A recombinant human collagen peptide lubricant and preparation method thereof

Abstract

The present invention relates to a recombinant human collagen peptide lubricant and a preparation method thereof. The recombinant human collagen peptide lubricant is composed of the following raw materials in weight percentage: 0.05-1.00% recombinant human collagen peptide, 5.00-10.00% glycerol, 0.05-0.10% hydrophilic surfactant, and the balance is water; wherein the amino acid sequence of the recombinant human collagen peptide comprises an amino acid sequence shown in SEQ ID No: 1, and / or an amino acid sequence having at least 85% homology with SEQ ID No: 1. The lubricant of the present invention has the advantages of preventing and / or treating pelvic floor diseases, moisturizing, anti-inflammatory, antibacterial, antiviral, and spermicidal, and the method of the present invention has the advantages of simple preparation and high yield of recombinant human collagen peptide.

Description

Technical Field

[0001] The invention relates to the technical field of lubricants, and in particular to a recombinant human collagen peptide lubricant and a preparation method thereof. Background Art

[0002] Condoms are barrier devices that can be used during sexual intercourse, and they cannot completely reduce the risk of sexually transmitted infections and unwanted pregnancies in women. This is because during the wearing and use of condoms, they will come into contact with the external environment for a long time, causing bacteria to form on the surface of the condoms. In severe cases, women may be infected, and existing condoms generally have poor antibacterial effects. In addition, there are an increasing number of condoms on the market, with a variety of colors and smells. Although this can improve people's aversion to condoms and increase sexual appeal, the large amount of spices, lubricants, antibacterial agents, fungicides, antiviral agents and other substances added to them may cause chronic inflammation of the female vagina. Existing lubricants are mainly composed of silicone oil or mineral oil, which are greasy and difficult to clean. Long-term use will destroy the balance of the vaginal microecological system, leading to changes in the extracellular matrix, and then cause gynecological diseases.

[0003] The fascia, ligaments and muscles of the female pelvic floor form a complex pelvic floor support system to support the pelvic floor organs. Extracellular matrix (ECM) is the main component of the pelvic floor fascia, ligaments and vaginal wall. ECM is mainly composed of long-chain proteins such as collagen, elastin and proteoglycans. Collagen plays an important role in maintaining the elasticity and toughness of the pelvic floor connective tissue (F, Al-Azzawi, AA A, et al. Human Reproduction, 2003.). Among them, type I collagen has tensile properties and plays a supporting role, and type III collagen maintains the elasticity of the tissue (μlI H, et al. Heart Fail Rev, 2021, 26(5):1231-1248.). Studies have shown that the pathogenesis of pelvic floor dysfunction (FPFD) is closely related to abnormal ECM metabolism and remodeling (Vashaghian M, et al. Acta Biomater, 2019, 84: 159-168.), and type I collagen and type III collagen bind to integrin receptors and discoidin domain receptors to exert multiple biological functions. At present, animal tissues from livestock and poultry and aquatic animal tissues are the main ways for people to obtain natural collagen and its collagen peptides. They are mainly extracted from animal tissues such as pig skin and fish skin, and are a mixture mainly composed of type I collagen.

[0004] A Chinese patent with authorization announcement number CN107789611B discloses a collagen peptide lubricant and a preparation method thereof, which is made from the following raw materials in parts by weight: 5 to 20 parts of kombucha fermentation liquid, 0.5 to 1.2 parts of hyaluronic acid, 1 to 2 parts of kombucha fermentation membrane modified material, 8 to 12 parts of collagen peptides, 0.3 to 0.5 parts of ylang-ylang essential oil, 3 to 5 parts of water-soluble camellia seed oil, 0.5 to 1 parts of hydroxyethyl urea, 0.5 to 1 parts of hexylene glycol and 60 to 80 parts of deionized water. Although the above-mentioned lubricant has good water solubility and biocompatibility, and improves the problem that silicone oil or mineral oil is difficult to clean, its collagen peptide is taken from fish skin. Due to the difference in the gene sequence between animal collagen and human collagen, it has uncertain immunogenicity, which may cause abnormal immune response in the human body. Even if enzyme degradation can remove most potential immunogenic components in animal tissues and reduce the risk of allergic reactions, its immunogenicity and allergic reactions cannot be completely avoided. In addition, due to the complex microbial flora of the human cavity and the fragility of the skin, especially for users with high sensitivity and gynecological diseases, the inflammatory response may be aggravated, resulting in local chronic erosion, persistent pain, immune response and other problems in FPFD patients.

