A connecting polypeptide targeting bFGF and its application

Abstract

The present invention provides a connecting polypeptide targeting bFGF and its application, relating to the field of biomedicine technology, comprising a connecting polypeptide and a collagen protein bound to the C-terminus of the connecting polypeptide; the amino acid sequence of the connecting polypeptide is shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3. The C-terminus of the connecting polypeptide is covalently cross-linked to the collagen protein via transglutaminase; the connection between the N-terminus of the connecting polypeptide and bFGF is via non-covalent van der Waals forces, which does not destroy the structure of bFGF; the fusion polypeptide formed by the connecting polypeptide and collagen can target bFGF and maintain the structure of bFGF. This solves the technical problem in the prior art that direct cross-linking of bFGF and collagen affects the molecular structure of bFGF, resulting in a decrease in bFGF activity and thus poor therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a connecting polypeptide targeting bFGF and applications thereof. Background Art

[0002] The endometrium is the foundation for embryo development. A relatively thick endometrium provides better support. A thin endometrium compromises both implantation and post-implantation pregnancy rates. Endometrial thickness is a recognized marker of endometrial receptivity. The minimum endometrial thickness for successful embryo transfer is 7 mm. Below this value, embryo transfer success is significantly compromised. In particular, an endometrial thickness less than 5 mm generally precludes embryo transfer, and embryo survival is significantly reduced. Endometrial damage is reported to occur in approximately one-fifth of infertile women. Currently, progestogen-derived estrogens are commonly used to repair endometrial damage, but this treatment is ineffective. Basic fibroblast growth factor (bFGF) is a polypeptide that transmits developmental signals and promotes cell division in the mesoderm and neuroectoderm. It has strong angiogenic effects and, in vitro, stimulates cell proliferation and migration, induces plasminogen activator and collagenase activity, and is a mitogen with a high affinity for heparin. Reports indicate that bFGF plays a crucial role in endometrial angiogenesis, endometrial re-epithelialization, vascular function, and stromal cell proliferation during the menstrual cycle. Exogenous administration of bFGF can promote rapid tissue vascularization and re-epithelialization. However, simple bFGF solutions or gel formulations are unable to adhere to the endometrial surface for extended periods, resulting in limited efficacy and difficulty in achieving endometrial repair. Reports suggest that cross-linking bFGF with collagen can extend its duration of action, but direct cross-linking can affect bFGF's molecular structure, leading to decreased bFGF activity and consequently poor therapeutic efficacy. Therefore, finding a method to enhance bFGF's ability to repair endometrial damage and promote the thickening of the damaged endometrium to normal levels is a pressing technical issue for those skilled in the art.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a connecting polypeptide targeting bFGF to solve the technical problem in the prior art that direct cross-linking of bFGF and collagen affects the molecular structure of bFGF, resulting in decreased bFGF activity and thus poor therapeutic effect.

[0005] A 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 bFGF.

[0006] A third object of the present invention is to provide a fusion polypeptide targeting bFGF.

[0007] A fourth object of the present invention is to provide a method for preparing the above-mentioned fusion polypeptide.

[0008] A fifth object of the present invention is to provide a fusion protein.

[0009] A sixth object of the present invention is to provide a method for preparing the above-mentioned fusion protein.

[0010] A 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.

[0011] An eighth object of the present invention is to provide a drug for promoting the repair of endometrial damage.

[0012] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0013] In a first aspect, the present invention provides a connecting polypeptide targeting bFGF, wherein the amino acid sequence of the connecting polypeptide is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0014] In a second aspect, the present invention provides use of the aforementioned connecting polypeptide in the preparation of a substance for targeting bFGF.

[0015] In a third aspect, the present invention provides a fusion polypeptide targeting bFGF, comprising the aforementioned connecting polypeptide and collagen connected to the C-terminus of the connecting polypeptide.

[0016] In a fourth aspect, the present invention provides a method for preparing a fusion polypeptide targeting bFGF, comprising heating a buffer solution containing a linker polypeptide, collagen, and a protein cross-linking agent at 50°C for 2-3 hours, then heating at 70°C for 15-20 minutes, cooling, purifying by chromatography, and freeze-drying to obtain the fusion polypeptide;

[0017] The preparation method of the buffer solution containing the connecting polypeptide, collagen and protein cross-linking agent comprises stirring the buffer solution containing the connecting polypeptide, collagen and protein cross-linking agent at 180 rpm for 20 to 30 minutes;

[0018] The mass ratio of the linker peptide, collagen, and protein cross-linker is 5-8:3:0.1;

[0019] The concentration of the linked polypeptide in the buffer is 5-8 mg / mL;

[0020] The protein cross-linking agent includes transglutaminase;

[0021] The pH of the buffer solution is 7.2-7.5;

[0022] The buffer solution is 10 mM PBS buffer solution;

[0023] The collagen includes human collagen.

