Crosslinked recombinant collagen gel as well as preparation method and application thereof

Abstract

The invention provides cross-linked recombinant collagen gel. The cross-linked recombinant collagen gel consists of the following components in percentage by mass: 0.4-1.6% of recombinant III-type humanized collagen; 0.2%-2% of a D-core; 0.1% to 0.5% of carbomer; 0.1% to 0.6% of triethanolamine; 1.0%-10% of glycerol; the collagen gel disclosed by the invention has the advantages of low immunogenicity, high safety, no toxicity, no stimulation, good biocompatibility, structural stability, collagen enzymolysis resistance and the like, and meanwhile, the invention also provides a preparation method and application of the cross-linked recombinant collagen gel.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a cross-linked recombinant collagen gel, a preparation method and an application thereof. Background Art

[0002] Sensitive skin is a manifestation of metastable skin. The skin is exposed to certain external stimuli and causes discomfort such as stinging, burning, itching, and tingling, with or without persistent erythema. The International Pruritus Research Forum defines sensitive skin as a syndrome. Epidemiological studies have shown that the incidence of sensitive skin is very high worldwide, accounting for about 50% of the total population; in my country, the number of people with sensitive skin is about 40.8%, with more women than men.

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

[0004] At present, 29 types of human collagen have been discovered, the most common types are type I, type II and type III. Type I collagen determines the firmness and strength of the human body's collagen elastic network, and type III collagen can keep organ tissues soft and elastic, determine the delicateness of the skin and promote scar-free healing of wounds. In infancy, type III collagen accounts for about 60%, but with age, the ratio of type I and type III in the skin changes, and the proportion of type III collagen will continue to decrease. In adults, type I collagen accounts for about 80%, and type III only accounts for about 20%.

[0005] Animal-derived collagen can vary significantly between batches and carries potential risks such as viral infection and immune rejection. Recombinant collagen offers the advantages of consistent composition, stable quality, and good safety. Recombinant humanized type III collagen can quickly penetrate the skin's underlying layers, directly replenishing lost collagen and enhancing the skin's self-repair ability. It can also alleviate symptoms of sensitive skin, such as redness and dryness, repair a damaged skin barrier, and improve the skin's tolerance. However, recombinant collagen suffers from poor thermal stability and is easily degraded.

[0006] By interacting with collagen through the functional groups of chemical crosslinkers to form covalent bonds, the structural stability and mechanical properties of collagen can be improved, its degradation rate in the body can be improved, and its existence time in tissues can be prolonged. Currently, commonly used chemical crosslinkers such as formaldehyde, glutaraldehyde, chitosan, etc. are exogenous substances in human tissues, which have certain toxicity to cells or tissues and affect biocompatibility. D-ribose is a natural component present in biological cells. It is a relatively safe crosslinker with better biocompatibility and repair synergy.

[0007] The domestic patent CN202411095415.1 provides a method for preparing recombinant humanized collagen hydrogel by using a dialdehyde crosslinking agent, although the crosslinking efficiency is high, but there is a risk of cytotoxicity and calcification. The domestic patent CN202211621549.3 provides a method for preparing recombinant human type III collagen hydrogel by using water-soluble crosslinking agents EDC and NHS, which has better cell compatibility than glutaraldehyde crosslinking products, but needs to be thoroughly washed to remove water-soluble by-products to avoid causing allergic reactions, the domestic patent 202310214474.5 provides a chitosan recombinant humanized collagen gel, which has good biocompatibility and anti-calcification ability, but the in vivo degradation and absorption time is longer, and the domestic patent CN202510479450.1 discloses a preparation method of collagen and hyaluronic acid co-crosslinked gel, but the preparation process conditions are harsh.