[0005] Chinese patent publication number CN113730557A discloses a use of recombinant type I humanized collagen in pelvic floor repair, wherein the recombinant type I humanized collagen comprises n repeats of the sequence shown in SEQ ID No. 1 (GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS), wherein when n is an integer greater than or equal to 2, each repeated sequence is directly connected, and the N-terminus of the recombinant type I humanized collagen comprises the sequence shown in SEQ ID No. 2 (ENLYFQ). The above-mentioned recombinant type I humanized collagen mainly provides plastic support for the vaginal skin and stimulates the reconstruction of collagen scaffolds to improve the decreased mechanical resistance of connective tissues such as pelvic floor muscles, ligaments, and fascia, and promote the normal metabolism of ECM, thereby achieving the purpose of preventing and / or treating FPFD. Although this humanized collagen eliminates the immune response and other problems existing in the original animal-derived collagen, its tissue components are large, tightly arranged, have poor anti-inflammatory factor performance, and insufficient lubrication properties. It is not easily absorbed by the vaginal skin. If used in large quantities in lubricants, it may cause vaginal skin stiffness and easily form scars after the inflamed skin heals. Summary of the invention

[0006] In view of the shortcomings of the prior art, the first purpose of the present invention is to provide a recombinant human collagen peptide lubricant, which solves the problems of poor antibacterial properties and immune response of existing animal-derived collagen, as well as the problems of poor anti-inflammatory factor performance, insufficient lubrication performance, and inability to take into account the tension and elasticity of the vaginal skin of recombinant type I human collagen, and has the advantages of preventing and / or treating pelvic floor diseases, moisturizing, anti-inflammatory, antibacterial, antiviral, and spermicidal.

[0007] The second object of the present invention is to provide a method for preparing a recombinant human collagen peptide lubricant, which has the advantages of simple preparation and high yield of recombinant human collagen peptide.

[0008] To achieve the above first object, the present invention provides the following technical solutions:

[0009] A recombinant human collagen peptide lubricant, composed of the following raw materials in weight percentage: 0.05-1.00% recombinant human collagen peptide, 5.00-10.00% glycerol, 0.05-0.10% hydrophilic surfactant, and the balance is water; wherein the amino acid sequence of the recombinant human collagen peptide comprises the amino acid sequence shown in SEQ ID No: 1, and / or an amino acid sequence having at least 85% homology with SEQ ID No: 1.

[0010] By adopting the above technical scheme, firstly, the recombinant human collagen peptide is genetically constructed using the mRNA sequence of the α1 chain of human type III collagen (Accession Number: X14420) as a template, and by analyzing the types, proportions and special sequences of the amino acids that make up the α1 chain, the characteristic sequence of the collagen domain, Gly-XY tripeptide repeat, is selected as the minimum repeating unit of the design sequence during artificial construction, and Trp, Tyr, Phe, Leu, lie, Val, Met and other amino acids are removed and replaced with Asp, Glu, Gln, Asn and other amino acids to improve the moisturizing, lubricating, anti-inflammatory and spermicidal properties of the recombinant type III human collagen, as well as the ability to promote normal metabolism of ECM;

[0011] Secondly, through the independently designed Linker (EGKSSGSGSEGKSS), the recombinant type III human collagen and the functional peptide (GLFGVLGSIAKHVLPHVVPVIAEKL) are fused and expressed to ensure that the two do not interfere with each other in terms of spatial structure. On the one hand, since the recombinant type III human collagen is a highly hydrophilic polypeptide, it can promote the soluble expression of the functional peptide, and the recombinant type III human collagen has lubricating and moisturizing properties, which can promote the effect of skin repair. On the other hand, the functional peptide connected by the Linker has strong anti-bacterial, anti-HSV and HIV virus properties, and can be used to prevent and treat the spread of gynecological diseases and epidemic sexually transmitted diseases.

