of recombinant erythropoietin. Curr Opin Pharmacol. 2006. Vol. 6 (2), P. 184-189.], It is logical to assume that with damage to tissues and organs, the therapeutic effect will also manifest itself with topical application of EPO (applications).

, Bendtsen M., Jensen J., Stiehler M, Foldager C.B., Hellfritzsch M.B.,

Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model. J. Orthop. Res. 2012. Vol. 30(7), P. 1083-1088.]. По способу применения этот способ (местный) является наиболее близким прототипом настоящего изобретения.The discovery of such a fact as stimulation of the formation of EPO bone served as a prerequisite for studying the possibility of topical application of EPO [Sun N., Jung Y., Shiozawa Y., Taichman RS, Krebsbach PH Erythropoietin modulates the structure of bone morphogenetic protein 2-engineered cranial bone. Tissue Eng Part A. 2012. Vol. 18 (19-20), P.2095-2105;

, Bendtsen M., Jensen J., Stiehler M, Foldager CB, Hellfritzsch MB,

Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model. J. Orthop. Res. 2012. Vol. 30 (7), P. 1083-1088.]. According to the method of application, this method (local) is the closest prototype of the present invention.

et al., 2012, используя модель спинальных дефектов позвоночника кролика, вводили Еро (Epoetin beta, NeoRecormon, Roche, Hvidovre, Denmark) с помощью регулярных локальных инъекций (20 инъекций ежедневно) из расчета 250 ед./кг веса животного (для снижения побочного системного действия использовали низкие дозы цитокина) в 0,3 мл физраствора, и наблюдали эффект Еро на остеогенез, выражающийся в статистически достоверном повышении объема костной ткани на 18%. Эта же группа проводила исследования изменений посттравматического остеогенеза под воздействием Еро на модели краниальных дефектов у свиней (

et al., 2012), но схема введения препарата значительно отличалась - однократное введение 900 ед. Еро на дефект на различных носителях и без носителя (всего 2700 ед. Еро на животное), т.о. доза на вес была многократно снижена (до ~18 ед. на кг веса). В вариантах с Еро так же, как и на кроличьей модели, наблюдалась положительная динамика (прирост объема костной ткани в среднем был на 6% больше), однако результаты этого эксперимента не были статистически достоверны.Authors of the work

et al., 2012, using a rabbit vertebral spine defects model, Epo (Epoetin beta, NeoRecormon, Roche, Hvidovre, Denmark) was injected with regular local injections (20 injections daily) at a rate of 250 units / kg of animal weight (to reduce the side systemic effects used low doses of cytokine) in 0.3 ml of saline, and the effect of EPO on osteogenesis was observed, which is expressed in a statistically significant increase in bone volume by 18%. The same group conducted studies of changes in post-traumatic osteogenesis under the influence of EPO on models of cranial defects in pigs (

et al., 2012), but the administration schedule was significantly different - a single injection of 900 units. EPO for the defect on various media and without media (total 2700 units of EPO per animal), i.e. dose to weight was many times reduced (up to ~ 18 units per kg of weight). In the variants with EPO, as well as in the rabbit model, a positive dynamics was observed (the increase in the volume of bone tissue was on average 6% more), but the results of this experiment were not statistically significant.

The authors of Sun et al., 2012, after creating critical-size cranial defects in the mouse skull, introduced EPO locally by injection into the defect area every other day for 2 weeks (dose 1000 units / ml EPO-EPO alpha, EPOGEN, Amgen, the dose was calculated on the weight of animals at the rate of 1000 units / kg). According to microcomputer tomography, EPO significantly enhanced BMP-2-induced bone formation in the defect area (i.e., a synergy of BMP-2 and EPO effects was observed).

The additive effect of EPO was also shown with simultaneous administration with BMP2. Patel et al., 2015 evaluated the effect on osteogenesis with the simultaneous administration of two cytokines that stimulate this process - EPO (200 IU / mL) and BMP2 (65 μg / mL), administered ectopically (subcutaneously) on a polyepsiloncaprolactone carrier. Implants with EPO + BMP2 and only with BMP2 were administered subcutaneously. At weeks 4 and 8, bone growth in the implant volume was analyzed using tomography and histology. Moreover, with the joint administration of cytokines, the volume of newly formed bone tissue exceeded by 31% this indicator in the variant with the introduction of BMP2 only.

