A double protein complex and a synergistic fermentation preparation method, preparation and application thereof

Abstract

The application discloses a double-protein complex and a synergistic fermentation preparation method, preparation and application thereof, wherein the double-protein complex is composed of recombinant type III humanized collagen microspheres and mussel mucin; the double-protein complex is composed of the recombinant type III humanized collagen microspheres and the mussel mucin, and the molecular quantity ratio of the recombinant type III humanized collagen microspheres and the mussel mucin is 1:1-8:1; the double-protein complex can increase the skin permeation rate of the mussel mucin through the action of the recombinant type III humanized collagen microspheres, and prevent the recombinant type III humanized collagen microspheres from being rapidly absorbed and transported through the adhesion of the mussel mucin. In addition, hyaluronic acid oligosaccharide is introduced as a system stabilizer, so that the double-protein complex is further stabilized, the anti-inflammatory repair function level is increased, the skin repair and wet adhesion are efficiently synergized, the excellent anti-inflammatory repair effect can be achieved at an extremely low concentration, and the double-protein complex has good application value and economic value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a dual-protein complex and its synergistic fermentation preparation method, formulation, and application. Background Technology

[0002] Skin inflammation is a common skin disease, and the number of patients is increasing year by year, with eczema, contact dermatitis, and seborrheic dermatitis being particularly prominent. Currently, measures to combat skin inflammation mainly fall into three categories: basic care, lifestyle interventions, and drug treatment. Basic care primarily involves using gentle medical moisturizers to protect and repair the skin barrier. Lifestyle interventions mainly involve avoiding contact with known allergens, reducing external irritants, and adjusting diet and lifestyle. However, these two measures can only prevent or intervene in the progression of inflammation; once inflammation occurs, drug treatment is the fundamental solution.

[0003] Existing medications for treating skin inflammation include nonsteroidal anti-inflammatory drugs (NSAIDs), calcineurin inhibitors (Calprophosphatase inhibitors), corticosteroids, and antihistamines. Mild inflammation can be treated with topical NSAIDs or Calcineurin inhibitors; moderate to severe inflammation may require short-term use of topical corticosteroids; and severe inflammation may require oral antihistamines to relieve itching or short-term oral corticosteroids. For chronic / autoimmune refractory inflammations, such as psoriasis and severe eczema, in addition to conventional medications, oral immunosuppressants or biologics are needed to precisely block inflammatory factors at specific immune targets. However, while these existing medications can effectively control inflammation, they still have significant limitations. For example, long-term use of topical corticosteroids can lead to side effects such as skin atrophy, telangiectasia, and pigmentation; oral immunosuppressants and biologics can suppress systemic immune function, increasing the risk of infection or liver damage; and highly effective biologics are not only expensive but also have low availability, placing a significant financial burden on patients with long-term use. In addition, some chronic inflammations, such as atopic dermatitis, are difficult to cure completely. Existing drugs can only control symptoms. After patients stop taking the medication, they are very likely to relapse when exposed to environmental stimuli. Therefore, long-term maintenance treatment is required. However, due to the side effects and usage restrictions of the existing drugs mentioned above, they are not conducive to long-term treatment and have poor accessibility.

[0004] Recombinant type III humanized collagen microspheres (rhCol III microspheres) are innovative recombinant type III humanized collagen microspheres with an innovative spatial structure developed by the applicant. They are formed by folding and cyclizing linear recombinant type III humanized collagen to create microspheres with a hollow structure. Their particle size is small, reaching 7-8 nanometers, making them very easy to cross the skin barrier. Furthermore, due to the structural modification, the degradation effect of proteases is delayed, effectively improving the stability and durability of collagen in the human body, showing promising application prospects in the medical aesthetics field. However, regarding skin inflammation, although reports show that recombinant type III humanized collagen (rhCol III) has anti-inflammatory activity, its effect in practical applications is weak, limited to regulating immune cell function and improving the inflammatory microenvironment, with limited efficacy in treating and repairing severe barrier damage. The application effect of rhCol III microspheres on skin inflammation is still unknown; moreover, due to the small particle size of rhCol III microspheres, they are easily absorbed and transported, which is unfavorable for the repair of inflamed epidermis.

[0005] Mussel adhesive protein (MAP) is a natural protein derived from the byssal threads of marine mussels, renowned for its strong adhesive properties, biocompatibility, and ability to promote repair. Studies show that mussel adhesive protein has anti-inflammatory, moisturizing, and cell migration-promoting effects. When it adheres to the skin surface, it not only reduces inflammation but also keeps the skin moist and promotes wound healing, making it an ideal anti-inflammatory and repairing ingredient. However, mussel adhesive protein is difficult to obtain, and natural extraction is extremely expensive, earning it the nickname "marine soft gold." Limited raw material availability leads to high pharmaceutical costs, which is highly disadvantageous for treating skin inflammations requiring long-term medication.

[0006] Hyaluronic acid oligosaccharides (o-Has) include hyaluronic acid disaccharide (o-Ha2), hyaluronic acid tetrasaccharide (o-Ha4), hyaluronic acid hexasaccharide (o-Ha6), hyaluronic acid octasaccharide (o-Ha8), and hyaluronic acid decasaccharide (o-Ha10), which are formed by the enzymatic degradation of hyaluronic acid (HA) by hyaluronidase (HAase). Studies have shown that all members of the hyaluronic acid oligosaccharide family, except for o-Ha2, have certain anti-inflammatory effects, with o-Ha4 and o-Ha6 showing the most significant effects. However, o-Ha6 is an immunomodulatory oligosaccharide, and o-Ha4 is the preferred choice for treating skin inflammation accompanied by severe itching and severe barrier dysfunction. Due to the low substrate affinity of existing HAases, feedback inhibition occurs. The applicant previously developed a hyaluronic acid fusion enzyme that can specifically bind to the substrate hyaluronic acid, solving the problem of substrate inhibition in the enzymatic catalytic reaction. However, experiments have shown that this hyaluronic acid fusion enzyme is more effective for large hyaluronic acid molecules with a molecular weight of 200,000 Da or higher, but its cleavage rate is poor for hyaluronic acid molecules with a molecular weight of less than 200,000 Da. Furthermore, the cleavage of excessively large hyaluronic acid molecules makes it difficult to control oligosaccharide fragments, resulting in more byproducts. These byproducts compete with the target product o-Ha4 for enzyme action sites, affecting the yield of o-Ha4. Therefore, when used for the treatment of skin inflammation, it is difficult to achieve the expected results.

[0007] Therefore, how to effectively and quickly repair inflammatory damage and prevent recurrence of inflammation has always been an urgent problem to be solved in the field of dermatitis treatment. Summary of the Invention

[0008] To address the aforementioned problems and achieve the aforementioned objectives, this invention provides a dual-protein complex, its synergistic fermentation preparation method, formulation, and application. First, a bicistronic expression plasmid is constructed to synergistically ferment and co-express the active fragments of recombinant type III humanized collagen and mussel adhesive protein, obtaining a dual-protein complex possessing the excellent properties of both, achieving a synergistic anti-inflammatory and repairing effect. Furthermore, hyaluronic acid oligosaccharides are used as a system stabilizer for the dual-protein complex, constructing a stable, multi-layered skin inflammation repair formulation to rapidly repair inflammatory damage and prevent inflammation recurrence. The specific technical solution is as follows:

[0009] First, the present invention provides a dual-protein complex, which is assembled from recombinant type III humanized collagen microspheres and mussel adhesive protein; the molecular weight of the dual-protein complex is 28-155 kDa, wherein the molecular ratio of recombinant type III humanized collagen microspheres to mussel adhesive protein is 1:1 to 8:1, preferably 4:1.

[0010] The aforementioned dual-protein complex further contains hyaluronic acid oligosaccharides as a system stabilizer; the amount of hyaluronic acid oligosaccharides added is 25-50 wt% of the mass of the dual-protein complex; and the content of hyaluronic acid tetrasaccharide in the hyaluronic acid oligosaccharides accounts for not less than 30 wt% of the total mass of all hyaluronic acid oligosaccharides.

[0011] Secondly, the present invention provides a method for preparing the aforementioned dual-protein complex, comprising the following steps:

[0012] 1) Construction of bicistronic expression plasmid: The recombinant type III humanized collagen active gene fragment and the mussel adhesive protein active gene fragment were tandemly linked to form a fusion gene, which was then inserted into a prokaryotic expression vector to construct a bicistronic expression plasmid;

[0013] 2) Preparation of fermentation engineered bacteria: The bicistronic expression plasmid constructed in step 1) is introduced into Escherichia coli competent cells together with the expression vectors for the cutting enzyme gene and the cyclase gene. After resistance screening and PCR identification, the positive bacteria obtained are the fermentation engineered bacteria; wherein, the cutting enzyme is TEV cutting enzyme, and the cyclase is Butelase ligase.

[0014] 3) Seed culture: The positive engineered bacteria prepared in step 2) were inoculated into seed culture medium and cultured at 37℃ with shaking at 200 rpm for 12–16 h until OD... 600 A value of 1.0 to 3.0 is used to obtain fermentation seed liquid;

[0015] 4) Batch feeding fermentation: Introduce the fermentation seed liquid cultivated in step 3) into the fermenter, and control the initial fermentation parameters as follows: temperature 30-37℃, pH 6.5-7.5, dissolved oxygen greater than 30%; when the dissolved oxygen value is greater than 60%-70%, start batch feeding culture, and maintain the dissolved oxygen value of the fermentation medium greater than 30% by adjusting the aeration rate and stirring speed, and continue to increase the cell density;

[0016] 5) Induced expression: When the OD of the fermentation broth... 600 When the dissolved oxygen value reaches 100-110, the culture temperature is lowered, and isopropyl-β-D-thiogalactopyranoside is added to induce the expression and cyclization of recombinant type III humanized collagen. The expression level of recombinant type III humanized collagen microspheres is detected at certain intervals. When the content of recombinant type III humanized collagen microspheres reaches the predetermined amount, the dissolved oxygen value is controlled to be less than 5%, the culture temperature is kept constant, and arabinose is added to induce the expression of mussel adhesive protein.

