A cross-linked reconstituted collagen gel, method of preparation and use

Abstract

The application provides a cross-linked recombinant collagen gel, which is composed of the following components in mass percentage: 0.4-1.6% of recombinant humanized collagen type III; 0.2-2% of D-nucleus; 0.1-0.5% of carbomer; 0.1-0.6% of triethanolamine; 1.0-10% of glycerol; and the balance of purified water. The collagen gel has the advantages of low immunogenicity, high safety, no toxicity, no irritation, good biocompatibility, structural stability and anti-collagenase digestion capacity, etc. Meanwhile, the application further provides a preparation method and application of the cross-linked recombinant collagen gel.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, and in particular relates to a cross-linked recombinant collagen gel, its preparation method, and its application. Background Technology

[0002] Sensitive skin is a manifestation of skin metastability. It is characterized by discomfort such as stinging, burning, itching, and tingling caused by certain external stimuli, with or without persistent erythema. The International Forum for the Study of Itch defines sensitive skin as a syndrome. Epidemiological studies show that the incidence of sensitive skin is very high worldwide, accounting for about 50% of the total population; in my country, the population with sensitive skin is about 40.8%, with more women than men.

[0003] Currently, there is no specific treatment for sensitive skin. Generally, personalized skin management plans are provided to patients based on their corresponding mechanisms. Collagen is the most abundant structural protein in the human body, which can improve the skin's ability to retain moisture, promote the repair of damaged tissues, reduce dryness and redness, and decrease the frequency of skin sensitivity.

[0004] Currently, 29 types of human collagen have been identified, with the most common types being type I, type II, and type III. Type I collagen determines the tightness and strength of the body's collagen elastic network, while type III collagen helps maintain the softness and elasticity of organs and tissues, determines skin smoothness, and promotes scarless wound healing. In infancy, type III collagen accounts for about 60%, but as we age, the ratio of type I to type III in the skin changes, with type III collagen gradually decreasing. By adulthood, type I collagen accounts for about 80%, while type III accounts for only about 20%.

[0005] Animal-derived collagen exhibits significant batch-to-batch variability and carries potential risks such as viral infection and immune rejection. Recombinant collagen offers advantages such as consistent composition, stable quality, and good safety. Recombinant type III humanized collagen, in particular, can rapidly penetrate the skin's deeper layers, directly replenishing lost collagen, enhancing the skin's self-repair capabilities, alleviating discomfort symptoms like redness and dryness in sensitive skin, repairing damaged skin barriers, and improving the tolerance of sensitive skin. However, recombinant collagen's drawbacks include poor thermal stability and susceptibility to degradation.

[0006] By using the functional groups of chemical cross-linking agents to interact with collagen and form covalent bonds, the structural stability and mechanical properties of collagen can be improved, its degradation rate in vivo can be enhanced, and its existence time in tissues can be extended. Currently, commonly used chemical cross-linking agents such as formaldehyde, glutaraldehyde, and chitosan are exogenous substances in human tissues and have certain toxicity to cells or tissues, affecting biocompatibility. D-ribose, on the other hand, is a natural component found in biological cells and is a relatively safe cross-linking agent with superior biocompatibility and repair synergy.

[0007] Domestic patent CN202411095415.1 discloses a method for preparing recombinant humanized collagen hydrogel using dialdehyde crosslinking agents. Although the crosslinking efficiency is high, there are risks of cytotoxicity and calcification. Domestic patent CN202211621549.3 discloses a method for preparing recombinant human type III collagen hydrogel using water-soluble crosslinking agents EDC and NHS. The cell compatibility is better than that of glutaraldehyde crosslinking products, but thorough washing is required to remove water-soluble byproducts to avoid allergic reactions. Domestic patent 202310214474.5 discloses a chitosan recombinant humanized collagen gel, which has good biocompatibility and anti-calcification ability, but its degradation and absorption time in vivo is relatively long. Domestic patent CN202510479450.1 discloses a method for preparing collagen-hyaluronic acid co-crosslinked gel, but the preparation process conditions are quite stringent.