[0008] The domestic patent CN201810580957.6 discloses an injection collagen prepared by crosslinking reaction of animal collagen with D-ribose as a crosslinking agent, which improves the safety after implantation and prolongs the degradation time after implantation, and the domestic patent CN202110898891.7 discloses a method for preparing a biological valve by crosslinking mammalian pericardium with D-ribose, which can effectively improve the mechanical strength and anti-calcification ability of the biological material, and significantly reduce the cytotoxicity and improve the biocompatibility. The above-mentioned collagen raw material is extracted from animal tissues, which has the safety risks of virus infection and immune rejection, and there is no related report on D-ribose crosslinked recombinant humanized collagen in China. SUMMARY

[0009] One of the problems to be solved by the present application is to provide a crosslinked recombinant collagen gel which is low in immunogenicity, high in safety, non-toxic, non-irritating, has good biocompatibility, structural stability and anti-collagenase ability.

[0010] The second purpose of the present application is to provide a preparation method and application of the crosslinked recombinant collagen gel.

[0011] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a crosslinked recombinant collagen gel, which is composed of the following components in mass percentage: recombinant type III humanized collagen 0.4%-1.6%; D-ribose 0.2%-2%; carbopol 0.1%-0.5%; triethanolamine 0.1%-0.6%; glycerol 1.0%-10%; the balance is purified water.

[0012] Further, consisting of the following ingredients by mass percentage: Recombinant humanized collagen type III 1%; D-ribose 0.8%; Carbomer 0.2%; Triethanolamine 0.3%; Glycerol 6%; The balance is purified water.

[0013] The present application provides a preparation method of cross-linked recombinant collagen gel, comprising the following steps: Step one: preparing cross-linked recombinant protein: after D-ribose is fully dissolved in 20ml purified water, add recombinant humanized collagen type III, mix uniformly, filter membrane sterilization, and incubate at 37℃ for 4 hours to prepare cross-linked recombinant protein; Step two: preparing carbomer-glycerol mixed solution: add carbomer to 40ml purified water, stand at room temperature for 2-4 hours to fully swell, add glycerol to the swollen carbomer solution, stir uniformly, sterilize at 121℃ high pressure steam for 20min, and then cool to room temperature to obtain carbomer-glycerol mixed solution; Step three: preparing triethanolamine solution: add triethanolamine to 40ml purified water, filter membrane sterilization to obtain triethanolamine solution; Step four: in a sterile environment, mix cross-linked recombinant protein and carbomer-glycerol mixed solution uniformly, slowly add triethanolamine solution to pH 6.5-7.0 to prepare cross-linked recombinant collagen gel.

[0014] Further, the preparation method of the recombinant humanized collagen type III comprises the following steps: Step one: screening human collagen type III fragments with the biological activity of promoting sensitive skin repair, and the amino acid sequence is shown as SEQ ID NO: 1; Step two: according to the codon bias of Pichia pastoris, the gene of human collagen type III fragments in step one is encoded, and the nucleotide sequence is shown as SEQ ID NO: 2; Step three: the gene obtained after encoding in step two is directionally connected to the vector to construct a recombinant plasmid; Step four: after linearizing the recombinant plasmid in step three with Pmel endonuclease, the host cell is introduced to obtain a recombinant genetic engineering bacteria; Step five: the recombinant genetic engineering bacteria obtained in step four is subjected to high-density fermentation culture, and after three stages of bacterial proliferation, feed flow addition and methanol induction, the recombinant humanized collagen type III is secreted into the fermentation broth; Step six: the recombinant humanized collagen type III in step five is separated and purified to obtain recombinant humanized collagen type III with a purity of more than 98%.

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

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

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

[0018] Further, one or a combination of several of salting-out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation or membrane separation is used for isolating and purifying the recombinant humanized type III collagen protein.

[0019] The application provides application of a cross-linked recombinant collagen protein gel in a product for soothing skin, relieving inflammation, fading redness, moisturizing and repairing sensitive skin.