[0017] 6) Primary protein complexation: Maintain the pH of the culture medium at 6.5-7.5 and the temperature at 28℃. Stir at 250-350 rpm for a certain period of time to allow the recombinant type III humanized collagen microspheres and mussel adhesive protein to assemble and form a dual protein precomplex. After the cell density no longer increases, continue to culture for 6 hours to end the fermentation culture.

[0018] 7) Secondary protein complex: After fermentation, the engineered bacteria cells are collected by centrifugation, broken, and the pH is adjusted to maintain at 6.5-7.5 and the temperature at 28℃. The mixture is stirred at 250-350 rpm for a certain period of time to allow the pre-complex of the two proteins and the free protein to fully assemble and combine again to form a wheat-ear-shaped protein complex. Then, centrifugation and purification are performed to obtain the two protein complex.

[0019] 8) Stabilize the system: If further stabilization is required, replace the prepared dual-protein complex with sodium phosphate buffer at pH 7.0, add 25-50 wt% hyaluronic acid oligosaccharide solution (pH 7.0), and stir until homogeneous.

[0020] As a preferred technical solution, in step 1), the recombinant type III humanized collagen active gene sequence is shown in SEQ NO.1, the mussel adhesive protein active gene sequence is shown in SEQ NO.2, the two are linked together by a flexible linker peptide gene, the gene sequence of which is shown in SEQ NO.3; the fusion gene sequence formed by the three is shown in SEQ NO.4.

[0021] As a preferred technical solution, in step 4), the batch-fed fermentation involves the following: the inoculation amount of the fermentation seed liquid is 0.1% (v / v); the initial fermentation medium is TB medium, with the following formula: yeast extract 10g / L, corn starch 1g / L, soybean meal powder 2g / L, potassium dihydrogen phosphate 12g / L, magnesium sulfate 0.6g / L, glycerol 15g / L, ammonium sulfate 5g / L, bone peptone 5g / L, citric acid monohydrate 3g / L, diammonium hydrogen phosphate 3g / L, sodium chloride 1g / L, polyether defoamer 1ml / L, and trace element solution 10ml / L; a significant increase in dissolved oxygen levels, meaning dissolved oxygen is greater than 60%–70%, is considered when the batch fermentation begins.

[0022] The culture medium formula for the supplement is: glycerol 500g / L, trace elements 20ml / L, magnesium sulfate 2g / L, ammonium sulfate 7g / L;

[0023] The feeding rate is as follows: initially, add 5 ml per liter of fermentation liquid per hour, and after 2 hours, increase the feeding rate to 7 ml / L / h until fermentation is complete.

[0024] As a preferred technical solution, in step 5), the induction of expression involves using isopropyl-β-D-thiogalactopyranoside at a concentration of 0.1 g / L, added in 200 ml, and having a concentration of 10%. The expression level of the recombinant type III humanized collagen microspheres is detected every 1 hour, and the target content of the recombinant type III humanized collagen microspheres in the culture medium is 0.5–4 mg / ml.

[0025] The concentration of arabinose used to induce mussel adhesive protein expression was 0.5 g / L, the amount added was 3.3 L, and the concentration was 3%.

[0026] As a preferred technical solution, in step 8), the hyaluronic acid oligosaccharide is obtained by hyaluronic acid with a molecular weight of less than 200,000 Da under the enzymatic cleavage of modified hyaluronidase;

[0027] The method for preparing the modified hyaluronidase is as follows:

[0028] Culture medium formulation: Each 1000ml of culture medium contains 200ml of 6×M9 salt solution, 2ml of 1M MgSO4•7H2O solution, 20ml of 20% glucose solution, 0.1ml of 1M CaCl2•6H2O solution, and the remainder is DD water.

[0029] Fermentation process: An engineered bacterial suspension containing the hyaluronic acid fusion enzyme gene with a viable concentration of 10^8 to 10^9 CFU / ml was inoculated into the modified M9 medium, and chloramphenicol was added simultaneously. The fermentation conditions were 33℃ and 180 rpm. After 10 h of fermentation, isopropyl-β-D-thiogalactopyranoside was added as an inducer to induce the expression of hyaluronic acid fusion enzyme. After fermentation, the fermentation broth was centrifuged and ultrafiltered, and the retentate was collected to obtain the modified hyaluronidase.

[0030] As a preferred technical solution, the enzymatic digestion is performed as follows: 25-30 μL of modified hyaluronidase is added to a sodium hyaluronate solution with a concentration of 20-25 g / L per milliliter, the digestion temperature is 40℃, and the mixture is stirred at 60 rpm for 15 h to obtain a hyaluronic acid oligosaccharide mixture, wherein the mass of the hyaluronic acid tetrasaccharide accounts for not less than 30 wt% of the total mass of all hyaluronic acid oligosaccharides.

[0031] Furthermore, the present invention provides an application of the aforementioned dual-protein complex in the preparation of products for post-medical aesthetic repair and adjuvant treatment of skin inflammation; the products include masks, gels and liquid dressings; the dual-protein complex is used directly or as the main active ingredient of the product, and its content is 0.1 to 0.2% of the total mass of the product.

[0032] In addition, the present invention also provides a dressing for post-medical aesthetic repair and adjunctive treatment of skin inflammation. This dressing is composed of the aforementioned dual-protein complex and excipients, wherein the excipients include a thickener, a moisturizer, and a pH adjuster; the specific components, by mass fraction, are as follows:

[0033] The aforementioned dual-protein complex is present at a concentration of 0.1–0.2%.

[0034] Thickener 0.15-0.3%,

[0035] Moisturizer content: 3.6%–7.5%

[0036] pH adjuster 0.02-0.05%,

[0037] The remainder is purified water.

[0038] The humectant is one or more of trehalose, glycerin, propylene glycol, butylene glycol, pentanediol, 1,2-hexanediol, sorbitol, polyethylene glycol, betaine, and sodium hyaluronate, preferably a combination of trehalose, glycerin, and 1,2-hexanediol, with mass ratios in the system of 0.5–1.5%, 3–5%, and 0.1–1%, respectively. The thickener is one or more of carbomer, sodium polyacrylate, methylcellulose, ethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, soluble starch, xanthan gum, and gelatin, preferably a combination of carbomer and hydroxyethylcellulose, with mass ratios in the system of 0.05–0.1% and 0.1–0.2%, respectively. The pH adjuster is one of triethanolamine, aminomethylpropanol, sodium hydroxide, and potassium hydroxide, preferably triethanolamine.

[0039] The beneficial effects of this invention are:

[0040] 1) This invention assembles recombinant type III humanized collagen microspheres and mussel adhesive protein. The chain-like mussel adhesive protein is rich in DOPA groups. The catechol hydroxyl group (-OH) of this group can form numerous hydrogen bonds with the amino (-NH2), hydroxyl (-OH), and carboxyl (-COOH) groups on the collagen microspheres, thereby adsorbing the recombinant type III humanized collagen microspheres onto specific sites to form a wheat-ear-shaped dual-protein complex. Furthermore, the lysine (Lys) and arginine (Arg) in the mussel adhesive protein carry a positive charge and can form a cation-π interaction with the π electron cloud of the DOPA benzene ring. The basic amino acid residues in the recombinant type III humanized collagen microspheres also participate in this interaction, stabilizing the composite effect of the two. In addition, the ring portion of the recombinant type III humanized collagen microspheres is a hydrophobic region, which can interact with hydrophobic amino acid residues (such as alanine and valine) in mussel adhesive protein and the benzene ring structure of DOPA, further enhancing the stability of the composite system and making the obtained dual-protein complex have good application value.

[0041] 2) In the dual-protein complex of the present invention, the molecular ratio of recombinant type III humanized collagen microspheres to mussel adhesive protein is 1:1 to 8:1, preferably 4:1, and the molecular weight of the dual-protein complex is controlled to be 28 to 155 kDa. At this time, the wheat-ear-shaped protein complex structure formed by the recombinant type III humanized collagen microspheres and mussel adhesive protein increases the transdermal permeability of mussel adhesive protein through the action of the recombinant type III humanized collagen microspheres. At the same time, the adhesion of mussel adhesive protein prevents the rapid absorption and transport of recombinant type III humanized collagen microspheres. The two work together to construct a multi-layered anti-inflammatory and repairing effect on the skin, greatly reducing the amount of mussel adhesive protein used while improving the product's efficacy.

[0042] 3) The dual-protein complex of this invention uses hyaluronic acid oligosaccharides as a system stabilizer. Hyaluronic acid oligosaccharides are rich in hydroxyl, carboxyl, and amide groups, which can be well linked to the protein complex, making the structure of the dual-protein complex more stable through hydrogen bonds. At the same time, hyaluronic acid oligosaccharides, especially hyaluronic acid tetrasaccharides, can promote the repair of damaged dermal and stratum corneum of skin, and have the functions of promoting the proliferation of human epidermal blastocysts and human skin fibroblasts, as well as moisturizing. Therefore, the introduction of hyaluronic acid oligosaccharides further increases the level of anti-inflammatory and repair effects, not only accelerating the recovery of skin barrier function, but also improving skin cell condition and enhancing immune resistance, inhibiting the recurrence of skin inflammation, and better exerting a synergistic anti-inflammatory and repair effect.

[0043] 4) The method for preparing the dual-protein complex of this invention involves constructing a bicistronic expression plasmid to co-express recombinant type III humanized collagen microspheres and the active fragment of mussel adhesive protein through synergistic fermentation. Then, in situ assembly allows the two to combine and form a dual-protein complex. This not only significantly reduces raw material costs but also ensures that the DOPA group of the mussel adhesive protein remains in a reduced state to the maximum extent, binding with the recombinant type III humanized collagen microspheres, thus facilitating the acquisition of a wheat-ear-shaped complex conformation with optimal synergistic function. Furthermore, the self-assembly after co-expression by engineered bacteria significantly reduces fermentation batches, purification steps, and related material consumption, achieving a reduction in production costs and extreme simplification of the process. Moreover, the induced expression of recombinant type III humanized collagen microspheres and mussel adhesive protein is initially combined in cells by engineered bacteria, and then further combined after cell wall disruption, ensuring a more complete and controllable combination process, a more uniform product structure, and guaranteeing product quality and efficacy.

