A targeting G-CSF linked polypeptide and its application

Abstract

The present invention provides a linking polypeptide targeting G-CSF and its application, which relates to the field of biomedical technology and includes a linking polypeptide and collagen bound to the C-terminus of the linking polypeptide; the amino acid sequence of the linking polypeptide is as shown in SEQ ID NO.1. The C-terminus of the linking polypeptide is covalently crosslinked to collagen through transglutaminase; the N-terminus of the linking polypeptide is linked to G-CSF through non-covalent van der Waals forces, which will not damage the structure of G-CSF; the fusion polypeptide formed by the linking polypeptide and collagen can target G-CSF and maintain the structure of G-CSF. It solves the technical problem in the prior art that a simple G-CSF solution or gel preparation cannot adhere to the endometrial surface for a long time and has limited effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a linking polypeptide targeting G-CSF and its applications. Background Art

[0002] The endometrium is the basis for embryo implantation. A relatively thick endometrium has a better bearing effect. If the endometrium is thin, both the implantation rate and the pregnancy rate after implantation will be affected. A recognized marker of endometrial receptivity is the thickness of the endometrium. The minimum endometrial thickness for successful embryo transfer is 7 mm. When it is less than this value, the success rate of embryo transfer will be greatly affected. Especially when the endometrial thickness is less than 5 mm, embryo transfer usually cannot be performed, and the survival rate of the embryo after transfer will also be significantly reduced. Currently, estrogen and progesterone therapy is mostly used for endometrial injury repair, but the effect is poor. Granulocyte Colony-Stimulating Factor (G-CSF), a glycoprotein synthesized by vascular endothelial cells, monocytes, and fibroblasts, can promote the maturation of neutrophils, stimulate the release of mature granulocytes from the bone marrow, and enhance the chemotaxis and phagocytosis functions of neutrophils. It has been reported that G-CSF can be used to treat thin endometrium, promote the synthesis and secretion of various angiogenic factors, accelerate the formation of capillaries, improve endometrial blood supply, and increase endometrial thickness. However, a simple G-CSF solution or gel preparation cannot adhere to the endometrial surface for a long time, and its effect is limited, making it difficult to achieve endometrial repair. Therefore, finding a method to enhance the repair of endometrial injury by G-CSF and promoting the thickening of the damaged endometrium to the normal level is a technical problem that needs to be urgently solved by those skilled in the art. In view of this, the present invention is specifically proposed. Summary of the Invention

[0003] One of the purposes of the present invention is to provide a linking polypeptide targeting G-CSF to solve the technical problem in the prior art that a simple G-CSF solution or gel preparation cannot adhere to the endometrial surface for a long time and has limited effects.

[0004] Another purpose of the present invention is to provide the application of the above linking polypeptide in the preparation of substances targeting G-CSF.

[0005] A third purpose of the present invention is to provide a fusion polypeptide targeting G-CSF.

[0006] A fourth purpose of the present invention is to provide a preparation method for a fusion polypeptide targeting G-CSF.

[0007] A fifth purpose of the present invention is to provide a fusion protein.

[0008] The sixth object of the present invention is to provide a method for preparing the above-mentioned fusion protein.

[0009] The seventh object of the present invention is to provide the use of the above-mentioned fusion protein or the fusion protein prepared by the above-mentioned preparation method in the preparation of a drug for promoting the repair of endometrial injury.

[0010] The eighth object of the present invention is to provide a drug for promoting the repair of endometrial injury.

[0011] In order to achieve the above objects of the present invention, the following technical solutions are specifically adopted:

[0012] In the first aspect, the present invention provides a linking polypeptide targeting G-CSF, and the amino acid sequence of the linking polypeptide is as shown in SEQ ID NO.1.

[0013] In the second aspect, the present invention provides the use of the above-mentioned linking polypeptide in the preparation of a substance for targeting G-CSF.

[0014] In the third aspect, the present invention provides a fusion polypeptide targeting G-CSF, which includes the above-mentioned linking polypeptide and collagen linked to the C-terminus of the linking polypeptide.

[0015] In the fourth aspect, the present invention provides a method for preparing the above-mentioned fusion polypeptide, which includes heating a buffer solution containing a linking polypeptide, collagen and a protein cross-linking agent at 50 °C for 2 - 3 h, then heating at 70 °C for 15 - 20 min, cooling, purifying by chromatography separation, and freeze-drying to obtain the fusion polypeptide;

[0016] The preparation method of the buffer solution containing a linking polypeptide, collagen and a protein cross-linking agent includes stirring the buffer solution containing a linking polypeptide, collagen and a protein cross-linking agent at 180 rpm for 20 - 30 min;

[0017] The mass ratio of the linking polypeptide, collagen and the protein cross-linking agent is 5 - 8:3:0.1;

[0018] The concentration of the linking polypeptide in the buffer solution is 5 - 8 mg / mL;

[0019] The protein cross-linking agent includes transglutaminase;

[0020] The pH of the buffer solution is 7.2 - 7.5;

[0021] The buffer solution is 10 mM PBS buffer solution;

[0022] The collagen includes human collagen.