Li et al., 2015 investigated the effect of a single local injection of 10 and 20 units. EPO in a cylindrical maxillary defect with a diameter of ~ 1 mm in rats. The most pronounced difference from the control was observed at 4 weeks after surgery - the option of adding 20 units of EPO was more than 3 times different from the control.

Very good results on the restoration of bone tissue in a model of femoral fracture in mice were obtained by Holstein et al., 2011 also with systemic administration of EPO, with daily interperitoneal administration of cytokine at the rate of 500 units / kg of animal weight. The distance between the bone segments was 1.8 mm, and was fixed with a metal bracket. Three options were investigated - control, fracture with removal of the periosteum and without removal of the periosteum. In the two-week period, the difference between the experimental and control options was 71.4%, and after 10 weeks, the result of the increase in bone volume when using EPO exceeded the control option by more than 2 times.

et al., 2012, Sun et al., 2012, Patel et al., 2015) следует, что наличие локально высокой концентрации цитокина имеет ключевое значение, однако системное введение высоких концентраций может приводить к серьезным побочным эффектам (вплоть до тромбоэмболии) и не гарантирует достижения локально высокой концентрации (например, в месте повреждения кости).From the examples of prototypes (

et al., 2012, Sun et al., 2012, Patel et al., 2015) it follows that the presence of a locally high concentration of cytokine is key, however, systemic administration of high concentrations can lead to serious side effects (up to thromboembolism) and not ensures that a locally high concentration is achieved (e.g. at the site of bone damage).

J.H., Bendtsen M., Jensen J., Stiehler M., Foldager C.B., Hellfritzsch M.B.,

. Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model. J. Orthop. Res. 2012. Vol. 30(7), P. 1083-1088; Patel J.J., Modes J.E., Flanagan C.L., Krebsbach P.H., Edwards S.P, Hollister S.J. Dual delivery of EPO and BMP2 from a Novel Modular Poly-ε-Caprolactone Construct to Increase the Bone Formation in Prefabricated Bone Flaps. Tissue Eng. Part С Methods. 2015. Vol. 21(9), P. 889-897.] состоит в том, что, во-первых, используется рекомбинантный Еро, полученный синтезом в прокариотических клетках и, во-вторых, Еро удерживается на носителе (ДКМ) благодаря связыванию с гепариновыми участками ДКМ гепарин-связывающего домена (HBD), который введен в состав аминокислотной последовательности рекомбинантного Еро. Носитель Еро - ДКМ, используемый в настоящем изобретении, получают по методу, разработанному в патенте Лунина и др. [Патент РФ №2456003] и описанному в работе Громова и др. [Громов А.В., Никитин К.Е., Карпова ТА., Зайцев В.В., Сидорова Е.И., Андреева Е.В., Бартов М.С., Мишина Д.М., Субботина М.Е., Шевлягина Н.В., Сергиенков М.А., Соболева Л.А., Котнова А.П., Шарапова Н.Е., Семихин А.С., Диденко Л.В., Карягина А.С., Лунин В.Г. Разработка методики получения остеопластического материала на основе деминерализованного костного матрикса с максимальным содержанием нативных факторов роста костной ткани. Биотехнология. 2012. №5. С. 66-75.] с модификациями, описанными в работе Barov et al. [Bartov M.S., Gromov A.V, Poponova M.S., Savina D.M., Nikitin K.E., Grunina T.M., Manskikh V.N., Gra O.A., Lunin V.G, Karyagina A.S., Gintsburg A.L. Modern approaches to research of new osteogenic biomaterials on the model of regeneration of cranial critical-sized defects in rats. Bulletin of Experimental Biology and Medicine. 2016. V. 162(2), P. 273-276.]. В настоящем изобретении HBD-Epo наносили на ДКМ отдельно и совместно с рекомбинантным костным морфогенетическим белком ВМР-2, полученным в клетках Е. coli по методу, описанному в статье Karyagina et al., 2016 [Karyagina A.S., Boksha I.S., Grunina T.M., Demidenko A.V, Poponova M.S., Sergienko O.V., Lyaschuk A.M., Galushkina Z.M., Soboleva L.A., Osidak E.O., Semikhin A.S., Gromov A.V., Lunin V.G. Optimization of rhBMP-2 active-form production in a heterologous expression system using microbiological and molecular genetic approaches. Molecular genetics, microbiology and virology. 2016. V. 31(4) P. 208-213.]. Метод нанесения ВМР-2 на ДКМ описан в работе Barov et al., 2016 [Bartov M.S., Gromov A.V., Poponova M.S., Savina D.M., Nikitin K.E., Grunina T.M., Manskikh V.N., Gra O.A., Lunin V.G, Karyagina A.S., Gintsburg A.L. Modern approaches to research of new osteogenic biomaterials on the model of regeneration of cranial critical-sized defects in rats. Bulletin of Experimental Biology and Medicine. 2016. V. 162(2). P. 273-276.].To increase the retention period of the active protein, including EPO, at the place of application, you can use an active protein immobilized on a biologically compatible material, for example, DCM. In the present invention, this approach is solved by obtaining a hybrid EPO protein with the HBD polypeptide heparin binding domain (HBD-Epo constructs). The difference of the present invention from prototypes [Sun N., Jung Y., Shiozawa Y., Taichman RS, Krebsbach PH Erythropoietin modulates the structure of bone morphogenetic protein 2-engineered cranial bone. Tissue Eng Part A. 2012. Vol. 18 (19-20), P.2095-2105;