[0012] Based on this, recombinant human collagen peptide can form a stable triple helix conformation and possess the characteristics of a biomaterial. Its structure is highly similar to that of human collagen and has good biocompatibility, affinity and permeability with vaginal skin. It can penetrate into the epidermis of the skin with an absorption rate of more than 99%. The absorbed recombinant human collagen peptide can fill in the skin matrix to form a reticular fiber and form a thin film layer on the skin surface. While enhancing the early tension and late plasticity of the skin, it can also stretch wrinkles, erosions and other inflammatory reactions caused by gynecological diseases and improve skin elasticity.

[0013] Finally, water is used as the main solvent, a small amount of glycerol is used to replace the silicone oil or mineral oil in the original lubricant, and a hydrophilic surfactant is added. Since each component in the raw material contains a hydrophilic group, it can absorb moisture in the air to achieve the purpose of moisturizing and lubricating. The lubricant obtained has a small odor and little irritation to the vaginal mucosa. It can be used to promote the adhesion, extension, and migration of HULFs of FPFD patients, and promote the expression of type I and III collagen, the main components of ECM. It has excellent biocompatibility, appropriate mechanical strength, flexibility and cell adhesion activity, and also has a good hemostatic effect, can protect the mucosa, promote the normal metabolism of ECM, can be used to prevent and / or treat pelvic floor diseases, and can play a moisturizing, anti-inflammatory, antibacterial, antiviral, and spermicidal role.

[0014] Furthermore, the amino acid sequence of the recombinant human collagen peptide comprises n repeated sequences of the amino acid sequence shown in SEQ ID No: 1, and / or an amino acid sequence having at least 85% homology with SEQ ID No: 1, where n≥1.

[0015] Furthermore, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32, wherein when n is an integer greater than or equal to 2, each repeated sequence is directly connected.

[0016] Furthermore, the amino acid sequence of the recombinant human collagen peptide has at least 88%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homology with the amino acid sequence of SEQ ID No:1.

[0017] Furthermore, the hydrophilic surfactant is Tween-80.

[0018] Furthermore, the lubricant also includes 0-4.00% of Inonotus obliquus (Fr) Pilat extract. Inonotus obliquus extract (INoNotus obliquus (Fr) Pilat) contains polysaccharides, inonotin, inonotinol and other active ingredients, which can effectively activate the immune system and inhibit the proliferation of viruses.

[0019] Furthermore, the lubricant also includes 0-8.00% of Ajuga extract. Ajuga extract (CAS: 41451-87-0) not only has an anti-inflammatory effect, but also can increase the activity of water channel proteins in skin cells, and promote recombinant human collagen peptides to fill in the skin matrix to form reticular fibers, and promote recombinant human collagen peptides to form a thin film layer on the skin surface to protect the skin and mucous membranes.

[0020] Furthermore, the lubricant also includes 0-1.00% of Echinacea extract. Echinacea extract (CAS: 70831-56-0) is used to assist recombinant human collagen peptides in immune regulation and prevention and treatment of gynecological diseases to prevent various infectious diseases and immune diseases such as HSV and HIV, and can promote vaginal mucosal repair of FPFD patients, maintain normal ECM metabolism, and inhibit inflammatory response, so that the wound surface after healing is smooth and scar-free.

[0021] Furthermore, the lubricant also includes 0-0.20% catechu extract. Catechu extract (CAS: 8001-48-7) can stabilize vaginal flora and prevent oxidation of recombinant human collagen peptides, and is preferably added in combination with echinacea extract to achieve better anti-pathogenic microorganism, anti-inflammatory and immunomodulatory effects.

[0022] Furthermore, the lubricant also includes 0-5.00% carnosine. Carnosine (CAS: 305-84-0) is a dipeptide composed of alanine and histidine, which can capture free oxygen radicals, improve fibroblast activity, prevent recombinant human collagen peptide oxidation, and promote recombinant human collagen peptide penetration and absorption by the skin. It can also significantly improve the plasticity of the skin after tension recovery, ensure the mechanical resistance of connective tissues such as pelvic floor muscles, ligaments, and fascia, and is preferably added in combination with the extract of tendon grass to avoid imbalance in the tension and plasticity of the skin after repair.