[0023] Fifth aspect, the present invention provides a fusion protein, comprising the above-mentioned fusion polypeptide or the fusion polypeptide prepared by the above-mentioned preparation method, and G-CSF that binds to the N-terminus of the linking polypeptide in the fusion polypeptide.

[0024] Furthermore, it also includes a chitosan-hyaluronic acid composite carrier.

[0025] Sixth aspect, the present invention provides a preparation method of the above-mentioned fusion protein, comprising dissolving the fusion polypeptide and G-CSF in physiological saline to obtain the fusion protein;

[0026] The mass ratio of the fusion polypeptide to G-CSF is 40-60:1;

[0027] The concentration of the fusion polypeptide in the physiological saline is 0.4-0.6 g / L;

[0028] The conditions for dissolution include stirring at 80 rpm for 30-40 min.

[0029] Furthermore, it also includes adding modified chitosan to the physiological saline in which the fusion polypeptide and G-CSF are dissolved, stirring at 110 rpm for 30-50 min to form a chitosan-fusion protein gel, freeze-drying, redissolving in physiological saline, adding modified hyaluronic acid gel, and stirring at 80 rpm for 15-25 min to obtain the fusion protein;

[0030] The mass ratio of the modified chitosan to the fusion polypeptide is 1-3:1;

[0031] The modified chitosan is prepared by dissolving chitosan in an acid solution, subjecting it to repeated freeze-thaw cycles 3-5 times, and then freeze-drying to obtain the modified chitosan;

[0032] The acid solution includes acetic acid and citric acid, and the volume ratio of acetic acid to citric acid is 1-2:8-9;

[0033] The chitosan is hydroxypropyltrimethylammonium chloride chitosan;

[0034] The addition amount of the modified hyaluronic acid gel is 30-50 ml per 1 L of physiological saline;

[0035] The preparation method of the modified hyaluronic acid gel includes emulsifying an aqueous solution of sodium hyaluronate by stirring at 1000 r / min for 40-60 min, and adding sodium tripolyphosphate for crosslinking modification to obtain the modified hyaluronic acid gel;

[0036] The concentration of sodium hyaluronate in the aqueous solution of sodium hyaluronate is 1-2% w / v;

[0037] The addition amount of sodium tripolyphosphate is 1%-1.5% w / v;

[0038] The time for crosslinking modification is 4 to 24 hours.

[0039] In a seventh aspect, the present invention provides the use of the above-mentioned fusion protein or the fusion protein prepared by the above-mentioned preparation method in the preparation of a drug for promoting the repair of endometrial injury.

[0040] In an eighth aspect, the present invention provides a drug for promoting the repair of endometrial injury, comprising the above-mentioned fusion protein or the fusion protein prepared by the above-mentioned preparation method.

[0041] The present invention provides a linking polypeptide targeting G-CSF. A linking polypeptide targeting G-CSF is designed and synthesized according to granulocyte colony-stimulating factor (abbreviated as G-CSF). The C-terminus of the linking polypeptide is rich in lysine residues and can be covalently crosslinked to collagen through transglutaminase; the N-terminus of the linking polypeptide mimics the structure of the G-CSF receptor and can bind to G-CSF. The connection between the linking polypeptide and G-CSF is through non-covalent van der Waals forces, which will not destroy the structure of G-CSF. This solves the technical problem in the prior art that a simple G-CSF solution or gel preparation cannot adhere to the endometrial surface for a long time and has limited effects.

[0042] In another aspect, the present invention provides a fusion protein. By binding the N-terminus of the fusion polypeptide to G-CSF, the formed fusion protein not only has a long attachment ability in the endometrium, but also can maintain the activity of G-CSF and improve the effect of repairing endometrial injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0044] Figure 1 It is the cytotoxicity test results of different fusion proteins provided in Experimental Example 1 of the present invention;

[0045] Figure 2 It is the test results of the retention concentration of different fusion proteins on the endometrium at different times provided in Experimental Example 2 of the present invention;

[0046] Figure 3 It is the staining comparison diagram of uterine sections treated with the fusion protein provided in Experimental Example 3 of the present invention;

[0047] Figure 4 It is the comparison diagram of the number of endometrial glands treated with the fusion protein provided in Experimental Example 3 of the present invention;

[0048] Figure 5 The comparative diagram of the proportion of endometrial fibrosis treated with the fusion protein provided in Experimental Example 3 of the present invention;

[0049] Figure 6 The test results of the retention concentration of different fusion proteins on the endometrium at different times provided in Experimental Example 4 of the present invention. Detailed implementation manners

[0050] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non-restrictive.

[0051] Unless otherwise specified, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0052] On the one hand, the present invention provides a linking polypeptide targeting G-CSF, and the amino acid sequence of the linking polypeptide is as shown in SEQ ID NO.1.

[0053] A linking polypeptide capable of targeting G-CSF was designed and synthesized according to granulocyte colony-stimulating factor (abbreviation G-CSF). The C-terminus of the linking polypeptide is rich in lysine residues and can be covalently cross-linked to collagen through transglutaminase; the N-terminus of the linking polypeptide mimics the structure of the G-CSF receptor and can bind to G-CSF. The connection between the linking polypeptide and G-CSF is through non-covalent van der Waals forces and does not destroy the structure of G-CSF. This solves the technical problem in the prior art that a simple G-CSF solution or gel preparation cannot adhere to the endometrial surface for a long time and has limited effects.