JH, Bendtsen M., Jensen J., Stiehler M., Foldager CB, Hellfritzsch MB,

. Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model. J. Orthop. Res. 2012. Vol. 30 (7), P. 1083-1088; Patel JJ, Modes JE, Flanagan CL, Krebsbach PH, Edwards SP, Hollister SJ Dual delivery of EPO and BMP2 from a Novel Modular Poly-ε-Caprolactone Construct to Increase the Bone Formation in Prefabricated Bone Flaps. Tissue Eng. Part With Methods. 2015. Vol. 21 (9), P. 889-897.] Consists in the fact that, firstly, the recombinant EPO obtained by synthesis in prokaryotic cells is used and, secondly, EPO is retained on a carrier (DCM) due to binding to DCM heparin sites heparin binding domain (HBD), which is introduced into the amino acid sequence of recombinant EPO. The carrier EPO - DCM used in the present invention is obtained according to the method developed in the patent of Lunin et al. [RF Patent No. 2456003] and described in the work of Gromov et al. [Gromov A.V., Nikitin K.E., Karpova TA ., Zaitsev V.V., Sidorova E.I., Andreeva E.V., Bartov M.S., Mishina D.M., Subbotina M.E., Shevlyagina N.V., Sergienkov M.A., Soboleva L.A., Kotnova A.P., Sharapova N.E., Semikhin A.S., Didenko L.V., Karyagina A.S., Lunin V.G. Development of a technique for producing osteoplastic material based on demineralized bone matrix with a maximum content of native bone growth factors. Biotechnology. 2012. No5. S. 66-75.] With the modifications described in Barov et al. [Bartov MS, Gromov AV, Poponova MS, Savina DM, Nikitin KE, Grunina TM, Manskikh VN, Gra OA, Lunin VG, Karyagina AS, Gintsburg AL Modern approaches to research of new osteogenic biomaterials on the model of regeneration of cranial critical- sized defects in rats. Bulletin of Experimental Biology and Medicine. 2016. V. 162 (2), P. 273-276.]. In the present invention, HBD-Epo was applied to DCM separately and together with the recombinant BMP-2 bone morphogenetic protein obtained in E. coli cells according to the method described in Karyagina et al., 2016 [Karyagina AS, Boksha IS, Grunina TM, Demidenko AV, Poponova MS, Sergienko OV, Lyaschuk AM, Galushkina ZM, Soboleva LA, Osidak EO, Semikhin AS, Gromov AV, Lunin VG Optimization of rhBMP-2 active-form production in a heterologous expression system using microbiological and molecular genetic approaches. Molecular genetics, microbiology and virology. 2016. V. 31 (4) P. 208-213.]. The method of applying BMP-2 to DCM is described in Barov et al., 2016 [Bartov MS, Gromov AV, Poponova MS, Savina DM, Nikitin KE, Grunina TM, Manskikh VN, Gra OA, Lunin VG, Karyagina AS, Gintsburg AL Modern approaches to research of new osteogenic biomaterials on the model of regeneration of cranial critical-sized defects in rats. Bulletin of Experimental Biology and Medicine. 2016. V. 162 (2). P. 273-276.].