[0024] In a fifth aspect, the present invention provides a fusion protein comprising the fusion polypeptide prepared by the above-mentioned preparation method and bFGF bound to the N-terminus of the linker polypeptide in the fusion polypeptide.

[0025] Furthermore, it also includes a chitosan-hyaluronic acid composite carrier.

[0026] In a sixth aspect, the present invention provides a method for preparing the above-mentioned fusion protein, comprising dissolving the fusion polypeptide and bFGF in physiological saline to obtain the fusion protein;

[0027] The mass ratio of the fusion polypeptide to bFGF is 40-60:1;

[0028] The concentration of the fusion polypeptide in the physiological saline is 0.4-0.6 g / L;

[0029] The dissolution conditions include stirring at 80 rpm for 30-40 min.

[0030] Furthermore, the method further comprises adding modified chitosan to the physiological saline containing the fusion polypeptide and bFGF, stirring at 110 rpm for 30 to 50 minutes to form a chitosan-fusion protein gel, freeze-drying, redissolving in physiological saline, adding modified hyaluronic acid gel, stirring at 80 rpm for 15 to 25 minutes to obtain a fusion protein;

[0031] The mass ratio of the modified chitosan to the fusion polypeptide is 1-3:1;

[0032] 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;

[0033] 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;

[0034] The chitosan is hydroxypropyltrimethylammonium chloride chitosan;

[0035] The amount of the modified hyaluronic acid gel added is 30-50 ml per 1 L of normal saline;

[0036] The preparation method of the modified hyaluronic acid gel comprises emulsifying a sodium hyaluronate aqueous solution by stirring at 1000 r / min for 40 to 60 minutes, and adding sodium tripolyphosphate for cross-linking modification to obtain a modified hyaluronic acid gel;

[0037] The concentration of sodium hyaluronate in the sodium hyaluronate aqueous solution is 1-2% w / v;

[0038] The addition amount of the sodium tripolyphosphate is 1% to 1.5% w / v;

[0039] The cross-linking modification time is 4 to 24 hours.

[0040] 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.

[0041] 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.

[0042] The present invention provides a bFGF-targeting linker polypeptide. This bFGF-targeting linker polypeptide was designed and synthesized based on basic fibroblast growth factor (bFGF). The C-terminus of the linker polypeptide is rich in lysine residues and can be covalently cross-linked to collagen via transglutaminase. The N-terminus of the linker polypeptide mimics the bFGF receptor structure, allowing it to bind to bFGF. The linker polypeptide and bFGF are linked via non-covalent van der Waals forces, which do not disrupt the bFGF structure. This solves the existing technical problem that direct cross-linking of bFGF with collagen affects the molecular structure of bFGF, leading to decreased bFGF activity and thus poor therapeutic effects.

[0043] Another aspect of the present invention provides a fusion protein, which is formed by combining the N-terminus of the fusion polypeptide with bFGF. The fusion protein not only has a long adhesion ability in the endometrium, but also can maintain the activity of bFGF, thereby improving the effect of repairing endometrial damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 The cytotoxicity test results of different fusion proteins provided in Experimental Example 1 of the present invention;

[0046] 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;

[0047] Figure 3This is a comparison diagram of uterine section staining treated with the fusion protein provided in Experimental Example 3 of the present invention;

[0048] Figure 4 This is a comparison chart of the number of endometrial glands treated with the fusion protein provided in Experimental Example 3 of the present invention;

[0049] 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;

[0050] Figure 6 The test results of the retention concentration of different fusion proteins in the endometrium at different times provided in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0051] 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 event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0052] 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.