[0054] Experiments have proved that the fusion polypeptide formed by the linking polypeptide and collagen can target G-CSF and maintain the structure of G-CSF. It can not only extend the attachment time on the endometrium but also does not affect the activity of G-CSF. On the other hand, the present invention also provides the application of the above-mentioned linking polypeptide in the preparation of substances for targeting G-CSF.

[0055] On the other hand, the present invention also provides a fusion polypeptide targeting G-CSF, including the above-mentioned linking polypeptide and collagen connected to the C-terminus of the linking polypeptide.

[0056] The C-terminus of the linking polypeptide is rich in lysine residues and can be covalently cross-linked to collagen through transglutaminase to form a fusion protein in the form of linker-collagen, which can prolong the attachment time of G-CSF to the endometrium without affecting the activity of G-CSF, effectively promoting endometrial thickening and the repair of endometrial injury.

[0057] According to another aspect of the present invention, there is also provided a method for preparing a fusion polypeptide targeting G-CSF, which includes heating a buffer solution containing a linking polypeptide, collagen, and a protein cross-linking agent at 50 °C for 2 - 3 h, then heating at 70 °C for 15 - 20 min, cooling, purifying by chromatography separation, and freeze-drying to obtain the fusion polypeptide.

[0058] The C-terminus of the linking polypeptide is covalently cross-linked to collagen through a protein cross-linking agent to form a fusion protein in the form of linker-collagen, which can prolong the attachment time of G-CSF to the endometrium without affecting the activity of G-CSF, effectively promoting endometrial thickening and the repair of endometrial injury.

[0059] Among them, the chromatography separation and purification can specifically be carried out by G25 molecular sieve chromatography separation and purification.

[0060] In some specific embodiments, the preparation method of the buffer solution containing a linking polypeptide, collagen, and a protein cross-linking agent includes stirring the buffer solution containing a linking polypeptide, collagen, and a protein cross-linking agent at 180 rpm for 20 - 30 min, so that the linking polypeptide and collagen are in full contact, and covalent cross-linking is completed under the action of the protein cross-linking agent.

[0061] Among them, the stirring time can be, but is not limited to, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, or 30 min, or any time between 20 - 30 min.

[0062] In some specific embodiments, the mass ratio of the linking polypeptide, collagen, and protein cross-linking agent is 5 - 8:3:0.1, which can improve the success rate of the connection between collagen and the linking polypeptide and avoid the occurrence of collagen that is not covalently linked to the linking polypeptide.

[0063] Among them, the mass ratio of the linking polypeptide, collagen, and protein cross-linking agent can be, but is not limited to, 5:3:0.1, 5.5:3:0.1, 6:3:0.1, 6.5:3:0.1, 7:3:0.1, 7.5:3:0.1, or 8:3:0.1, or any ratio between 5 - 8:3:0.1.

[0064] In some specific embodiments, the concentration of the linking polypeptide in the buffer solution is 5 - 8 mg / mL.

[0065] Among them, the concentration of the linking polypeptide in the buffer can be, but is not limited to, 5 mg / mL, 5.4 mg / mL, 5.8 mg / mL, 6 mg / mL, 6.4 mg / mL, 6.8 mg / mL, 7 mg / mL, 7.4 mg / mL, 7.8 mg / mL, or 8 mg / mL, and can also be any value between 5 and 8 mg / mL.

[0066] In some specific embodiments, the protein cross-linking agent includes transglutaminase.

[0067] In some specific embodiments, the pH of the buffer is 7.2 - 7.5. In some specific embodiments, the buffer is 10 mM PBS buffer.

[0068] Among them, the pH of the buffer can be, but is not limited to, 7.2, 7.3, 7.4, or 7.5, and can also be any value between 7.2 and 7.5, preferably 7.4.

[0069] In some specific embodiments, the collagen includes human collagen.

[0070] Human collagen includes at least one of human type I collagen, human type III collagen, human type IV collagen, human type IV collagen, or human type V collagen. Human collagen is preferably human type III collagen.

[0071] According to another aspect of the present invention, there is also provided a fusion protein, including the above-mentioned fusion polypeptide or the fusion polypeptide prepared by the above-mentioned preparation method, and G-CSF that binds to the N-terminus of the linking polypeptide in the fusion polypeptide.

[0072] By binding the N-terminus of the fusion polypeptide to G-CSF, the formed fusion protein not only has a long attachment ability in the endometrium, but also can maintain the activity of G-CSF and improve the effect of repairing endometrial injury.

[0073] In order to further improve the activity and adhesion of the fusion protein to the endometrium, in some specific embodiments, it also includes a chitosan-hyaluronic acid composite carrier.

[0074] Chitosan-hyaluronic acid can form a gel network structure to load the fusion protein, so as to improve the adhesion of the fusion protein. Through gradual release, the time for the fusion protein to act on the endometrium is prolonged; the gel network wraps the fusion protein macromolecule to avoid degradation and maintain its activity.

[0075] According to another aspect of the present invention, there is also provided a method for preparing the above-mentioned fusion protein, which includes dissolving the fusion polypeptide and G-CSF in physiological saline to obtain the fusion protein.