[0020] Compared with the prior art, the application has the advantages and beneficial effects that: 1. An active functional region of human type III collagen protein for improving the tolerance of sensitive skin is selected, a gene thereof is encoded, and the target gene is inserted downstream of a Kex2 protease recognition site of a signal peptide of a vector, so that the N-terminal signal peptide of the recombinant target protein is removed by using Kex2 protease of pichia pastoris itself, the prepared recombinant humanized type III collagen protein does not contain non-human collagen protein amino acid sequences, 100% of the corresponding part of human type III collagen protein amino acid sequences is the same, immunogenicity is low, and safety is high. 2. The recombinant collagen protein is expressed by using a pichia pastoris system, the expression amount is high, there is no endotoxin hidden danger, and no additional purification tag such as a histidine marker needs to be removed, so that the purification step is greatly shortened, industrialized large-scale production is suitable, potential risks such as virus infection and immune rejection of traditional animal-derived collagen protein are overcome, and the shortcomings such as purification difficulty and easy production of pyrogen of an escherichia coli expression system are avoided. 3. A D-ribose contained in human cells is selected as a cross-linking agent to perform a cross-linking reaction with the recombinant collagen protein, the structural stability is increased, and the toxic side effects of a commonly used chemical cross-linking agent on human cells can be effectively avoided. BRIEF DESCRIPTION OF DRAWINGS

[0021] The above and / or additional aspects and advantages of the application will become apparent and be readily understood from the following description, taken in connection with the accompanying drawings, in which:

[0022] Figure 1 is the electrophoresis map of enzyme digestion of recombinant plasmid pPICZalphaA-rhCOL III.

[0023] Figure 2 is the PCR identification map of recombinant genetically engineered bacterium pPICZalphaA-rhCOL III / X-33.

[0024] Figure 3 is the SDS-PAGE electrophoresis detection map of purified sample of recombinant type III humanized collagen.

[0025] Figure 4 is the chromatogram of size exclusion chromatography for determining the purified sample of recombinant type III humanized collagen.

[0026] Figure 5 is the comparison chart of cell migration experiment, wherein (a) is the negative control group, (b) is the positive control group, and (c) is the experimental group. DETAILED DESCRIPTION

[0027] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the present application will be further described in detail below in conjunction with the Figures 1-5 and specific embodiments. It should be noted that the technical solutions and features in the technical solutions of the present application can be combined with each other without conflict.

[0028] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, however, the present application can also be implemented in other ways different from those described herein, and therefore, the protection scope of the present application is not limited by the specific technical solutions disclosed below.

[0029] The technical solutions of the present application will be described below in conjunction with specific embodiments. The described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments obtained by those skilled in the art without creative labor based on the present application are within the protection scope of the present application.

[0030] In the following examples, the experimental methods are all conventional methods unless otherwise specified. In the following examples, the materials, reagents, instruments, etc. used are all commercially available unless otherwise specified. In the following examples, the 1st nucleotide of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA, and the last nucleotide is the 3' terminal nucleotide of the corresponding DNA unless otherwise specified.

[0031] The culture medium formula applied in the present application is as follows:

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

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

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

[0035] Fermentation medium formula (1 L): 85% phosphoric acid 26.7 ml / L, calcium sulfate dehydrate 0.93 g / L, potassium sulfate 18.2 g / L, magnesium sulfate dehydrate 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 medium formula (1 L): copper sulfate pentahydrate 6.0 g, potassium iodide 0.088 g, manganese sulfate monohydrate 3.0 g, sodium molybdate dihydrate 0.2 g, boric acid 0.02 g, cobalt chloride hexahydrate 0.5 g, zinc chloride 20.0 g, ferrous sulfate heptahydrate 65.0 g, biotin 0.2 g, concentrated sulfuric acid 5.0 ml.

[0037] Example

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

[0039] 1. By analyzing the functional domain of human collagen type III (accession number: NP_000081.2) in the NCBI database, a human collagen type III fragment with the biological activity of promoting sensitive skin repair was screened out, named rhCOLIII, and the amino acid sequence is shown as SEQ ID NO: 1. The screened human collagen type III 594aa-1085aa fragment has a length of 492 amino acids, which contains 1 LpxTG-like motif surface protein LPGTG and 7 integrin recognition sites GER. These active sites have anti-inflammatory and cell migration-promoting functions, which are beneficial to repair damaged skin barrier and improve the tolerance of sensitive skin. According to the codon bias of Pichia pastoris, the gene of rhCOLIII is encoded, and the nucleotide sequence of the synthesized gene is shown as SEQ ID NO: 2.

