Medical recombinant protein gynecological gel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of gynecological gel, and provides medical recombinant protein gynecological gel and a preparation method and application thereof.The medical recombinant protein gynecological gel is prepared from, by mass, 0.05%-0.3% of recombinant collagen, 0.01%-0.05% of recombinant fibronectin, 0.05%-0.3% of recombinant elastin, 0.1%-1% of isomaltooligosacharide, 0.01%-0.1% of sodium hyaluronate, 21%-26% of poloxamer 407, 0.03%-0.1% of lactic acid, 0.08%-0.2% of preservative and the balance water. And 2-8% of glycerol and the balance of purified water. The gel provided by the invention particularly has a good repairing effect on cervicitis, and is long in retention time, high in retention rate and not easy to flow out.

Description

Technical Field

[0001] This invention belongs to the field of gynecological gel technology, and relates to a medical recombinant protein gynecological gel, its preparation method and application. Background Technology

[0002] The female vagina is naturally warm and moist with abundant secretions, making it highly susceptible to microbial growth. Therefore, vaginal care is crucial for maintaining women's health. Currently, commonly used vaginal preparations for treating gynecological inflammation include: washes, suppositories, gels, effervescent tablets, and films. Washes are liquid preparations with a short local action time and poor local anti-inflammatory and bactericidal effects. Suppositories have good local retention characteristics, but their effective treatment area is limited to 3-5 cm from the vaginal opening, while the vagina is 8-12 cm long. Gels, delivered via applicator, can reach 8 cm into the vagina, but due to their solid gel form, the drug is unevenly dispersed in contact with the affected tissue. Furthermore, current gels have low gel strength and poor bioadhesion, resulting in insufficient local action time and significantly impacting efficacy. Effervescent tablets address the uneven dispersion issue of gels, but their local action time is too short, and they produce a large amount of discharge that easily stains clothing. Films address the issue of drug action time, but they can cause a foreign body sensation and poor patient compliance. The novel thermosensitive gel, when the temperature is below a certain gelling temperature, is in liquid form and solidifies within 1-2 minutes upon contact with body temperature. This successfully solves the problem of uneven dispersion of ordinary gels at the affected area, while also avoiding the issue of suppositories failing to reach the deepest parts of the vagina. It ensures that the drug concentration is maintained for a certain period, providing protection to the mucosa. The thermosensitive gel is convenient and hygienic to use, does not stain clothing, and has high patient compliance.

[0003] Collagen is a major component of the extracellular matrix (ECM), assisting in signal transduction and participating in the transport of cytokines and growth factors. Fibronectin (FN) is also an important ECM component, involved in fibroblast initiation and promoting target cell proliferation, migration, adhesion, and differentiation. Compared to natural proteins, artificially prepared recombinant proteins have advantages such as low immunogenicity, wide availability, low price, and high activity. Recombinant collagen, obtained through synthetic biology techniques, is highly similar in structure to human collagen, exhibiting good biocompatibility and repair-promoting effects.

[0004] Elastin is also an important structural protein in the extracellular matrix, playing a crucial role in maintaining the elasticity of skin, tendons, and ligaments. High-purity recombinant elastin prepared using synthetic biology techniques exhibits high temperature sensitivity, rapidly responding to external temperatures under specific conditions through a reversible phase transition. During this reversible phase transition, elastin remains active, undergoing adaptive conformational changes to form a morphology that more closely adheres to the original tissue, effectively supporting skin elasticity and thus exerting a sustained and long-lasting effect in promoting tissue regeneration. It is currently commonly used in aesthetic medicine and skin repair, as well as in surgical assistance and wound healing.

[0005] The current drawbacks of thermosensitive gels include short drug retention time and low drug absorption, affecting efficacy. For example, Chinese patent application CN 118059215 A discloses a gynecological thermosensitive in-situ gel, which, by percentage, comprises the following raw materials: collagen 0.05–5% (w / v), sodium hyaluronate 0.05–10% (w / v), mussel adhesive protein 0.01–2% (w / v), plant polysaccharides 0.5–5% (w / v), probiotic growth promoter 0.5–30% (w / v), thermosensitive gel matrix 0.1–20% (w / v), penetration enhancer 1.2–25% (w / v), preservative 0.1–3% (w / v), pH adjuster 0.01–3% (w / v), and water as the balance. This gel does not investigate the gel retention rate during its retention time. For example, Chinese patent application CN 113940915 A discloses a vaginal gel containing the following ingredients by mass percentage: 1%–5% active ingredient carrier, 0.01%–5% moisturizer, 15%–37% gelling agent, 0.01%–2.5% preservative, 0.1%–1.5% regulator, with the remainder being purified water. This gel provides long-lasting, sustained-release medication. However, its effectiveness in repairing cervicitis was not investigated.

[0006] In summary, there is an urgent need to research a gynecological gel that has a long drug retention time, a high retention rate, and good repair effects. Summary of the Invention

[0007] This invention proposes a medical recombinant protein gynecological gel, its preparation method and application. The gel has good cell adhesion effect, long retention time and high retention rate, and has a good repair effect, especially for cervicitis.

[0008] The technical solution of this invention is implemented as follows: Technical Topic 1 This invention provides a medical recombinant protein gynecological gel, which, by mass percentage, is composed of the following raw materials: 0.05-0.3% recombinant collagen, 0.01-0.05% recombinant fibronectin, 0.05-0.3% recombinant elastin, 0.1-1% isomaltooligosaccharide, 0.01-0.1% sodium hyaluronate, 21-26% poloxamer 407, 0.03-0.1% lactic acid, 0.08-0.2% preservative, 2-8% glycerin, and purified water to make up to 100%.

[0009] Preferably, the recombinant collagen is recombinant human type III collagen with a molecular weight of 10kDa to 90kDa.

[0010] Preferably, the molecular weight of the recombinant fibronectin is 40kDa to 55kDa.

[0011] Preferably, the recombinant elastin has a molecular weight of 10kDa to 50kDa.

[0012] Preferably, the recombinant collagen is recombinant human type III collagen, and its amino acid sequence is shown in SEQ ID No. 1 of Chinese Patent 202011366731.X.

[0013] Preferably, the recombinant fibronectin has the amino acid sequence shown in SEQ ID No. 1 of Chinese Patent 2025108130374.

[0014] Preferably, the recombinant elastin has the amino acid sequence shown in SEQ ID No. 1 of Chinese Patent 2025112802533.

[0015] Preferably, the preservative is selected from one or more of methylparaben, ethylparaben, and propylparaben.

[0016] Preferably, the molecular weight of the sodium hyaluronate is 1,000,000 to 1,400,000 Da.

[0017] Preferably, the gelation temperature of the medical recombinant protein gynecological gel is 32℃~37℃.

