Targeted G-CSF connecting polypeptide and application thereof

Abstract

The invention provides a G-CSF targeting connecting polypeptide and application thereof, and relates to the technical field of biological medicine, the G-CSF targeting connecting polypeptide comprises a connecting polypeptide and collagen combined with the C terminal of the connecting polypeptide; the amino acid sequence of the connecting polypeptide is as shown in SEQ ID NO. 1. The C end of the connecting polypeptide is covalently cross-linked to collagen through glutamine transaminase; the N end of the connecting polypeptide is connected with the G-CSF through non-covalent Van der Waals force, so that the structure of the G-CSF cannot be damaged; the fusion polypeptide formed by the connecting polypeptide and the collagen can target the G-CSF and maintain the structure of the G-CSF. The technical problems that in the prior art, a pure G-CSF solution or gel preparation cannot be attached to the surface of the endometrium for a long time, and the action effect is limited are solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a G-CSF-targeted connecting polypeptide and application thereof. Background Art

[0002] The endometrium is the basis for embryo development. A relatively thick endometrium has a better bearing effect. If the endometrium is thin, the implantation rate and pregnancy rate after implantation will be affected. A recognized sign of endometrial receptivity is the thickness of the endometrium. The minimum endometrial thickness for successful embryo transplantation is 7 mm. When it is less than this value, the success rate of embryo transplantation will be greatly affected. In particular, when the endometrial thickness is less than 5 mm, embryo transplantation is usually not possible, and the survival rate of the embryo after transplantation will also be significantly reduced. At present, progesterone is mostly used for the repair of endometrial damage, but the effect is poor. Granulocyte Colony-Stimulating Factor (G-CSF) is a glycoprotein synthesized by vascular endothelial cells, monocytes and fibroblasts. It can promote the maturation of neutrophils, stimulate the release of mature granulocytes from the bone marrow, and enhance the chemotaxis and phagocytosis 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 the blood supply of the endometrium, and increase the thickness of the endometrium. However, a simple G-CSF solution or gel preparation cannot adhere to the surface of the endometrium for a long time, has limited effect, and is difficult to achieve endometrial repair. Therefore, finding a method that can enhance G-CSF to repair endometrial damage and promote the thickening of the damaged endometrium to a normal level is a technical problem that needs to be solved urgently by those skilled in the art. In view of this, the present invention is proposed. Summary of the invention

[0003] One of the purposes of the present invention is to provide a G-CSF-targeted connecting polypeptide to solve the technical problem in the prior art that simple G-CSF solutions or gel preparations cannot adhere to the endometrial surface for a long time and have limited effects.

[0004] The second object of the present invention is to provide the use of the above-mentioned connecting polypeptide in the preparation of a substance for targeting G-CSF.

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

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

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

[0008] A 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 damage.

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

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: In a first aspect, the present invention provides a connecting polypeptide targeting G-CSF, wherein the amino acid sequence of the connecting polypeptide is shown in SEQ ID NO.1.

[0012] In a second aspect, the present invention provides use of the above-mentioned connecting polypeptide in the preparation of a substance for targeting G-CSF.

[0013] In a third aspect, the present invention provides a fusion polypeptide targeting G-CSF, comprising the above-mentioned connecting polypeptide and collagen connected to the C-terminus of the connecting polypeptide.

[0014] In a fourth aspect, the present invention provides a method for preparing the above-mentioned fusion polypeptide, comprising heating a buffer solution containing a connecting polypeptide, collagen and a protein cross-linking agent at 50°C for 2-3 hours, heating at 70°C for 15-20 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 5-8:3:0.1; The concentration of the linked polypeptide in the buffer is 5-8 mg / mL; The protein cross-linking agent includes transglutaminase; The pH of the buffer solution is 7.2-7.5; The buffer is 10mM PBS buffer; The collagen includes human collagen.

[0015] In a 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 bound to the N-terminus of the linker polypeptide in the fusion polypeptide.

[0016] Furthermore, it also includes a chitosan-hyaluronic acid composite carrier.