[0053] In one aspect, the present invention provides a connecting polypeptide targeting bFGF, wherein the amino acid sequence of the connecting polypeptide is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0054] Based on basic fibroblast growth factor (bFGF), a linker peptide targeting bFGF was designed and synthesized. The C-terminus of the linker peptide is rich in lysine residues and can be covalently cross-linked to collagen via transglutaminase. The N-terminus of the linker peptide mimics the structure of the bFGF receptor, allowing it to bind to bFGF. The connection between the linker peptide and bFGF is achieved through non-covalent van der Waals forces, which do not disrupt the bFGF structure. This solves the technical problem in existing technologies where direct cross-linking of bFGF with collagen affects the molecular structure of bFGF, resulting in decreased bFGF activity and poor therapeutic effects.

[0055] Experiments have demonstrated that a fusion polypeptide formed by linking a polypeptide and collagen can target bFGF while maintaining its structure, prolonging its attachment time to the endometrium without affecting bFGF activity. Another aspect of the present invention provides the use of the aforementioned linking polypeptide in the preparation of a substance for targeting bFGF.

[0056] According to another aspect of the present invention, a fusion polypeptide targeting bFGF is provided, comprising the above-mentioned connecting polypeptide and collagen connected to the C-terminus of the connecting polypeptide.

[0057] The C-terminus of the connecting polypeptide is rich in lysine residues and can be covalently cross-linked to collagen through transglutaminase to form a linker-collagen fusion protein, which can prolong the attachment time of bFGF in the endometrium without affecting the activity of bFGF, effectively promoting endometrial thickening and endometrial damage repair.

[0058] According to another aspect of the present invention, a method for preparing the above-mentioned fusion polypeptide is also provided, comprising heating a buffer solution containing a connecting polypeptide, collagen and a protein cross-linking agent at 50°C for 2-3 hours, then heating at 70°C for 15-20 minutes, cooling, purifying by chromatography, and freeze-drying to obtain the fusion polypeptide.

[0059] The C-terminus of the connecting polypeptide is covalently cross-linked to collagen through a protein cross-linker to form a linker-collagen fusion protein, which can prolong the attachment time of bFGF in the endometrium without affecting the activity of bFGF, effectively promoting endometrial thickening and endometrial damage repair.

[0060] Specifically, the chromatographic separation and purification can be performed by G25 molecular sieve chromatographic separation and purification.

[0061] In some specific embodiments, the method for preparing the buffer solution containing the linker polypeptide, collagen, and protein cross-linking agent comprises stirring the buffer solution containing the linker polypeptide, collagen, and protein cross-linking agent at 180 rpm for 20-30 minutes to allow the linker polypeptide to fully contact the collagen and achieve covalent cross-linking under the action of the protein cross-linking agent.

[0062] 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.

[0063] In some specific embodiments, the mass ratio of the linker polypeptide, collagen, and protein cross-linker is 5-8:3:0.1, thereby increasing the success rate of collagen-linking linker polypeptides and preventing the occurrence of collagen that is not covalently linked to the linker polypeptides.

[0064] The mass ratio of the connecting 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 and 8:3:0.1.

[0065] In some specific embodiments, the concentration of the linked polypeptide in the buffer is 5-8 mg / mL.

[0066] 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, and may be any value between 5 and 8 mg / mL.

[0067] In some specific embodiments, the protein cross-linking agent comprises transglutaminase.

[0068] 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.

[0069] 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.

[0070] In some specific embodiments, the collagen comprises human collagen.

[0071] 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.

[0072] According to another aspect of the present invention, a fusion protein is provided, comprising the above-mentioned fusion polypeptide or the fusion polypeptide prepared by the above-mentioned preparation method, and bFGF bound to the N-terminus of the linker polypeptide in the fusion polypeptide.

[0073] By combining the N-terminus of the fusion polypeptide with bFGF, the resulting fusion protein not only has a longer attachment ability in the endometrium, but can also maintain the activity of bFGF, thereby improving the effect of repairing endometrial damage.

[0074] In order to further improve the activity and adhesion of the fusion protein to the endometrium, in some specific embodiments, a chitosan-hyaluronic acid composite carrier is also included.

[0075] Chitosan-hyaluronic acid can form a gel network structure to load the fusion protein to improve the adhesion of the fusion protein and prolong the time the fusion protein acts on the endometrium through gradual release; the gel network wraps the fusion protein macromolecules to avoid degradation and maintain its activity.

[0076] According to another aspect of the present invention, a method for preparing the above-mentioned fusion protein is also provided, comprising dissolving the fusion polypeptide and bFGF in physiological saline to obtain the fusion protein.