The difference from the prototype [Wang Y.J., Liu Y.D., Chen J., Hao S.J., Hu T., Ma G.H., Su Z. G Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli. Int. J. Pharm. 2010. Vol. 386 (1-2), P. 156-164.] And the other listed methods of derivatization of EPO is that instead of obtaining the pegylated derivatives of EPO, as proposed in the prototype, and other methods of derivatization of EPO described above, the inclusion of HBD in the sequence The hybrid protein allows you to bind (immobilize) the HBD-Epo hybrid protein on heparin-containing carriers (for example, demineralized bone matrix, DCM), which lengthens the half-life of EPO from the body.

The technical solution of the invention is expressed in the preparation of a recombinant HBD-Epo protein in cells of non-pathogenic prokaryotic microorganisms (Escherichia coli strains), thereby reducing the cost of the resulting product, and in increasing the bioavailability of EPO by obtaining a chimeric variant of EPO with a heparin binding domain and its immobilization on DCM with topical application in the affected area (insertion into a bone defect during a fracture or other damage to bone tissue, application to a wound surface or introduction into surrounding soft tissues in case of wounds eve process).

The essence of the invention lies in the fact that receive a plasmid containing the recombinant gene HBD-Epo. Synthetic DNA with a sequence corresponding to the gene encoding the HBD-Epo protein, designed so that the nucleotide composition of the codons is optimized for heterologous expression in a non-pathogenic laboratory strain of E. coli, is flanked at the 5'-end by the NcoI site, and at the 3'-end - Kpn2I website. This synthetic gene is inserted into the pQE6 plasmid at the NcoI and Kpn2I sites and the plasmid pL610 is obtained. After transformation of the E. coli M15 strain [pREP4] with the obtained plasmid pL610, the E. coli M15 producer strain [pREP4, pL610] is produced with the production of the recombinant HBD-Epo protein in inclusion bodies. The estimated molecular weight of the recombinant HBD-Epo protein is ~ 20.5 kDa. The HBD-Epo protein is reduced in a solution containing dithiothreitol or mercaptoethanol and purified by column chromatography on a cation exchange sorbent as a stationary phase under denaturing conditions, after which the active form of HBD-Epo is concentrated on a cation exchange and / or affinity sorbent (heparin-sepharose).

A recombinant biologically active EPO protein is obtained containing a heparin binding domain (HBD-Epo) of a high degree of purity, capable of sorption on DCM containing heparin.

For this, a synthetic DNA sequence encoding the HBD-Epo protein is obtained. The present method differs from the prototype [Wang Y.J., Liu Y.D., Chen J., Nao S.J., Hu T., Ma G.H., Su Z.G. Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli. Int J Pharm. 2010. Vol. 386 (1-2), P. 156-164.] In that, to ensure a high level of production of the recombinant HBD-Epo protein in the heterologous system, codons are optimized. The planned coding DNA sequence differs from the native human EPO DNA sequence in its nucleotide composition. This technique makes it possible to obtain highly efficient production of the recombinant HBD-Epo protein in the insoluble fraction in an amount of about 30% of the total cell protein.

The claimed group of inventions allows to obtain the optimal synthetic DNA sequence for heterologous expression encoding the recombinant HBD-Epo protein, providing a high level of production of HBD-Epo protein in non-pathogenic laboratory expression strains of E. coli; to carry out simple and effective purification of the HBD-Epo protein in a biologically active form, and in this form to bind to DCM and effectively and specifically accelerate bone regeneration; to create active biocompatible compositions based on a recombinant protein using DCM (including DCM containing other active components, for example, recombinant BMP-2) as the matrix carrier of the biologically active protein HBD-Epo.

This result is achieved due to the synthesis of recombinant HBD-Epo protein in cells of the laboratory E. coli strain carrying the recombinant plasmid pL610 (example 1, figure 1) with the nucleotide sequence of SEQ ID NO 1, as well as by creating a complex preparation of HBD-Epo, immobilized on DCM and on DCM containing BMP-2 (example 4).

The recombinant HBD-Epo protein has the amino acid sequence of SEQ ID NO 2, including the sequence of amino acid residues of the heparin binding domain from Danio rerio (SEQ ID NO 3), the sequence of amino acid residues of the spacer (SEQ ID NO 4), the sequence of amino acid residues of the EPO protein from Homo sapiens (SEQ ID NO 5). The calculated value of the molecular weight of the protein 20520.51 Yes; the estimated value of the IET is 9.62.

The technical result achieved by carrying out the invention is to obtain a high level of production of HBD-Epo protein (Example 2, Fig. 2), which, after purification, forms a stable active form capable of binding to DCM heparin and inducing accelerated bone tissue regeneration.