[0017] In a sixth aspect, the present invention provides a method for preparing the above-mentioned fusion protein, comprising dissolving the fusion polypeptide and G-CSF in physiological saline to obtain the fusion protein; The mass ratio of the fusion polypeptide to G-CSF is 40-60:1; The concentration of the fusion polypeptide in the physiological saline is 0.4-0.6 g / L; The dissolution conditions include stirring at 80 rpm for 30-40 min.

[0018] Furthermore, the method further comprises adding modified chitosan to the physiological saline containing the fusion polypeptide and G-CSF, stirring at 110 rpm for 30 to 50 min 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 min 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.

[0019] 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 damage.

[0020] In an eighth aspect, the present invention provides a drug for promoting the repair of endometrial damage, comprising the above-mentioned fusion protein or the fusion protein prepared by the above-mentioned preparation method.

[0021] The present invention provides a G-CSF-targeting connecting polypeptide. A connecting polypeptide that can target G-CSF is designed and synthesized based on granulocyte colony stimulating factor (abbreviated as G-CSF). The C-terminus of the connecting polypeptide is rich in lysine residues and can be covalently cross-linked to collagen by transglutaminase; the N-terminus of the connecting polypeptide simulates the G-CSF receptor structure and can bind to G-CSF. The connection between the connecting polypeptide and G-CSF is through non-covalent van der Waals force, which will not destroy the structure of G-CSF. The technical problem that a simple G-CSF solution or gel preparation in the prior art cannot be attached to the endometrial surface for a long time and has limited effect is solved.

[0022] Another aspect of the present invention provides a fusion protein, which is formed by combining the N-terminus of the fusion polypeptide with G-CSF. The fusion protein not only has a longer adhesion ability in the endometrium, but also can maintain the activity of G-CSF, thereby improving the effect of repairing endometrial damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The cytotoxicity test results of different fusion proteins provided in Experimental Example 1 of the present invention; Figure 2 The test results of the retention concentration of different fusion proteins in the endometrium at different times provided in Experimental Example 2 of the present invention; Figure 3 This is a staining comparison diagram of uterine sections treated with the fusion protein provided in Experimental Example 3 of the present invention; Figure 4 A comparison chart of the number of endometrial mitochondria treated with the fusion protein provided in Experimental Example 3 of the present invention; Figure 5 This is a comparison chart of the endometrial fibrosis ratio treated with the fusion protein provided in Experimental Example 3 of the present invention; Figure 6 The test results of the retention concentrations of different fusion proteins in the endometrium at different times provided in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0025] 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 meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0026] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0027] In one aspect, the present invention provides a G-CSF-targeting connecting polypeptide, the amino acid sequence of the connecting polypeptide is shown in SEQ ID NO.1.

[0028] Based on granulocyte colony stimulating factor (abbreviated as G-CSF), a connecting polypeptide that can target G-CSF was designed and synthesized. The C-terminus of the connecting polypeptide is rich in lysine residues and can be covalently cross-linked to collagen through glutamine transaminase; the N-terminus of the connecting polypeptide simulates the G-CSF receptor structure and can bind to G-CSF. The connection between the connecting polypeptide and G-CSF is through non-covalent van der Waals force, which will not destroy the structure of G-CSF. This solves the technical problem that the simple G-CSF solution or gel preparation in the prior art cannot adhere to the surface of the endometrium for a long time and has limited effect.

[0029] Experiments have shown that the fusion polypeptide formed by connecting the polypeptide and collagen can target G-CSF and maintain the structure of G-CSF, which can not only prolong the attachment time in the endometrium, but also does not affect the activity of G-CSF. According to another aspect of the present invention, the use of the above-mentioned connecting polypeptide in the preparation of a substance for targeting G-CSF is also provided.

[0030] According to another aspect of the present invention, there is also provided a fusion polypeptide targeting G-CSF, comprising the above-mentioned connecting polypeptide and collagen connected to the C-terminus of the connecting polypeptide.