[0018] Technical Theme Two This invention also provides a method for preparing the above-mentioned medical recombinant protein gynecological gel, comprising the following steps: S1: Mix poloxamer 407, recombinant elastin, sodium hyaluronate and purified water, and let stand at 4~8℃ for 12-18h to obtain solution-1; S2: Mix the preservative, glycerin and purified water, dissolve at 70~80℃, cool to room temperature and add to solution-1, stir at 4-10℃ to obtain solution-2; S3: Mix recombinant collagen, recombinant fibronectin, isomaltooligosaccharide and purified water, stir to dissolve, filter to remove bacteria and add to solution-2, stir and mix at 4-10℃ to obtain solution-3; S4: While stirring in solution-3, add lactic acid dropwise to adjust the pH value, and add the remaining purified water to obtain medical recombinant protein gynecological gel.

[0019] Preferably, the stirring rate in S2-S4 is 200-300 rpm and the stirring time is 20-30 min.

[0020] Preferably, the pH value of the medical recombinant protein gynecological gel is 3.8 to 4.8.

[0021] Technical Theme 3 The present invention also provides the use of the above-mentioned medical recombinant protein gynecological gel in the preparation of a drug for the prevention or treatment of vaginitis or cervicitis.

[0022] The beneficial effects of the present invention using the above technical solution are as follows: 1. The medical recombinant protein gynecological gel provided by this invention, through the addition of recombinant collagen, recombinant fibronectin and recombinant elastin, the three work together to improve the cell repair effect, especially for gynecological inflammations such as vaginitis or cervicitis.

[0023] Among them, recombinant collagen promotes collagen synthesis, inhibits inflammatory factors, and regulates cell proliferation and migration; recombinant fibronectin strengthens the skin's foundation, maintains the skin barrier function, and significantly promotes wound healing; recombinant collagen and recombinant fibronectin can jointly stimulate the proliferation of cervical mucosal epithelial cells, accelerating the healing of damaged areas. Upon contact with vaginal tissue, these three recombinant proteins can fully adhere to the vaginal wall, stimulating the production of endogenous collagen, thereby promoting the healing and repair of vaginal tissues, while also improving dryness symptoms and increasing vaginal lubrication and tightness.

[0024] 2. The medical recombinant protein gynecological gel provided by this invention is thermosensitive. By combining recombinant elastin and poloxamer 407 in a specific ratio, they produce a synergistic effect. The interaction between recombinant elastin and poloxamer 407 constructs a dual-network thermosensitive hydrogel that rapidly gels at body temperature. When injected deep into the vagina in liquid form, this invention penetrates into any deep folds of the vagina and uterus, dispersing evenly at the affected area, thus enhancing therapeutic efficacy. After injection, the interaction between recombinant elastin and poloxamer 407 rapidly forms a dual-network hydrogel, creating a protective film on the cervical mucosa surface. This effectively blocks the invasion of external bacteria, prolonging the action time of the active ingredients and promoting wound repair and regeneration. Simultaneously, the gel formed by the interaction of recombinant elastin and poloxamer 407 improves the gel's adhesion within the vagina, increasing retention time and rate, preventing leakage, and making administration convenient and hygienic. Attached Figure Description

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0026] Figure 1 The images show the state diagrams of Example 1 of the present invention at room temperature and 35°C, and the gel state diagrams of Comparative Example 2 and Comparative Example 4.

[0027] Figure 2 This is an in vitro cell activity evaluation diagram of the medical recombinant protein gynecological gel, which is an example of the efficacy of this invention (Example 1).

[0028] Figure 3 This is an evaluation diagram of the in vitro cell repair efficacy of the medical recombinant protein gynecological gel, Example 2 of the present invention.

[0029] Figure 4 This is a graph showing the retention time of the recombinant protein gynecological gel used in animal bodies in Example 3 of the present invention.

[0030] Figure 5 This is an evaluation diagram of the repair effect of the medical recombinant protein gynecological gel on rat cervicitis, which is Example 4 of the present invention.

[0031] Figure 6 This is a histological staining image of rats after treatment with recombinant protein gynecological gel for cervicitis, as shown in Example 4 of the present invention.

[0032] Figure 7 This is a schematic diagram of the formation of the dual-network hydrogel of the present invention.

[0033] Figure 8 This is a scanning electron microscope image of Embodiment 1 of the present invention.

[0034] Figure 9 This is a scanning electron microscope image of the gel formed by poloxamer 407.

[0035] Figure 10 This is a scanning electron microscope image of a gel formed from recombinant elastin.

[0036] Figure 1 In the text, a represents the gynecological gel prepared in Example 1 at room temperature, b represents the gynecological gel prepared in Example 1 at 35°C, c represents the gynecological gel prepared in Comparative Example 2 at 55°C, and d represents the gynecological gel prepared in Comparative Example 4 at 65°C. Detailed Implementation

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. Unless otherwise specified, the experimental or testing methods involved in the embodiments of this invention are conventional methods in the prior art, and their names and / or abbreviations are conventional names in the art, clearly defined in their respective fields of application. Those skilled in the art can understand the conventional process steps based on these names and apply the corresponding equipment, implementing them under conventional conditions or conditions recommended by the manufacturer. The various instruments, equipment, raw materials, or reagents used in the embodiments of this invention are not subject to any special restrictions on their source; they are all conventional products that can be purchased through legitimate commercial channels and can be prepared according to conventional methods well known to those skilled in the art.

[0039] In the following embodiments and comparative examples: Poloxamer 407 is a commercially available pharmaceutical excipient purchased from Nanjing Well Pharmaceutical Group Co., Ltd.

[0040] The isomaltooligosaccharide was commercially available in IMO-90 form and was purchased from Shandong Bailong Chuangyuan Biotechnology Co., Ltd.

[0041] Sodium hyaluronate was purchased from Shandong Zhongshan Biotechnology Co., Ltd., with a molecular weight of 1,000,000-1,400,000 Da.

[0042] The recombinant collagen used is recombinant type III collagen. Specifically, the recombinant type III collagen is prepared using the method disclosed in Examples 1-4 and 7 of Chinese Patent 202011366731.X, entitled "A recombinant human type III collagen and its preparation method and uses". Specifically, the amino acid sequence of the recombinant human type III collagen is shown in SEQ ID No. 1 of Chinese Patent 202011366731.X.

[0043] The recombinant fibronectin was prepared using the method disclosed in the embodiments of Chinese Patent 2025108130374, entitled "A Recombinant Human Fibronectin and its Preparation Method and Application"; specifically, the amino acid sequence of the recombinant fibronectin is shown in SEQ ID No. 1 of Chinese Patent 2025108130374, and the preparation method includes the following steps: S1 gene design and synthesis (1) Gene design: Based on the sequence characteristics of human fibronectin, a fibronectin sequence FN was designed, with its amino acid sequence shown in SEQ ID NO. 1. Its N-terminus begins with methionine to reduce the protein degradation rate, and a histidine tag is added to facilitate identification and purification. The amino acid sequence of the recombinant human fibronectin fragment is shown below: SEQ ID NO.1 MGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDA PAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMK EINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKI YLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTHHHHHH The encoding nucleic acid sequence was reverse-engineered using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for expression in the host *E. coli*. After the above optimization, the corresponding nucleic acid fragment encoding recombinant human fibronectin was obtained, and the nucleotide sequence corresponding to the above amino acid sequence is shown below: SEQ ID NO.2 (2) Gene synthesis: Based on the nucleic acid sequence shown above, Genscript Biotech Inc. synthesized a nucleic acid fragment encoding a recombinant human fibronectin fragment.