[0008] Domestic patent CN201810580957.6 discloses an injectable collagen prepared by cross-linking animal collagen with D-ribose as a cross-linking agent, which improves the safety after implantation and prolongs the degradation time after implantation. Domestic patent CN202110898891.7 discloses a method for preparing bioprosthetic valves by cross-linking mammalian pericardium with D-ribose, which can effectively improve the mechanical strength and anti-calcification ability of biomaterials, while significantly reducing cytotoxicity and improving biocompatibility. The collagen raw materials of the above inventions are extracted from animal tissues, which poses safety risks of viral infection and immune rejection. There are no reports in China on D-ribose cross-linked recombinant humanized collagen. Summary of the Invention

[0009] One of the problems to be solved by this invention is to provide a cross-linked recombinant collagen gel that is low in immunogenicity, high in safety, non-toxic, non-irritating, and has good biocompatibility, structural stability, and resistance to collagen enzymatic degradation.

[0010] The second objective of this invention is to provide a method for preparing and applying cross-linked recombinant collagen gel.

[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a cross-linked recombinant collagen gel, composed of the following components by mass percentage: Recombinant type III humanized collagen 0.4%-1.6%; D-ribose 0.2%-2%; Carbomer 0.1%-0.5%; Triethanolamine 0.1%-0.6%; Glycerin 1.0%-10%; The remainder is purified water.

[0012] Furthermore, it is composed of the following components by mass percentage: 1% recombinant type III humanized collagen; D-ribose 0.8%; Carbomer 0.2%; Triethanolamine 0.3%; Glycerin 6%; The remainder is purified water.

[0013] This invention provides a method for preparing cross-linked recombinant collagen gel, comprising the following steps: Step 1: Preparation of cross-linked recombinant protein: After fully dissolving D-ribose in 20ml of purified water, add recombinant type III humanized collagen, mix evenly, filter through a filter membrane for sterilization, and incubate at 37℃ for 4 hours to prepare cross-linked recombinant protein; Step 2: Preparation of carbomer-glycerol mixture: Add carbomer to 40ml of purified water and let it stand at room temperature for 2-4 hours to fully swell. Add glycerol to the swollen carbomer solution, stir well, autoclave at 121℃ for 20min, and then cool to room temperature to obtain carbomer-glycerol mixture. Step 3: Preparation of triethanolamine solution: Dilute triethanolamine to 40 ml of purified water, filter through a membrane to remove bacteria, and obtain triethanolamine solution; Step 4: In a sterile environment, mix the cross-linked recombinant protein and carbomer-glycerol mixture evenly, and slowly add triethanolamine solution to adjust the pH to 6.5-7.0 to prepare cross-linked recombinant collagen gel.

[0014] Furthermore, the preparation method of the recombinant type III humanized collagen includes the following steps: Step 1: Screen for human type III collagen fragments with bioactivity that promotes the repair of sensitive skin, the amino acid sequence of which is shown in SEQ ID NO: 1; Step 2: Based on the codon bias of Pichia pastoris, the gene encoding the human type III collagen fragment from Step 1 is encoded, and its nucleotide sequence is shown in SEQ ID NO: 2; Step 3: Directly ligate the gene obtained after encoding in Step 2 into the vector to construct a recombinant plasmid; Step 4: After linearizing the recombinant plasmid Pmel endonuclease from Step 3, the plasmid is introduced into the host cell to obtain recombinant genetically engineered bacteria; Step 5: The recombinant genetically engineered bacteria obtained in Step 4 are subjected to high-density fermentation culture. Through three stages of cell proliferation, fed-batch feeding and methanol induction, the recombinant type III humanized collagen is secreted into the fermentation broth. Step 6: Separate and purify the recombinant type III humanized collagen from step 5 to obtain recombinant type III humanized collagen with a purity greater than 98%.