[0031] The C-terminus of the connecting polypeptide is rich in lysine residues and can be covalently cross-linked to collagen through glutamine transaminase to form a linker-collagen fusion protein, which can prolong the attachment time of G-CSF in the endometrium without affecting the activity of G-CSF, and effectively promote endometrial thickening and endometrial damage repair.

[0032] According to another aspect of the present invention, a method for preparing a fusion polypeptide targeting G-CSF is also provided, comprising 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.

[0033] The C-terminus of the connecting polypeptide is covalently cross-linked to collagen through a protein cross-linking agent to form a linker-collagen fusion protein, which can prolong the attachment time of G-CSF in the endometrium without affecting the activity of G-CSF, and effectively promote endometrial thickening and endometrial damage repair.

[0034] Specifically, the chromatographic separation and purification can be carried out by G25 molecular sieve chromatographic separation and purification.

[0035] In some specific embodiments, the method for preparing the buffer containing the linker polypeptide, collagen and protein crosslinker comprises stirring the buffer containing the linker polypeptide, collagen and protein crosslinker at 180 rpm for 20 to 30 minutes, so that the linker polypeptide and collagen are fully in contact, and covalent crosslinking is completed under the action of the protein crosslinker.

[0036] The stirring time may 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 and 30 min.

[0037] In some specific embodiments, the mass ratio of the connecting polypeptide, collagen and protein cross-linking agent is 5-8:3:0.1, thereby improving the success rate of connecting collagen to the connecting polypeptide and avoiding the occurrence of collagen that is not covalently connected to the connecting polypeptide.

[0038] The mass ratio of the connecting polypeptide, collagen and protein cross-linking agent may 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 and 8:3:0.1.

[0039] In some specific embodiments, the concentration of the linked polypeptide in the buffer is 5-8 mg / mL.

[0040] The concentration of the linked polypeptide in the buffer may 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, or any value between 5 and 8 mg / mL.

[0041] In some specific embodiments, the protein cross-linking agent comprises transglutaminase.

[0042] In some specific embodiments, the pH of the buffer is 7.2 to 7.5. In some specific embodiments, the buffer is 10 mM PBS buffer.

[0043] The pH of the buffer solution may be, but is not limited to, 7.2, 7.3, 7.4 or 7.5, or any value between 7.2 and 7.5, preferably 7.4.

[0044] In some specific embodiments, the collagen comprises human collagen.

[0045] The 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. The human collagen is preferably human type III collagen.

[0046] According to another aspect of the present invention, a fusion protein is provided, comprising the above fusion polypeptide or the fusion polypeptide prepared by the above preparation method, and G-CSF bound to the N-terminus of the linker polypeptide in the fusion polypeptide.

[0047] By combining the N-terminus of the fusion polypeptide with G-CSF, the resulting fusion protein not only has a longer attachment ability in the endometrium, but can also maintain the activity of G-CSF, thereby improving the effect of repairing endometrial damage.

[0048] In order to further improve the activity and adhesion of the fusion protein on the endometrium, in some specific embodiments, a chitosan-hyaluronic acid composite carrier is also included.

[0049] Chitosan-hyaluronic acid can form a gel network structure to load the fusion protein to improve the adhesion of the fusion protein. Through gradual release, the time for the fusion protein to act on the autologous inner membrane is prolonged; the gel network wraps the fusion protein macromolecules to avoid degradation and maintain its activity.

[0050] According to another aspect of the present invention, there is also provided a method for preparing the above-mentioned fusion protein, comprising dissolving the fusion polypeptide and G-CSF in physiological saline to obtain the fusion protein.

[0051] The fusion protein obtained by this preparation method can exist in the form of a protein solution or in the form of a fusion protein solid through freeze-drying. It can form a stable repair microenvironment on the surface of the endometrium, promote the repair of endometrial damage, restore the number of endometrial glands with intrauterine adhesions, and reduce the proportion of fibrosis. It has the beneficial effects of high tissue compatibility, simple preparation, obvious improvement effect, etc., has no potential toxic effects on cells, and has good clinical application prospects.

[0052] In some specific embodiments, the mass ratio of the fusion polypeptide to G-CSF is 40-60:1.