[0076] The fusion protein obtained by this preparation method can exist in the form of a protein solution or can be freeze-dried to exist in the form of a solid fusion protein. It can form a stable repair microenvironment on the endometrial surface, promote the repair of endometrial injury, restore the number of endometrial glands with intrauterine adhesions, reduce the fibrosis ratio, and has beneficial effects such as high tissue compatibility, simple preparation, and obvious improvement effect. It has no potential toxic effect on cells and has good clinical application prospects.

[0077] In some specific embodiments, the mass ratio of the fusion polypeptide to G-CSF is 40-60:1.

[0078] Among them, the mass ratio of the fusion polypeptide to G-CSF can be, but is not limited to, 40:1, 42:1, 44:1, 46:1, 48:1, 50:1, 52:1, 54:1, 56:1, 58:1 or 60:1, or can also be any value between 40-60:1.

[0079] In some specific embodiments, the concentration of the fusion polypeptide in the physiological saline is 0.4-0.6 g / L.

[0080] Among them, the concentration of the fusion polypeptide in the physiological saline can be, but is not limited to, 0.4 g / L, 0.42 g / L, 0.44 g / L, 0.46 g / L, 0.48 g / L, 0.5 g / L, 0.52 g / L, 0.54 g / L, 0.56 g / L, 0.58 g / L or 0.6 g / L, or can also be any value between 0.4-0.6 g / L.

[0081] In some specific embodiments, the dissolution conditions include stirring at 80 rpm for 30-40 min.

[0082] In some specific embodiments, it also includes adding modified chitosan to the physiological saline dissolved with the fusion polypeptide and G-CSF, stirring at 110 rpm for 30-50 min to form a chitosan-fusion protein gel, freeze-drying, redissolving in physiological saline, and adding modified hyaluronic acid gel, stirring at 80 rpm for 15-25 min to obtain the fusion protein.

[0083] In some specific embodiments, the mass ratio of the modified chitosan to the fusion polypeptide is 1-3:1.

[0084] In some specific embodiments, the modified chitosan includes dissolving chitosan in an acid solution, repeatedly freezing and thawing 3-5 times, and freeze-drying to obtain the modified chitosan.

[0085] In some specific embodiments, the acid solution includes acetic acid and citric acid, and the volume ratio of acetic acid to citric acid is 1-2:8-9.

[0086] In some specific embodiments, the chitosan is hydroxypropyltrimethylammonium chloride chitosan.

[0087] In some specific embodiments, the addition amount of the modified hyaluronic acid gel is 30-50 ml per 1 L of normal saline.

[0088] In some specific embodiments, the preparation method of the modified hyaluronic acid gel includes emulsifying an aqueous solution of sodium hyaluronate with stirring at 1000 r / min for 40-60 min, and adding sodium tripolyphosphate for cross-linking modification to obtain the modified hyaluronic acid gel.

[0089] In some specific embodiments, the concentration of sodium hyaluronate in the aqueous solution of sodium hyaluronate is 1-2% w / v.

[0090] In some specific embodiments, the addition amount of sodium tripolyphosphate is 1%-1.5% w / v.

[0091] In some specific embodiments, the time for cross-linking modification is 4-24 h.

[0092] According to another aspect of the present invention, there is also provided the use of the above-mentioned fusion protein or the fusion protein prepared by the above-mentioned preparation method in the preparation of a drug for promoting the repair of endometrial injury.

[0093] According to another aspect of the present invention, there is also provided a drug for promoting the repair of endometrial injury, including the above-mentioned fusion protein or the fusion protein prepared by the above-mentioned preparation method.

[0094] Next, the technical solutions of the present invention will be described clearly and completely in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0095] It should be noted that unless otherwise specified, the chemical reagents involved in the present invention are all purchased through commercial channels.

[0096] Example 1

[0097] According to G-CSF, linker polypeptides -1 to -6 capable of linking G-CSF and collagen were designed and synthesized. Each linker polypeptide was directionally synthesized according to the polypeptide sequence using solid-phase synthesis method with amino acids as raw materials.

[0098] Amino acid sequence of Linker Peptide-1 (SEQ ID NO.1):

[0099] NTGLAIPPPSDFHNNRYPGVSNVPGGHSTSMFFRVVESAYLNLSSEPNLIPEVTVGEGLNLGVMVEAYPGLPGFNWTYLGFSDHPPEPGLANATTGDTYEPFHGVTVPSLLTVETLEHNAFGLGGEDAVLGVAKGKLGSTAHKDEKEALGAKTHKKKVLKKTEYKKYKDLLNFLKKGAE。

[0100] Amino acid sequence of Linker Peptide-2 (SEQ ID NO.2):

[0101] ECGHISVSAPIVHLGDPITPPSASNSNTWRMENGRATGFASCIINCSHLDPEPILWRLPPSASNSNTWRMENGRATGFGAELPGGRRLSDGTESIITLLILDVELRAGYPPAIPHNLSCLMNLT。

[0102] Amino acid sequence of Linker Peptide-3 (SEQ ID NO.3):

[0103] TSSLICWEPGPETHLPTSFTPHLNHTAFLSCCLNWGNSLSFSHCCIPRHLLLYNMGIWVAENALGTPHLNHTAFLSCCLNWGNSSMSPLCLDPMDAPPA。

[0104] Amino acid sequence of Linker Peptide-4 (SEQ ID NO.4):