[0015] Furthermore, the screened human type III collagen fragments contain one LPxTG-like motif surface protein LPGTG and seven integrin recognition sites GER.

[0016] Furthermore, the carrier is one of pPICZalphaA, pPICZA, pPIC9, or pPIC3.5K.

[0017] Furthermore, the host cell is one of Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71, or Pichia pastoris SMD1168.

[0018] Furthermore, the separation and purification of recombinant type III humanized collagen can be achieved by one or a combination of several of the following methods: salting out, chromatographic chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, or membrane separation.

[0019] This invention provides an application of cross-linked recombinant collagen gel in products that soothe skin, relieve allergies and inflammation, reduce redness, moisturize, and repair sensitive skin.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. Select the active functional region of human type III collagen that enhances the tolerance of sensitive skin, encode its gene, and insert the target gene downstream of the recognition site of the vector signal peptide Kex2 protease. Use the Kex2 protease of Pichia pastoris to cleave the N-terminal signal peptide of the recombinant target protein. The resulting recombinant type III humanized collagen does not contain non-human collagen amino acid sequences and is 100% identical to the corresponding part of the human type III collagen amino acid sequence. It has low immunogenicity and high safety. 2. Recombinant collagen is expressed using the Pichia pastoris system, which has high expression levels and no endotoxin risk. It also does not require additional purification tags such as histidine label removal, greatly shortening the purification steps and making it suitable for large-scale industrial production. It overcomes the potential risks of viral infection and immune rejection associated with traditional animal-derived collagen, and avoids the disadvantages of E. coli expression systems such as purification difficulties and the easy generation of pyrogens. 3. The cross-linking agent is D-ribose, which is naturally present in human cells, and it undergoes a cross-linking reaction with recombinant collagen to increase its structural stability. At the same time, it can effectively avoid the toxic side effects of commonly used chemical cross-linking agents on human cells. Attached Figure Description

[0021] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0022] Figure 1 This is an electrophoresis image of the recombinant plasmid pPICZalphaA-rhCOLⅢ after restriction enzyme digestion.

[0023] Figure 2 This is a PCR identification diagram of the recombinant genetically engineered bacterium pPICZalphaA-rhCOLⅢ / X-33.

[0024] Figure 3 This is an SDS-PAGE electrophoresis image of a purified sample of recombinant type III humanized collagen.

[0025] Figure 4 This is a chromatogram of a purified sample of recombinant type III humanized collagen determined by size exclusion chromatography.

[0026] Figure 5 These are comparative images of cell migration experiments, where (a) is the negative control group, (b) is the positive control group, and (c) is the experimental group. Detailed Implementation

[0027] To better understand the above-mentioned objects, features, and advantages of the present invention, the following description is provided. Figures 1-5 The present invention will be further described in detail below with reference to specific embodiments. It should be noted that, unless otherwise specified, the technical solutions and features of the present application can be combined with each other.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific technical solutions disclosed below.

[0029] The technical solution of the present invention will now be described with reference to specific embodiments. These embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the present invention without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA.

[0031] The culture medium formulation used in this invention is as follows:

[0032] LLB medium (1L): 5g yeast extract, 10g peptone, 10g sodium chloride (solid medium contains 2% agar powder).

[0033] YPD medium (1L): 10g yeast extract, 20g peptone, 20g glucose.

[0034] YPDS medium (1L): 10g yeast extract, 20g peptone, 20g glucose, 1M sorbitol (solid medium containing 2% agar powder).

[0035] Fermentation medium formula (1L): 85% phosphate 26.7 ml / L, calcium sulfate dihydrate 0.93 g / L, potassium sulfate 18.2 g / L, magnesium sulfate dihydrate 14.9 g / L, potassium hydroxide 4.13 g / L, glycerol 40 g / L, PTM1 4.0 ml / L, ascorbic acid 120 mg / L, lysine 100 mg / L, calcium pantothenate 50 mg / L.