[0053] The mass ratio of the fusion polypeptide to G-CSF may 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 any value between 40 and 60:1.

[0054] In some specific embodiments, the concentration of the fusion polypeptide in the physiological saline is 0.4-0.6 g / L.

[0055] The concentration of the fusion polypeptide in the physiological saline may 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, and may be any value between 0.4 and 0.6 g / L.

[0056] In some specific embodiments, the dissolution conditions include stirring at 80 rpm for 30 to 40 min.

[0057] In some specific embodiments, the method further comprises adding modified chitosan to the physiological saline containing the fusion polypeptide and G-CSF, stirring at 110 rpm for 30 to 50 min 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 min to obtain a fusion protein.

[0058] In some specific embodiments, the mass ratio of the modified chitosan to the fusion polypeptide is 1-3:1.

[0059] In some specific embodiments, 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.

[0060] In some specific embodiments, the acid solution includes acetic acid and citric acid, and the volume ratio of the acetic acid to the citric acid is 1-2:8-9.

[0061] In some specific embodiments, the chitosan is hydroxypropyltrimethylammonium chloride chitosan.

[0062] In some specific embodiments, the modified hyaluronic acid gel is added in an amount of 30 to 50 ml per 1 L of normal saline.

[0063] In some specific embodiments, the preparation method of the modified hyaluronic acid gel comprises stirring the 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 the modified hyaluronic acid gel.

[0064] In some specific embodiments, the concentration of sodium hyaluronate in the sodium hyaluronate aqueous solution is 1-2% w / v.

[0065] In some specific embodiments, the amount of sodium tripolyphosphate added is 1% to 1.5% w / v.

[0066] In some specific embodiments, the cross-linking modification time is 4 to 24 hours.

[0067] According to another aspect of the present invention, there is also provided the use of the above fusion protein or the fusion protein prepared by the above preparation method in the preparation of a drug for promoting the repair of endometrial damage.

[0068] According to another aspect of the present invention, there is also provided a drug for promoting the repair of endometrial damage, comprising the above-mentioned fusion protein or the fusion protein prepared by the above-mentioned preparation method.

[0069] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0070] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0071] Example 1 Based on G-CSF, connecting peptides-1 to -6 that can connect G-CSF and collagen were designed and synthesized. Each connecting peptide was synthesized in a directional manner according to the peptide sequence using amino acids as raw materials and solid phase synthesis.

[0072] Amino acid sequence of connecting polypeptide-1 (SEQ ID NO.1): NTGAIPPPSDFHNNRYPGVSNVPGGHSTSMFFRVVESAYLNLSSEPNLIPEVTVGEGLNLGVMVEAYPGLPGFNWTYLGFSDHPPEPGLANATTGDTYEPFHGVTVPSLLTVETLEHNAFGLGGEDAVLGVAKGKLGSTAHKDEKEALGAKTHKKKVLKKTEYKKYKDLLNFLKKGAE.

[0073] Amino acid sequence of connecting polypeptide-2 (SEQ ID NO.2): ECGHISVSAPIVHLGDPITPPSASNSNTWRMENGRATGFASCIINCSHLDPEPILWRLPPSASNSNTWRMENGRATGFGAELPGGRRLSDGTESIITLLILDVELRAGYPPAIPHNLSCLMNLT.

[0074] Amino acid sequence of connecting polypeptide-3 (SEQ ID NO.3): TSSLICWEPGPETHLPTSFTPHLNHTAFLSCCLNWGNSLSFSHCCIPRHLLLYNMGIWVAENALGTPHLNHTAFLSCCLNWGNSSMSPLCLDPMDAPPA.

[0075] Amino acid sequence of connecting polypeptide-4 (SEQ ID NO.4): CLLCWYVVSWRPSGAGAILPLCNTTELSCTFHLWALVGCIRWPLPGHWPSGAGAILPLCNTTELSKKCTFHKKKLPSTTETKKWRRLDKKKLF.