[0023] Furthermore, the lubricant comprises a composition of at least two of Inonotus obliquus extract, Ajuga oleifera extract, Catechu catechu extract and carnosine, and the content of the composition is controlled at 8.00-18.00%.

[0024] Furthermore, the recombinant human collagen peptide lubricant can be used to prepare human lubricants, condoms, etc. that prevent and / or treat pelvic floor diseases, and have moisturizing, anti-inflammatory, antibacterial, antiviral and spermicidal effects.

[0025] Among them, the condom of the present invention is provided with a plurality of evenly distributed micro grooves and protruding particles on the surface of the outer layer, which can increase the friction during use and improve the user experience; and the grooves are filled with recombinant human collagen peptide lubricant, so that the recombinant human collagen peptide lubricant can be continuously transported and evenly applied to the external space, thereby playing a role in continuous moisturizing, antibacterial and vaginal repair.

[0026] Preferably, the pelvic floor disease is a pelvic floor dysfunction disease; the female pelvic floor dysfunction disease has at least one of the following symptoms: decreased urethral dynamics, decreased vaginal biomechanical properties, local tissue structure damage, decreased expression of extracellular matrix components, and abnormal sacral ligament function.

[0027] More preferably, the decreased urethral dynamics is manifested as at least one of the following characteristics: decreased bladder urine storage function and decreased abdominal urine leakage point pressure.

[0028] More preferably, the local tissue structure damage is manifested as at least one of the following characteristics: thinning of the epithelial layer of the vaginal and urethral perivascular tissue, reduction in the number of blood vessels and collagen fibers in the connective tissue of the lamina propria, and atrophy and rupture of the muscular layer.

[0029] More preferably, the extracellular matrix component comprises at least one of type I collagen and type III collagen.

[0030] More preferably, the sacral ligament dysfunction is manifested as at least one of the following characteristics: decreased early adhesion ability of sacral ligament fibroblasts, and reduced cell spreading area during the early adhesion process of sacral ligament fibroblasts.

[0031] Preferably, the antibacterial activity is mainly manifested as a broad-spectrum and highly effective antibacterial and bactericidal effect, and has resistance to Gram-positive bacteria, Gram-negative bacteria and fungi, such as Aeromonas caviae, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Neisseria gonorrhoeae, Nocardia brasiliensis, Candida albicans, Aspergillus niger, Aspergillus fumigatus, Arthomyces sagoensis, Microsporum fumigatus, Cryptococcus neoformans, etc., all of which have a strong inhibitory and killing effect.

[0032] Preferably, the antiviral activity is mainly manifested in a strong inhibitory effect on herpes simplex virus (HSV) and human immunodeficiency virus (HIV).

[0033] To achieve the above second purpose, the present invention provides the following technical solutions:

[0034] A method for preparing a recombinant human collagen peptide lubricant comprises the following steps:

[0035] S1 artificially synthesizing the nucleotide sequence of the recombinant human collagen peptide SEQ ID No: 2, and inserting it between the Xho I and EcoR I restriction enzyme sites of the pPIC9K vector to obtain an expression vector;

[0036] S2 linearizes the expression vector obtained in S1 by single enzyme digestion with Sal I, introduces it into Pichia pastoris through electroporation, and obtains the recombinant human collagen peptide by high-density fermentation and post-treatment;

[0037] S3: preparing raw materials including recombinant human collagen peptide, glycerol, a hydrophilic surfactant and water according to a ratio, and mixing them to obtain the lubricating liquid.

[0038] By adopting the above technical scheme, the preparation steps are simple, and using the above method, a recombinant human collagen peptide with a yield of ≥900 mg and a purity of ≥98% can be obtained in every 1L of fermentation liquid.

[0039] Furthermore, in S2, the Pichia pastoris cell is Pichia pastoris GS115. Pichia pastoris GS115 is firstly screened on an MD plate, and then the clones grown on the MD plate are picked to a YPD plate containing different concentrations of the antibiotic G418 (0.1-20 mg / mL) for resistance screening. Finally, highly resistant colonies are selected and identified using PCR technology.