[0036] PTM1 culture medium formula (1L): 6.0g copper sulfate pentahydrate, 0.088g potassium iodide, 3.0g manganese sulfate monohydrate, 0.2g sodium molybdate dihydrate, 0.02g boric acid, 0.5g cobalt chloride hexahydrate, 20.0g zinc chloride, 65.0g ferrous sulfate heptahydrate, 0.2g biotin, 5.0ml concentrated sulfuric acid.

[0037] Example

[0038] I. Constructing a recombinant plasmid containing the target gene, specifically including the following steps:

[0039] 1. By analyzing the functional domains of human type III collagen (accession number: NP_000081.2) in the NCBI database, a human type III collagen fragment with bioactivity promoting the repair of sensitive skin was screened and named rhCOLⅢ. Its amino acid sequence is shown in SEQ ID NO: 1. The screened human type III collagen 594aa-1085aa fragment is 492 amino acids in length and contains one LPxTG-like motif surface protein LPGG and seven integrin recognition sites GER. These active sites have anti-inflammatory and cell migration-promoting functions, which are beneficial for repairing damaged skin barriers and improving the tolerance of sensitive skin. Based on the codon preference of Pichia pastoris, the rhCOLⅢ gene was encoded, and the nucleotide sequence of the synthesized gene is shown in SEQ ID NO: 2.

[0040] 2. Using conventional molecular biology techniques, the target gene was directionally ligated downstream of the Kex2 protease recognition site in the vector pPICZalphaA. Single colonies were picked, plasmids were extracted, and enzyme digestion was performed for identification. Figure 1Agarose gel electrophoresis results showed that the single enzyme digestion product fragment was 5011bp, and the double enzyme digestion product fragments were 1493bp and 3518bp, respectively, and the positive recombinant plasmid pPICZalphaA-rhCOLⅢ was identified.

[0041] 3. After linearizing the Pmel endonuclease, it was introduced into the host cell Pichia pastoris X-33 to construct a recombinant Pichia pastoris genetically engineered strain. The specific steps included are as follows:

[0042] (1) Pick a single colony of X-33 into a test tube containing 5 mL of YPD liquid medium and culture overnight at 30°C and 200 rpm with shaking. Take 500 μl of the overnight culture and inoculate it into an Erlenmeyer flask containing 50 ml of fresh YPD liquid medium. Culture overnight at 30°C and 200 rpm with shaking. Centrifuge the culture at 4°C and 1500 × g for 5 min. Resuspend the cells in 50 ml of pre-cooled sterile double-distilled water and centrifuge. Resuspend the cells in 25 ml of pre-cooled sterile double-distilled water and centrifuge. Resuspend the cells in 20 ml of pre-cooled 1 M sorbitol solution and centrifuge. Resuspend the cells in 0.5 ml of pre-cooled 1 M sorbitol solution to obtain the prepared competent cells.

[0043] (2) Use the restriction enzyme PmeI to cut 10 μg of recombinant plasmid in a 100 μl reaction system. Add the linearized plasmid to 80 μl of Pichia pastoris X-33 competent cells, mix well and perform electroporation transformation. Take 200 μl of bacterial culture and spread it on a Zeocin resistant (100 μg / ml) YPDS plate. Incubate at 30℃ for 2-5 days until single colonies appear.

[0044] (3) Genomic DNA was extracted from single colonies grown on YPDS plates and verified by PCR using primers 5′AOX1 and 3′AOX1. The PCR reaction system was 50 μl, and the reaction components included: 5 μl of 10×Taq buffer, 4 μl of dNTPs, 1 μl each of forward and reverse primers, 2 μl of template, 1 μl of Taq polymerase, and 36 μl of sterile double-distilled water. The PCR amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 120 s, 30 cycles; 72℃ extension for 10 min. Figure 2 As shown, the agarose gel electrophoresis results show that the amplified band is about 2000 bp, indicating that the target gene has been integrated into the Pichia pastoris chromosome.