[0040] 2. By conventional molecular biology techniques, the target gene is directionally connected downstream of the Kex2 protease recognition site of the vector pPICZalphaA, and a single colony is picked to extract the plasmid for enzyme digestion identification, such as Figure 1The agarose gel electrophoresis result shows that the single enzyme cutting product fragment is 5011 bp, and the double enzyme cutting product fragments are 1493 bp and 3518 bp respectively, and the positive recombinant plasmid pPICZalphaA-rhCOLIII is identified.

[0041] 3. After linearization treatment of Pmel endonuclease, the recombinant Pichia pastoris genetic engineering bacteria is constructed by introducing into host cell Pichia pastoris X-33, which specifically includes the following steps:

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

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

[0044] (3) Pick the single colonies grown on the YPDS plate to extract genomic DNA, and perform PCR verification using primers 5'AOX1 and 3'AOX1. The PCR reaction system is 50 μl, and the reaction components include: 10 x Taq buffer 5 μl, dNTPs 4 μl, upper and lower primers each 1 μl, template 2 μl, Taq polymerase 1 μl, and sterilized double distilled water 36 μl. The PCR amplification conditions are: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 120 s, 30 cycles; and 72°C extension for 10 min. As shown in the agarose gel electrophoresis result, the amplification band is about 2000 bp, indicating that the target gene has been integrated into the Pichia pastoris chromosome. Figure 2

[0045] 4. Induced expression and fermentation of the recombinant genetic engineering bacteria, which specifically includes the following steps:

[0046] ​(1) The glycerol bacteria liquid preserved at 1% inoculation amount was transferred to a 50ml BMGY medium in a triangular flask, and cultured overnight at 30°C, 200rpm in a constant temperature shaker, and the bacteria were collected by centrifugation at 1500g for 5min, and then suspended in 50ml BMMY medium in a triangular flask, and induced to express at an induction temperature of 25°C, and 1.0% methanol was added every 24h to induce expression, and after primary screening, rescreening and verification screening, a high-yield recombinant engineering bacteria was obtained.

[0047] (2) The preserved recombinant engineering bacteria was inoculated into 1000ml YPD medium, and cultured at 30°C, 200rpm until OD=2-6 to prepare a primary seed, and the primary seed liquid was transferred to a 30L fermenter containing fermentation medium at a 5% inoculation amount, and the initial fermentation temperature was set to 30°C, the rotation speed was 500rpm, the aeration was 0.5-2vvm, and the pH was 5.0, and then fermentation was started, when the dissolved oxygen suddenly rose, 50% glycerol containing 12mL / L PTM1 was added to enter the glycerol feeding stage, and the dissolved oxygen was maintained in the range of 20%-30%. When the wet bacteria weight of the fermentation broth reached more than 200g / L, the feeding was stopped until the glycerol was consumed, and then the methanol induction stage was started, in which the induction temperature was 28°C, the pH was 5.0, and the fermentation was stopped after 48h, and the target protein was secreted into the fermentation broth.

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

[0049] (1) The fermentation supernatant was added with saturated ammonium sulfate solution to a saturation degree of 30%, and then placed at room temperature for 90min, and then fully mixed by stirring, and then the target protein-containing precipitate was collected by centrifugation.

[0050] (2) The crude protein liquid obtained by precipitation with ultrapure water was subjected to liquid exchange with a 30KD ultrafiltration membrane package, and was replaced into 25mM citrate, 50mM sodium chloride, pH6.2 citric acid buffer to obtain the chromatography sample liquid.