[0044] 5) The hyaluronidase obtained by this invention through the modification of M9 medium has higher purity and is more suitable for the enzymatic digestion of hyaluronic acid below 200,000 Da, and can significantly increase the content of tetrasaccharides in the enzymatically digested hyaluronic acid oligosaccharides. Using the enzymatically digested hyaluronic acid oligosaccharide solution as a system stabilizer for the dual-protein complex is more conducive to exerting a synergistic anti-inflammatory and repairing effect, and is suitable for skin inflammation.

[0045] 6) The dual protein complex of the present invention can be used as an effective ingredient in products for post-medical aesthetic repair and adjuvant treatment of skin inflammation (including masks, gels and liquid dressings, etc.). Its content in the product is low, only about 0.1 to 0.2%, which can achieve good anti-inflammatory and repair effects.

[0046] In summary, this invention not only creates a high-performance collagen-mussel adhesive protein ear-shaped complex structure, but also achieves comprehensive optimization from molecular design and production process to final function through a co-expression in-situ assembly preparation process and a system construction that stabilizes and enhances the effect with hyaluronic acid oligosaccharides. It successfully integrates multiple functions such as structural support, wet adhesion, cell repair, and immune regulation, providing a groundbreaking solution for the field of skin repair that is innovative, efficient, and economical. It achieves highly efficient synergy between skin repair and wet adhesion, exhibiting excellent anti-inflammatory and repairing effects even at extremely low concentrations, demonstrating significant application and economic value. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the dual-protein complex structure of recombinant type III humanized collagen microspheres and mussel adhesive protein of the present invention.

[0048] Figure 2 This is a schematic diagram of the cationic-π interaction in the recombinant type III humanized collagen microsphere-mussel adhesive protein complex of the present invention.

[0049] Figure 3 This is a schematic diagram of the stable system of the recombinant type III humanized collagen microsphere-mussel adhesive protein dual protein complex and hyaluronic acid oligosaccharide of the present invention.

[0050] Figure 4 The molecular weight and purity of the recombinant type III humanized collagen microsphere-mussel adhesive protein dual-protein complex of the present invention are as follows:

[0051] Figure 5 The distribution of hyaluronidase digestion products after the improvement in Example 2 of this invention;

[0052] Figure 6 The distribution of the original patent hyaluronidase digestion products in Comparative Example 1 of Example 2 of this invention;

[0053] Figure 7 The results of the transdermal permeability of mussel adhesive protein in each sample at different time points in Example 3 of this invention;

[0054] Figure 8 This illustrates the effect of each sample in Example 3 of the present invention on HSF cell migration;

[0055] Figure 9This describes the effect of each sample in Example 3 of the present invention on the expression of the inflammatory factor TNF-α in RAW264.7 cells;

[0056] Figure 10 This demonstrates the wound repair effect of each sample in Example 3 of the present invention on the rat trauma infection model. Detailed Implementation

[0057] To address the problems of existing drugs for treating skin inflammation, such as side effects, usage limitations, difficulty in long-term treatment, and poor accessibility; and the small particle size of recombinant type III humanized collagen microspheres, which are easily absorbed and transported, hindering their application in repairing inflamed epidermis; the difficulty in obtaining mussel adhesive protein, resulting in high drug costs; and the high oligosaccharide byproducts and low yield of hyaluronidase in the current preparation of hyaluronidase, which are also detrimental to the treatment of skin inflammation, this invention provides a dual-protein complex and its co-fermentation preparation method, formulation, and application. Firstly, a bicistronic expression plasmid is constructed. This invention involves co-fermenting and co-expressing recombinant type III humanized collagen with active fragments of mussel adhesive protein to obtain a dual-protein complex that combines the excellent properties of both, thereby achieving a synergistic anti-inflammatory and repairing effect. Furthermore, the invention optimizes the hyaluronidase preparation method, increasing the yield of hyaluronic acid tetrasaccharide in the product, making it suitable for the treatment of skin inflammation. The hyaluronic acid tetrasaccharide obtained by enzymatic digestion is used as a stabilizer for the dual-protein complex, strengthening the dual-protein complex system while further constructing a multi-layered therapeutic and repairing effect for skin inflammation, achieving the effect of rapidly repairing inflammatory damage and preventing inflammation recurrence.

[0058] This invention provides a dual-protein complex assembled from recombinant type III humanized collagen microspheres and mussel adhesive protein. The chain-like mussel adhesive protein is rich in DOPA groups, whose catechol hydroxyl (-OH) groups can form numerous hydrogen bonds with the amino (-NH2), hydroxyl (-OH), and carboxyl (-COOH) groups on the collagen microspheres. This adsorbs the recombinant type III humanized collagen microspheres onto specific sites, forming a wheat-ear-shaped dual-protein complex. Figure 1 As shown. Furthermore, the lysine (Lys) and arginine (Arg) in mussel adhesive proteins carry a positive charge, which can form a cation-π interaction with the π electron cloud of the DOPA benzene ring. The basic amino acid residues in the recombinant type III humanized collagen microspheres also participate in this interaction, stabilizing the complex interaction between the two, such as... Figure 2 As shown. In addition, the ring portion of the recombinant type III humanized collagen microspheres is a hydrophobic region, which can interact with hydrophobic amino acid residues (such as alanine and valine) in mussel adhesive protein and the benzene ring structure of DOPA, further enhancing the stability of the complex system and making the obtained dual-protein complex have good application value.

[0059] The molecular weight of the dual-protein complex of the present invention is 28-155 kDa, wherein the molecular ratio of recombinant type III humanized collagen microspheres to mussel adhesive protein is 1:1 to 8:1, preferably 4:1. In this case, the wheat-ear-shaped protein complex structure formed by the recombinant type III humanized collagen microspheres and mussel adhesive protein increases the transdermal permeability of mussel adhesive protein through the action of the recombinant type III humanized collagen microspheres, while simultaneously preventing the rapid absorption and transport of the recombinant type III humanized collagen microspheres through the adhesion of the mussel adhesive protein. The two work synergistically to construct a multi-layered anti-inflammatory and repairing effect on the skin, significantly reducing the amount of mussel adhesive protein required while maintaining the product's effectiveness.

[0060] In addition, the dual-protein complex also contains hyaluronic acid oligosaccharides as a system stabilizer; the amount of hyaluronic acid oligosaccharides added is 25-50 wt% of the mass of the dual-protein complex; and the content of hyaluronic acid tetrasaccharides in the hyaluronic acid oligosaccharides accounts for no less than 30 wt% of the total mass of all hyaluronic acid oligosaccharides. Hyaluronic acid oligosaccharides are rich in hydroxyl, carboxyl, and amide groups, which can be well linked to the protein complex, making the structure of the dual-protein complex more stable through hydrogen bonds, such as... Figure 3 As shown. Simultaneously, hyaluronic acid oligosaccharides, especially hyaluronic acid tetrasaccharides, can promote the repair of damaged dermis and stratum corneum, and also have the effect of promoting the proliferation of human epidermal morphocytes and human skin fibroblasts, as well as moisturizing functions. Therefore, the introduction of hyaluronic acid oligosaccharides further enhances the anti-inflammatory and repairing effects, not only accelerating the recovery of skin barrier function, but also improving skin cell condition and enhancing immune resistance, inhibiting the recurrence of skin inflammation, and better exerting a synergistic anti-inflammatory and repairing effect.

[0061] The preferred method for preparing the dual-protein complex of the present invention includes the following steps:

[0062] 1) Construction of bicistronic expression plasmid: The recombinant type III humanized collagen active gene fragment and the mussel adhesive protein active gene fragment were tandemly linked to form a fusion gene, which was then inserted into a prokaryotic expression vector to construct a bicistronic expression plasmid; the sequence of the recombinant type III humanized collagen active gene is shown in SEQ NO.1, the sequence of the mussel adhesive protein active gene is shown in SEQ NO.2, and the two are tandemly linked by a flexible linker peptide gene, the sequence of which is shown in SEQ NO.3; the sequence of the fusion gene formed by the three is shown in SEQ NO.4.

[0063] SEQ NO.1: Recombinant type III humanized collagen active gene sequence:

[0064] 5'-GGCATCCCCGGCGAGAAGGGCCCCGCCGGCGAGAGGGGCCCCCGGCCCCGCCGGCCCCAGGGGCGAGAGGGGCGCCCCCGGCTTCAGGGGCCCCGCCGGCCCCAACCACGTG-3';

[0065] Its encoded amino acid sequence is: GIPGEKGPAGERGAPGPAGPRGERGAPGFRGPAGPN (SEQ NO.5).

[0066] SEQ NO.2: Mussel adhesive protein active gene sequence:

[0067] 5'-ATGAGCAGCGAGGAATATAAGGGGTGGCTACTATCCGGGCAACACCTATCACTATCATAGCGGTGGCAGCTACCACGGAAGCGGTTATCACGGTGGCTACAAGGGCAAGTACTACGGCAAGGCGAAGAAATACTACTACAAGTACAAGAACAGCGGCAAGTATAAATACCTGAAGAAAGCGCGTAAGTATCACCGTAAAGGTTACAAGAAATATTACGGTGGCGGTAGCAGCTAA-3';

[0068] Its encoded amino acid sequence is as follows:

[0069] MSSEEYKGGYYPGNAVHYHSGGSYNGSGYHGGYKGYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKYYGGSS (SEQ NO. 6).

[0070] SEQ NO.3: Flexible linker peptide gene sequence:

[0071] 5'-CCGCCGTCGCCGCCG-3';

[0072] This flexible linker peptide gene can be directly embedded into the ends of the target genes (recombinant type III humanized collagen activity gene and mussel adhesive protein activity gene), allowing the two to be linked to construct a co-expression plasmid.