[0105] CLLCWYVVSWRPSGAGAILPLCNTTELSCTFHLWALVGCIRWPLPGHWPSGAGAILPLCNTTELSKKCTFHKKKLPSTTETKKWRRLDKKKLF。

[0106] Amino acid sequence of Linker Peptide-5 (SEQ ID NO.5):

[0107] PVPLEEDSGRIGYVVLSCASSRGFVLHGLEPTFHLPSEAEVGTSRASSRGFVLHGLEPPTPHAMARDPHSLKKWVGKKWEPKKPNPKKKWPG。

[0108] Amino acid sequence of Linker polypeptide-6 (SEQ ID NO.6):

[0109] VIEWGLGPPENGRAASSRGFVLHGLEPTGFLLENIRPFLYEIIVTPLYDTMAPSHAPELHLHIGTWALEWVPEPPELGSPKKLTHYKKTIFKKWTKKN。

[0110] Amino acid sequence of Linker polypeptide-7 (SEQ ID NO.7):

[0111] NSFSAILNASSRGFATGFASCIINCVLHGLEPASLYHIHLMAASAATGFASCIINCSSRGFVLHGLEPAGATNSTVLKTKKKLMKKKTLTPEKKKGSEKKLG。

[0112] Examples 2 to 8

[0113] A fusion polypeptide comprising a linker polypeptide and collagen bound to the C-terminus of the linker polypeptide. Linker polypeptides -1 to -6 provided in Example 1 were respectively selected and the fusion polypeptide was prepared according to the following steps:

[0114] (1) Add the linker polypeptide, human type III collagen and transglutaminase to a 10 mM PBS buffer at pH 7.4. The dosage ratio of the PBS buffer, linker polypeptide, human type III collagen and transglutaminase is 1 mL: 7 mg: 3 mg: 0.1 mg, and stir at 180 rpm for 30 min to obtain a mixed solution;

[0115] (2) Heat the mixed solution obtained in step (1) to 50 °C and maintain for 3 h, then heat to 70 °C and maintain for 20 min, and naturally cool to room temperature to obtain a post-reaction solution;

[0116] (3) Subject the post-reaction solution described in step (2) to separation and purification by G25 molecular sieve chromatography and freeze-dry to obtain linker polypeptide - collagen (fusion polypeptide).

[0117] Among them, human type III collagen was obtained by the method disclosed in Chinese Patent CN103102407A.

[0118] Examples 9 to 15

[0119] A fusion protein comprising a fusion polypeptide and G-CSF bound to the N-terminus of the linker polypeptide in the fusion polypeptide. Among them, the fusion polypeptides prepared in Examples 2 to 7 were respectively selected and the fusion protein was prepared according to the following steps:

[0120] Add the fusion polypeptide and G-CSF to physiological saline, stir at 80 rpm for 30 min. The dosage ratio of physiological saline, fusion polypeptide and G-CSF is 1 L: 0.5 g: 0.01 g to obtain a protein solution for promoting the repair of endometrial injury.

[0121] Comparative Example 1

[0122] The difference from Example 7 is that human type III collagen not linked to the linking polypeptide is used instead of the fusion polypeptide. Add human type III collagen and G-CSF to physiological saline, stir at 80 rpm for 30 min. The dosage ratio of physiological saline, human type III collagen and G-CSF is 1 L: 0.5 g: 0.01 g to obtain a protein solution for promoting the repair of endometrial injury.

[0123] Experimental Example 1

[0124] In this experimental example, the methods for preparing the protein solutions for promoting the repair of endometrial injury in Examples 9 - 15 and Comparative Example 1 were selected. Replace physiological saline with MEM medium containing 10% fetal bovine serum to obtain experimental solutions, and operate according to the following steps respectively:

[0125] Culture L-929 cells (NCTC clone 929) in MEM medium containing 10% fetal bovine serum and antibiotics (penicillin 100 U / mL, streptomycin 100 μg / mL), and culture in an incubator at 37°C and 5% CO2. Digest the cells with 0.25% trypsin, centrifuge at 200 g for 3 min, and then redisperse the cells in fresh medium and adjust the cell density to 2×10 5Cell suspension at cells / mL; Inoculate the above cell suspension into a 96-well culture plate, 100 μL per well, and culture it in a carbon dioxide incubator (5% CO2, 37°C, humidity > 90%) for 24 h. Then add 100 μL of the experimental solution to each well, which serves as the experimental group. Additionally, set up wells with only 200 μL of MEM medium containing 10% fetal bovine serum as the blank control group, and wells with 100 μL of medium + 100 μL of cells as the negative control. After adding the experimental solution, continue to culture for 5 days. Every day, add 20 μL of MTT (prepared with PBS at 5 mg / mL) to each well in the dark. On the last day, after adding MTT, continue to culture for 4 h. Then discard all the liquid in the wells and add 150 μL of DMSO solution to each well. Vortex at low speed in an enzyme-linked immunosorbent assay (ELISA) reader for 15 min, and measure the optical density (OD) value at λ = 490 nm. The cytotoxicity of the cells is determined by calculating the relative growth rate (RGR, %) of the cells: RGR = [(ODexperimental group - ODblank group) / (ODnegative control group - ODblank group)] × 100%. The specific grading criteria for toxicity are as follows: Grade 0: RGR ≥ 100%; Grade 1: 99% ≥ RGR ≥ 75; Grade 2: 74 ≥ RGR ≥ 50; Grade 3: 49 ≥ RGR ≥ 25; Grade 4: 24 ≥ RGR ≥ 1; Grade 5 = 0. Among them, a toxicity level of 2 or above can be regarded as a cytotoxic reaction. The results are as Figure 1 shown.