[0040] Furthermore, in the high-density fermentation process of S2, the dosage of the first-level seed liquid BMGY is controlled to be 1.0~2.0L, the dosage of the second-level seed liquid BMGY is controlled to be 10~20L, the fermentation medium is BMMY medium, the fermentation container is a 300L fermenter, the actual loading volume is 100~200L, the inoculation ratio is 1:(8~12), the culture temperature is 25~35°C, the inducer is 1% methanol, the induction time is 70~75h, the pH is adjusted to 5.5~6.5 with ammonia water, and 8~12L of 10% peptone is added as feed every 24h.

[0041] Furthermore, in the post-treatment process of S2, the fermentation broth obtained by high-density fermentation is first centrifuged, the centrifugation temperature is controlled to be 3-5°C, the rotation speed is 3500-4500 rpm, and the time is 25-35 min. The supernatant after centrifugation is collected, and then sequentially subjected to Ni-NTA affinity chromatography, cation exchange chromatography, and ultrafiltration desalting concentration to obtain the recombinant human collagen peptide.

[0042] In summary, the beneficial technical effects of the present invention are:

[0043] 1. The lubricant of the present invention has the advantages of preventing and / or treating pelvic floor diseases, moisturizing, anti-inflammatory, antibacterial, antiviral, and spermicidal;

[0044] 2. The method of the present invention has the advantages of simple preparation and high yield of recombinant human collagen peptide BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a flow chart of the method provided in Example 1 of the present invention.

[0046] Figure 2 : is a graph of the expression level of the recombinant human collagen peptide in Example 1 of the present invention in Pichia pastoris; wherein M is a protein marker, and columns 1 to 3 are Pichia pastoris fermentation supernatants (72 h);

[0047] Figure 3 3 is a graph showing the antibacterial effect of the recombinant human collagen peptide of Example 1 of the present invention on Staphylococcus aureus; wherein, * indicates a significant difference, P<0.01.

[0048] Figure 4 This is a graph showing the antibacterial effect of the recombinant human collagen peptide of Example 1 of the present invention on Neisseria lactosus; where * indicates a significant difference, P<0.01

[0049] Figure 5 This is a graph showing the infection rate of the recombinant human collagen peptide of Example 1 of the present invention in inhibiting HIV infection in T cells. DETAILED DESCRIPTION

[0050] In order to make the technical means, creative features, objectives and functions achieved by the present invention clearer and easier to understand, the present invention is further explained below in conjunction with the accompanying drawings and specific implementation methods.

[0051] Example

[0052] Example 1: A recombinant human collagen peptide lubricant disclosed in the present invention, the weight percentage of each component in the raw material is shown in Table 1. The amino acid sequence of the recombinant human collagen peptide comprises the amino acid sequence shown in SEQ ID No:1.

[0053] SEQ ID No:1 (HHHHHHGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGA PGQRGEPGPQGHAGAQGPPGPPGGPGSDGKPGPPGSQGESGRPGPPGSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDK GDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGGPGSDGKPGPPGSQGESGRPGPPGSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNG ETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGP EGKSS GSGSEGKSS GLFGVLGSIAKHVLPHVVPVIAEKL)

[0054] Reference Figure 1 and Figure 2 The present invention also discloses a method for preparing the above-mentioned recombinant human collagen peptide lubricant, comprising the following steps:

[0055] S1 artificially synthesizes the nucleotide sequence of recombinant human collagen peptide SEQ ID No: 2, and inserts it between the Xho I and EcoR I restriction enzyme sites of the pPIC9K vector to obtain an expression vector;

[0056]

[0057] S2 linearizes the expression vector obtained in S1 by single enzyme digestion with Sal I, introduces it into Pichia pastoris through electroporation, and obtains recombinant human collagen peptide through high-density fermentation and post-treatment; wherein,

[0058] The S21 Pichia pastoris cells were Pichia pastoris GS115. Pichia pastoris GS115 was first screened on MD plates, and then the clones grown on the MD plates were picked for resistance screening on YPD plates containing different concentrations of antibiotic G418 (0.1-20 mg / mL). Finally, highly resistant colonies were selected and identified using PCR technology.