[0044] Construction of expression vector pET30a(+)-FN The nucleic acid fragment SEQ ID NO.2 obtained in Example 1 (via NdeI and XhoI multiple cloning sites) was ligated to the pET30a(+) plasmid to obtain the recombinant plasmid pET30a(+)-FN.

[0045] Construction of expression strain BL21(DE3) / pET30a(+)-FN The expression strain was constructed according to the method described in *Molecular Cloning: A Laboratory Manual (3rd Edition)* (by J. Sambrook et al.), and the specific steps are as follows: A single colony of *E. coli* BL21(DE3) was picked and inoculated into an LB tube and cultured overnight at 37°C with shaking. 0.5 ml of overnight culture medium was added to a 50 ml LB flask, and the tube was incubated at 37°C with vigorous shaking for approximately 2 hours to allow the cells to reach the pre-logarithmic growth phase. Under aseptic conditions, the bacteria were transferred to a 50 ml pre-chilled polypropylene tube and placed on ice for 10 minutes. The tube was centrifuged at 4°C and 4000 rpm, the supernatant was discarded, and the tube was inverted to allow as much residual liquid as possible to flow out. The precipitate was resuspended in 6 ml of pre-chilled 0.1 mol / L CaCl2 and placed on ice for 30 minutes. The tube was centrifuged at 4°C and 3000 rpm, the supernatant was discarded, and the tube was inverted to allow as much residual liquid as possible to flow out. The precipitate was resuspended in 1.2 ml of pre-chilled 0.1 mol / L CaCl2 (if preparing competent cells for storage at -70°C, add 0.1 mol / L CaCl2 containing 20% ​​glycerol). After incubating the cells in CaCl2 suspension for 5-24 hours at 4°C, take 200 μl of competent cell suspension and add the recombinant plasmid pET30a(+)-FN (volume <10 μl, DNA <50 ng) prepared in Example 2. Mix gently and place on ice for 30 minutes. Perform static heat shock in a 42°C water bath for 90 seconds and immediately place on ice to cool. Add 500 μl of liquid LB medium, mix well, and place in a 37°C shaker with low-speed shaking for 45 minutes to revive (or add LB and place directly in a 37°C water bath for 1 hour, shaking the tube in between to suspend the cells). Spread the transformed cells on a plate containing antibiotics (kanamycin) and incubate upside down in a 37°C incubator. The colonies that grow are the expression strain BL21(DE3) / pET30a(+)-FN.

[0046] Inducible expression of strain BL21(DE3) / pET30a(+)-FN Single colonies of the expression strain BL21(DE3) / pET30a(+)-FN prepared in Example 3 were picked and cultured overnight at 37°C and 200 rpm in LB liquid medium containing 50 μg / mL Kan to activate the seed culture. This seed culture was then inoculated at a rate of 3% into a 5L fermenter containing 3L of complete culture medium. The fermentation process was controlled at 37°C, dissolved oxygen 30%, and pH 7.0. When OD... 600 When the concentration reached 60, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM to induce expression. The cells were cultured for another 12 hours and then collected by centrifugation.

[0047] Purification of recombinant human fibronectin FN The theoretical isoelectric point of FN is 9.44, and the pH of all buffer solutions is 7.5. The specific purification steps are as follows: 1. Sterilization The bacterial cells prepared in Example 4 were resuspended in an appropriate amount of lysis buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, 1 M urea, pH 7.5), with a bacterial cell to lysis buffer ratio (m / v) of 1 g: 5-1: 20 ml. In this example, 1 g: 10 ml was selected. The bacterial cells were homogenized using a high-pressure homogenizer. The homogenized bacterial solution was centrifuged at 12000 g for 1 h, and the supernatant was collected and filtered through a 0.45 μm filter membrane.

[0048] 2. Affinity chromatography (1) Column balance: The Ni Sepharose FF column was equilibrated for 5 CVs until the baseline stabilized using Buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5).

[0049] (2) Sample loading: Load the supernatant into the chromatography column and control the column retention time to be no less than 5 minutes.

[0050] (3) Column flushing: Rinse the column with Buffer A until the baseline is stable and not less than 7 CV.

[0051] (4) Target protein elution: Elute with 60% Buffer A and 40% Buffer B (20mM Tris, 500mM NaCl, 500mM imidazole, pH 7.5), collect the eluent into a new centrifuge tube to obtain a solution containing recombinant human fibronectin FN.

[0052] 3. Ion exchange chromatography (1) Sample preparation: The affinity chromatography eluent was diluted with ion exchange solution A (20 mM Tris, pH 7.5) or replaced by ultrafiltration until the conductivity was less than 5 mS / cm.

[0053] (2) Column balance: The SP Chrpmstar FF chromatography column was equilibrated for 5 CVs with ion exchange buffer A until the baseline stabilized.

[0054] (3) Sample loading: Load the replacement solution into the chromatography column and control the column retention time to be no less than 5 minutes.

[0055] (4) Column flushing: Wash the column with ion exchange buffer A until the baseline is stable and not less than 7 CV.

[0056] (5) Target protein elution: Elute with 50% ion exchange solution A and 50% ion exchange solution B (20mM Tris, 1M NaCl, pH 7.5), collect the eluent into a new centrifuge tube to obtain a solution containing recombinant human fibronectin FN.

[0057] 4. Desalination The FN solution obtained in step 3 was replaced with purified water using a 3kDa ultrafiltration membrane device with an equal volume displacement method to remove the salt in the solution. After filtration through a 0.45μm filter membrane, the solution was freeze-dried to obtain the recombinant fibronectin freeze-dried product.