[0051] (3) The Capto SP ImpRes (Cytiva company) chromatography column was equilibrated with 20mM citrate, 50mM sodium chloride, pH6.2 buffer for 4-5 column volumes (CV); after sample loading, the equilibration buffer was used for washing for 2CV; and the target protein chromatography peak liquid was obtained by elution with 25mM citric acid, 250mM sodium chloride, pH6.2 buffer for 4CV.

[0052] (4) The collected chromatography peak liquid was desalted and exchanged with a 30KD ultrafiltration membrane package, and was replaced into purified water, and was concentrated into a 10% stock solution, and was subjected to SDS-PAGE electrophoresis detection, as shown in Figure 3 , the results showed that the size of the target band was consistent with the expectation, as shown in Figure 4 , size exclusion chromatography determination showed that the purity of the target protein reached 99.28%.

[0053] And the target protein is detected by fibroblast biological activity, and a control group is set, which specifically includes the following steps:

[0054] The fibroblasts are diluted to 3x10 5 cells / ml and inoculated into a 6-well plate for culture until the cells grow to 80%-90% confluence.

[0055] A sterile pipette tip is used to gently draw uniform scratches on the cell monolayer, and the scratches intersect with the marker lines to form several intersection points as fixed detection points. The cells are washed with PBS twice to remove unattached cells.

[0056] 2mL of the test sample prepared in DMEM serum-free medium is added, and the final concentration is 0.5mg / ml. The negative control is DMEM serum-free medium, the positive control is a certain marketed recombinant humanized collagen type III, and the experimental group is the recombinant humanized collagen type III prepared by the application. The cells are placed in a 37℃, 5% CO2 incubator for culture, and the scratch width at the same position is observed under a microscope at 0, 24 hours and photographed, Figure 5 showing that the scratch area has migrated cells, among which the negative control group ( Figure 5 (a)) still has obvious scratch area, the positive control group ( Figure 5 (b)) and the experimental group ( Figure 5 (c)) have significantly reduced scratch area. Compared with the positive control, the recombinant collagen protein of the application shows better cell migration promoting effect.

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

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

[0059] (2) 0.4g-1.6g of recombinant humanized collagen type III, preferably 1.0g, is added, mixed uniformly, filtered with a filter membrane to remove bacteria, and incubated 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 protein are determined. Compared with the control recombinant humanized collagen type III, the differential scanning calorimetry (DSC) determination result shows that cross-linking gives the collagen protein matrix additional stability, and the size exclusion chromatography (SEC) analysis shows that the cross-linked recombinant collagen protein gel prepared by the application has good anti-degradation effect.

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

[0062] (1) Take 0.1g-0.5g carbomer, preferably 0.2g, add 40mL purified water at room temperature and stand for 2-4 hours to swell fully; add 1g-10g glycerol, preferably 6g, to the swollen carbomer solution, stir evenly, sterilize by high-pressure steam (121℃, 20 min) and cool to room temperature for standby.

[0063] (2) Take 0.1g-0.6g triethanolamine, preferably 0.3g, make up to 40mL, filter sterilize.

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

[0065] (4) The viscosity of the cross-linked recombinant collagen medical gel is 370546 / mpa·s measured by a viscometer, which shows that the gel has good viscoelasticity and can withstand a certain external tensile force.

[0066] (5) The rheological parameters of the cross-linked recombinant collagen medical gel are tested by a TA rheometer (HR30, WATERS, USA), time scanning is carried out at room temperature, the elastic storage modulus (G' / Pa) is 53.26, the viscous loss modulus (G" / Pa) is 27.61, and the detection result G"<G'shows that the gel forms a relatively stable network structure.

[0067] (6) Take 0.5g of the collagen gel and place it in PBS buffer, etc., which quickly reaches swelling equilibrium within 4 hours, while the control group gel of non-cross-linked D-ribose only starts to reach swelling equilibrium after 4 hours, and the swelling rates are 12.6% and 20.7% respectively. It is shown that after cross-linking of D-ribose collagen, the gap between molecules in the entire gel network is shortened, the pore size is reduced, and thus the swelling rate is reduced. The gel with high water content and stable structure of the present application has the advantage of immediate filling.