[0059] The S22 high-density fermentation process adopted secondary fermentation, and the dosage of the first-level seed liquid BMGY was controlled to be 1.5L, the dosage of the second-level seed liquid BMGY was 15L, the fermentation medium was BMMY medium, the fermentation container was a 300L fermenter, the actual loading volume was 150L, ​​the inoculation ratio was 1:10, the culture temperature was 30℃, the inducer was 1% methanol, the induction time was 72h, the pH was adjusted to 6.0 with ammonia water, and 10L of 10% peptone was added every 24h;

[0060] After the S23 high-density fermentation is completed, the fermentation broth is first centrifuged, and the centrifugation temperature is controlled to be 4°C, the speed is 4000 rpm, and the time is 30 min. The supernatant after centrifugation is collected and then sequentially subjected to Ni-NTA affinity chromatography, cation exchange chromatography, and ultrafiltration desalting concentration to obtain recombinant human collagen peptides;

[0061] S3 prepares raw materials including recombinant human collagen peptide, glycerin, a hydrophilic surfactant, inonotus birch extract, ajuga extract, catechu extract, carnosine, and water according to a ratio, and mixes them to obtain a lubricating liquid.

[0062] In addition, the raw materials can be made of natural latex and other raw materials, and the mold can be immersed in the raw material liquid to make it evenly spread on the surface of the mold into a thin latex film, and then go through thinness and drying control, cleaning and packaging, cleaning, and testing of compression resistance, and then apply the lubricant prepared by the above method. Finally, after product packaging, random quality inspection, air blasting and stretching inspection, pinhole test, and functional test, you can get a condom that has the effects of preventing and / or treating pelvic floor diseases, moisturizing, anti-inflammatory, antibacterial, antiviral, and spermicidal.

[0063] Example 2: A recombinant human collagen peptide lubricant disclosed in the present invention. The difference from Example 1 is that the weight percentage of each component in the raw material is as shown in Table 1.

[0064] Example 3: A recombinant human collagen peptide lubricant disclosed in the present invention. The difference from Example 1 is that the weight percentage of each component in the raw material is as shown in Table 1.

[0065] Example 4: A recombinant human collagen peptide lubricant disclosed in the present invention. The difference from Example 1 is that the weight percentage of each component in the raw material is as shown in Table 1.

[0066] Example 5: A recombinant human collagen peptide lubricant disclosed in the present invention. The difference from Example 1 is that the weight percentage of each component in the raw material is as shown in Table 1.

[0067] Comparative Example

[0068] Comparative Example 1 is a recombinant human collagen peptide lubricant disclosed in the present invention. The difference from Example 1 is that the weight percentages of the components in the raw materials are as shown in Table 1.

[0069] Comparative Example 2 is a recombinant human collagen peptide lubricant disclosed in the present invention. The difference from Example 1 is that the weight percentages of the components in the raw materials are as shown in Table 1.

[0070] Comparative Example 3 is a recombinant human collagen peptide lubricant disclosed in the present invention. The difference from Example 1 is that the weight percentages of the components in the raw materials are as shown in Table 1.

[0071] Comparative Example 4 is a recombinant human collagen peptide lubricant disclosed in the present invention. The difference from Example 1 is that the weight percentages of the components in the raw materials are as shown in Table 1.

[0072] Comparative Example 5 is a recombinant human collagen peptide lubricant disclosed in the present invention. The difference from Example 1 is that the weight percentages of the components in the raw materials are as shown in Table 1.

[0073] Comparative Example 6: A recombinant human collagen peptide lubricant disclosed in the present invention, which is different from Example 1 in that the weight percentages of the components in the raw materials are as shown in Table 1.