[0045] 4. Induction and fermentation of recombinant genetically engineered bacteria, specifically including the following steps:

[0046] (1) Transfer the preserved glycerol bacterial culture to a 50ml Erlenmeyer flask of BMGY medium with a 1% inoculation amount, culture overnight at 30℃ and 200rpm in a constant temperature shaker, collect the bacterial cells by centrifugation at 1500g for 5min, and suspend them in Erlenmeyer flasks of 50ml BMMY medium. The induction temperature is 25℃, and methanol is added every 24h to induce expression. After primary screening, secondary screening and verification screening, high-yield recombinant engineered bacteria are obtained.

[0047] (2) The recombinant engineered bacteria were inoculated into 1000 ml of YPD medium and cultured at 30℃ and 200 rpm until OD=2-6 to prepare primary seed culture. The primary seed culture was transferred to a 30 L fermenter containing fermentation medium at an inoculation rate of 5%. The initial fermentation temperature was set at 30℃, the fermentation speed at 500 rpm, the aeration rate at 0.5-2 vvm, and the pH at 5.0. Fermentation began when the dissolved oxygen level suddenly increased. When the dissolved oxygen level suddenly increased, 50% glycerol containing 12 mL / L PTM1 was added to start the glycerol feeding stage, maintaining the dissolved oxygen level in the range of 20%-30%. When the wet bacterial weight of the fermentation broth reached more than 200 g / L, the feeding was stopped and the glycerol was depleted before starting the methanol induction stage. The induction stage temperature was 28℃ and the pH was 5.0. The induction was carried out for 48 h and then the fermenter was removed. The target protein was secreted into the fermentation broth.

[0048] 5. Isolation and purification of recombinant type III humanized collagen, specifically including the following steps:

[0049] (1) Add saturated ammonium sulfate solution to the fermentation supernatant until the saturation is 30%, let it stand at room temperature for 90 min, stir to mix thoroughly, and then centrifuge to collect the precipitate containing the target protein.

[0050] (2) The crude protein solution was obtained by precipitation with ultrapure water and replaced with a 30KD ultrafiltration membrane to a citrate buffer solution containing 25mM citrate, 50mM sodium chloride, and pH 6.2 to obtain the chromatographic loading solution.

[0051] (3) Equilibrate the Capto SP ImpRes (Cytiva) chromatography column to 4-5 column volumes (CV) using a buffer of 20 mM citrate, 50 mM sodium chloride, and pH 6.2; after loading the sample, wash with equilibration buffer for 2 CV; and elute with buffer of 25 mM citrate, 250 mM sodium chloride, and pH 6.2 for 4 CV to obtain the target protein chromatographic peak.

[0052] (4) The collected chromatographic peak was desalted and replaced with purified water using a 30KD ultrafiltration membrane, and concentrated to a 10% stock solution for SDS-PAGE electrophoresis detection. Figure 3 As shown, the results indicate that the target band size meets expectations, as... Figure 4 As shown, size exclusion chromatography determined that the purity of the target protein reached 99.28%.

[0053] The target protein was then subjected to fibroblast bioactivity assays, and a control group was established. The specific steps included the following:

[0054] Dilute fibroblasts to 3 × 10 5 Cells / ml were seeded into 6-well plates and cultured until the cells reached 80%-90% confluence.

[0055] Using a sterile pipette tip, gently draw uniform scratches on the cell monolayer, creating several intersections between the scratches and the marker lines to serve as fixed detection points. Rinse twice with PBS to remove any unattached cells.