[0073] Its encoded amino acid sequence is: Gly-Gly-Ser-Gly-Gly. The main purpose of the flexible linker peptide is to avoid interference between the folding of the two proteins. It can also be cleaved along with the tag by a specific protease, thus obtaining a "clean" target protein.

[0074] SEQ NO.4: Fusion gene sequence:

[0075] 5'-GGCATCCCCGGCGAGAAGGGCCCCGCCGGCGAGAGGGGCCCCCGGCCCCGCCGGCCCCAGGGGCGAGAGGGGCGCCCCCGGCTTCAGGGGCCCCGCCGGCCCCAACCACGTGCCGCCGTCGCCGCCGATGAGCAGCGAGGAATATAAGGGTGGCTACTATCCGGGCAACACCTATCACTA TCATAGCGGTGGCAGCTACCACGGAAGCGGTTATCACGGTGGCTACAAGGGCAAGTACTACGGCAAGGCGAAGAAATACTACTACAAGTACAAGAACAGCGGCAAGTATAAATAACCTGAAGAAAGCGCGTAAGTATCACCGTAAAGGTTACAAGAAATATTACGGTGGCGGTAGCAGCTAA-3'.

[0076] 2) Preparation of fermentation engineered bacteria: The bicistronic expression plasmid constructed in step 1) is introduced into Escherichia coli competent cells together with the expression vectors for the cutting enzyme gene and the cyclase gene. After resistance screening and PCR identification, the positive bacteria obtained are the fermentation engineered bacteria. The cutting enzyme is TEV cutting enzyme and the cyclase is Butelase ligase. This is the prior art. Refer to the previously filed patent technology (patent number: 202311780528.0).

[0077] 3) Seed culture: The positive engineered bacteria prepared in step 2) are inoculated into seed culture medium and cultured at 37℃ and 200 rpm for 12-16 h until the OD600 value is 1.0-3.0 to obtain the fermentation seed liquid;

[0078] 4) Fed-batch fermentation: Introduce the fermentation seed culture from step 3) into the fermenter, controlling the initial fermentation parameters as follows: temperature 30–37℃, pH 6.5–7.5, dissolved oxygen greater than 30%; when the dissolved oxygen level is greater than 60%–70%, begin fed-batch culture, maintaining the dissolved oxygen level of the fermentation medium above 30% by adjusting the aeration rate and stirring speed, and continue to increase the cell density; the inoculum size of the fed-batch fermentation seed culture is 0. 0.1% (v / v); the initial fermentation medium was TB medium, with the following formula: yeast extract 10 g / L, corn starch 1 g / L, soybean meal powder 2 g / L, potassium dihydrogen phosphate 12 g / L, magnesium sulfate 0.6 g / L, glycerol 15 g / L, ammonium sulfate 5 g / L, bone peptone 5 g / L, citric acid monohydrate 3 g / L, diammonium hydrogen phosphate 3 g / L, sodium chloride 1 g / L, polyether defoamer 1 ml / L, and trace element solution 10 ml / L. The addition of corn starch and soybean meal powder serves to protect the bacteria, allowing the engineered bacteria to adapt to high-density fermentation, thereby increasing protein yield.

[0079] The culture medium formula for feeding is: 500 g / L glycerol, 20 ml / L trace elements, 2 g / L magnesium sulfate, and 7 g / L ammonium sulfate; the feeding rate is: initially 5 ml / L / hour per liter of fermentation broth, then increasing to 7 ml / L / h after 2 hours until fermentation is complete.

[0080] 5) Induced expression: When the OD of the fermentation broth... 600 When the dissolved oxygen level reached 100–110, the culture temperature was lowered, and isopropyl-β-D-thiogalactopyranoside was added to induce the expression and cyclization of recombinant type III humanized collagen. The expression level of recombinant type III humanized collagen microspheres was detected at certain time intervals. When the content of recombinant type III humanized collagen microspheres reached the predetermined amount, the dissolved oxygen level was controlled to be less than 5%, the culture temperature was kept constant, and arabinose was added to induce the expression of mussel adhesive protein. The concentration of isopropyl-β-D-thiogalactopyranoside used to induce the expression of recombinant type III humanized collagen was 0.1 g / L, the amount added was 200 ml, and the concentration was 10%. The concentration of arabinose used to induce the expression of mussel adhesive protein was 0.5 g / L, the amount added was 3.3 L, and the concentration was 3%.

[0081] 6) Primary protein complexation: Maintain the pH of the culture medium at 6.5-7.5 and the temperature at 28℃. Stir at 250-350 rpm for a certain period of time to allow the recombinant type III humanized collagen microspheres and mussel adhesive protein to assemble and form a dual protein precomplex. After the cell density no longer increases, continue to culture for 6 hours to end the fermentation culture.

[0082] 7) Secondary protein complex: After fermentation, the engineered bacteria cells are collected by centrifugation, broken, and the pH is adjusted to maintain at 6.5-7.5 and the temperature at 28℃. The mixture is stirred at 250-350 rpm for a certain period of time to allow the pre-complex of the two proteins and the free protein to fully assemble and combine again to form a wheat-ear-shaped protein complex. Then, centrifugation and purification are performed to obtain the two protein complex.

[0083] 8) Stabilize the system: If further stabilization is required, replace the prepared dual-protein complex with sodium phosphate buffer at pH 7.0, add 25-50 wt% hyaluronic acid oligosaccharide solution (pH 7.0), and stir until homogeneous.

[0084] The hyaluronic acid oligosaccharide is obtained by enzymatic digestion of hyaluronic acid with a molecular weight of less than 200,000 Da using a modified hyaluronidase. The preparation method of the modified hyaluronidase is as follows: Culture medium formulation: per 1000 ml of culture medium, there are 200 ml of 6×M9 salt solution, 2 ml of 1M MgSO4•7H2O solution, 20 ml of 20% glucose solution, 0.1 ml of 1M CaCl2•6H2O solution, and the remainder is DD water; Fermentation process: an engineered bacterial suspension containing the hyaluronic acid fusion enzyme gene with a viable bacterial concentration of 10^8~10^9 CFU / ml is inoculated into the modified M9 culture medium, and chloramphenicol is added at the same time. The fermentation culture conditions are 33℃ and 180 rpm; after 10 h of fermentation, isopropyl-β-D-thiogalactopyranoside is added to induce the expression of hyaluronic acid fusion enzyme; after fermentation, the fermentation broth is centrifuged and ultrafiltered, and the retentate is collected to obtain the modified hyaluronidase.

[0085] The enzymatic digestion process is as follows: 25-30 μL of modified hyaluronidase is added to each milliliter of sodium hyaluronate solution with a concentration of 20-25 g / L. The digestion temperature is 40℃, and the mixture is stirred at 60 rpm for 15 h to obtain a mixture of hyaluronic acid oligosaccharides, wherein the mass of hyaluronic acid tetrasaccharide accounts for no less than 30 wt% of the total mass of all hyaluronic acid oligosaccharides.

[0086] This invention controls the amount of collagen expression by controlling the collagen induction time (2, 3, 4, 5, 6 h). Specifically, collagen expression is induced for 2 hours by adding 0.1 g / L isopropyl-β-D-thiogalactoside (IPTG), and the fermentation broth is then tested. At this time, the content of recombinant type III humanized collagen is about 0.5-0.8 mg / ml. After extending to 3 hours, the content is about 1.0-1.3 mg / ml. The protein complexes obtained at different induction times were analyzed by SDS-PAGE gel electrophoresis. The results showed that the molecular weight of the protein composition after 2 hours of IPTG induction was mainly about 28 kDa (at this time, the ratio of rhCol III microspheres to mussel adhesive protein molecules was 1:1); after 3 hours of induction, the molecular weight of the complex was mainly about 80 kDa, suggesting that the ratio of collagen to mussel adhesive protein molecules was 4:1 at this time; after 5 and 6 hours of induction, the molecular weight of the complex was the largest, about 150 kDa, suggesting that the maximum adsorption ratio of collagen to mussel adhesive protein was about 8:1. Thereafter, with further extension of the culture time, the molecular weight did not increase further, which is speculated to be due to the steric hindrance of the recombinant type III humanized collagen microspheres, preventing them from binding to the DOPA sites on mussel adhesive protein. The purified dual-protein complexes in various proportions were then tested to evaluate their transdermal performance and biological activity. The protein complex with a 4:1 ratio of collagen to mussel adhesive protein showed the best transdermal performance and biological activity. Therefore, the molecular ratio of recombinant type III humanized collagen microspheres and mussel adhesive protein in the dual-protein complex of the present invention is 1:1 to 8:1, with a preferred ratio of 4:1.

[0087] The dual-protein complex provided by this invention can be used to prepare products for post-medical aesthetic repair and adjuvant treatment of skin inflammation, including masks, gels and liquid dressings. The dual-protein complex is used directly or as the main active ingredient in the product. Its content in the product is low, at 0.1-0.2% of the total product mass, but it can achieve good anti-inflammatory and repair effects.

[0088] For example, a dressing for post-medical aesthetic repair and adjunctive treatment of skin inflammation is composed of the aforementioned dual-protein complex and excipients. The excipients include thickeners, humectants, pH adjusters, etc., and by mass fraction, the dressing components include: 0.1–0.2% of the aforementioned dual-protein complex, 0.15–0.3% of the thickener, 3.6–7.5% of the humectant, 0.02–0.05% of the pH adjuster, with the balance being purified water.

[0089] The moisturizing agent is one or more of trehalose, glycerin, propylene glycol, butylene glycol, pentanediol, 1,2-hexanediol, sorbitol, polyethylene glycol, betaine, and sodium hyaluronate, preferably a combination of trehalose, glycerin, and 1,2-hexanediol, with mass ratios of 0.5-1.5%, 3-5%, and 0.1-1% respectively in the system. The thickener is one or more of carbomer, sodium polyacrylate, methylcellulose, ethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, soluble starch, xanthan gum, and gelatin, preferably a combination of carbomer and hydroxyethylcellulose, with mass ratios of 0.05-0.1% and 0.1-0.2% respectively in the system. The pH adjuster is one of triethanolamine, aminomethylpropanol, sodium hydroxide, and potassium hydroxide, preferably triethanolamine. This dressing can be used for post-medical cosmetic repair and as an adjunct treatment for skin inflammation.