[0126] As Figure 1 can be seen, the toxicity grades of the protein solutions in Examples 9 - 13 and Comparative Example 1 are all 0, indicating that the protein solutions in Examples 9 - 13 have no cytotoxicity and high biosafety. However, the toxicity of Examples 14 and 15 is Grade 1, indicating that the protein solutions prepared using Linker Peptide - 6 and Linker Peptide - 7 have cytotoxicity.

[0127] Experimental Example 2

[0128] Respectively select the protein solutions prepared in Examples 9 - 13 and Comparative Example 1 with biosafety, and conduct the following operations for rat experiments to detect the attachment duration of the fusion protein in a rat model of endometrial injury.

[0129] 1. Experimental animals: Select 60 female Sprague - Dawley (SD) rats at 8 - 10 weeks of age and weighing 220 - 260 g, and adaptively raise them for one week.

[0130] 2. Experimental grouping: Randomly divide the rats into 6 groups, with 10 rats in each group.

[0131] 3. Establishment of a rat model of endometrial injury: The mechanical injury and ethanol perfusion method was used to induce the model. The rats were fasted and watered for 12 h before surgery. After anesthesia with intraperitoneal injection of 2% sodium pentobarbital, they were fixed in the supine position. A transverse abdominal incision was made to expose the uterine horns. A longitudinal incision about 4 mm long was made at about 0.5 cm below the uterine horns. An endometrial curette with a diameter of 2.5 mm was used to scrape the middle and upper endometrium until the four walls of the uterus felt rough. The uterine horns were clamped with a vascular clamp. A 1 mL syringe was used to draw 95% ethanol and inject it downward from the incision until the uterine cavity was filled. Then the incision was also temporarily clamped with a vascular clamp. After 3 min, the vascular clamp at the incision was released, and the residual ethanol was discharged. The uterus was rinsed and aspirated with normal saline and dried with a sterile gauze to absorb the liquid on the uterine surface.

[0132] 4. Administration of drugs: Immediately after modeling, 0.5 mL of protein solution was perfused into the uterine cavity. After the operation, the abdominal cavity was rinsed with sterile normal saline. After the uterus was repositioned, the abdomen was closed layer by layer. The incision was disinfected with 75% alcohol. After the operation, 2 mL of gentamicin was given intramuscularly every day to prevent infection. One rat in each group was sacrificed at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after the operation, and the uterus was collected. The drug concentration of G-CSF in the uterus was detected by ELISA method. The results were as Figure 2 shown.

[0133] As Figure 2 can be seen, the concentration of G-CSF in the experimental group 9 was significantly higher than that in experimental groups 10 - 13 and comparative example 1, and the decline rate was also lower, indicating that the linker polypeptide-1 could effectively prolong the action time of G-CSF and promote the repair of uterine injury.

[0134] Experimental Example 3

[0135] In this experimental example, the protein solutions prepared in experimental example 9 and comparative example 1 were selected and the following steps were carried out to verify the repair effect of the protein solution on endometrial injury.

[0136] 1. Experimental animals: Forty female SD rats aged 8 - 10 weeks and weighing 220 - 260 g were selected and adaptively fed for one week.

[0137] 2. Experimental grouping: The rats were randomly divided into 4 groups with 10 rats in each group, namely 2 experimental groups, 1 sham operation group, and 1 model group. The experimental groups and the model group were induced to model by mechanical injury and ethanol perfusion method. The modeling method was the same as that in experimental example 2. The sham operation group was anesthetized and laparotomy was performed. After the uterine horns were fully exposed outside the body, they were replaced and no other operations were taken. The abdomen was rinsed and aspirated with normal saline and dried with a sterile gauze to absorb the liquid on the uterine surface.

[0138] 3. Administration: Immediately after modeling, each experimental group was respectively perfused with 0.5 mL of the protein solutions of Example 9 and Comparative Example 1 into the uterine cavity. The sham operation group and the model group were given an equal amount of normal saline. After the operation was completed, the abdominal cavity was rinsed with sterile normal saline. After the uterus was repositioned, the abdomen was closed layer by layer. The incision was disinfected with 75% alcohol. Gentamicin 2 mL was intramuscularly injected for 3 consecutive days after the operation to prevent infection. Each group was sacrificed on the 14th day after the operation, the uterine tissue was taken, fixed with paraformaldehyde, paraffin sectioned, HE stained, and Masson stained, as Figure 3 shown. The number of endometrial glands and the proportion of endometrial fibrosis were statistically analyzed, and the statistical results were as Figure 4 , Figure 5 shown.