[0077] 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 beneficial effects such as high tissue compatibility, simple preparation, and obvious improvement effect. It has no potential toxic effects on cells and has good clinical application prospects.

[0078] In some specific embodiments, the mass ratio of the fusion polypeptide to bFGF is 40-60:1.

[0079] The mass ratio of the fusion polypeptide to bFGF 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.

[0080] In some specific embodiments, the concentration of the fusion polypeptide in the physiological saline is 0.4-0.6 g / L.

[0081] 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 also be any value between 0.4 and 0.6 g / L.

[0082] In some specific embodiments, the dissolution conditions include stirring at 80 rpm for 30 to 40 minutes.

[0083] In some specific embodiments, the method further comprises adding modified chitosan to the physiological saline containing the fusion polypeptide and bFGF, stirring at 110 rpm for 30 to 50 minutes 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 to 25 minutes to obtain the fusion protein.

[0084] In some specific embodiments, the mass ratio of the modified chitosan to the fusion polypeptide is 1-3:1.

[0085] In some specific embodiments, the modified chitosan comprises dissolving chitosan in an acid solution, repeatedly freezing and thawing the chitosan 3 to 5 times, and freeze-drying the chitosan to obtain the modified chitosan.

[0086] In some specific embodiments, 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.

[0087] In some specific embodiments, the chitosan is hydroxypropyltrimethylammonium chloride chitosan.

[0088] In some specific embodiments, the modified hyaluronic acid gel is added in an amount of 30-50 ml per 1 L of normal saline.

[0089] In some specific embodiments, the preparation method of the modified hyaluronic acid gel comprises emulsifying a sodium hyaluronate aqueous solution by stirring at 1000 r / min for 40 to 60 minutes, and adding sodium tripolyphosphate for cross-linking modification to obtain the modified hyaluronic acid gel.

[0090] In some specific embodiments, the concentration of sodium hyaluronate in the sodium hyaluronate aqueous solution is 1-2% w / v.

[0091] In some specific embodiments, the amount of sodium tripolyphosphate added is 1% to 1.5% w / v.

[0092] In some specific embodiments, the cross-linking modification time is 4 to 24 hours.

[0093] 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 damage.

[0094] 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.

[0095] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0096] It should be noted that, unless otherwise specified, all chemical reagents involved in the present invention were purchased through commercial channels.

[0097] Example 1

[0098] Based on bFGF, connecting peptides 1 to 5 were designed and synthesized to connect bFGF and collagen. Each connecting peptide was synthesized using solid-phase synthesis with amino acids as raw materials and according to the peptide sequence.

[0099] Amino acid sequence of connecting polypeptide-1 (SEQ ID NO.1):

[0100] RPSPTLPEAPWGAPVEVESINWLRDGVLAESNRTRITGEEVEVDSVPADSGLSDTTYFSVNVSDALPSETDNTVPAATVRWLNGEFPPSDGNYTCIVENEYGILAGLPVALGSNVEIEVLTAGVNTTKKMEKKYTCLAGKSIKKSKKSAWLKKLEALEKKKKKMTKPLKKE.

[0101] Amino acid sequence of connecting polypeptide-2 (SEQ ID NO.2):

[0102] RPSPTLPESASPWGAPKEMEVLHLRNVSFVEVESFLVHPGDLLKEMEVLHLRNVSFSLRCRLRDDVSSINWLRDGVSLAESNRTRITGEEVEVKKKVPADKKKLYKKKVTKK.

[0103] Amino acid sequence of connecting polypeptide-3 (SEQ ID NO.3):

[0104] SSPSGSDTTYFSVNVSDALPSSEDDDDDDSSSEEETDNTPNRMPVAPYWTSPEMELHAVPAATVFKCPSSGTPNPTLRWLKNGKEFKPDHRIGKKKVRYATWSIIMVKKKVPADKKKLYKKKVTK.

[0105] Amino acid sequence of connecting polypeptide-4 (SEQ ID NO.4):

[0106] DSVVPSDGNYTCIVENEYGSINHTYSLDVVERSPHRPILSAGLPANKTVALGSNVEFMCKVYSDPSPHISWLKHIEKKGSKIKKKKLPKKKSI.

[0107] Amino acid sequence of connecting polypeptide-5 (SEQ ID NO.5):

[0108] LTAGVNTTDEMEVLHLRNVSFEDAGEYTCLADDEMEVLHLRNVSFVEVESFLDDSSSEEETDNTPNRMPGNSIGLSHHSAWLTVLEKKKALEERPAVKKSPLKKE.