[0058] The recombinant elastin was prepared using the method disclosed in Examples 1-5 of Chinese Patent 2025112802533, entitled "A Recombinant Human Elastin and Its Preparation Method and Application". Specifically, the amino acid sequence of the recombinant elastin is shown in SEQ ID No. 1 of Chinese Patent 2025112802533, and the preparation method includes the following steps: S1 gene design and synthesis (1) Gene design Based on the sequence characteristics of human elastin, a recombinant human elastin sequence EL06 was designed. The amino acid sequence of the recombinant human elastin is shown below: SEQ ID NO.1 MGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPAAAAAAAKAAAKFGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPAAAAAAAKAAAKFGVGVAPGVGVAPGVGVAPGVGVAPGVG VAPGVGVAPAAAAAAAKAAAKFGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPAAAAAAAKAAAKFGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPAAAAAAAKAAAKFHHHHHH The encoding nucleic acid sequence was designed using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for expression in the host *E. coli*. After the above optimization, the corresponding nucleic acid fragment encoding recombinant human elastin was obtained, and the nucleic acid sequence corresponding to the above amino acid sequence is shown below: SEQ ID NO.2 CATATGGGTGTAGGAGTTGCTCCCGGGGTCGGAGTGGCACCGGGTTGTTGGTGTTGCGCCAGGCGTGGGCGTTGCGCCGGGTGTAGGCGTGGCGCCGGGTGTTGGTGTGGCTCCGGCAGCCGCGGCGGCGGCGGCCAAGGCAGCGGCCAAGTTTGGTGTTGGCGTGGCCCCGGGTGTGGGCGTGGCCCCGGGT GTAGGCGTTGCGCCGGGTGTGGGCGTGGCTCCGGGCGTTGGTGTGGCACCGGGGGTAGGCGTGGCCCCTGCAGCGGCTGCGGCGGCGGCGAAAGCCGCTGCGAAGTTCGGTGTGGGCGTGGCACCGGGCGTGGGCGTTGCCCCGGGCGTTGGCGTTGCGCCGGGTGTGGGCGTGGCCCCGGGCGTTGGCGTC GCTCCGGGTGTTGGCGTTGCGCCAGCAGCGGCGGCGGCTGCTGCTAAGGCAGCGGCAAAATTCGGCGTCGGTGTCGCTCCGGGTGTTGGTGTCGCCCCAGGTGTTGGCGTGGCACCGGGTGTGGGTTGCTCCGGGCGTTGGTGTCGCTCCGGGTGTGGGCGTGGCGCCGGCAGCGGCTGCGGCGGCGGCG AAAGCGCGGCGAAATTCGGTGTCGGGGTTGCGCCGGGTGTCGGCGTGGCCGGGTGTTGGTGTGGCGCCAGGTGTGGGTGTCGCCCCGGGCGTTGGTGTTGCCGGGTGTTGGTGTGGCCCCGGCAGCTGCTGCGGCTGCGGCGAAAGCAGCGGCGAAGTTTCATCACCACCACCATCACTAAGGTACC (2) Gene synthesis: Based on the nucleic acid sequence shown above, Genscript Biotech Inc. synthesized a nucleic acid fragment encoding a recombinant human elastin fragment.

[0059] Construction of expression vector pET30a(+)-EL06 The nucleic acid fragment SEQ ID NO.2 obtained in Example 1 (via Nde I and Kpn I multiple cloning sites) was ligated and transformed into the pET30a(+) plasmid to obtain the recombinant plasmid pET30a(+)-EL06.

[0060] Construction of expression strain BL21(DE3) / pET30a(+)-EL06 The expression strain was constructed according to the method described in *Molecular Cloning: A Laboratory Manual (3rd Edition)* (by J. Sambrook et al.), and the specific steps are as follows: A single colony of *E. coli* BL21(DE3) was picked and inoculated into an LB tube and cultured overnight at 37°C with shaking. 0.5 ml of overnight culture medium was added to a 50 ml LB flask, and the tube was incubated at 37°C with vigorous shaking for approximately 2 hours to allow the cells to reach the pre-logarithmic growth phase. Under aseptic conditions, the bacteria were transferred to a 50 ml pre-chilled polypropylene tube and placed on ice for 10 minutes. The tube was centrifuged at 4°C and 4000 rpm, the supernatant was discarded, and the tube was inverted to allow as much residual liquid as possible to flow out. The precipitate was resuspended in 6 ml of pre-chilled 0.1 mol / L CaCl2 and placed on ice for 30 minutes. The tube was centrifuged at 4°C and 3000 rpm, the supernatant was discarded, and the tube was inverted to allow as much residual liquid as possible to flow out. The precipitate was resuspended in 1.2 ml of pre-chilled 0.1 mol / L CaCl2 (if preparing competent cells for storage at -70°C, add 0.1 mol / L CaCl2 containing 20% ​​glycerol). After incubating the cells in CaCl2 suspension for 5-24 hours at 4°C, take 200 μl of competent cell suspension and add the recombinant plasmid pET30a(+)-EL06 (2 μl, DNA < 50 ng) prepared in Example 2. Mix gently and place on ice for 30 minutes. Perform static heat shock in a 42°C water bath for 90 seconds, then immediately place on ice to cool. Add 500 μl of liquid LB medium, mix well, and place in a 37°C shaker at 200 rpm for 45 minutes to revive (or add LB and place directly in a 37°C water bath for 1 hour, shaking the tube occasionally to suspend the cells). Spread the transformed cells onto plates containing antibiotics (kanamycin) and incubate upside down in a 37°C incubator. The resulting colonies are the expression strain BL21(DE3) / pET30a(+)-EL06.

[0061] Inducible expression of strain BL21(DE3) / pET30a(+)-EL06 Single colonies of the expression strain BL21(DE3) / pET30a(+)-EL06 prepared in Example 3 were picked and cultured overnight at 37°C and 200 rpm in LB liquid medium containing 50 μg / mL Kan to activate the seed culture. Then, 3% of the seed culture was inoculated into a 5L fermenter containing 3L of complete culture medium. Three groups were set up: fermenter 1, fermenter 2, and fermenter 3. The fermentation process was controlled at 37°C, dissolved oxygen 30%, and pH 7.0. When OD... 600When the concentration reaches 60, IPTG (isopropyl-β-D-thiogalactopyranoside) with a final concentration of 0.5 mM is added to induce expression. After culturing for another 10 hours, a portion of the culture medium is taken out and centrifuged to collect some bacterial cells. After culturing for another 2 hours, the remaining bacterial cells are collected by centrifugation.

[0062] Purification of recombinant human elastin EL06 The theoretical isoelectric point of EL06 is 10.9, and the pH of the buffer solution is 7.5. The specific purification steps are as follows: 1. Sterilization The bacterial cells prepared in Example 4 were resuspended in an appropriate amount of lysis buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5). The ratio of bacterial cells to lysis buffer (m / v) was 1 g:5 ml to 1 g:20 ml; in this example, 1 g:10 ml was selected. The bacterial cells were homogenized using a high-pressure homogenizer. The lysed bacterial solution was adjusted to pH 3 with hydrochloric acid and centrifuged at 12000 g for 0.5 h. The supernatant was collected and filtered through a 0.45 μm filter membrane.

[0063] 2. Affinity chromatography (1) Column balance: The Ni Focurose FF (IMAC) chromatography column was equilibrated to a stable baseline using affinity chromatography solution A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5).

[0064] (2) Sample loading: The supernatant after filtration through the filter membrane is loaded into the chromatography column, and the column retention time is controlled to be no less than 5 minutes.

[0065] (3) Column flushing: Wash the column with affinity chromatography solution A until the baseline is stable and not less than 5 CVs.

[0066] (4) Target protein elution: Elute with 70% affinity chromatography solution A and 30% affinity chromatography solution B (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5), collect the eluent into a new centrifuge tube to obtain a solution containing recombinant human elastin.