[0074] Table 1

[0075] Components (wt%) Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Recombinant human collagen peptide 0.30 1.00 0.10 0.25 0.50 0 0.30 0.30 0.30 0.30 0.30 glycerin 8.00 5.00 10.00 6.00 9.00 8.00 8.00 8.00 8.00 8.00 8.00 Hydrophilic surfactant 0.08 0.09 0.05 0.10 0.06 0.40 0.40 0.40 0.40 0.40 0.08 Inonotus obliquus extract 3.28 3.50 4.00 2.00 2.80 3.28 0 3.28 3.28 3.28 3.28 Ajuga sibiricum Extract 5.20 5.00 6.00 8.00 3.00 5.20 5.20 0 5.20 5.20 5.20 Echinacea Extract 0.75 0.80 0.60 1.00 0.50 0.75 0.75 0.75 0 0.75 0.75 Catechu Extract 0.12 0.13 0.15 0.20 0.10 0.12 0.12 0.12 0.12 0 0.12 Carnosine 4.35 3.50 3.00 5.00 4.00 4.35 4.35 4.35 4.35 4.35 0 water margin margin margin margin margin margin margin margin margin margin margin

[0076] Performance testing

[0077] (1) Pick a single bacterial colony from the agar plate (G + The bacteria is Neisseria lactosus, G - The bacteria was Staphylococcus aureus), cultured in MH liquid medium for 16-18 hours, and then the bacterial solution was diluted to 1×10 6CFU / ml, added to a 96-well plate (50 μl / well, three replicates for each sample), and then 50 μl of culture medium was added to each well to a total volume of 100 μl. The recombinant human collagen peptide obtained in Example 1 was continuously diluted with PBS to a final concentration of 0.4, 0.8, 1.6, 3.12, 6.25, 12.5, 25 and 50 μM. For the positive control, 50 μl of sterile water was added to 50 μl of bacterial solution. After gently shaking with an oscillator for 1 min, it was placed in a constant temperature and humidity incubator and cultured for 24 hours. Using a Biotek ELx808 plate reader, the OD of each well before and after 24 hours was recorded. 630nm The data of the experiment are expressed as the mean OD ± standard error of the mean at each peptide concentration (one-way ANOVA with Tukey's post hoc test). Figure 3 and Figure 4 As shown. Figure 3 and Figure 4 It can be seen that at a concentration of 10-50 μM, recombinant human collagen peptide has an + , G - It has a significant inhibitory effect on sexual bacteria.

[0078] (2) HIV-VSV-G-GFP virus (vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped replication-incompetent HIV particles) was cultured with activated T cells in the presence of different concentrations (0.2-25 µM) of recombinant human collagen peptide. Specifically, the recombinant collagen peptide was incubated with 3×10 4 Activated human CD4+ T cells were incubated with virus at 37°C for 3 days. Cells were harvested and analyzed for GFP expression as an indicator of viral infection using a four-color flow cytometer and CelQuest software (BD Biosciences). 2 × 10 cells were removed from the culture 2 h after infection and addition of recombinant human collagen peptide. 4 T cells were stained with propidium iodide (PI; 25 µg / ml, Sigma) and analyzed for viability by flow cytometry. Phosphate-buffered saline (PBS) treatment served as a control group, and the results were as follows Figure 5 As shown. Figure 5 It can be seen that at a concentration of 10~50 μM, recombinant human collagen peptide has a strong inhibitory effect on HIV infection.

[0079] (3) 80 female SD rats aged 6 to 8 weeks were selected and randomly divided into 8 groups. Among them, the control group (n=10); the model group (n=10): only modeling (FPFD), without any treatment; the experimental group (6 groups, n=10), after 28 days of modeling, 0.5 ml of the recombinant human collagen peptide lubricant prepared in Example 1 and Comparative Examples 1 to 5 was vaginally administered daily for 14 consecutive days. The relative expression levels of TIMP-1, MMP-1, VEGF, and IL-6 factors were determined by qRT-PCR, and the results are shown in Table 2.