[0109] Example 2 to Example 6

[0110] A fusion polypeptide comprises a connecting polypeptide and a collagen protein bound to the C-terminus of the connecting polypeptide. Connecting polypeptides 1 to 5 provided in Example 1 are selected, respectively, and the fusion polypeptides are prepared according to the following steps:

[0111] (1) Add the linker peptide, human type III collagen, and transglutaminase to a 10 mM PBS buffer solution at pH 7.4. The ratio of PBS buffer, linker peptide, human type III collagen, and transglutaminase is 1 mL:7 mg:3 mg:0.1 mg. Stir at 180 rpm for 30 min to obtain a mixed solution.

[0112] (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;

[0113] (3) The reaction solution of step (2) is separated and purified by G25 molecular sieve chromatography and freeze-dried to obtain a linked polypeptide-collagen (fusion polypeptide).

[0114] Among them, human type III collagen is obtained by the method disclosed in Chinese patent CN103102407A.

[0115] Examples 7-11

[0116] A fusion protein comprising a fusion polypeptide and bFGF bound to the N-terminus of a linker polypeptide in the fusion polypeptide. The fusion polypeptides are selected from the fusion polypeptides prepared in Examples 2 to 6, respectively, and the fusion protein is prepared according to the following steps:

[0117] The fusion polypeptide and bFGF were added to normal saline, and stirred at 80 rpm for 30 min. The dosage ratio of normal saline, fusion polypeptide and bFGF was 1 L:0.5 g:0.01 g to obtain a protein solution that promotes the repair of endometrial damage.

[0118] Comparative Example 1

[0119] The difference from Example 7 is that human type III collagen not connected to the linker polypeptide is used instead of the fusion polypeptide, human type III collagen and bFGF 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 bFGF is 1 L:0.5 g:0.01 g to obtain a protein solution that promotes the repair of endometrial damage.

[0120] Experimental Example 1

[0121] This experimental example uses the method of preparing a protein solution for promoting the repair of endometrial damage using Examples 7 to 11 and Comparative Example 1. The physiological saline is replaced with MEM culture medium containing 10% fetal bovine serum to obtain an experimental solution. The following steps are performed:

[0122] 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 and centrifuged at 200 g for 3 min. The cells were then redispersed in fresh medium to adjust the cell density to 2 × 10 5 Cells were plated in a 96-well culture plate with a 100 μL cell suspension of 100 cells / mL. The cells were incubated in a CO2 incubator (5% CO2, 37°C, humidity >90%) for 24 hours. Afterward, 100 μL of the experimental solution was added to each well to serve as the experimental group. A blank control group consisted of wells containing only 200 μL of MEM medium supplemented with 10% fetal bovine serum, and a negative control group contained 100 μL of medium plus 100 μL of cells. Following the addition of the experimental solution, the cells were cultured for 5 days. Daily, 20 μL of MTT (5 mg / mL in PBS) was added to each well in the dark. On the final day, MTT was added and the culture continued for 4 hours. After all liquid was removed from the wells, 150 μL of DMSO solution was added to each well. The plates were shaken at low speed for 15 minutes in a microplate reader, and the OD value at λ = 490 nm was measured. 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. Level 2 and above can be considered as a cytotoxic reaction. The results are as follows: Figure 1 shown.

[0123] Depend on Figure 1It can be seen that the toxicity levels of the protein solutions of Examples 7 to 11 and Comparative Example 1 are all 0, indicating that the fusion protein provided by the present invention has no cytotoxicity and high biosafety.

[0124] Experimental Example 2

[0125] The protein solutions prepared in Examples 7 to 11 and Comparative Example 1 were respectively selected to conduct rat experiments according to the following procedures to detect the attachment duration of the fusion protein in the endometrial injury rat model.

[0126] 1. Experimental animals: 60 female SD rats aged 8-10 weeks and weighing 220-260 g were selected and acclimated for one week.

[0127] 2. Experimental groups: Rats were randomly divided into 6 groups, with 10 rats in each group.