[0067] 3. Ion exchange chromatography (1) Sample preparation: The affinity chromatography eluent was replaced by ultrafiltration with ion exchange solution A (20 mM Tris, pH 7.5) until the conductivity was less than 4 mS / cm, thus obtaining the replacement solution.

[0068] (2) Column balance: The SP Sepharose FF column was equilibrated with ion exchange chromatography solution A until the baseline was stable.

[0069] (3) Sample loading: Load the replacement solution into the chromatography column and control the column retention time to be no less than 5 minutes.

[0070] (4) Column flushing: Wash the column with ion exchange chromatography solution A until the baseline is stable and not less than 5 CVs.

[0071] (5) Target protein elution: Elute with 70% ion-exchange chromatography solution A and 30% ion-exchange chromatography solution B (20 mM Tris, 1 M NaCl, pH 7.5), collect the eluent into a new centrifuge tube to obtain a solution containing recombinant human elastin.

[0072] 4. Desalination The solution containing recombinant human elastin obtained from the above ion exchange chromatography step was replaced with purified water using a 5 kDa ultrafiltration membrane device in an equal volume displacement manner to remove the salt in the solution. After filtration through a 0.45 μm filter membrane, the solution was lyophilized to obtain the lyophilized recombinant elastin product.

[0073] The recombinant elastin used in this invention possesses unique temperature phase transition properties due to its amino acid sequence: under certain conditions, when the temperature is below its gelation temperature, the molecular chains hydrophilically extend and dissolve in water; when the temperature rises to the gelation temperature, a gel network is rapidly formed. By precisely controlling its sequence and molecular weight using synthetic biology techniques, stable temperature-sensitive gelation behavior is ensured.

[0074] Example 1 A medical recombinant protein gynecological gel, by mass percentage, is composed of the following raw materials: 0.3% recombinant collagen, 0.03% recombinant fibronectin, 0.1% recombinant elastin, 0.5% isomaltooligosaccharide, 0.05% sodium hyaluronate, 24% poloxamer 407, 0.05% lactic acid, 0.1% methylparaben, 5% glycerin, and purified water to make up to 100%.

[0075] The preparation method of the above-mentioned medical recombinant protein gynecological gel includes the following steps: S1: Weigh out the prescribed amounts of poloxamer 407, recombinant elastin and sodium hyaluronate, add 2 times the mass of purified water, and let stand at 5°C for 15 hours to dissolve, to obtain solution-1; S2: Weigh the prescribed amounts of methylparaben and glycerin into a beaker, add purified water equal to the sum of the masses of the two components, heat to 75°C to dissolve, cool to room temperature and add to solution-1, stir at 5°C (stirring speed 250 rpm, stirring time 25 min) to obtain solution-2; S3: Weigh recombinant collagen, recombinant fibronectin and isomaltooligosaccharide, add 10 times the mass of purified water (the sum of the masses of the three components), stir to dissolve (stirring speed 250 rpm, stirring time 25 min), filter to remove bacteria, add to solution-2, stir and mix at 5℃ (stirring speed 250 rpm, stirring time 25 min) to obtain solution-3. S4: While stirring, add lactic acid dropwise to solution-3 (stirring speed 250 rpm, stirring time 25 min), and add the remaining purified water to obtain solution 4. The pH value of solution 4 is measured to be in the range of 3.8~4.8. S5: Fill solution 4 into a disposable applicator and seal it with an aluminum foil bag to obtain the medical recombinant protein gynecological gel product.

[0076] Example 2 A medical recombinant protein gynecological gel, by mass percentage, is composed of the following raw materials: 0.15% recombinant collagen, 0.01% recombinant fibronectin, 0.05% recombinant elastin, 0.2% isomaltooligosaccharide, 0.01% sodium hyaluronate, 26% poloxamer 407, 0.03% lactic acid, 0.2% methylparaben, 2% glycerin, and purified water to make up to 100%.

[0077] The preparation method of the above-mentioned medical recombinant protein gynecological gel includes the following steps: S1: Weigh out the prescribed amounts of poloxamer 407, recombinant elastin and sodium hyaluronate, add 2 times the mass of purified water, and let stand at 4℃ for 18 hours to dissolve, to obtain solution-1; S2: Weigh the prescribed amounts of methylparaben and glycerin into a beaker, add 1 times the mass of purified water, heat to 70°C to dissolve, cool to room temperature and add to solution-1, stir at 5°C (stirring speed 250 rpm, stirring time 25 min) to obtain solution-2; S3: Weigh recombinant collagen, recombinant fibronectin and isomaltooligosaccharide, add 10 times the mass of purified water (the sum of the masses of the three components), stir to dissolve (stirring speed 250 rpm, stirring time 25 min), filter to remove bacteria, add to solution-2, stir and mix at 5℃ (stirring speed 250 rpm, stirring time 25 min) to obtain solution-3. S4: While stirring, add lactic acid dropwise to solution-3 (stirring speed 250 rpm, stirring time 25 min), and add the remaining purified water to obtain solution 4. The pH value of solution 4 is measured to be in the range of 3.8~4.8. S5: Fill solution 4 into a disposable applicator and seal it with an aluminum foil bag to obtain the medical recombinant protein gynecological gel product.

[0078] Example 3 A medical recombinant protein gynecological gel, by mass percentage, is composed of the following raw materials: 0.1% recombinant collagen, 0.03% recombinant fibronectin, 0.3% recombinant elastin, 0.1% isomaltooligosaccharide, 0.1% sodium hyaluronate, 21% poloxamer 407, 0.1% lactic acid, 0.08% methylparaben, 8% glycerin, and purified water to make up to 100%.

[0079] The preparation method of the above-mentioned medical recombinant protein gynecological gel includes the following steps: S1: Weigh out the prescribed amounts of poloxamer 407, recombinant elastin and sodium hyaluronate, add 2 times the mass of purified water, and let stand at 8°C for 12 hours to dissolve, to obtain solution-1; S2: Weigh the prescribed amounts of methylparaben and glycerin into a beaker, add 1 times the mass of purified water, heat to 80°C to dissolve, cool to room temperature and add to solution-1, stir and mix at 5°C (stirring speed 200 rpm, stirring time 30 min) to obtain solution-2; S3: Weigh recombinant collagen, recombinant fibronectin and isomaltooligosaccharide, add 10 times the mass of purified water (the sum of the masses of the three components), stir to dissolve (stirring speed 300 rpm, stirring time 20 min), filter to remove bacteria, add to solution-2, stir and mix at 5℃ (stirring speed 200 rpm, stirring time 30 min) to obtain solution-3. S4: While stirring, add lactic acid dropwise to solution-3 (stirring speed 300 rpm, stirring time 20 min), and add the remaining purified water to obtain solution 4. The pH value of solution 4 is measured to be in the range of 3.8~4.8. S5: Fill solution 4 into a disposable applicator and seal it with an aluminum foil bag to obtain the medical recombinant protein gynecological gel product.