The activity of the recombinant HBD-Epo protein in solution was studied in vitro on the human erythroleukemia cell line TF-1 (ATCC CRL-2003). The result was manifested in the form of specific stimulation of cell proliferation and color development when a specific substrate was added to the cells (example 3, Fig. 3).

In terms of formulating the preparation of the recombinant HBD-Epo protein immobilized on DCM, the technical result is achieved by creating a recombinant HBD-Epo protein with the sequence SEQ ID NO 2, as well as by obtaining a composition in which the recombinant HBD-Epo protein is contained in the form of a complex with heparin in the DCM and in the form of a complex with heparin in the DCM containing BMP-2 (example 4).

In addition, the technical result is that these biologically active compositions specifically and effectively induce bone tissue regeneration.

The specificity and effectiveness of the complex preparations of HBD-Epo - DKM and HBD-Epo - BMP-2 - DKM were investigated on a mouse model of cranial defects of critical size (DKR), (example 5, Fig. 4).

The invention also includes a method for specific induction of bone tissue regeneration, comprising filling in bone defects with fragments of a demineralized bone matrix with recombinant HBD-Epo protein immobilized on them or fragments of a demineralized bone matrix with recombinant HBD-Epo and BMP 2 recombinant proteins immobilized on them.

Description of figures

In FIG. 1 shows Recombinant plasmid DNA pL610, which has a size of 3583 bp and contains the artificial bacterial operon of the recombinant HBD-Epo protein, including: the promoter region of the early promoter of the bacteriophage T5 (7 - 87 n.p.), the gene for the recombinant protein HBD-Epo (117 - 674 n.p.), the transcription terminator (695 - 812 n.p.); the bacterial operon beta-lactamase (resistance to ampicillin) (3368 - 2518 n.p. complementary chain); ColE1 type bacterial replication initiation site, which provides plasmid replication in E. coli strains (1755 bp), encodes the synthesis of recombinant HBD-Epo protein.

In FIG. 2 shows an Electrophoregram of HBD-Epo recombinant protein samples at various stages of purification. Lanes: 1 — producer cell extract before IPTG induction, 2 — producer cell extract after IPTG induction, 3 — supernatant (readily soluble producer cell proteins), 4 — washed inclusion bodies, 5 — eluate from a column with a cation exchange sorbent, 6 — molecular markers masses.

In FIG. Figure 3 presents a graph of the dependence of the optical density in the well on the concentration of the sample when determining specific activity of HBD-Epo in vitro. A broken line with diamond-shaped markers shows a graph obtained with eukaryotic erythropoietin, a solid line with filled circles - with HBD-Epo.

Fig. 4 shows tomograms of defects 9 weeks after surgery (control-1 - unfilled DKR; control-2 - DKM; experience-1 - DKM + BMP-2; experience-2 - DKM + HBD-Epo; experience-3 - DKM + BMP-2 + HBD-Epo).

The invention is illustrated by the following examples below.

Example 1

Obtaining plasmid pL610 carrying the HBD-Epo gene

Genetic engineering and microbiological manipulations, amplification and DNA sequencing were carried out according to standard methods [Maniatis T., Fritsch E., Sambrook J. Molecular cloning, M .: Mir. 1984. DNA cloning. Methods Ed. D. Glover, Per. from English, M.: Mir. 1988; Saiki R.K., Gelfand D.H., Stoffel S., Scharf S.J., Higuchi R., Horn G.T., Mullis K.B., Erlich H.A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988. Vol. 239, P. 487-491; Sanger F., Nicklen S., Coulson A.R. DNA sequencing with chain-terminating inhibitors (DNA polymerase / nucleotide sequences / bacteriophage FX174). Proc. Nat. Acad. Sci. 1977. Vol. 74, P. 5463-5467.].

The synthetic DNA sequence corresponding to the gene encoding the HDB-Epo protein was designed so that the nucleotide composition of the codons was optimized for heterologous expression in a non-pathogenic laboratory E. coli strain. This synthetic HBD-Epo gene was flanked at the 5'-end by the NcoI site, and at the 3'-end by the Kpn2I site. The synthetic gene was inserted into the pQE6 plasmid at the NcoI and Kpn2I sites; start and termination codons were provided to initiate and stop translation.

a) Chemical synthesis of a gene.