[0090] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0091] Example 1

[0092] This embodiment describes the preparation of a dual-protein complex. Specifically, it includes the following steps:

[0093] First, construct the bicistronic expression plasmid as follows:

[0094] Based on the type III collagen α1 chain gene sequence (SEQ NO.1, accession number: NM_000090.4) and the mussel adhesive protein Mfp-5 gene sequence (SEQ NO.2, accession number: AY222877) published in GenBank, primers were designed to amplify the type III collagen active gene fragment (encoding amino acid 36) and the mussel adhesive protein active gene fragment (encoding amino acid 79). The amino acid sequences and primer sequences are as follows:

[0095] COL3 amino acid sequence:

[0096] GIPGEKGPAGERGAPGPAGPRGERGAPGFRGPAGPN (SEQ NO. 5);

[0097] MFP5 amino acid sequence:

[0098] MSSEEYKGGYYPGNAVHYHSGGSYNGSGYHGGYKGYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKYYGGSS (SEQ NO. 6);

[0099] Primer sequences:

[0100] Col3-F: 5'-CGGGATCCATGAAGGTGATGCTGGTG-3' (BamHI restriction site, SEQ NO.7);

[0101] Col3-R: 5'-GGTGGTGGTGGTGGTGCTTCTTGTAGTCCTCG-3' (Copyright linker peptide encoding sequence, SEQ NO. 8);

[0102] Mfp-F: 5'-CGCGGATCCATGAAGAAGAAGAAGAAGAAG-3' (Copyright linker peptide coding sequence, BamHI restriction site, SEQ NO.9);

[0103] Mfp-R: 5'-CCGCTCGAGTTAGTGGTGGTGGTGGTGGTGTTATTTGTTCGTCTTTTTG-3' (XhoⅠ restriction site, SEQ NO. 10).

[0104] Two gene fragments were obtained by PCR amplification, recovered by agarose gel electrophoresis, and then subjected to overlap extension PCR. The two fragments were tandemly linked through a flexible linker peptide encoding gene (SEQ NO.3) to obtain the Col3-linker-Mfp fusion gene (SEQ NO.4). The fusion gene and the pET-28a vector were double-digested with BamHI and XhoI, respectively. The digestion products were recovered and ligated with T4-DNA ligase, transformed into E. coli DH5α competent cells, plated on LB agar plates containing kanamycin, and single colonies were picked for enzyme digestion identification and sequencing to obtain the correctly constructed bicistronic expression plasmid pET-28a-Col3-Mfp.

[0105] Then, engineered bacteria were prepared and fermentation seed culture was cultivated. The constructed plasmid pET-28a-Col3-Mfp was transformed into Escherichia coli BL21(DE3) competent cells. The specific steps are as follows: 10 μL of plasmid was added to 100 μL of competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, and then immediately incubated on ice for 2 min. 900 μL of antibiotic-free LB medium was added, and the cells were cultured at 37℃ and 200 rpm for 1 h with shaking. 100 μL of the bacterial culture was spread on an LB agar plate containing 50 μg / mL kanamycin and cultured at 37℃ for 12 h. Single colonies were picked and inoculated into 5 mL of LB medium containing kanamycin and cultured at 37℃ and 200 rpm for 12 h with shaking. The plasmid was extracted for PCR identification. Positive bacterial cultures were the engineered bacteria seed culture. 600 The value is 1.5.

[0106] Next, fed-batch fermentation and induced expression were carried out. The seed culture was inoculated into the fermenter at a rate of 0.1% (v / v). The initial fermentation medium was TB medium with the following formula: yeast extract 10 g / L, corn starch 1 g / L, soybean meal powder 2 g / L, potassium dihydrogen phosphate 12 g / L, magnesium sulfate 0.6 g / L, glycerol 15 g / L, ammonium sulfate 5 g / L, bone peptone 5 g / L, citric acid monohydrate 3 g / L, diammonium hydrogen phosphate 3 g / L, sodium chloride 1 g / L, polyether defoamer 1 ml / L, and trace element solution 10 ml / L. During fermentation, the temperature was controlled within the range of 30–37℃, and the pH was adjusted to 6.5–7.5 using ammonia. The dissolved oxygen level in the initial medium was calibrated to 100% by maximizing the stirring and aeration speeds. The dissolved oxygen level was maintained above 30% by adjusting the stirring speed and aeration rate. Once the basal culture medium is completely consumed (i.e., with constant aeration and stirring speed), the dissolved oxygen level rises significantly, exceeding 60%–70%. At this point, fed-batch culture begins (feed culture medium: 500 g / L glycerol, 20 ml / L trace elements, 2 g / L magnesium sulfate, 7 g / L ammonium sulfate), continuously increasing the cell density. Initially, the feeding rate is 5 ml / L / h per liter of fermentation broth, fed uniformly into the fermenter using a peristaltic pump. After 2 hours, the feeding rate is increased to 7 ml / L / h, and then remains constant. Throughout the process, the dissolved oxygen level in the fermentation medium is maintained above 30% by adjusting the aeration and stirring speed.

[0107] When fermentation broth OD 600 When the dissolved oxygen value reached the range of 100-110, the culture temperature was lowered to 28℃, and 0.1 g / L of IPTG (filtered and sterilized) was added to induce the expression and cyclization of recombinant type III humanized collagen. During this period, samples were taken every hour to detect the expression level of recombinant type III humanized collagen microspheres. After 4 hours of culture, the content of recombinant type III humanized collagen microspheres was found to be approximately 2 mg / ml. Then, the dissolved oxygen value was controlled to be less than 5%, the culture temperature was kept constant, and 0.5 g / L of arabinose was added to induce the expression of mussel adhesive protein.

[0108] Next, the protein complexation step was carried out. First, during the induction of mussel adhesive protein expression, the pH was maintained in the range of 6.5 to 7.5 and the temperature was 28°C. The mixture was stirred at 300 rpm for 1 hour to allow the recombinant type III humanized collagen microspheres and mussel adhesive protein to spontaneously assemble into a protein pre-complex. After the cell density stopped increasing, the culture was continued for 6 hours to end the fermentation culture. Then, the bacterial cells were collected by centrifugation (17,000 rpm, until clear) using a tubular centrifuge. The collected bacterial cells were then lysed using a high-pressure homogenizer at 800 Bar for three cycles. Next, the cells were centrifuged using a refrigerated centrifuge (8,000 rpm, 20 min), and the supernatant was collected. The pH was adjusted to maintain the value within the range of 6.5–7.5, and the temperature was kept at 28°C. The mixture was stirred at 300 rpm for 30 min to allow the recombinant type III humanized collagen microspheres and mussel adhesive protein to fully assemble again into a protein complex. The complex was then purified by salting out with 90 g / L ammonium sulfate for 3–5 precipitation cycles to obtain the dual-protein complex of recombinant type III humanized collagen microspheres and mussel adhesive protein.

[0109] The supernatant collected after purification and centrifugation was analyzed by SDS-PAGE electrophoresis to determine the molecular weight of the target protein. Figure 4 As shown, the results indicate that the target protein has a molecular weight of approximately 80 kDa and its expression level accounts for 97% of the total protein.

[0110] Example 2

[0111] This embodiment is for preparing a mixture of hyaluronic acid and oligosaccharides. Specifically, it includes the following steps:

[0112] I. Preparation of the modified M9 culture medium;

[0113] ① 2.46g of 1M MgSO4•7H2O solution, added to 10ml of DD water, sterilized and ready for use;

[0114] ② 2.191 g of 1M CaCl2•6H2O solution, added to 10 ml of DD water, sterilized and ready for use;

[0115] ③ 6×M9 salt solution: 12.8g Na2HPO4•7H2O, 3.0g KH2PO4, 0.5g NaCl, 1.0g NH4Cl, dissolved in 200ml DD water, sterilized at 121℃ for 15min.

[0116] ④ Prepare a 20% glucose solution: Dissolve 4g of glucose in 20ml of DD water and filter to sterilize using a 0.22-micron filter.

[0117] ⑤ Prepare M9 culture medium under aseptic conditions: Take 200ml of 6×M9 salt solution, 2ml of 1M MgSO4•7H2O solution, 20ml of 20% glucose solution, 0.1ml of 1M CaCl2•6H2O solution, and add DD water to 1000ml.

[0118] II. Induction of hyaluronidase expression through inoculation and fermentation

[0119] Take 100ml of the modified M9 medium and place it in a 250mL Erlenmeyer flask with a baffle. Inoculate 50μL of a bacterial suspension containing the hyaluronic acid fusion enzyme gene (this is prior art, refer to the previously filed patent technology, patent number: 202210200683.X), with a viable cell concentration of 10^9 CFU / ml and an inoculum-to-liquid ratio of 0.05% (w / v). Simultaneously, add chloramphenicol to make the chloramphenicol concentration in the fermentation broth 5mg / ml. The shake-flask fermentation conditions are 33℃ and 180rpm. At the 10th hour of fermentation, add the inducer IPTG to make the IPTG concentration in the fermentation broth 0.1mM. After 6 hours of induction, end the fermentation.

[0120] III. Hyaluronidase Collection and Purification

[0121] Centrifuge the fermentation broth at 10,000 rpm for 10 min, then perform ultrafiltration using a 50 kDa filter membrane. Collect the retentate and dilute it to one-tenth of the fermentation broth volume with 0.05 M pH 5.5 phosphate buffer, thus concentrating the fermentation broth 10 times as the enzyme digestion solution.