[0080] Example 4 A medical recombinant protein gynecological gel, by mass percentage, is composed of the following raw materials: 0.05% recombinant collagen, 0.05% recombinant fibronectin, 0.2% recombinant elastin, 0.8% isomaltooligosaccharide, 0.08% sodium hyaluronate, 23% poloxamer 407, 0.08% lactic acid, 0.15% methylparaben, 4% glycerin, and purified water to make up to 100%.

[0081] The preparation method of the above-mentioned medical recombinant protein gynecological gel includes the following steps: S1: Weigh out the prescribed amounts of poloxamer 407, recombinant elastin and sodium hyaluronate, add 2 times the mass of purified water, and let stand at 5°C for 15 hours to dissolve, to obtain solution-1; S2: Weigh the prescribed amounts of methylparaben and glycerin into a beaker, add 1 times the mass of purified water, heat to 75°C to dissolve, cool to room temperature and add to solution-1, stir at 5°C to mix (stirring speed 300 rpm, stirring time 20 min) to obtain solution-2; S3: Weigh recombinant collagen, recombinant fibronectin and isomaltooligosaccharide, add 10 times the mass of purified water, stir to dissolve (stirring speed 200 rpm, stirring time 30 min), filter to remove bacteria and add to Solution-2, stir to mix at 5℃ (stirring speed 300 rpm, stirring time 20 min) to obtain Solution-3; S4: While stirring, add lactic acid dropwise to solution-3 (stirring speed 200 rpm, stirring time 30 min), and add the remaining purified water to obtain solution 4. The pH value of solution 4 is measured to be in the range of 3.8~4.8. S5: Fill solution 4 into a disposable applicator and seal it with an aluminum foil bag to obtain the medical recombinant protein gynecological gel product.

[0082] Comparative Example 1 A gynecological gel, differing from Example 1 only in that the recombinant elastin content is 0.01%. Specifically, it is composed of the following ingredients: 0.3% recombinant collagen, 0.03% recombinant fibronectin, 0.01% recombinant elastin, 0.5% isomaltooligosaccharide, 0.05% sodium hyaluronate, 24% poloxamer 407, 0.05% lactic acid, 0.1% methylparaben, 5% glycerin, and purified water to make up to 100%.

[0083] The preparation method of this gynecological gel is the same as in Example 1.

[0084] Comparative Example 2 A gynecological gel, differing from Example 1 only in that it does not contain recombinant elastin. Specifically, it is composed of the following ingredients: 0.3% recombinant collagen, 0.03% recombinant fibronectin, 0.5% isomaltooligosaccharide, 0.05% sodium hyaluronate, 24% poloxamer 407, 0.05% lactic acid, 0.1% methylparaben, 5% glycerin, and purified water to make up to 100%.

[0085] The preparation method of this gynecological gel is the same as in Example 1.

[0086] Comparative Example 3 A gynecological gel, differing from Example 1 only in that it contains 18% poloxamer 407. Specifically, it is composed of the following ingredients: 0.3% recombinant collagen, 0.03% recombinant fibronectin, 0.1% recombinant elastin, 0.5% isomaltooligosaccharide, 0.05% sodium hyaluronate, 18% poloxamer 407, 0.05% lactic acid, 0.1% methylparaben, 5% glycerin, and purified water to make up to 100%.

[0087] The preparation method of this gynecological gel is the same as in Example 1.

[0088] Comparative Example 4 A gynecological gel, differing from Example 1 only in that it does not contain poloxamer 407. Specifically, it is composed of the following ingredients: 0.3% recombinant collagen, 0.03% recombinant fibronectin, 0.1% recombinant elastin, 0.5% isomaltooligosaccharide, 0.05% sodium hyaluronate, 0.05% lactic acid, 0.1% methylparaben, 5% glycerin, and purified water to make up to 100%.

[0089] The preparation method of this gynecological gel is the same as in Example 1.

[0090] Comparative Example 5 A gynecological gel, with the same raw materials as in Example 1.

[0091] The preparation method of this gynecological gel differs from that of Example 1 only in that step S1, which involves standing and dissolving at 5°C for 15 hours, is replaced by stirring and dissolving at room temperature for 15 hours.

[0092] Comparative Example 6 A gynecological gel, with the same raw materials as in Example 1.

[0093] The preparation method of this gynecological gel differs from that of Example 1 only in that step S4, adjusting the pH value, is moved to after step S1 and before step S2, as detailed below: S1: Weigh out the prescribed amounts of poloxamer 407, recombinant elastin and sodium hyaluronate, add 2 times the mass of purified water, and let stand at 5°C for 15 hours to dissolve, to obtain solution-1; S2: Add lactic acid dropwise to solution-1 while stirring (stirring speed 250 rpm, stirring time 25 min) to adjust the pH value and obtain solution-2; S3: Weigh the prescribed amounts of methylparaben and glycerin into a beaker, add purified water equal to the sum of the masses of the two components, heat to 75°C to dissolve, cool to room temperature and add to solution-2, stir and mix at 5°C (stirring speed 250 rpm, stirring time 25 min) to obtain solution-3; S4: Weigh recombinant collagen, recombinant fibronectin and isomaltooligosaccharide, add 10 times the mass of purified water (the sum of the masses of the three components), stir to dissolve (stirring speed 250 rpm, stirring time 25 min), filter to remove bacteria, add to solution -3, 5℃ and stir to mix (stirring speed 250 rpm, stirring time 25 min), add the remaining purified water to obtain solution 4. S5: Fill solution 4 into a disposable applicator and seal it with an aluminum foil bag to obtain the medical recombinant protein gynecological gel product.

[0094] Comparative Example 7 A gynecological gel, differing from Example 1 only in that the recombinant elastin content is 0.4%. Specifically, it is composed of the following ingredients: 0.3% recombinant collagen, 0.03% recombinant fibronectin, 0.4% recombinant elastin, 0.5% isomaltooligosaccharide, 0.05% sodium hyaluronate, 24% poloxamer 407, 0.05% lactic acid, 0.1% methylparaben, 5% glycerin, and purified water to make up to 100%.

[0095] The preparation method of this gynecological gel is the same as in Example 1.

[0096] Comparative Example 8 A gynecological gel, differing from Example 1 only in that it contains 28% poloxamer 407. Specifically, it is composed of the following ingredients: 0.3% recombinant collagen, 0.03% recombinant fibronectin, 0.1% recombinant elastin, 0.5% isomaltooligosaccharide, 0.05% sodium hyaluronate, 28% poloxamer 407, 0.05% lactic acid, 0.1% methylparaben, 5% glycerin, and purified water to make up to 100%.

[0097] The preparation method of this gynecological gel is the same as in Example 1.

[0098] Comparative Example 9 A gynecological gel, differing from Example 1 only in that it contains 0.33% recombinant collagen and no recombinant fibronectin. Specifically, it is composed of the following ingredients: 0.33% recombinant collagen, 0.1% recombinant elastin, 0.5% isomaltooligosaccharide, 0.05% sodium hyaluronate, 24% poloxamer 407, 0.05% lactic acid, 0.1% methylparaben, 5% glycerin, and purified water to make up to 100%.