[0080] Table 2

[0081] Control group Model Group Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 MMP-1 (fold) 1.22 3.68 1.42 2.75 1.74 3.28 3.80 1.77 2.08 IL-6 (fold) 0.87 3.46 1.03 3.16 1.22 1.54 0.92 3.24 1.87 TIMP-1 (fold) 1.21 0.13 1.33 0.42 0.55 0.64 0.38 1.20 0.95 VEGF (fold) 0.98 0.23 0.95 0.33 0.70 0.52 0.35 0.85 0.68

[0082] As can be seen from Table 2, compared with the control group, in the model group, the relative expression levels of IL-6 and MMP-1 increased. After treatment with the recombinant human collagen peptide lubricant of Example 1, the relative expression levels of TIMP-1 and VEGF (vascular endothelial growth factor) increased compared with the model group, and the relative expression level of IL-6 decreased compared with the model group, indicating that the recombinant human collagen peptide lubricant of Example 1 can promote cell proliferation, blood vessel growth, and reduce the generation of inflammatory factors (such as IL-6). TIMP-1 is an extracellular matrix generation factor, and MMP-1 is an extracellular matrix degradation factor. After treatment with the recombinant human collagen peptide lubricant of Example 1, TIMP-1 expression increased and MMP-1 expression decreased, indicating that type III collagen can promote the generation of extracellular matrix, and the recombinant human collagen peptide lubricant treatment of Example 1 can increase the content of extracellular matrix in vaginal tissue.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A recombinant human collagen peptide lubricant, characterized in that: The invention is composed of the following raw materials in weight percentage: 0.05-1.00% recombinant human collagen peptide, 5.00-10.00% glycerol, 0.05-0.10% hydrophilic surfactant, and the balance is water; wherein the amino acid sequence of the recombinant human collagen peptide comprises the amino acid sequence shown in SEQ ID No: 1, and / or an amino acid sequence having at least 85% homology with SEQ ID No:

1.

2. A recombinant human collagen peptide lubricant according to claim 1, characterized in that: The hydrophilic surfactant is Tween-80.

3. The recombinant human collagen peptide lubricant according to claim 1, characterized in that: The lubricant also includes 0-4.00% of Inonotus obliquus extract.

4. A recombinant human collagen peptide lubricant according to claim 3, characterized in that: The lubricant also includes 0-8.00% of Ajuga sibiricum extract.

5. The recombinant human collagen peptide lubricant according to claim 4, characterized in that: The lubricant also includes 0-1.00% of Echinacea extract.

6. The recombinant human collagen peptide lubricant according to claim 5, characterized in that: The lubricant also includes catechu extract 0-0.20%.

7. The recombinant human collagen peptide lubricant according to claim 6, characterized in that: The lubricant also includes 0-5.00% carnosine.

8. The method for preparing a recombinant human collagen peptide lubricant according to any one of claims 1 to 7, characterized in that: The following steps are included: S1 artificially synthesizing the nucleotide sequence of the recombinant human collagen peptide SEQ ID No: 2, and inserting it between the Xho I and EcoR I restriction enzyme sites of the pPIC9K vector to obtain an expression vector; S2 linearizes the expression vector obtained in S1 by single enzyme digestion with Sal I, introduces it into Pichia pastoris through electroporation, and obtains the recombinant human collagen peptide by high-density fermentation and post-treatment; S3: preparing raw materials including recombinant human collagen peptide, glycerol, a hydrophilic surfactant and water according to a ratio, and mixing them to obtain the lubricant.

9. The method for preparing a recombinant human collagen peptide lubricant according to claim 8, characterized in that: In the high-density fermentation process of S2, secondary fermentation is adopted, the dosage of the first-level seed liquid BMGY is controlled to be 1.0~2.0L, the dosage of the second-level seed liquid BMGY is controlled to be 10~20L, the fermentation medium is BMMY medium, the fermentation container is a 300L fermenter, the actual loading volume is 100~200L, the inoculation ratio is 1:(8~12), the culture temperature is 25~35°C, the inducer is 1% methanol, the induction time is 70~75h, the pH is adjusted to 5.5~6.5 with ammonia water, and 8~12L of 10% peptone is added as feed every 24h.

10. The method for preparing a recombinant human collagen peptide lubricant according to claim 8, characterized in that: In the post-treatment process of S2, the fermentation broth obtained by high-density fermentation is first centrifuged, and the centrifugation temperature is controlled to be 3-5°C, the rotation speed is 3500-4500 rpm, and the time is 25-35 min. The supernatant after centrifugation is collected, and then sequentially subjected to Ni-NTA affinity chromatography, cation exchange chromatography, and ultrafiltration desalting concentration to obtain the recombinant human collagen peptide.

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