[0056] The test samples were prepared using 2 mL of DMEM serum-free medium, with a final concentration of 0.5 mg / mL. The negative control was DMEM serum-free medium, the positive control was a commercially available recombinant type III humanized collagen, and the experimental group was the recombinant type III humanized collagen prepared according to this invention. Cells were cultured at 37 °C in a 5% CO2 incubator. After 0 and 24 hours, the cells were removed and the width of the scratches at the same location was observed under a microscope and photographed. Figure 5 The underlined areas all showed migrating cells, including the negative control group ( Figure 5 (a) The marked area is still clearly visible, and the positive control group ( Figure 5 (b) and the experimental group ( Figure 5 (c) The underlined area was significantly reduced, and the recombinant collagen of the present invention showed better cell migration promotion compared with the positive control.

[0057] 6. Preparation and determination of cross-linked recombinant collagen, specifically including the following steps:

[0058] (1) Dissolve 0.2g-1g of D-ribose, preferably 0.8g of D-ribose, in 20mL of purified water.

[0059] (2) Add 0.4g-1.6g of recombinant type III humanized collagen, preferably 1.0g, mix evenly, filter through a filter membrane for sterilization, and keep warm at 37℃ for 4 hours to prepare cross-linked recombinant protein.

[0060] (3) The thermal stability and anti-collagenase degradation performance of the cross-linked recombinant collagen were determined respectively. Compared with the control recombinant type III humanized collagen, the differential scanning calorimetry (DSC) results showed that cross-linking gave the collagen matrix additional stability. Size exclusion chromatography (SEC) analysis showed that the cross-linked recombinant collagen gel prepared in this invention had a better anti-degradation effect.

[0061] 7. Preparation and performance testing of cross-linked recombinant collagen gel, specifically including the following steps:

[0062] (1) Weigh 0.1 g-0.5 g of carbomer, preferably 0.2 g, add 40 mL of purified water and let stand at room temperature for 2-4 hours for full swelling; add 1 g-10 g of glycerol, preferably 6 g, to the swollen carbomer solution, stir uniformly, sterilize by high-pressure steam (121°C, 20 min) and cool to room temperature for use.

[0063] (2) Weigh 0.1 g-0.6 g of triethanolamine, preferably 0.3 g, dilute to a constant volume of 40 mL, and sterilize by filtration through a filter membrane.

[0064] (3) In a sterile environment, mix the cross-linked recombinant collagen solution and the carbomer-glycerol solution uniformly, slowly add the triethanolamine solution until the pH reaches 6.5, to prepare the cross-linked recombinant collagen gel.

[0065] (4) A viscometer is used to determine that the viscosity of the cross-linked recombinant collagen medical gel is 370546 mpa·s, which shows that the gel has good viscoelasticity and can withstand a certain degree of external force stretching.

[0066] (5) A TA rheometer (HR30, WATERS, USA) is used to test the rheological parameters of the cross-linked recombinant collagen medical gel. Time scanning is performed at room temperature, the measured elastic storage modulus (G' / Pa) is 53.26, and the viscous loss modulus (G" / Pa) is 27.61. The test result that G"<G' indicates that the gel forms a relatively stable network structure.

[0067] (6) Weigh 0.5 g of the collagen gel and place it in PBS buffer etc., it reaches swelling equilibrium rapidly within 4 hours, while the control gel without cross-linked D-ribose only starts to reach swelling equilibrium after 4 hours. The calculated swelling rates are 12.6% and 20.7% respectively. This shows that after D-ribose cross-links collagen, the intermolecular gaps in the entire gel network are shortened and the pore size is reduced, resulting in a decreased swelling rate. The gel of the present invention with high water content and stable structure has more advantages for immediate filling.

[0068] The cross-linked recombinant collagen gel of the present invention has significant advantages in structural stability, swelling rate, rheological properties, collagenase resistance and thermal stability, and is used in products for soothing skin, relieving sensitivity and anti-inflammation, reducing redness and moisturizing, and repairing sensitive skin.