[0099] The preparation method of this gynecological gel is the same as in Example 1.

[0100] Test Example 1: In vitro gelation test of gynecological gel The gynecological gels prepared in the examples and comparative examples were poured into vials and heated in a water bath. The phase transition temperature was set to the point where the gel no longer flowed. The initial temperature of the water bath was set to 30 °C, and the gelation time was set to 3 min. After gelation failed, the water bath temperature was increased by 1 °C, and gelation was detected after 3 minutes. The results are shown in Table 1 below.

[0101] Table 1 As shown in Table 1, in Examples 1-4, gel formation occurred within 30 seconds to 1 minute at 32℃-37℃. The gel prepared in Example 1 showed comparable results at room temperature and 35℃. Figure 1 Comparative Example 1 formed a hydrogel at 42℃ for 5 min; Comparative Example 3 formed a hydrogel at 48℃ for 20 min; Comparative Example 2 showed gelation after prolonged high-temperature standing, but remained slightly fluid. Comparative Example 5 was prepared at room temperature; the components were viscous, and stirring produced a lot of white foam, affecting the gelation effect at 37℃. Comparative Example 6, after adjusting the pH and adding recombinant collagen and recombinant fibronectin, showed flocculent precipitation, indicating that the protein components lost the protection of the micelles and flocculated. Comparative Examples 2, 4, and 6 all showed milky white precipitation, indicating the loss of the protective effect of the double-network hydrogel and the denaturation of the active protein components. In Table 1, 10 mg of recombinant elastin dissolved in 0.5 M NaCl solution self-assembled into a translucent hydrogel after standing at 52℃ for 7 min.

[0102] This result indicates that both recombinant elastin and poloxamer 407 play a crucial role in controlling gelation time and temperature. Only when both are used together and within the dosage range specified in this invention can optimal gelation time and gel state be achieved. Comparative Example 6, by altering the order of addition, caused a conformational change in the recombinant protein component, resulting in decreased stability and precipitation. The test results of the examples confirm that the recombinant elastin and poloxamer 407, in a specific ratio and process, successfully constructed a hydrogel structure in this invention, protecting the biological activity of the recombinant protein within the component. It rapidly forms a temperature-sensitive gel under body temperature conditions, and its gelation temperature and gelation time meet clinical requirements.

[0103] like Figure 8 This is a scanning electron microscope image of the gel prepared in Example 1. Figure 9 , Figure 10 The images show scanning electron microscopy (SEM) images of the gels prepared from poloxamer 407 and recombinant elastin (10 mg of recombinant elastin dissolved in 0.5 M NaCl solution and placed at 52 °C for 7 min to self-assemble into a translucent hydrogel). It can be seen that both recombinant elastin and poloxamer 407 can form network gels. In Example 1, the two interacted to form a double network gel with interwoven structures.

[0104] Example 1: Evaluation of in vitro cell activity and repair efficacy Cell proliferation activity: Select L929 cells in good growth condition, digest them with trypsin, and then dilute the cells to 0.8 × 10⁻⁶. 5 The culture medium (MEM containing 10% fetal bovine serum) was seeded at 100 μL per well in 96-well plates and incubated at 37°C for 24 h. The MEM medium was then removed. 100 μL of the blank control and experimental group test solutions were added to each well, and the plates were incubated at 37°C for another 24 h. 10 μL of CCK8 reagent was added to each well, and the plates were incubated for 2 h. The absorbance at OD=450 nm was measured using a microplate reader.

[0105] Cell proliferation rate = (absorbance value of sample wells - absorbance value of blank culture medium wells) / (absorbance value of blank control wells - absorbance value of blank culture medium wells) * 100%.

[0106] The inoculated L929 cells were divided into a blank control group and a sample group, and the specific treatments for each group were as follows: Blank control group: MEM culture medium (containing 10% fetal bovine serum); Test solutions for the experimental group: Extracts of samples from Example 1, Comparative Example 2, and Comparative Example 4 were prepared according to the method specified in GB / T16886.

[0107] The specific extraction method is as follows: Take samples from each group and extract them at a ratio of 0.1g sample to 1ml MEM culture medium (containing 10% fetal bovine serum). The extraction conditions are 37℃±1℃ for 24h±2h (37℃ for 24h is used in this effective example). After extraction, take 100 μL of each group's extract as the test solution.

[0108] After 24 hours of cell plating, the culture medium was replaced with sample extraction medium and cultured for another 24 hours.

[0109] The results are as follows Figure 2 As shown, the cell proliferation rate in the blank control group was 100.00%, the cell proliferation rate in the Example 1 sample group was 130.00%, the cell proliferation rate in the Comparative Example 2 sample group was 99.33%, and the cell proliferation rate in the Comparative Example 4 sample group was 108.67%. The possible reason is that Comparative Examples 2 and 4 lost the protective effect of the double-network hydrogel, resulting in partial denaturation of the protein active components, thus affecting the product's effect on cell proliferation. Compared with the blank control group, the Example 1 sample significantly promoted cell proliferation, increasing it by approximately 1.30 times (**). P <0.01). Among the sample groups, the sample group of Example 1 showed a more significant promoting effect on cell proliferation. The above results confirm that the gel prepared in Example 1 of this invention has excellent bioactivity and can improve cell proliferation.

[0110] Cell migration ability: Select L929 cells in good growth condition, digest with trypsin, and dilute to 1×10⁻⁶. 5 Cells per ml were counted, and 2 ml of the solution was added to each well of a 6-well plate for culture. After 24 h of culture, when the cell density reached 85%, cell scratching was performed, and serum-free culture medium corresponding to each group was added. The results were then photographed. The cell culture plates were placed in a 37°C, 5% CO2 cell culture incubator for 24 h. The scratched area was then photographed. The cell migration rate was calculated as follows: Cell migration rate (%) = (0 h scratch area - 24 h scratch area) / 0 h scratch area.

[0111] The sample groups are as follows: Blank control group: serum-free MEM culture medium; Sample group: Sample extracts of Example 1, Comparative Example 2 and Comparative Example 4 were prepared according to the method specified in GB / T16886.

[0112] The specific extraction method is as follows: Take samples from each group and extract them at a ratio of 0.1g sample to 1ml serum-free MEM medium. The extraction conditions are 37℃±1℃ for 24h±2h (37℃ for 24h is used in this example). After extraction, take 2ml of the extract from each group as the test solution.

[0113] The results are as follows Figure 3 As shown, the cell migration rate in the blank control group was 22.33%, the cell migration rate in the Example 1 sample group was 48.56%, the cell migration rate in the Comparative Example 2 sample group was 24.67%, and the cell migration rate in the Comparative Example 4 sample group was 31.00%. The possible reason is that Comparative Examples 2 and 4 lost the protective effect of the double-network hydrogel, resulting in partial denaturation of the protein active components, thus affecting the product's effect on cell migration. Compared with the blank control group, the Example 1 sample significantly promoted cell migration ability, increasing it by approximately 2.17 times (**P<0.01). Among the sample groups, the Example 1 sample group showed a more significant promoting effect on cell proliferation. The above results confirm that the gel prepared in Example 1 of this invention has excellent bioactivity, significantly promotes cell migration, and is more conducive to the repair of damaged tissues.