The HBD-Epo gene was synthesized by Eurogen (Russia). By codon composition, the HBD-Epo gene was optimized for expression in E. coli and was flanked at the 5'-end by the NcoI site, and at the 3'-end by the Kpn2I site. The codon composition of the synthetic gene was optimized using the JCat program (http://www.jcat.de/), and the secondary structure of transcribed RNA was adjusted using the DINAMelt web server (http://mfold.rna.albany.edu/?q = DINAMelt / Two-state-folding).

b) Obtaining and cloning of the recombinant HBD-Epo gene into pQE6 vector.

The synthetic gene was inserted into the pQE6 plasmid at the NcoI and Kpn2I sites. The obtained construct pL610 (Fig. 1) with the nucleotide sequence of SEQ ID NO 1 was used to transform the E. coli M15 strain [pREP4] and the producer strain E. coli M15 [pREP4, pL610] was obtained with the HBD-Epo protein production level equal to 30% of the total cell protein.

Example 2

The producer biomass was resuspended in a 5-10-fold volume of buffer solution: 10-100 mM Tris-HCl, pH 7.0-8.0, 50 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF) and lysozyme was added at the rate of 100 μg / ml suspension cells. Incubated for 1 h at room temperature, stirring occasionally. The resulting suspension was treated with ultrasound to disintegrate cells using an ultrasonic disintegrator. Centrifuged for 30 min at 10,000 g and a temperature of 5 ° C. The target protein was in inclusion bodies (TB), i.e. in sedimentary fraction. The supernatant was decanted and the precipitate was washed with a 10-fold volume of buffer solution: 10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1% Triton X-100. The washed TB was dissolved in a 5-fold volume of a solution of 8 M urea in a buffer of 10-100 mM Tris-HCl, pH 8.0, with 50-100 mM dithiothreitol (DTT). The content of the target protein in the washed TVs was about 80% (estimated from the densitogram of the electrophoregram obtained after electrophoresis in 15% polyacrylamide gel and Coomassie blue R-250 staining). Purification of the HBD-Epo protein was carried out by column cation exchange or affinity (on heparin-containing sorbent - heparin-sepharose) chromatography under denaturing conditions in 10-100 mM Tris-HCl, pH 8.0, containing 6-8 M urea. The electrophoregram of HBD-Epo recombinant protein samples at various stages before and during purification is shown in FIG. 2.

The resulting purified protein fractions were dialyzed against 10 mM Na-phosphate buffer, pH 4.5-5.5, and frozen at -70 ° C.

Example 3

Assessment of the biological activity of HBD-Epo in vitro. The biological activity of HBD-Epo preparations was evaluated in vitro in a proliferative test on the human erythroleukemia cell line TF-1 (ATCC CRL-2003). Cells were cultured at 37 ° C, in an atmosphere of 5% CO ₂ , in RPMI 1640 medium (Paneko, Russia) with the addition of 10% fetal calf serum (HyClone, USA), gentamicin (Paneko, Russia) to a concentration 10 μg / ml and granulocyte-macrophage colony stimulating factor (GM-CSF) (Paneko, Russia) at a final concentration of 2 ng / ml. Before using in the test to determine the specific activity, the cells were incubated for 22-26 hours in an environment with a minimum (0.5%) serum content without the addition of GM-CSF and other growth factors at 37 ° C, in an atmosphere of 5% CO ₂ . After that, the cells were washed from the culture medium by centrifugation at 800 rpm, selection of the supernatant and resuspension of the cell pellet in cold RPMI 1640 medium. The procedure was repeated 3 times. After the third centrifugation procedure, the cell pellet was suspended in such a volume of the test medium (RPMI 1640 with the addition of 5% fetal calf serum, 10 μg / ml gentamicin, without GM-CSF) so that the resulting cell concentration was in the range of 600-800 thousand cells per 1 ml HBD-Epo samples were sterilized using a 0.2 μm filter head (Corning, Germany) and diluted in a test medium. 96-well plates were prepared with various concentrations of samples diluted in test medium (well volume 50 μl, binary dilutions from 1000 ng / ml to 4 ng / ml and from 10 ng / ml to 0.04 ng / ml). Medium without HBD-Epo was used as a negative control, and recombinant EPO from mammalian cells, Epostim (epoetin beta) (Pharmapark, Russia) was used as a comparison drug. Prepared cells were added 50 μl (30-40 thousand cells) in the wells of the tablet containing samples. The plate was incubated for 68-72 hours at 37 ° C, in an atmosphere of 5% CO ₂ . After incubation, 10 μl of the substrate mixture WST-1 (Roche, Switzerland) was added to each well of the plate. Then the plate was incubated at 37 ° C, in an atmosphere of 5% CO ₂ for 5-6 hours with visual monitoring of the development of staining. The optical density in the wells of the plate was measured at a wavelength of 450 nm using a Tecan Infinite M 200 Pro plate spectrophotometer (Tecan Group Ltd., Switzerland). A plot of the optical density in the well versus sample concentration in determining the specific activity of HBD-Epo in vitro is shown in FIG. 3. The specific activity of HBD-Epo, calculated by the regression model, was 12.4% of the activity of eukaryotic erythropoietin.