[0139] As Figures 3 to 5 can be seen, the endometrial structure of the sham operation group was normal, the number of glands was relatively large, and the fibrosis proportion was normal; severe intrauterine adhesions occurred in the model group, the number of glands decreased significantly, and the fibrosis proportion increased; the intrauterine adhesion situation in the experimental group was reduced compared with the model group, the number of glands recovered, and the fibrosis proportion was lower than that of the model group and was close to that of the sham operation group. It can be seen that the protein solution of Example 9 has a good repair effect on intrauterine adhesions, and the effect is better than that of using G-CSF and collagen alone. The linking polypeptide can effectively improve the repair effect of endometrial injury and promote the repair of endometrial injury.

[0140] Example 16

[0141] A fusion protein, comprising a fusion polypeptide, G-CSF bound to the N-terminus of the linking polypeptide in the fusion polypeptide, and a chitosan-hyaluronic acid composite carrier. Among them, the fusion polypeptide is the fusion polypeptide provided in Example 2, and it is prepared according to the following steps:

[0142] 1. Modified chitosan: Hydroxypropyltrimethylammonium chloride chitosan was dissolved in an acid solution to obtain a 4 wt% chitosan solution, which was placed in a refrigerator at -20 °C and frozen for 12 h, then thawed at room temperature for 12 h, and freeze-thawed 5 times repeatedly. It was pre-frozen at -20 °C for 4 h and freeze-dried at -80 °C for 24 h to obtain modified chitosan, where the acid solution was prepared by mixing acetic acid and citric acid according to a volume ratio of 1.4:8.6.

[0143] 2. Modified hyaluronic acid gel: A 2% sodium hyaluronate aqueous solution was emulsified at 1000 r / min for 40 - 60 min, and 1.3% sodium tripolyphosphate was added for cross-linking modification for 12 h to obtain a modified hyaluronic acid gel.

[0144] 3. Add the fusion polypeptide and G-CSF to physiological saline, stir at 80 rpm for 30 min to dissolve. The dosage ratio of physiological saline, fusion polypeptide and G-CSF is 1 L: 0.5 g: 0.01 g. Add modified chitosan to the physiological saline dissolved with the fusion polypeptide and G-CSF. The dosage of modified chitosan is 1 g per 1 L of physiological saline. Stir at 110 rpm for 40 min to form a chitosan-fusion protein gel. Pre-freeze at -20 °C for 4 h and freeze-dry at -80 °C for 24 h. Re-dissolve in physiological saline, and add modified hyaluronic acid gel. The dosage of modified hyaluronic acid gel is 40 ml per 1 L of physiological saline. Stir at 80 rpm for 20 min to obtain a protein solution for promoting the repair of endometrial injury.

[0145] Comparative Example 2

[0146] Differing from Example 4, in step 3, add the fusion polypeptide and G-CSF to physiological saline, stir at 80 rpm for 30 min to dissolve. The dosage ratio of physiological saline, fusion polypeptide and G-CSF is 1 L: 0.5 g: 0.01 g. Add modified chitosan to the physiological saline dissolved with the fusion polypeptide and G-CSF. The dosage of modified chitosan is 1 g per 1 L of physiological saline. Stir at 110 rpm for 40 min to form a chitosan-fusion protein gel. Pre-freeze at -20 °C for 4 h and freeze-dry at -80 °C for 24 h. Re-dissolve in physiological saline to obtain a protein solution for promoting the repair of endometrial injury.

[0147] Comparative Example 3

[0148] Differing from Example 4, add the fusion polypeptide and G-CSF to physiological saline, stir at 80 rpm for 30 min to dissolve. The dosage ratio of physiological saline, fusion polypeptide and G-CSF is 1 L: 0.5 g: 0.01 g. Pre-freeze at -20 °C for 4 h and freeze-dry at -80 °C for 24 h. Re-dissolve in physiological saline, and add modified hyaluronic acid gel. The dosage of modified hyaluronic acid gel is 40 ml per 1 L of physiological saline. Stir at 80 rpm for 20 min to obtain a protein solution for promoting the repair of endometrial injury.

[0149] Comparative Example 4

[0150] Differing from Example 4, add modified hyaluronic acid gel to the physiological saline dissolved with the fusion polypeptide and G-CSF. The dosage of modified hyaluronic acid gel is 40 ml per 1 L of physiological saline. Stir at 110 rpm for 40 min. Pre-freeze at -20 °C for 4 h and freeze-dry at -80 °C for 24 h. Re-dissolve in physiological saline, and add modified chitosan. The dosage of modified chitosan is 1 g per 1 L of physiological saline. Stir at 80 rpm for 20 min to obtain a protein solution for promoting the repair of endometrial injury.

[0151] Experimental Example 4

[0152] To prove that the protein solutions prepared in Example 16 and Comparative Examples 2 to 4 can further extend the attachment time of G-CSF to the endometrium, the protein solutions prepared in Example 9, Example 16 and Comparative Examples 2 to 4 were used to conduct rat experiments according to the method of Experimental Example 2.