[0128] 3. Establishment of a rat model of endometrial injury: The model was induced using mechanical injury and ethanol perfusion. Rats were fasted for 12 hours before surgery and anesthetized with intraperitoneal injection of 2% sodium pentobarbital. The rats were then immobilized in the supine position. A transverse abdominal incision was made to expose the uterine horns. A longitudinal incision approximately 4 mm in length was made approximately 0.5 cm below the uterine horns. The mid- and upper endometrium was scraped with a 2.5 mm diameter endometrial scraper. Curettage was stopped when the uterine wall felt rough. The uterine horns were clamped with vascular clamps. 95% ethanol, drawn with a 1 mL syringe, was injected downward through the incision until the uterine cavity was filled. The incision was then temporarily clamped with a vascular clamp. After 3 minutes, the clamp was released, and any residual ethanol was expelled. The uterus was then rinsed and aspirated with normal saline. The surface fluid of the uterus was then blotted dry with sterile gauze.

[0129] 4. Drug administration: 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. 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 bFGF in the uterus was detected by ELISA. The results are as follows: Figure 2 shown.

[0130] Depend on Figure 2 It can be seen that the bFGF concentrations of Examples 7 to 9 are all higher than those of Examples 10, 11, and Comparative Example 1, and the rate of decline is also relatively slow, indicating that the fusion polypeptides formed by connecting polypeptides-1 to -3 and collagen can effectively prolong the action time of bFGF and promote the repair of uterine damage. Among them, the fusion polypeptide formed by connecting polypeptide-1 and collagen has the best effect of prolonging bFGF attachment, and its bFGF concentration is the highest and the rate of decline is the slowest.

[0131] Experimental Example 3

[0132] This experimental example selected the protein solutions prepared in Example 7 and Comparative Example 1, and performed the following steps to verify the repair effect of the protein solutions on endometrial damage.

[0133] 1. Experimental animals: 40 female SD rats aged 8-10 weeks and weighing 220-260 g were selected and acclimated for one week.

[0134] 2. Experimental Grouping: Rats were randomly divided into 4 groups, with 10 rats in each group, including 2 experimental groups, 1 sham operation group, and 1 model group. The experimental and model groups were induced to establish models 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 underwent laparotomy. The uterine horns were fully exposed to the body and then retracted. No other operations were performed. The uterus was rinsed and aspirated with physiological saline, and the liquid on the surface of the uterus was dried with sterile gauze.

[0135] 3. Administration: Immediately after modeling, each experimental group was given 0.5 mL of the protein solution of Example 7 and Comparative Example 1 to perfuse the uterine cavity. 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. 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. The uterine tissue was obtained, fixed with paraformaldehyde, and paraffin sectioned. HE staining and Masson staining were performed. Figure 3 The number of endometrial glands and the proportion of endometrial fibrosis were statistically analyzed, and the statistical results are shown in Figure 2. Figure 4 、 Figure 5 shown.

[0136] Depend on Figures 3 to 5 As can be seen, the sham-operated 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 less intrauterine adhesions than the model group, with a restored number of glands and a lower fibrosis ratio than the model group, and close to that of the sham-operated group. It can be seen that the protein solution of Example 7 has a good repair effect on intrauterine adhesions, and the effect is better than that of bFGF and collagen alone. The linked polypeptide can effectively improve the repair effect of endometrial damage and promote endometrial damage repair.

[0137] Example 12

[0138] A fusion protein comprising a fusion polypeptide, bFGF bound to the N-terminus of a linker 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:

[0139] 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 for 12 h, then thawed at room temperature for 12 h, and repeatedly frozen and thawed 5 times. The solution was 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.

[0140] 2. Modified hyaluronic acid gel: 2% sodium hyaluronate aqueous solution was stirred at 1000 r / min for 40-60 min to emulsify, and 1.3% sodium tripolyphosphate was added for cross-linking modification for 12 h to obtain modified hyaluronic acid gel.

[0141] 3. Add the fusion polypeptide and bFGF to normal saline and stir at 80 rpm for 30 minutes to dissolve. The ratio of normal saline, fusion polypeptide, and bFGF is 1 L: 0.5 g: 0.01 g. Add modified chitosan to the normal saline containing the fusion polypeptide and bFGF at a rate of 1 g per 1 L of normal saline. Stir at 110 rpm for 40 minutes to form a chitosan-fusion protein gel. Prefreeze at -20°C for 4 hours, freeze-dry at -80°C for 24 hours, and redissolve in normal saline. Add modified hyaluronic acid gel at a rate of 40 ml per 1 L of normal saline. Stir at 80 rpm for 20 minutes to obtain a protein solution that promotes endometrial damage repair.