[0069] The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Various modifications and changes can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a cross-linked recombinant collagen gel, characterized in that: The cross-linked recombinant collagen gel is composed of the following components by weight percentage: Recombinant type III humanized collagen 0.4%-1.6%; D-ribose 0.2%-2%; Carbomer 0.1%-0.5%; Triethanolamine 0.1%-0.6%; Glycerin 1.0%-10%; The remainder is purified water; The preparation method includes the following steps: Step 1: Preparation of cross-linked recombinant protein: After fully dissolving D-ribose in 20ml of purified water, add recombinant type III humanized collagen, mix evenly, filter through a filter membrane for sterilization, and incubate at 37℃ for 4 hours to prepare cross-linked recombinant protein; Step 2: Preparation of carbomer-glycerol mixture: Add carbomer to 40ml of purified water and let it stand at room temperature for 2-4 hours to fully swell. Add glycerol to the swollen carbomer solution, stir well, autoclave at 121℃ for 20min, and then cool to room temperature to obtain carbomer-glycerol mixture. Step 3: Preparation of triethanolamine solution: Dilute triethanolamine to 40 ml of purified water, filter through a membrane to remove bacteria, and obtain triethanolamine solution; Step 4: In a sterile environment, mix the cross-linked recombinant protein and carbomer-glycerol mixture evenly, and slowly add triethanolamine solution to pH 6.5-7.0 to prepare cross-linked recombinant collagen gel; The preparation method of the recombinant type III humanized collagen includes the following steps: Step 1: Screening out human type III collagen fragments with bioactivity that promotes the repair of sensitive skin, the amino acid sequence of which is shown in SEQ ID NO: 1; Step 2: Based on the codon bias of Pichia pastoris, obtain the gene for the human type III collagen fragment from Step 1, the nucleotide sequence of which is shown in SEQ ID NO: 2; Step 3: Directly ligate the gene from Step 2 into the vector to construct the recombinant plasmid; Step 4: After linearizing the recombinant plasmid Pmel endonuclease from Step 3, the plasmid is introduced into the host cell to obtain recombinant genetically engineered bacteria; Step 5: The recombinant genetically engineered bacteria obtained in Step 4 are subjected to high-density fermentation culture. Through three stages of cell proliferation, fed-batch feeding and methanol induction, the recombinant type III humanized collagen is secreted into the fermentation broth. Step 6: Separate and purify the recombinant type III humanized collagen from step 5 to obtain recombinant type III humanized collagen with a purity greater than 98%.

2. The method for preparing cross-linked recombinant collagen gel according to claim 1, characterized in that: The cross-linked recombinant collagen gel is composed of the following components by weight percentage: 1% recombinant type III humanized collagen; D-ribose 0.8%; Carbomer 0.2%; Triethanolamine 0.3%; Glycerin 6%; The remainder is purified water.

3. The method for preparing cross-linked recombinant collagen gel according to claim 1, characterized in that: The selected human type III collagen fragments contain one LPxTG-like motif surface protein LPGTG and seven integrin recognition sites GER.

4. The method for preparing cross-linked recombinant collagen gel according to claim 1, characterized in that: The carrier is either pPICZalphaA or pPICZA.

5. The method for preparing cross-linked recombinant collagen gel according to claim 1, characterized in that: The host cell is one of Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71 or Pichia pastoris SMD1168.

6. The method for preparing cross-linked recombinant collagen gel according to claim 1, characterized in that: The separation and purification of recombinant type III humanized collagen can be achieved by one or a combination of salting out, chromatographic chromatography, acid-base precipitation, or membrane separation.

7. The cross-linked recombinant collagen gel prepared by the preparation method according to any one of claims 1-6 is used in the preparation of products that soothe the skin, relieve allergies, and reduce inflammation.

8. The cross-linked recombinant collagen gel prepared by the preparation method according to any one of claims 1-6 is used in the preparation of products for reducing redness and moisturizing.

9. The cross-linked recombinant collagen gel prepared by the preparation method according to any one of claims 1-6 is used in the preparation of products for sensitive skin repair.

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