[0076] Amino acid sequence of connecting polypeptide-5 (SEQ ID NO.5): PVPLEEDSGRIGYVVLSCASSRGFVLHGLEPTFHLPSEAEVGTSRASSRGFVLHGLEPPTPHAMARDPHSLKKWVGKKWEPKKPNPKKKWPG.

[0077] Amino acid sequence of connecting polypeptide-6 (SEQ ID NO.6): VIEWGLGPPENGRAASSRGFVLHGLEPTGFLLENIRPFLYEIIVTPLYDTMAPSHAPELHLHIGTWALEWVPEPPELGSPKKLTHYKKTIFKKWTKKN.

[0078] Amino acid sequence of connecting polypeptide-7 (SEQ ID NO.7): NSFSAILNASSRGFATGFASCIINCVLHGLEPASLYHIHLMAASAATGFASCIINCSSRGFVLHGLEPAGATNSTVLKTKKKLMKKKTLTPEKKKGSEKKLG.

[0079] Embodiment 2 to Embodiment 8 A fusion polypeptide comprises a connecting polypeptide and a collagen protein bound to the C-terminus of the connecting polypeptide. The connecting polypeptides-1 to -6 provided in Example 1 are respectively selected to prepare the fusion polypeptide according to the following steps: (1) Add the linker peptide, human type III collagen and transglutaminase to a pH 7.4 10 mM PBS buffer, wherein the amount ratio of PBS buffer, linker peptide, 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; (2) heating the mixed solution obtained in step (1) to 50°C for 3 h, then heating to 70°C for 20 min, and naturally cooling to room temperature to obtain a post-reaction solution; (3) The reaction solution of step (2) is separated and purified by G25 molecular sieve chromatography and freeze-dried to obtain a connected polypeptide-collagen (fusion polypeptide).

[0080] Among them, human type III collagen is obtained by the method disclosed in Chinese patent CN103102407A.

[0081] Embodiments 9 to 15 A fusion protein includes a fusion polypeptide and G-CSF bound to the N-terminus of a connecting polypeptide in the fusion polypeptide. The fusion polypeptides are selected from the fusion polypeptides prepared in Examples 2 to 7, respectively, and the fusion protein is prepared according to the following steps: The fusion polypeptide and G-CSF were added to the physiological saline, and stirred at 80 rpm for 30 min. The dosage ratio of the physiological saline, the fusion polypeptide and G-CSF was 1 L:0.5 g:0.01 g to obtain a protein solution that promotes the repair of endometrial damage.

[0082] Comparative Example 1 The difference from Example 7 is that human type III collagen not connected to the connecting polypeptide is used instead of the fusion polypeptide, human type III collagen and G-CSF are added to normal saline, and the mixture is stirred at 80 rpm for 30 min. The dosage ratio of normal saline, human type III collagen and G-CSF is 1 L:0.5 g:0.01 g, and a protein solution that promotes the repair of endometrial damage is obtained.

[0083] Experimental Example 1 This experimental example uses the method of preparing a protein solution for promoting the repair of endometrial damage according to Examples 9 to 15 and Comparative Example 1, and replaces the physiological saline with a MEM culture medium containing 10% fetal bovine serum to obtain an experimental solution, and performs the following operations respectively: L-929 cells (NCTC clone 929) were cultured in MEM medium containing 10% fetal bovine serum and antibiotics (penicillin 100 U / mL, streptomycin 100 µg / mL) at 37°C in a 5% CO2 incubator. The cells were digested with 0.25% trypsin, centrifuged at 200 g for 3 min, and then redispersed in fresh medium to adjust the cell density to 2×10 5 cells / mL cell suspension; inoculate the above cell suspension into a 96-well culture plate, 100 μL per well, place 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 as the experimental group, set up a well with only 200 μL of MEM medium containing 10% fetal bovine serum as the blank control group, and a well with 100 μL of medium + 100 μL of cells as the negative control. After adding the experimental solution, continue to culture for 5 days, add 20 μL of MTT (5 mg / mL prepared in PBS) to each well in the dark every day, and continue to culture for 4 h after adding MTT on the last day, remove all the liquid in the well, add 150 μL of DMSO solution to each well, shake at low speed for 15 min in an ELISA reader, and measure its OD value at λ=490 nm. The cytotoxicity was determined by calculating the relative growth rate (RGR, %): RGR = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%. The specific toxicity grading standards are: Level 0: RGR ≥ 100%; Level 1: 99% ≥ RGR ≥ 75; Level 2: 74 ≥ RGR ≥ 50; Level 3: 49 ≥ RGR ≥ 25; Level 4: 24 ≥ RGR ≥ 1; Level 5 = 0. Levels 2 and above can be considered as cytotoxic reactions. The results are shown in Figure 1 shown.