Example 4

Obtaining disks from DCM with the recombinant protein HBD-EPO immobilized on them, as well as HBD-EPO and BMP-2.

To obtain membranes from DCM, the diaphysis of the femoral bones of cattle using a band saw was cut into 1 mm thick layers and degreased, decalcified, and deproteinized in this form to remove non-collagen proteins, after which 4 mm diameter disks were cut and lyophilized. To immobilize HBD-Epo, each disk was placed in 100 μl of a solution containing 10 μg of HBD-Epo in a buffer containing 0.5 M NaCl and 25 mM Tris-HCl, pH 7.5, incubated for 3 hours, after which they were washed three times with same buffer and lyophilization. The content of HBD-Epo was 10 μg per disk. After that, the disks were freeze-dried and sterilized by radiation (the absorbed dose was 20 ± 5 kGy). The disks thus obtained were used for implantation into the cranial defects of mice (invention example 5).

To immobilize HBD-Epo on DCM disks containing also BMP-2, a procedure similar to that described above was performed, but a solution containing 10 μg of recombinant BMP-2 was also used for incubation. Such discs contained 10 μg HBD-Epo and 10 μg BMP-2 per disk.

Example 5

Evaluation of the biological activity of HBD-Epo in a bone tissue regeneration model. The biological activity of HBD-Epo preparations was evaluated in vivo in a mouse model of critical size cranial defects. The study was performed on 30 outbred ICR mice (CD-I), males aged 38-47 days.

The animals were injected into general anesthesia using an intraperitoneal injection of the preparations Zoletil 100 (15 mg / kg body weight) and Rometar (6 mg / kg body weight). The operation was performed in a laminar box (Lamsystems, Russia) on a thermostatically controlled surgical table for laboratory animals (Medax, Germany) at a constant temperature of 37 ° C. Having prepared the surgical field, in the parietal region of the skull with a scalpel, a skin-muscle incision was made in the sagittal plane 6-7 mm long, after which 4-mm-diameter DCBs were created in the parietal bones using Surgic AR surgery and implantology apparatus (NSK Nakanishi Inc., Japan) at a trepan rotation speed of 800 rpm and a torque of 0.10-0.15 N⋅m. The contact area of trephine with bone was constantly cooled with sterile saline. At the end of the operation, the wound was sutured in layers with an atraumatic self-absorbable thread Vikril 4/0 (Ethicon, Belgium) and treated with the Terramycin antibiotic (Pfizer Animal Health, USA).

Animals were divided into 5 groups of 6 mice each: unfilled DCR (control-1), with implantation of DCM disks (control-2), DCM disks with bone morphogenetic protein 2 (Bone Morphogenetic Protein 2, BMP-2) (experiment-1 ), disks with HBD-Epo (experiment-2) and disks with BMP-2 and HBD-Epo (experiment-3).

Microcomputer tomography was performed on a SkyScan 1176 instrument (Bruker, USA) immediately after surgery, as well as 3, 6, and 9 weeks after surgery. Anesthetized animals were placed in the “bed” of the tomograph in the supine position, the body and head were fixed using paper tape for complete immobilization. Scanning was carried out in one field of view of the camera using an aluminum filter with a thickness of 0.5 mm at a voltage of 60 kV, a current of 575 mA, a resolution of 35 μm, an exposure of 240 ms, a step of 0.5 °, and a camera angle of 180 °. Data processing was performed in SkyScan software. The obtained data set was reconstructed with smoothing values “2”, correction of ring artifacts “2”, correction of radiation hardness “5”. In the set of reconstructed data for the analysis of DCR, the region of interest was chosen as a cylindrical shape with a diameter of 4 mm and a height of 15 sections, corresponding to 1.5 mm In the selected area of interest, the following morphological parameters were calculated: the volume of all tissues, the volume of bone tissue, the surface area of all tissues, the surface area of bone tissue, the proportion of bone tissue, bone density.