[0122] IV. Hyaluronic acid enzymatic digestion

[0123] Add 750ml of purified water to a 1L beaker, add 11.5g of sodium citrate, then add 2.5g of citric acid monohydrate, and stir to dissolve. Add 16.0g of sodium hyaluronate powder (molecular weight 20W), stir to dissolve, and heat to 100℃ and keep warm for 10min. After the temperature drops to 40℃, keep warm, add 20ml of the above enzyme solution, and stir at 40℃ (60rpm) for 15h to obtain a hyaluronic acid oligosaccharide mixture. High performance liquid chromatography (HPLC) analysis was performed using an anion exchange column, 5μm, 250×4.6mm; mobile phase A: pure water; mobile phase B: 0.1M sodium sulfate aqueous solution, pH adjusted to 3.5 with sulfuric acid; detection wavelength: 210nm; column temperature: 40℃; flow rate: 1.0ml / min. The hyaluronic acid oligosaccharide mixture contained 33.7% hyaluronic acid tetrasaccharide. Figure 5 As shown.

[0124] Comparative Example 1

[0125] Add 750ml of purified water to a 1L beaker, add 11.5g of sodium citrate, then add 2.5g of citric acid monohydrate, and stir to dissolve. Add 16.0g of sodium hyaluronate powder (molecular weight 20W), stir to dissolve, and heat to 100℃ and keep warm for 10min. After the temperature drops to 40℃, keep warm, add 20ml of enzyme solution obtained by existing technology (patent number: 202210200683.X), and stir at 40℃ (60rpm) for 15h to obtain a hyaluronic acid oligosaccharide mixture. Detected by high-performance liquid chromatography (HPLC), the chromatographic column was an anion exchange column, 5μm, 250×4.6mm; mobile phase A: pure water; mobile phase B: 0.1M sodium sulfate aqueous solution, pH adjusted to 3.5 with sulfuric acid; detection wavelength: 210nm; column temperature: 40℃; flow rate: 1.0ml / min. The content of hyaluronic acid tetrasaccharide in this hyaluronic acid oligosaccharide mixture was 13.2%. Figure 6 As shown.

[0126] Example 3

[0127] This embodiment examines the transdermal effect, cell migration rate, and anti-inflammatory effect of the dual-protein complex prepared in Example 1.

[0128] In addition to the dual-protein complex prepared in Example 1, the experiments also included control samples, specifically the following:

[0129] Sample 1: Pure recombinant type III humanized collagen microsphere solution (prepared using existing technology);

[0130] Sample 2: Pure mussel adhesive protein solution (purchased from Jiangyin Beiruisen Biochemical Technology Co., Ltd.);

[0131] Sample 3: A mixture of recombinant type III humanized collagen microsphere solution (prepared using existing technology) and purchased mussel adhesive protein (purchased from Jiangyin Beiruisen Biochemical Technology Co., Ltd.);

[0132] Preparation method: Weigh mussel adhesive protein and add it to the recombinant type III humanized collagen microsphere solution, stir and mix well;

[0133] Sample 4: The dual-protein complex prepared in Example 1 of this invention;

[0134] Sample 5: A dual-protein complex stabilized with hyaluronic acid oligosaccharides prepared in Example 2;

[0135] Preparation method: Add the hyaluronic acid oligosaccharide solution prepared in Example 2 to the sodium phosphate buffer of the dual protein complex prepared in Example 1, the concentration of which is 50 wt% of the dual protein complex, and stir until homogeneous;

[0136] Sample 6: A hyaluronic acid oligosaccharide-stable dual-protein complex obtained by hyaluronidase digestion using existing technology;

[0137] Preparation method: The hyaluronic acid oligosaccharide stabilized dual-protein complex prepared in Comparative Example 1 was used. The hyaluronic acid oligosaccharide solution prepared in Comparative Example 1 was added to the sodium phosphate buffer of the dual-protein complex prepared in Example 1, with a concentration of 50 wt% of the dual-protein complex, and stirred until homogeneous.

[0138] Sample 7: A mixture of uncyclized recombinant type III humanized collagen (purchased from Zhejiang Zhuji Juyuan Biotechnology Co., Ltd.) and mussel adhesive protein (purchased from Jiangyin Beiruisen Biochemical Technology Co., Ltd.);

[0139] Preparation method: Weigh a certain amount of recombinant type III humanized collagen and mussel adhesive protein into purified water, mix well and the product is obtained.

[0140] The examination method is as follows:

[0141] 1. Transdermal effect

[0142] Thawed pigskin was gently rinsed with 0.05M pH 7.4 phosphate buffer. The skin was then cut into circular pieces with the same effective area as the diffusion chamber using a skin punch. These pieces were pre-incubated in 0.05M pH 7.4 phosphate buffer at 32℃ for 30 minutes to reach the experimental temperature and restore physiological function. The pretreated skin was fixed between the donor and receiver chambers, with the epidermis facing upwards, ensuring a good seal between the skin and the chamber without air bubbles. Preheated receiving solution to 32℃ was added to the receiver chamber, ensuring the solution completely covers the dermal side of the skin. A magnetic stir bar was added and stirring was started (400 rpm). Each sample was evenly applied to the skin surface. Transdermal absorption times were set to 0h, 4h, 8h, 12h, and 24h. After the transdermal absorption time was completed, the stratum corneum of the skin was washed with phosphate buffer, and the volume was adjusted to 1.0mL. The content of residual mussel adhesive protein in the donor chamber was measured, and the transdermal absorption rate was calculated based on the content of the remaining mussel adhesive protein.

[0143] The concentration of mussel adhesive protein in each sample was 2.5 mg / ml, and the sample loading volume was 20 μL. After collection, liquid chromatography was performed. The chromatographic column was an Agilent Zorbax SB 300Å C8 (4.6 × 250 mm, 5 μm); the mobile phase was 0.1% TFA aqueous solution (A) - 0.1% TFA acetonitrile (B) with gradient elution; the detection wavelength was 280 nm; the column temperature was 25 ℃; the flow rate was 0.9 mL / min; and the injection volume was 20 μL. A mussel adhesive protein standard solution (0.05 mg / ml) was used as the standard. The collagen content in the experimental sample was 20 mg / ml, and the concentration of mussel adhesive protein was 2.5 mg / ml.

[0144] The results are shown in Table 1 and Appendix. Figure 7 As shown.

[0145] Table 1: Transdermal test results of mussel adhesive protein in each sample (μg)

[0146]

[0147] Note: In Table 1, the transmittance = 1 - content at each time point / initial addition amount * 100%.

[0148] The results in Table 1 show that 98% of mussel adhesive protein remained in the supply tank 24 hours after transdermal absorption, indicating that mussel adhesive protein was almost impossible to absorb transdermally (Sample 2). Physical mixing with collagen microspheres could promote the transdermal absorption of mussel adhesive protein to a certain extent, with a transdermal absorption rate of 39.4% at 24 hours (Sample 3). The amount of mussel adhesive protein absorbed transdermally by the fermented dual-protein complex was significantly increased, with a transdermal absorption rate exceeding 70% at 24 hours (Sample 4). Enzymatic cleavage of hyaluronic acid oligosaccharides with modified enzymes did not affect the transdermal absorption of mussel adhesive protein (Sample 5), while enzymatic cleavage of hyaluronic acid oligosaccharides using existing technology slightly affected the transdermal absorption of mussel adhesive protein (Sample 6), possibly due to the higher content and molecular weight of hexose, octose, and decaose. Mixing commercially available collagen with mussel adhesive protein had no effect on the absorption of mussel adhesive protein (Sample 7).

[0149] In summary, the dual-protein complex described in this invention can significantly promote the transdermal absorption of mussel adhesive protein. The addition of hyaluronic acid oligosaccharides has no significant effect on its absorption, and the higher the content of hyaluronic acid tetrasaccharides, the smaller the effect. Therefore, the improvement of hyaluronidase also has high application value.

[0150] 2. Cell migration rate

[0151] The principle of the scratch assay is to use a micropipette tip to streak lines on the cell growth area of ​​a monolayer of adherent cells cultured in an in vitro culture dish or plate. Cells in the streaked area are then aspirated, and the cells are cultured for the predetermined time (e.g., 24 hours). The cell growth and migration ability of peripheral cells is then observed. To assess this, a marker pen is used to draw evenly spaced horizontal lines on the back of a 6-well plate, approximately every 0.5–1 cm, passing through the wells. Three lines are drawn through each well. Approximately 5 × 10⁵ cells are then added to the wells. 5Cells were cultured in an incubator. Cell growth was observed the next day. Once the cells had filled the bottom of the wells, a pipette tip was used to make horizontal scratches, perpendicular to the back of the cell line. The cells were then washed three times with PBS to remove the scratched cells, and medium containing 1% FBS was added. A certain concentration of the test sample was then added, and the cells were incubated at 37°C with 5% CO2. After 24 hours, the degree of confluence of the scratches in each well was photographed and recorded. A control group was included, with only 1% FBS medium. Human skin fibroblasts (HSF) were used. The concentrations of collagen, mussel adhesive protein, and hyaluronic acid oligosaccharides were 0.5 mg / ml. Specific results are shown in Table 2 and Appendix. Figure 8 .

[0152] Table 2: Effects of each sample on HSF cell migration rate

[0153]

[0154] Note: In Table 2, the values ​​represent the distance L (mm) of the scratch, and the migration rate = 1 - L. 24h / L 0h *100%.

[0155] Table 2 shows that, compared to the control group, both collagen microspheres and mussel adhesive protein (samples 1 and 2) promoted HSF cell migration to a certain extent when used alone. Physical mixing of the two showed a synergistic effect (sample 3). The fermented dual-protein complex exhibited a stronger cell migration-promoting effect at the same concentration, reaching 81.4% (sample 4). The addition of hyaluronic acid oligosaccharides resulted in cell migration rates exceeding 90% (samples 5 and 6), with the modified hyaluronidase-digested hyaluronic acid oligosaccharides showing a more pronounced effect, achieving a nearly 100% HSF cell migration rate (sample 6), demonstrating a strong synergistic effect. The mixture of commercially available linear collagen and mussel adhesive protein showed a weaker effect on cell migration, with a migration rate of 45.1% (sample 7).