[0084] Depend on Figure 1It can be seen that the toxicity level of the protein solutions of Examples 9 to 13 and Comparative Example 1 is 0, indicating that the protein solutions of Examples 9 to 13 are non-cytotoxic and have high biological safety. However, the toxicity of Examples 14 and 15 is level 1, indicating that the protein solutions prepared using Connecting Peptide-6 and Connecting Peptide-7 are cytotoxic.

[0085] Experimental Example 2 The protein solutions prepared in Examples 9 to 13 and Comparative Example 1, which have biological safety, were respectively selected to conduct rat experiments according to the following operations to detect the attachment duration of the fusion protein in the endometrial injury rat model.

[0086] 1. Experimental animals: 60 female SD rats aged 8-10 weeks and weighing 220-260 g were selected and adaptively raised for one week.

[0087] 2. Experimental groups: Rats were randomly divided into 6 groups, with 10 rats in each group.

[0088] 3. Modeling of rats with endometrial injury: The modeling was induced by mechanical injury and ethanol perfusion. The rats were fasted and deprived of water for 12 h before the operation. After intraperitoneal injection of 2% sodium pentobarbital for anesthesia, they were fixed in the supine position. A transverse incision was made on the abdomen to expose the uterine horns. A longitudinal incision of about 4 mm was made about 0.5 cm below the uterine horns. The middle and upper endometrium was scraped with an endometrial scraper with a diameter of 2.5 mm. The curettage was stopped when the roughness of the uterine walls was felt. The uterine horns were clamped with a vascular clamp, and 95% ethanol was extracted with a 1 mL syringe and injected downward from the incision until the uterine cavity was filled. Then the incision was temporarily clamped with a vascular clamp. After 3 minutes, the vascular clamp at the incision was released to discharge the residual ethanol, and the uterine surface liquid was dried with sterile gauze.

[0089] 4. Drug administration: Immediately after modeling, 0.5 mL of protein solution was injected into the uterine cavity. After the operation, the abdominal cavity was flushed with sterile saline. After the uterus was repositioned, the abdomen was closed layer by layer. The incision was disinfected with 75% alcohol. After surgery, 2 mL of gentamicin was injected intramuscularly every day to prevent infection. One rat in each group was killed at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after surgery, and the uterus was collected. The drug concentration of G-CSF in the uterus was detected by ELISA. The results are as follows: Figure 2 shown.

[0090] Depend on Figure 2 It can be seen that the concentration of G-CSF in Example 9 group is significantly higher than that in Examples 10 to 13 and Comparative Example 1, and the rate of decrease is also lower, indicating that linker polypeptide-1 can effectively prolong the action time of G-CSF and promote the repair of uterine damage.

[0091] Experimental Example 3 This experimental example selected the protein solutions prepared in Example 9 and Comparative Example 1, and performed the following steps to verify the repair effect of the protein solutions on endometrial damage.

[0092] 1. Experimental animals: 40 female SD rats aged 8-10 weeks and weighing 220-260 g were selected and adaptively raised for one week.

[0093] 2. Experimental grouping: Rats were randomly divided into 4 groups, 10 rats in each group, including 2 experimental groups, 1 sham operation group, and 1 model group. The experimental and model groups were induced by mechanical injury and ethanol perfusion. The modeling method was the same as that in Experimental Example 2. The sham operation group was anesthetized and laparotomy was performed. The uterine horns were fully exposed to the body and then retracted. No other operations were performed. The uterus was rinsed and cleaned with saline, and the liquid on the surface of the uterus was dried with sterile gauze.