60 days after implantation of the materials, the animals were euthanized in a CO ₂ chamber (NPK Otkrytaya Nauka LLC, Russia) and necropsy was performed on the area of the cranial fornix with DCR. To perform a histomorphometric analysis, skull samples with DCR were divided into 2 parts in the frontal plane, one of them was decalcified. Samples were embedded in paraffin, demineralized sections were stained with hematoxylin and eosin, non-demineralized sections were stained with alizarin red and von Koss. The degree of neovascularization was evaluated in the preparations; the area of all tissues and bone tissue was measured in ImageJ.

Statistical data processing was performed using the non-parametric Kruskal-Wallis test in Statistica 12.0 (StatSoft, USA).

9 weeks after surgery, defects in group 1 (control-1) were filled with connective tissue with small foci of bone tissue, in group 2 (control-2) with fibrovascular connective tissue with osteoid, in groups 3, 4 and 5 (experiment-1, 2, 3) - fibrovascular connective tissue, osteoid and mature bone tissue. In all groups with implantation, discs from DCM were completely resorbed, stained with alizarin red and, according to von Koss, confirmed the partial mineralization of the osteoid, the periosteoid spaces contained different-sized blood vessels. Side effects were not recorded in any case. According to the results of histomorphometry, the volume and proportion of bone tissue in all groups with DCM implantation is significantly higher than in the group with unfilled DCR (control-1), while significant differences between groups with DCM were recorded in case of pairwise comparison of the experience-1 * control groups -2; experience-2 * - control-2; experience-3 * - control-2; experience-3 * - experience-1 (* significant increase with significance level p <0.05 when using the Kruskal-Wallis test).

According to the results of tomography (Fig. 4), for the volume, area, fraction and density of bone tissue, significant differences between the groups with implantation and control-1 (p <0.01) are observed at all periods of observation, and the maximum increase in indicators occurs within 3 weeks after operation. All three experimental groups showed significant differences in the volume, area, share and density of bone tissue also from control-2 (p <0.01) for a period of 3 weeks after surgery. Moreover, in the case of using disks with immobilization of BMP-2 and HBD-Epo (experiment-3), a maximum increase in the density of newly formed bone tissue was observed with a significant difference from experimental groups 1 and 2 (p <0.05), which indicates the stimulation of osteoinductive processes with the immobilization of HBD-Epo on DCM, especially when combined with BMP-2.

Sequence listing:

SEQ ID NO 1 The nucleotide sequence of recombinant plasmid DNA pL610

SEQ ID NO 2 The sequence of amino acid residues of the protein HBD-Epo

SEQ ID NO 3 The sequence of amino acid residues of the heparin binding domain from Danio rerio

SEQ ID NO 4 Sequence of amino acid residues of the spacer

SEQ ID NO 5 Sequence of amino acid residues of the EPO protein from Homo sapiens

Claims (9)

1. Recombinant gene with the sequence of SEQ ID NO 1 encoding a protein heparin binding domain-erythropoietin (HBD-Epo). 2. Recombinant plasmid DNA pL610 with the physical map shown in FIG. 1, carrying the recombinant gene according to claim 1. 3. A method of obtaining a recombinant protein HBD-Epo, including: - obtaining HBD-Epo protein in the form of inclusion bodies in expression strains of Escherichia coli transformed with the expression recombinant plasmid according to claim 2; - washing inclusion bodies, solubilization of HBD-Epo, chromatographic purification of the protein on an ion-exchange or heparin-containing sorbent. 4. Recombinant protein HBD-Epo with an estimated molecular weight of 20520.51 Yes and an estimated isoelectric point of 9.62, obtained by the method according to p. 3. 5. A composition for the specific induction of bone tissue regeneration, comprising the recombinant HBD-Epo protein according to claim 4 and the demineralized bone matrix, wherein the recombinant HBD-Epo protein according to claim 4 is immobilized on a demineralized bone matrix. 6. The composition according to p. 5, also containing a recombinant BMP-2 protein, which is also immobilized on a demineralized bone matrix. 7. A method for specific induction of bone tissue regeneration, comprising filling in bone defects with a composition according to claim 5 or. 6.

Images