[0114] Example 2: Evaluation of retention time in animals The recombinant protein gynecological gel from Example 1 of this invention was mixed with a fluorescent marker; a commercially available gynecological gel sample was mixed with a fluorescent marker as a control group. Two ml of the gynecological gel was drawn from a syringe with a blunt-tipped soft tube and injected into the deep cervix of the vagina of ordinary female Japanese white rabbits. Fluorescent signal images of the cervix were acquired at the same exposure intensity for 1 min under a small animal imaging system at 1 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, and 42 h.

[0115] The results are as follows Figure 4 As shown, after 24 hours, the fluorescence residue percentage in Example 1 group was 60.82%, and 2.1% fluorescence residue was still detected even after 42 hours. The commercially available ordinary gynecological gel sample group showed a fluorescence residue percentage of only 5.6% after 24 hours, and no fluorescence residue was detected at 30 hours, indicating a limited retention time. The medical recombinant protein gynecological gel prepared in Example 1 of this invention still showed significant retention after 24 hours and fluorescence residue after 42 hours, indicating a retention time in vivo greater than 42 hours. This gel maintains good adhesion in the vagina, has a long retention time, is not easily discharged, and can effectively exert the efficacy of its components, achieving the therapeutic goal of once-every-two-day administration. It was verified that Examples 2–4, similar to Example 1, all had good retention effects, showing significant retention after 24 hours and fluorescence residue after 42 hours.

[0116] Example 3: Evaluation of vaginal irritation test The recombinant protein gynecological gel sample from Example 1 was tested according to Part B6 of GB / T16886.10-2017 Medical Device Biological Evaluation Part 10: Irritation and Skin Sensitization Tests. The results confirmed that the sample prepared using the method of Example 1 was non-irritating to the vaginal mucosa and met regulatory requirements.

[0117] Example 4: Evaluation of the repair effect of cervicitis in rats SD rats were acclimatized to a standard diet for one week and then randomly divided into three groups of six rats each: a normal group (n=6, no treatment); a model group; sample group 1 (Example 1); and sample group 2 (Comparative Example 9) (n=6 each). A cervicitis model was established in SD rats using 25% phenol gel. Rats were first anesthetized with isoflurane. Then, a 12-gauge rat gavage needle was gently inserted approximately 2 cm deep into the rat's vagina, and 0.25 mL of 25% phenol gel was injected. The vaginal opening was fixed with a cotton ball, and the rat was kept in an inverted position for 2 minutes. The drug was administered every other day for a total of five times. Vaginal changes were observed during the modeling process. After five administrations of phenol gel, most rats showed redness and swelling at the vaginal opening, with purulent discharge, indicating successful establishment of the rat cervicitis model.

[0118] The day after successful model establishment, rats in both the normal control group and the model group were fed normally, with no treatment administered from days 10 to 19. Starting on day 10, SD rats in the sample group were placed in a fixed frame with their heads facing down. A treated injection needle was gently inserted approximately 2 cm deep into the rat's vagina to administer 0.25 mL of the sample from Example 1 and Comparative Example 9. The medication was administered once daily for 10 consecutive days. Afterward, the rats were rapidly anesthetized and euthanized for observation.

[0119] like Figure 6HE and Masson histological staining results showed that, compared with the model group, the sample group showed significantly reduced vaginal redness and swelling and discharge after treatment, significantly reduced inflammatory cell infiltration, and no obvious edema or congestion. Figure 6 The tissues showed collagen deposition and orderly arrangement, indicating that the treatment of the sample groups had a good therapeutic effect on cervicitis in rats. Example 1 showed a more significant repair effect than Comparative Example 9, with fewer inflammatory cells, greater collagen deposition, and a more orderly arrangement. This indicates that the recombinant protein gynecological gel of the present invention has a more significant therapeutic effect on cervicitis in rats, with recombinant collagen and recombinant fibronectin playing a synergistic repair role. Verification showed that, compared with the model group, Examples 2–4 were similar to Example 1, showing good therapeutic effects on cervicitis in rats after treatment, significantly reducing vaginal swelling and discharge, significantly decreasing inflammatory cell infiltration, and showing no obvious edema or congestion.

[0120] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A medical recombinant protein gynecological gel, characterized in that, By weight percentage, it consists of the following raw materials: recombinant collagen 0.05-0.3%, recombinant fibronectin 0.01-0.05%, recombinant elastin 0.05-0.3%, isomaltooligosaccharide 0.1-1%, sodium hyaluronate 0.01-0.1%, poloxamer 407 21-26%, lactic acid 0.03-0.1%, preservatives 0.08-0.2%, glycerin 2%-8%, and purified water to make up to 100%.

2. The medical recombinant protein gynecological gel according to claim 1, characterized in that, The recombinant collagen is recombinant human type III collagen with a molecular weight of 10kDa~90kDa.

3. The medical recombinant protein gynecological gel according to claim 1, characterized in that, The recombinant fibronectin has a molecular weight of 40kDa to 55kDa.

4. The medical recombinant protein gynecological gel according to claim 1, characterized in that, The recombinant elastin has a molecular weight of 10kDa to 50kDa.

5. The medical recombinant protein gynecological gel according to claim 1, characterized in that, The molecular weight of the sodium hyaluronate is 1,000,000 to 1,400,000 Da.

6. The medical recombinant protein gynecological gel according to claim 1, characterized in that, The preservative is selected from one or more of methylparaben, ethylparaben, and propylparaben.

7. The method for preparing the medical recombinant protein gynecological gel according to any one of claims 1-6, characterized in that, Includes the following steps: S1: Mix poloxamer 407, recombinant elastin, sodium hyaluronate and purified water, and let stand at 4~10℃ for 12-18h to obtain solution-1; S2: Mix the preservative, glycerin and purified water, dissolve at 70-80℃, cool to room temperature and add to solution-1, stir at 4-10℃ to obtain solution-2; S3: Mix recombinant collagen, recombinant fibronectin, isomaltooligosaccharide and purified water, stir to dissolve, filter to remove bacteria and add to solution-2, stir and mix at 4-10℃ to obtain solution-3; S4: While stirring, add lactic acid dropwise to solution-3, and replenish the remaining purified water to obtain medical recombinant protein gynecological gel.

8. The method for preparing the medical recombinant protein gynecological gel according to claim 7, characterized in that: The stirring rate in S2-S4 is 200-300 rpm and the stirring time is 20-30 min.

9. The method for preparing the medical recombinant protein gynecological gel according to claim 7, characterized in that: The pH value of the medical recombinant protein gynecological gel is 3.8~4.

8.

10. The use of the medical recombinant protein gynecological gel according to any one of claims 1-6 in the preparation of a medicament for the prevention or treatment of vaginitis or cervicitis.

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