[0153] Fifty female SD rats aged 8-10 weeks and weighing 220-260 g were selected and adaptively fed for one week. The rats were randomly divided into 5 groups, with 10 rats in each group. Immediately after modeling, 0.5 mL of the protein solution was perfused into the uterine cavity. After the operation was completed, the abdominal cavity was rinsed with sterile normal saline. After the uterus was reset, the abdomen was sutured layer by layer. The incision was disinfected with 75% alcohol. After the operation, 2 mL of gentamicin was injected intramuscularly every day to prevent infection. One rat in each group was sacrificed at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after the operation, and the uterus was collected. The G-CSF drug concentration in the uterus was detected by ELISA method, and the results are as Figure 6 shown.

[0154] As Figure 6 can be seen, compared with Example 9, the G-CSF drug concentration in Example 16 was increased during the period of 12 h to 72 h, indicating that the chitosan-hyaluronic acid carrier formed by adding modified chitosan and modified hyaluronic acid gel can further extend the attachment time of the fusion protein; the G-CSF drug concentration in Comparative Example 4 was slightly higher than that in Example 9 at 12 h, but showed a rapid downward trend in the subsequent detections after 12 h and was lower than that in Example 9; the overall level in Comparative Example 2 was lower than that in Example 9; the difference between Comparative Example 3 and Example 9 was not significant during the period of 12-60 h, but it decreased rapidly at 72 h. It can be seen that the preparation method of adding modified chitosan and modified hyaluronic acid gel plays a key role in the attachment time of the prepared fusion protein.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fusion protein, characterized in that comprising a fusion polypeptide and G-CSF bound to the N-terminus of a linker polypeptide in the fusion polypeptide; The fusion polypeptide comprises a connecting polypeptide and a collagen connected to the C-terminus of the connecting polypeptide; The amino acid sequence of the connecting polypeptide is shown in SEQ ID NO.1; The C-terminus of the connecting polypeptide is rich in lysine residues and is covalently cross-linked to collagen by transglutaminase; the N-terminus of the connecting polypeptide simulates the G-CSF receptor structure and binds to G-CSF, and the connection between the connecting polypeptide and G-CSF is through non-covalent van der Waals forces; The collagen is human type III collagen.

2. The fusion protein according to claim 1, characterized in that It also includes a chitosan-hyaluronic acid composite carrier.

3. The method for preparing the fusion protein according to claim 1 or 2, characterized in that: The method comprises dissolving the fusion polypeptide and G-CSF in physiological saline to obtain a fusion protein; The mass ratio of the fusion polypeptide to G-CSF is 45-55:2; The concentration of the fusion polypeptide in the physiological saline is 0.45-0.55 g / L; The dissolution conditions include stirring at 80 rpm for 30-40 min.

4. The preparation method according to claim 3, characterized in that: The method further comprises adding modified chitosan to physiological saline containing the fusion polypeptide and G-CSF, stirring at 110 rpm for 30 to 50 minutes to form a chitosan-fusion protein gel, freeze-drying, re-dissolving in physiological saline, adding modified hyaluronic acid gel, stirring at 80 rpm for 15 to 25 minutes to obtain a fusion protein; The mass ratio of the modified chitosan to the fusion polypeptide is 1-3:1; The modified chitosan comprises dissolving chitosan in an acid solution, repeatedly freezing and thawing for 3 to 5 times, and freeze-drying to obtain the modified chitosan; The acid solution comprises acetic acid and citric acid, and the volume ratio of the acetic acid to the citric acid is 1-2:8-9; The chitosan is hydroxypropyltrimethylammonium chloride chitosan; The modified hyaluronic acid gel is added in an amount of 30-50 ml per 1 L of normal saline; The preparation method of the modified hyaluronic acid gel comprises the steps of stirring a sodium hyaluronate aqueous solution at 1000 r / min for 40 to 60 minutes for emulsification, and adding sodium tripolyphosphate for cross-linking modification to obtain a modified hyaluronic acid gel; The concentration of sodium hyaluronate in the sodium hyaluronate aqueous solution is 1-2% w / v; The addition amount of the sodium tripolyphosphate is 1% to 1.5% w / v; The cross-linking modification time is 4 to 24 hours.

5. The preparation method according to claim 3, characterized in that: The preparation method of the fusion polypeptide comprises heating a buffer solution containing a connecting polypeptide, collagen and a protein cross-linking agent at 50° C. for 2 to 3 hours, heating at 70° C. for 15 to 20 minutes, cooling, chromatographic separation and purification, and freeze-drying to obtain the fusion polypeptide; The method for preparing the buffer solution containing the connecting polypeptide, collagen and the protein cross-linking agent comprises stirring the buffer solution containing the connecting polypeptide, collagen and the protein cross-linking agent at 180 rpm for 20 to 30 minutes; The mass ratio of the linker polypeptide, collagen, and protein cross-linker is 6-10:4:0.1; The concentration of the linked polypeptide in the buffer is 6-10 mg / mL; The protein cross-linking agent includes transglutaminase; The pH of the buffer solution is 7.2-7.5; The buffer is 10 mM PBS buffer.

6. Use of the fusion protein according to claim 1 or 2 or the fusion protein prepared by the preparation method according to any one of claims 3 to 5 in the preparation of a drug for promoting the repair of endometrial damage.

7. A drug for promoting the repair of endometrial damage, characterized in that: The invention comprises the fusion protein according to claim 1 or 2 or the fusion protein prepared by the preparation method according to any one of claims 3 to 5.

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