[0156] Therefore, it can be concluded that the fermented collagen microspheres / mussel adhesive protein dual protein complex and hyaluronic acid oligosaccharides in this invention exhibit a significant synergistic effect in promoting cell migration, which provides a theoretical basis for synergistic repair applications in clinical practice.

[0157] 3. Anti-inflammatory effect

[0158] 3.1 The anti-inflammatory activity of each sample was investigated using an in vitro RAW264.7 cell inflammation model.

[0159] Experimental Procedure: RAW264.7 cells were cultured and passaged twice before the experiment. Cells were digested with trypsin / EDTA, and the cell suspension was collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in culture medium. Cells were counted using a cell counter or hemocytometer. The cell suspension was then divided into two groups of 2 × 10⁻⁶ cells. 4 Cells / wells were seeded into 96-well plates at 200 μl per well and incubated for 24 h. The culture medium was discarded, and induction and drug administration began. Test wells were treated with culture medium containing a specific concentration of the test substance and lipopolysaccharide (LPS) (1 μg / ml). Negative control wells were treated with culture medium containing only LPS. Positive control wells were treated with culture medium containing the positive control (dexamethasone 100 μg / ml) and LPS. Blank / solvent control wells were treated with 200 μl of cell culture medium per well. Incubation was continued for 24 h. After incubation, 150 μl of cell culture medium was collected and centrifuged at 1000g for 20 min at 4°C. The supernatant was transferred to a 1.5 ml sterile centrifuge tube, and the TNF-α content was detected using an ELISA kit. The concentrations of collagen microspheres, mussel adhesive protein, and hyaluronic acid oligosaccharide were 1 mg / ml, 0.125 mg / ml, and 0.5 mg / ml, respectively. The specific results are shown in Table 3 and Appendix. Figure 9 .

[0160] Table 3: TNF-α content in cell sap of each group of samples after 24 hours of treatment (expressed as optical density value)

[0161]

[0162] Note: In Table 3, the concentration "1+0.125" in Sample 3 represents the concentration of collagen microspheres and mussel adhesive protein; "1.125+0.5" in Samples 5 and 6 represents the concentration of the dual protein complex and hyaluronic acid oligosaccharide.

[0163] Table 3 shows that collagen microspheres (sample 1) exhibited certain anti-inflammatory activity at 1 mg / ml, with an inhibition rate of 17.1% on TNF-α production in cells; mussel adhesive protein (sample 2) showed significant anti-inflammatory activity at 0.125 mg / ml, with an inhibition rate of 44.8% on TNF-α production in cells; the combination of the two (sample 3) enhanced anti-inflammatory activity, but did not show a significant synergistic effect; the dual-protein complex (sample 4) in the invention showed significantly higher anti-inflammatory activity than the combination of the two (sample 3), demonstrating a 1+1>2 effect; and the addition of hyaluronic acid oligosaccharides to the dual-protein complex further enhanced its anti-inflammatory activity. Furthermore, comparing the anti-inflammatory activities of samples 5 and 6, the higher the content of hyaluronic acid tetrasaccharides, the stronger the anti-inflammatory enhancement effect, with sample 5 showing an inhibition rate of 95.6% on TNF-α production in cells.

[0164] Therefore, it can be concluded that the dual-protein complex obtained by fermentation in this invention has significant anti-inflammatory activity, significantly enhances the anti-inflammatory activity of mussel adhesive protein, and exhibits a significantly stronger anti-inflammatory effect than the physical combination of collagen microspheres and mussel adhesive protein; the addition of hyaluronic acid oligosaccharides further enhances the anti-inflammatory activity of the dual-protein complex. To this end, animal experiments were also conducted to verify the anti-inflammatory and repairing activity of this complex, providing a solid foundation for its clinical application.

[0165] 3.2 Investigating the repair effect of the dual-protein complex on a rat skin injury infection model.

[0166] Experimental Procedure: Twenty-five healthy adult male SD rats (220g±20g) were acclimatized for one week and randomly divided into four groups: a model group, a mussel adhesive protein group (sample 2), a dual-protein complex group (sample 4), a dual-protein complex + hyaluronic acid oligosaccharide group (sample 5), a positive control group, and a recombinant type III humanized collagen microsphere composition group, with five rats in each group. The back hair was shaved with a razor and then removed with depilatory cream before anesthesia. After skin disinfection with povidone-iodine, circular wounds were prepared at the corresponding sites using a 6mm diameter punch. After hemostasis, 50μL of a 1×10⁻⁶ concentration was applied to each animal's wound. 8 Infection with CFU / ml E. coli solution, covered with sterile gauze, and the gauze was removed after 24 hours. Successful modeling was indicated by redness, swelling, and exudation at the wound site. The model group had 0.5 ml of physiological saline applied to the wound surface, the test sample group had an equal amount of the test sample applied, and the positive control group had gentamicin ointment applied. The wound was then fixed with medical gauze and tape. Administration was once daily for three days. Post-surgery, rats were housed individually in single cages with free access to food and water. Wound healing was observed daily with dressing changes. Wound healing was observed and the healing rate calculated on days 3, 7, and 14 post-surgery. The wound was photographed at a fixed focal length, and the wound area was measured using ImageJ software. The skin wound healing rate was calculated as (initial wound area - current wound area) / initial wound area × 100%. On day 14, animals were sacrificed, and newly formed skin tissue was collected to detect the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). A blank skin area was used as a control. Specific results are shown in Tables 4 and 5 and the appendix. Figure 10 .

[0167] Table 4: Skin healing rate (%) in each group of rats

[0168]

[0169] Note: In Table 4, the healing rate of each group is compared with the wound area at each time point and 0d; "*", "**", "***" are comparisons with the model group, P<0.05, P<0.01, P<0.001; "a" indicates comparison with sample 2, P<0.05; "b" indicates comparison with sample 4, P<0.05.

[0170] Table 5: Comparison of IL-6 and TNF-α levels in each group

[0171]

[0172] Note: In Table 5, “a” indicates comparison with the model group, P<0.05; “b” indicates comparison with sample 2, P<0.05; and “c” indicates comparison with sample 4, P<0.05.

[0173] As shown in Tables 4 and 5, compared with the mussel adhesive protein group (sample 2), the dual-protein complex described in this invention (sample 4) significantly accelerated the healing rate of infected wounds in rats and significantly reduced the levels of IL-6 and TNF-α in the skin, with statistically significant differences (P<0.05). This indicates that the dual-protein complex of collagen microspheres and mussel adhesive protein can increase the anti-inflammatory and repairing effects of mussel adhesive protein, which may be related to its promotion of the transdermal efficiency of mussel adhesive protein and the achievement of multi-level synergistic anti-inflammatory effects. In addition, the dual-protein complex with added hyaluronic acid oligosaccharides (sample 5) had a faster healing rate and lower inflammation level, indicating that hyaluronic acid oligosaccharides have a promoting effect on the anti-inflammatory and repairing effects of the dual-protein complex, which may be related to its participation in stabilizing the dual-protein complex and its own barrier repair function, and its effect is better than that of the positive control group.

[0174] Example 4 Stability Test

[0175] This experiment aims to investigate the stability of the dual-protein complex in a formulation. The formulation investigated in this experiment is a liquid dressing, specifically prepared using the dual-protein complex prepared in Example 1 and excipients. The dressing components, by mass fraction, are as follows:

[0176] Example 1 prepared a dual-protein complex of 0.1125%, carbomer 980 0.08%, trehalose 1%, glycerol 4%, hydroxyethyl cellulose 0.15%, 1,2-hexanediol 0.5%, triethanolamine 0.04%, and purified water to 100%.

[0177] The preparation method of this dressing includes the following steps:

[0178] (1) Add 800g purified water, 40g glycerol, 0.8g carbomer 980 and 1.5g hydroxyethyl cellulose to a beaker, turn on the stirrer, stir at 280rpm, heat to 80℃ (±5℃) and stir for 30min.

[0179] (2) Add 10g of trehalose and 5g of 1,2-hexanediol to a beaker, turn on the stirrer, and stir at 280 rpm for 30 minutes.

[0180] (3) Cool down to 40℃ (±5℃), add the dual protein complex prepared in Example 1, turn on the stirrer, stir at 280 rpm for 10 min.

[0181] (4) Add 0.4g of triethanolamine to the beaker, add purified water, turn on the stirrer, and stir for 10 minutes at a speed of 280 rpm.

[0182] (5) After preparation, observe the appearance and pH value. If there are no abnormalities, discharge the liquid dressing. This liquid dressing is designated as Dressing 1.

[0183] The following dressings were used for comparison in this embodiment:

[0184] Dressing 2: The preparation method is the same as that of Dressing 1, except that the dual protein complex prepared in Example 1 in the formulation is replaced with Sample 3 in Example 3.

[0185] Dressing 3: The preparation method is the same as that of Dressing 1, except that the dual protein complex prepared in Example 1 in the formulation is replaced with Sample 5 in Example 3.

[0186] Dressing 4: The difference between this dressing and dressing 1 is that the formula does not contain the dual protein complex. In step (3) of the preparation method, after cooling to 40℃ (±5℃), add an equal mass of purified water, start stirring, and stir at 280 rpm for 10 min. The remaining components and preparation methods are the same as those of dressing 1.

[0187] Experimental procedure: Take 3 pieces of each of dressing 1 to dressing 4 and place them in an accelerated stability incubator. Maintain relative humidity (RH 70±5%) and temperature (55±2℃) for 40 days. Take them out and detect the changes in protein molecular weight and bioactivity of each dressing. The detection method is the same as in Example 3.

[0188] The results are shown in Table 6:

[0189] Table 6: Comparison of molecular weight, cell migration-promoting and anti-inflammatory activity results before and after acceleration of dressings 1-4

[0190]

[0191] Table 6 shows that after accelerated stabilization testing, the proteins in dressing 2 underwent significant degradation, with a marked decrease in cell migration and anti-inflammatory activity. The molecular weight and biological activity of the protein complexes in dressings 1 and 3 remained largely unchanged, but the effect on the dual-protein complex in dressing 3 was smaller compared to dressing 1. Therefore, the dual-protein complex in this invention exhibits better stability than simple physical mixtures, and hyaluronic acid oligosaccharides also play a significant role in its stability.