[0094] 3. Administration: After modeling, each experimental group was immediately given 0.5 mL of the protein solution of Example 9 and Comparative Example 1 to perfuse the uterine cavity, and the sham operation group and the model group were given an equal amount of normal saline. After the operation, the abdominal cavity was flushed with sterile normal saline. After the uterus was repositioned, the abdomen was closed layer by layer, and the incision was disinfected with 75% alcohol. After the operation, 2 mL of gentamicin was injected intramuscularly for 3 consecutive days to prevent infection. Each group was sacrificed 14 days after the operation, and the uterine tissue was taken, fixed with paraformaldehyde, and paraffin sectioned, and stained with HE and Masson. Figure 3 The number of endometrial glands and the proportion of endometrial fibrosis were statistically analyzed, and the statistical results are shown in Figure 4 , Figure 5 shown.

[0095] Depend on Figure 3~Figure 5 It can be seen that the sham operation group had normal endometrial structure, a large number of glands, and a normal fibrosis ratio; the model group had severe intrauterine adhesions, a significantly reduced number of glands, and an increased fibrosis ratio; the experimental group had lower intrauterine adhesions than the model group, the number of glands recovered, and the fibrosis ratio was lower than the model group, and close to 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 using G-CSF and collagen alone. The connected polypeptide can effectively improve the repair effect of endometrial damage and promote the repair of endometrial damage.

[0096] Example 16 A fusion protein comprises a fusion polypeptide, G-CSF bound to the N-terminus of a connecting polypeptide in the fusion polypeptide, and a chitosan-hyaluronic acid composite carrier. The fusion polypeptide is selected from the fusion polypeptide provided in Example 2 and is prepared according to the following steps: 1. Modified chitosan: Hydroxypropyltrimethylammonium chloride chitosan was dissolved in an acid solution to obtain a 4 wt% chitosan solution, which was placed in a -20°C refrigerator and frozen for 12 h, then thawed at room temperature for 12 h, and repeated freezing and thawing for 5 times, pre-frozen at -20°C for 4 h, and freeze-dried at -80°C for 24 h to obtain modified chitosan, wherein the acid solution was prepared by mixing acetic acid and citric acid in a volume ratio of 1.4:8.6.

[0097] 2. Modified hyaluronic acid gel: 2% sodium hyaluronate aqueous solution was stirred at 1000 r / min for 40-60 min for emulsification, and 1.3% sodium tripolyphosphate was added for cross-linking modification for 12 h to obtain modified hyaluronic acid gel.

[0098] 3. Add fusion polypeptide and G-CSF to saline, stir at 80 rpm for 30 min to dissolve, the dosage ratio of saline, fusion polypeptide and G-CSF is 1 L: 0.5 g: 0.01 g. Add modified chitosan to the saline containing fusion polypeptide and G-CSF, the dosage of modified chitosan is 1 g per 1L saline, stir at 110 rpm for 40 min to form chitosan-fusion protein gel, pre-freeze at -20°C for 4 h, freeze-dry at -80°C for 24 h, redissolve in saline, add modified hyaluronic acid gel, the dosage of modified hyaluronic acid gel is 40 ml per 1L saline, stir at 80 rpm for 20 min, and obtain a protein solution that promotes the repair of endometrial damage.

[0099] Comparative Example 2 Different from Example 4, in step 3, the fusion polypeptide and G-CSF are added to the physiological saline, stirred at 80 rpm for 30 minutes to dissolve, and the amount ratio of physiological saline, fusion polypeptide and G-CSF is 1 L: 0.5 g: 0.01 g. Modified chitosan is added to the physiological saline containing the fusion polypeptide and G-CSF, and the amount of modified chitosan is 1 g per 1L of physiological saline, stirred at 110 rpm for 40 minutes to form a chitosan-fusion protein gel, pre-frozen at -20°C for 4 hours, freeze-dried at -80°C for 24 hours, and redissolved in physiological saline to obtain a protein solution that promotes the repair of endometrial damage.