[0068] The cross-linked recombinant collagen gel of the present application has significant advantages in structural stability, swelling rate, rheological properties, collagenase resistance and thermal stability, etc., and is applied in products for soothing skin, anti-inflammatory, redness reduction, moisturizing and sensitive skin repair.

[0069] The above is a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A cross-linked recombinant collagen gel, characterized by: It is composed of the following components in percentage by mass: Recombinant humanized type III collagen 0.4%-1.6%; D-ribose 0.2%-2%; Carbomer 0.1%-0.5%; Triethanolamine 0.1%-0.6%; Glycerol 1.0%-10%; The balance is purified water.

2. The cross-linked recombinant collagen gel according to claim 1, characterized in that: It is composed of the following components in percentage by mass: Recombinant humanized type III collagen 1%; D-ribose 0.8%; Carbomer 0.2%; Triethanolamine 0.3%; Glycerol 6%; The balance is purified water.

3. The method for preparing the cross-linked recombinant collagen gel according to any one of claims 1 to 2, characterized in that: The following steps are involved: Step 1: Preparation of cross-linked recombinant protein: Dissolve D-ribose in 20 ml of purified water, add recombinant humanized type III collagen, mix well, filter sterilize, and incubate at 37°C for 4 hours to prepare cross-linked recombinant protein; Step 2: Prepare a carbomer-glycerol mixture: add carbomer to 40 ml of purified water and let it stand at room temperature for 2-4 hours to fully swell. Add glycerol to the swollen carbomer solution and stir evenly. Sterilize with high-pressure steam at 121° C. for 20 minutes and then cool to room temperature to obtain a carbomer-glycerol mixture. Step 3: Prepare triethanolamine solution: dilute triethanolamine to 40 ml of purified water, filter and sterilize with a filter membrane to obtain a 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 a cross-linked recombinant collagen gel.

4. The method for preparing the cross-linked recombinant collagen gel according to claim 3, wherein: The preparation method of the recombinant humanized type III collagen comprises the following steps: Step 1: Screening a human type III collagen fragment with biological activity of promoting the repair of sensitive skin, the amino acid sequence of which is shown in SEQ ID NO: 1; Step 2: Based on the codon preference of Pichia pastoris, the gene encoding the human type III collagen fragment in step 1 is encoded, and the nucleotide sequence thereof is shown in SEQ ID NO: 2; Step 3: Directly connect the gene encoded in step 2 to the vector to construct a recombinant plasmid; Step 4: After linearization of the recombinant plasmid Pmel in step 3, it is introduced into the host cell to obtain a recombinant genetically engineered bacterium; Step 5: The recombinant genetically engineered bacteria obtained in step 4 are subjected to high-density fermentation culture, and the recombinant humanized type III collagen is secreted into the fermentation broth through three stages: bacterial proliferation, feed addition, and methanol induction; Step 6: Separate and purify the recombinant humanized type III collagen in step 5 to obtain recombinant humanized type III collagen with a purity greater than 98%.

5. The method for preparing the cross-linked recombinant collagen gel according to claim 4, wherein: The screened human type III collagen fragment contains a LPxTG-like motif surface protein LPGTG and 7 integrin recognition sites GER.

6. The method for preparing the cross-linked recombinant collagen gel according to claim 4, wherein: The vector is one of pPICZalphaA, pPICZA, pPIC9 or pPIC3.5K.

7. The method for preparing the cross-linked recombinant collagen gel according to claim 4, wherein: The host cell is one of Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71 or Pichia pastoris SMD1168.

8. The method for preparing the cross-linked recombinant collagen gel according to claim 4, wherein: The recombinant type III humanized collagen is separated and purified by using one or a combination of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation or membrane separation.

9. Use of the cross-linked recombinant collagen gel according to any one of claims 1 to 2 in products that soothe skin, relieve allergies and inflammation, reduce redness and moisturize, and repair sensitive skin.

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