[0192] In summary, this invention not only creates a high-performance collagen-mussel adhesive protein ear-shaped complex structure, but also achieves comprehensive optimization from molecular design and production process to final function through a co-expression in-situ assembly preparation process and a system construction that stabilizes and enhances the effect with hyaluronic acid oligosaccharides. It successfully integrates multiple functions such as structural support, wet adhesion, cell repair, and immune regulation, providing a groundbreaking solution for the field of skin repair that is innovative, efficient, and economical. It achieves highly efficient synergy between skin repair and wet adhesion, exhibiting excellent anti-inflammatory and repairing effects even at extremely low concentrations, demonstrating significant application and economic value.

[0193] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and not restrictive in all respects. Furthermore, it should be understood that although this specification describes embodiments, it does not encompass only one technical solution. This descriptive method is merely for clarity, and those skilled in the art should consider the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A dual-protein complex, characterized in that: This dual-protein complex is a wheat-ear-shaped protein complex formed by first inducing the production of recombinant type III humanized collagen microspheres in engineered bacteria, and then inducing the expression of mussel adhesive protein. The two are assembled in situ through non-covalent bonds before and after bacterial lysis. Wherein: the recombinant type III humanized collagen active gene sequence is shown in SEQ NO.1, and the mussel adhesive protein active gene sequence is shown in SEQ NO.2; The molecular weight of this dual-protein complex is 27–155 kDa, and the molecular ratio of recombinant type III humanized collagen microspheres to mussel adhesive protein is 4:

1.

2. The dual-protein complex according to claim 1, characterized in that: This dual-protein complex also contains hyaluronic acid oligosaccharides as a system stabilizer; The amount of hyaluronic acid oligosaccharide added is 25-50 wt% of the mass of the dual protein complex; and the content of hyaluronic acid tetrasaccharide in the hyaluronic acid oligosaccharide accounts for no less than 30 wt% of the total mass of all hyaluronic acid oligosaccharides.

3. A method for preparing the dual-protein complex according to claim 2, characterized in that: Includes the following steps: 1) Construction of bicistronic expression plasmid: The recombinant type III humanized collagen active gene fragment and the mussel adhesive protein active gene fragment were tandemly linked to form a fusion gene, which was then inserted into a prokaryotic expression vector to construct a bicistronic expression plasmid; 2) Preparation of fermentation engineered bacteria: The bicistronic expression plasmid constructed in step 1) is introduced into Escherichia coli competent cells together with the expression vectors for the cutting enzyme gene and the cyclase gene. After resistance screening and PCR identification, the positive bacteria obtained are the fermentation engineered bacteria; wherein, the cutting enzyme is TEV cutting enzyme, and the cyclase is Butelase ligase. 3) Seed culture: the positive engineering bacteria prepared in step 2) are inoculated into a seed culture medium, and cultured at 37°C under the condition of 200 rpm shaking for 12-16h, until the OD 600 value is 1.0-3.0, to obtain a fermentation seed liquid; 4) Batch feeding fermentation: Introduce the fermentation seed liquid cultivated in step 3) into the fermenter, and control the initial fermentation parameters as follows: temperature 30-37℃, pH 6.5-7.5, dissolved oxygen greater than 30%; when the dissolved oxygen value rises significantly, start batch feeding culture, and maintain the dissolved oxygen value of the fermentation medium greater than 30% by adjusting the aeration rate and stirring speed, and continue to increase the cell density; 5) Induced expression: When the OD of the fermentation broth... 600 When the dissolved oxygen value reaches 100-110, the culture temperature is lowered, and isopropyl-β-D-thiogalactopyranoside is added to induce the expression and cyclization of recombinant type III humanized collagen. The expression level of recombinant type III humanized collagen microspheres is detected at certain intervals. When the content of recombinant type III humanized collagen microspheres reaches the predetermined amount, the dissolved oxygen value is controlled to be less than 5%, the culture temperature is kept constant, and arabinose is added to induce the expression of mussel adhesive protein. 6) Primary protein complexation: Maintain the pH of the culture medium at 6.5-7.5 and the temperature at 28℃. Stir at 250-350 rpm for a certain period of time to allow the recombinant type III humanized collagen microspheres and mussel adhesive protein to assemble and form a dual protein precomplex. After the cell density no longer increases, continue to culture for 6 hours to end the fermentation culture. 7) Secondary protein complex: After fermentation, collect the engineered bacteria cells by centrifugation, break them up, adjust the pH to maintain at 6.5-7.5, and keep the temperature at 28℃. Continue stirring at 250-350 rpm for a certain period of time to allow the pre-complex of the two proteins and the free protein to fully assemble and combine again to form a wheat-ear-shaped protein complex. Then, centrifuge and purify to obtain a stable two-protein complex. 8) Stabilize the system: If further stabilization is required, replace the prepared dual-protein complex in sodium phosphate buffer at pH 7.0, add 25-50 wt% hyaluronic acid oligosaccharide solution (pH 7.0), and stir until homogeneous.

4. The method for preparing the dual-protein complex according to claim 3, characterized in that: In step 1), the recombinant type III humanized collagen active gene sequence is shown in SEQ NO.1, the mussel adhesive protein active gene sequence is shown in SEQ NO.2, the two are linked together by a flexible linker peptide gene, the gene sequence of which is shown in SEQ NO.3; the fusion gene sequence formed by the three is shown in SEQ NO.

4.

5. The method for preparing the dual-protein complex according to claim 3, characterized in that: In step 4), the batch fermentation involves the following: the inoculation amount of the fermentation seed liquid is 0.1% (v / v); the initial fermentation medium is TB medium, with the following formula: yeast extract 10g / L, corn starch 1g / L, soybean meal powder 2g / L, potassium dihydrogen phosphate 12g / L, magnesium sulfate 0.6g / L, glycerol 15g / L, ammonium sulfate 5g / L, bone peptone 5g / L, citric acid monohydrate 3g / L, diammonium hydrogen phosphate 3g / L, sodium chloride 1g / L, polyether defoamer 1ml / L, and trace element solution 10ml / L. A significant increase in dissolved oxygen levels indicates that feeding should be initiated when the dissolved oxygen level exceeds 60%–70%. The culture medium formula for the supplemental feed is: glycerol 500g / L, trace elements 20ml / L, magnesium sulfate 2g / L, and ammonium sulfate 7g / L; The feeding rate is as follows: initially, add 5 ml per liter of fermentation liquid per hour, and after 2 hours, increase the feeding rate to 7 ml / L / h until fermentation is complete.

6. The method for preparing the dual-protein complex according to claim 3, characterized in that: In step 5), the induction of expression uses isopropyl-β-D-thiogalactopyranoside at a concentration of 0.1 g / L, added in 200 ml, and at a concentration of 10%. The concentration of arabinose used to induce mussel adhesive protein expression was 0.5 g / L, the amount added was 3.3 L, and the concentration was 3%. The expression level of recombinant type III humanized collagen microspheres was detected every 1 hour, and the target concentration of recombinant type III humanized collagen microspheres in the culture medium was 2-4 mg / ml.

7. The method for preparing the dual-protein complex according to claim 3, characterized in that: In step 8), the hyaluronic acid oligosaccharide is obtained by hyaluronic acid with a molecular weight of less than 200,000 Da under the enzymatic cleavage of modified hyaluronidase; The preparation method of the modified hyaluronidase is as follows: The culture medium formula is as follows: per 1000ml of culture medium, there are 200ml of 6×M9 salt solution, 2ml of 1M MgSO4∙7H2O solution, 20ml of 20% glucose solution, 0.1ml of 1M CaCl2∙6H2O solution, and the remainder is DD water; The fermentation process is as follows: an engineered bacterial suspension containing the hyaluronic acid fusion enzyme gene with a viable bacterial concentration of 10^8 to 10^9 CFU / ml is inoculated into the modified M9 medium, and chloramphenicol is added simultaneously. The fermentation conditions are 33℃ and 180 rpm. After 10 h of fermentation, isopropyl-β-D-thiogalactopyranoside is added as an inducer to induce the expression of hyaluronic acid fusion enzyme. After fermentation, the fermentation broth is centrifuged and ultrafiltered, and the retentate is collected to obtain the modified hyaluronidase.

8. The method for preparing the dual-protein complex according to claim 7, characterized in that: The enzymatic digestion process is as follows: 25-30 μL of modified hyaluronidase is added to each milliliter of sodium hyaluronate solution with a concentration of 20-25 g / L. The digestion temperature is 40℃, and the mixture is stirred at 60 rpm for 15 h to obtain a hyaluronic acid oligosaccharide mixture, wherein the mass of the hyaluronic acid tetrasaccharide accounts for no less than 30 wt% of the total mass of all hyaluronic acid oligosaccharides.

9. The use of the dual protein complex according to claim 1 or 2 in the preparation of products for post-medical aesthetic repair and adjuvant treatment of skin inflammation; the products include masks, gels and liquid dressings; the dual protein complex is used directly or as the main active ingredient of the product, and its content is 0.1 to 0.2% of the total mass of the product.

10. A dressing for post-medical cosmetic repair and adjunctive treatment of skin inflammation, characterized in that: This dressing, by mass fraction, consists of the following components composition: The dual-protein complex according to claim 1 or 2, at 0.1-0.2%, Thickener 0.15-0.3%, Moisturizer content: 3.6%–7.5% pH adjuster 0.02-0.05%, The remainder is purified water; The moisturizer is selected from one or more of the following: trehalose, glycerin, propylene glycol, butylene glycol, pentanediol, 1,2-hexanediol, sorbitol, polyethylene glycol, betaine, and sodium hyaluronate. The thickener is selected from one or more of the following: carbomer, sodium polyacrylate, methylcellulose, ethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, soluble starch, xanthan gum, and gelatin. The pH adjuster is selected from triethanolamine, aminomethylpropanol, sodium hydroxide, and potassium hydroxide.

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