[0100] Comparative Example 3 Different from Example 4, the fusion polypeptide and G-CSF were added to the physiological saline, stirred at 80 rpm for 30 min to dissolve, and the dosage ratio of physiological saline, fusion polypeptide and G-CSF was 1 L: 0.5 g: 0.01 g. Pre-freeze at -20°C for 4 h, freeze-dry at -80°C for 24 h, redissolve in physiological saline, 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, and obtain a protein solution that promotes the repair of endometrial damage.

[0101] Comparative Example 4 The difference from Example 4 is that modified hyaluronic acid gel is added to the physiological saline containing the fusion polypeptide and G-CSF, the amount of modified hyaluronic acid gel is 40 ml per 1L of physiological saline, the mixture is stirred at 110 rpm for 40 min, pre-frozen at -20°C for 4 h, freeze-dried at -80°C for 24 h, redissolved in physiological saline, modified chitosan is added, the amount of modified chitosan is 1 g per 1L of physiological saline, and stirred at 80 rpm for 20 min to obtain a protein solution for promoting the repair of endometrial damage.

[0102] Experimental Example 4 In order to prove that the protein solutions prepared in Example 16 and Comparative Examples 2 to 4 can further prolong the attachment time of G-CSF on the endometrium, the protein solutions prepared in Example 9, Example 16 and Comparative Examples 2 to 4 were selected to carry out rat experiments according to the method of Experimental Example 2.

[0103] Fifty female SD rats aged 8-10 weeks and weighing 220-260 g were selected and adaptively raised for one week. The rats were randomly divided into 5 groups, with 10 rats in each group. Immediately after modeling, 0.5 mL of protein solution was perfused into the uterine cavity. After the operation, the abdominal cavity was flushed with sterile saline. After the uterus was repositioned, the abdomen was closed layer by layer, and the incision was disinfected with 75% alcohol. After surgery, 2 mL of gentamicin was injected intramuscularly every day to prevent infection. One rat in each group was killed at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after surgery, and the uterus was collected. The G-CSF drug concentration in the uterus was detected by ELISA. The results are as follows: Figure 6 shown.

[0104] Depend on Figure 6 It can be seen that compared with Example 9, the G-CSF drug concentration in Example 16 was improved during the period of 12h to 72h, indicating that the chitosan-hyaluronic acid carrier formed by adding modified chitosan and modified hyaluronic acid gel can further prolong 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 12h, but the detection after 12h showed a rapid downward trend, and was lower than that in Example 9; Comparative Example 2 was lower than Example 9 as a whole; Comparative Example 3 was not much different from Example 9 during the period of 12~60h, but rapidly decreased at 72h. 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.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A G-CSF-targeting connecting polypeptide, characterized in that: The amino acid sequence of the connecting polypeptide is shown in SEQ ID NO.

1.

2. Use of the connecting polypeptide according to claim 1 in preparing a substance for targeting G-CSF.

3. A fusion polypeptide targeting G-CSF, characterized in that: The invention comprises the connecting polypeptide according to claim 1 and collagen connected to the C-terminus of the connecting polypeptide.

4. The method for preparing the fusion polypeptide according to claim 3, characterized in that: The method 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 a 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 10mM PBS buffer; The collagen includes human collagen.

5. A fusion protein, characterized in that The method comprises the fusion polypeptide according to claim 3 or the fusion polypeptide prepared by the preparation method according to claim 4, and G-CSF bound to the N-terminus of the connecting polypeptide in the fusion polypeptide.

6. The fusion protein according to claim 5, characterized in that It also includes a chitosan-hyaluronic acid composite carrier.

7. The method for preparing the fusion protein according to claim 5 or 6, 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.

8. The preparation method according to claim 7, 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.

9. Use of the fusion protein according to claim 5 or 6 or the fusion protein prepared by the preparation method according to claim 7 or 8 in the preparation of a drug for promoting the repair of endometrial damage.

10. A drug for promoting the repair of endometrial damage, characterized in that: The invention comprises the fusion protein according to claim 5 or 6 or the fusion protein prepared by the preparation method according to claim 7 or 8.

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