[0142] Comparative Example 2

[0143] Different from Example 12, in step 3, the fusion polypeptide and bFGF were added to normal saline and stirred at 80 rpm for 30 minutes to dissolve. The ratio of normal saline, fusion polypeptide, and bFGF was 1 L: 0.5 g: 0.01 g. Modified chitosan was added to the normal saline containing the fusion polypeptide and bFGF at a rate of 1 g per 1 L of normal saline. The mixture was stirred at 110 rpm for 40 minutes to form a chitosan-fusion protein gel. The gel was pre-frozen at -20°C for 4 hours, freeze-dried at -80°C for 24 hours, and redissolved in normal saline to obtain a protein solution that promotes the repair of endometrial damage.

[0144] Comparative Example 3

[0145] Unlike Example 12, the fusion polypeptide and bFGF were added to normal saline and stirred at 80 rpm for 30 minutes to dissolve. The ratio of normal saline, fusion polypeptide, and bFGF was 1 L: 0.5 g: 0.01 g. The solution was pre-frozen at -20°C for 4 hours, freeze-dried at -80°C for 24 hours, and redissolved in normal saline. The modified hyaluronic acid gel was added at a rate of 40 ml per 1 L of normal saline and stirred at 80 rpm for 20 minutes to obtain a protein solution that promotes the repair of endometrial damage.

[0146] Comparative Example 4

[0147] Different from Example 12, modified hyaluronic acid gel was added to the physiological saline containing the fusion polypeptide and bFGF at a rate of 40 ml per 1 L of physiological saline. The mixture was 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, and modified chitosan was added at a rate of 1 g per 1 L of physiological saline. The mixture was stirred at 80 rpm for 20 min to obtain a protein solution that promotes the repair of endometrial damage.

[0148] Experimental Example 4

[0149] In order to prove that the protein solutions prepared in Example 12 and Comparative Examples 2-4 can further prolong the attachment time of bFGF on the endometrium, the protein solutions prepared in Example 7, Example 12 and Comparative Examples 2-4 were used to conduct rat experiments according to the method of Experimental Example 2.

[0150] 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 was completed, 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 bFGF drug concentration in the uterus was detected by ELISA. The results are as follows: Figure 6 shown.

[0151] Depend on Figure 6It can be seen that the bFGF drug concentrations in Example 12 and Comparative Example 4 were higher than those in Example 7 at 12 hours, and that in Comparative Example 4 decreased rapidly after 36 hours. The bFGF drug concentration in Example 12 was slightly lower than that in Example 7 at 36 hours, but was higher than that in Example 7 after 48 hours. The bFGF drug concentrations in Comparative Examples 2 and 3 were generally lower than those in Example 7 and Example 12. Therefore, it can be seen that the chitosan-hyaluronic acid carrier formed by adding modified chitosan and modified hyaluronic acid gel in Example 12 can further prolong the attachment time of the fusion protein.

[0152] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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 fusion protein, characterized in that comprising a fusion polypeptide and bFGF bound to the N-terminus of a linker polypeptide in the fusion polypeptide; The fusion polypeptide includes a connecting polypeptide and a collagen protein connected to the C-terminus of the connecting polypeptide; The amino acid sequence of the connecting polypeptide is as 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 mimics the bFGF receptor structure and binds to bFGF, and the connection between the connecting polypeptide and bFGF 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 Also included is 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 bFGF in physiological saline to obtain a fusion protein; The mass ratio of the fusion polypeptide to bFGF 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.

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 bFGF, stirring at 110 rpm for 30 to 50 minutes to form a chitosan-fusion protein gel, freeze-drying, redissolving 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 amount of the modified hyaluronic acid gel added is 30-50 ml per 1 L of normal saline; The preparation method of the modified hyaluronic acid gel comprises emulsifying a sodium hyaluronate aqueous solution by stirring at 1000 r / min for 40 to 60 minutes, 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 4, 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, then heating at 70° C. for 15 to 20 minutes, cooling, chromatographic separation and purification, and freeze-drying to obtain the fusion polypeptide; The preparation method of the buffer solution containing the connecting polypeptide, collagen and protein cross-linking agent comprises stirring the buffer solution containing the connecting polypeptide, collagen and protein cross-linking agent at 180 rpm for 20 to 30 minutes; The mass ratio of the linker peptide, 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 solution is 10 mM PBS buffer solution.

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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