Recombinant humanized III-type collagen gel as well as preparation method and application thereof

Abstract

The invention discloses recombinant humanized III-type collagen gel as well as a preparation method and application thereof, and belongs to the technical field of biology. The recombinant humanized III-type collagen gel is prepared from the following components: 0.5 to 2 percent (w / v) of recombinant humanized III-type collagen solution, 0.5 to 2 percent (w / v) of trehalose, 0.5 to 1 percent (w / v) of carbomer, 0.1 to 0.2 percent (w / v) of hydroxyethyl cellulose, 1 to 7 percent (w / v) of glycerol, 0.4 to 0.9 percent (w / v) of hexanediol, 0.05 to 0.6 percent (w / v) of beta-glucan, 0.07 to 0.09 percent (w / v) of triethanolamine and the balance of purified water. The concentration of the recombinant humanized III-type collagen solution is 18-22 mg / mL, and the amino acid sequence of the recombinant humanized III-type collagen is as shown in SEQ ID NO. 1. The recombinant humanized collagen gel prepared by the preparation method disclosed by the invention has remarkable moisturizing, isolating and repairing effects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant humanized type III collagen gel and a preparation method and application thereof. Background Art

[0002] As a new type of biomaterial, recombinant humanized type III collagen gel has shown broad application prospects in the field of medical skin care due to its unique biological characteristics and advantages. Recombinant humanized type III collagen gel can replenish collagen in the skin, increase skin elasticity and firmness, and reduce the formation of wrinkles. It can also be used for sensitive skin and skin repair after laser cosmetic surgery. It has a significant effect on improving skin moisture function, can significantly improve the effectiveness of hydration, and has a high anti-wrinkle effect and skin repair effect, etc., and has promotion value.

[0003] At present, there are two common methods for preparing recombinant humanized type III collagen gel:

[0004] (1) Genetic engineering technology: The human collagen gene is selected and optimized, introduced into a suitable expression system for expression, and then purified to obtain recombinant collagen. It is then mixed with a gelling agent to prepare a gel.

[0005] (2) Formulation optimization technology: Add a variety of ingredients with good biocompatibility with recombinant collagen to the gel formula to enhance its performance.

[0006] Problems with existing technologies:

[0007] (1) Quality control issues: Fluctuations in raw material quality may lead to unstable product performance, and each link in the production process needs to be precisely controlled and monitored to ensure product quality and consistency. For example, improper control of fermentation process conditions may affect the expression and quality of collagen, and improper purification process operation may introduce impurities.

[0008] (2) Cost issue: The production process is complex and requires advanced technology and equipment as well as high-quality raw materials, which leads to high production costs and limits its widespread application in some fields. Summary of the invention

[0009] One of the purposes of the present invention is to provide a recombinant humanized type III collagen gel, which is prepared from the following components: 0.5-2% w / v of recombinant humanized type III collagen solution, 0.5-2% w / v of trehalose, 0.5-1% w / v of carbomer, 0.1-0.2% w / v of hydroxyethyl cellulose, 1-7% w / v of glycerol, 0.4-0.9% w / v of hexylene glycol, 0.05-0.6% w / v of β-glucan, 0.07-0.09% w / v of triethanolamine, and the remainder of purified water; the concentration of the recombinant humanized type III collagen solution is 18-22 mg / mL, and the amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID NO.1.

[0010] Preferably, the recombinant humanized type III collagen gel is prepared from the following components: 1% w / v recombinant humanized type III collagen solution, 1% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 6% w / v glycerol, 0.5% w / v hexylene glycol, 0.1% w / v β-glucan, 0.08% w / v triethanolamine, and the remainder purified water.

[0011] Preferably, the recombinant humanized type III collagen gel is prepared from the following components: 1% w / v recombinant humanized type III collagen solution, 1.5% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 6% w / v glycerol, 0.8% w / v hexylene glycol, 0.1% w / v β-glucan, 0.08% w / v triethanolamine, and the remainder purified water.

[0012] Preferably, the recombinant humanized type III collagen gel is prepared from the following components: 1% w / v recombinant humanized type III collagen solution, 1% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 4% w / v glycerol, 0.5% w / v hexylene glycol, 0.5% w / v β-glucan, 0.08% w / v triethanolamine, and the remainder purified water.

[0013] Preferably, the recombinant humanized type III collagen gel is prepared from the following components: 1.5% w / v recombinant humanized type III collagen solution, 1% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 4% w / v glycerol, 0.8% w / v hexylene glycol, 0.1% w / v β-glucan, 0.08% w / v triethanolamine, and the remainder purified water.

[0014] Preferably, the recombinant humanized type III collagen gel is prepared from the following components: 1.5% w / v recombinant humanized type III collagen solution, 1% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 2% w / v glycerol, 0.5% w / v hexylene glycol, 0.1% w / v β-glucan, 0.08% w / v triethanolamine, and the remainder purified water.

[0015] The second object of the present invention is to provide a method for preparing the above-mentioned recombinant humanized type III collagen gel, the preparation method comprising the following steps:

[0016] (1) Add carbomer to purified water and stir until completely dissolved;

[0017] (2) Add hydroxyethyl cellulose to purified water and stir until completely dissolved;

[0018] (3) pouring the dissolved carbomer solution and hydroxyethyl cellulose solution into the same container and mixing them thoroughly to form a mixed thickener solution;

[0019] (4) adding the recombinant type III humanized collagen solution to the mixed thickener solution in the required proportion, continuing to stir, and then adding trehalose, glycerol, hexylene glycol, β-glucan, and triethanolamine in sequence;

[0020] (5) Add the remaining amount of purified water, adjust the pH, and finally make up to volume.

[0021] The third object of the present invention is to provide the use of the above-mentioned recombinant humanized type III collagen gel in the preparation of skin care products.

[0022] Preferably, the product is a facial mask.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention adds trehalose on the basis of recombinant humanized collagen, which can form a protective film on the cell surface, lock in moisture, prevent skin moisture loss, keep the skin hydrated, and help improve the skin's moisturizing ability. Carbomer helps to form a uniform thin film on the skin surface, which can play a certain role in isolation and moisturizing, and enhance the protective effect on the skin. Hydroxyethyl cellulose helps to stabilize the product formula system and prevent unstable phenomena such as stratification and precipitation due to interactions between different ingredients. β-glucan can also promote the repair and regeneration of skin cells, and has a certain role in helping damaged skin recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1a , 1b1c is the expression diagram of recombinant humanized type III collagen (COL01) under different conditions in Example 1.

[0026] Figure 2 This is a diagram of molecular sieve purification of recombinant humanized type III collagen (COL01) in Example 1.

[0027] Figure 3 This is a graph showing the protein concentration of recombinant humanized type III collagen (COL01) in Example 1.

[0028] Figure 4 This is the recombinant type III humanized collagen gel prepared in Examples 3-7 and Comparative Example 1. DETAILED DESCRIPTION

[0029] Example 1

[0030] 1. Vector Construction

[0031] The amino acid sequence of recombinant humanized type III collagen is COL01 (SEQ ID NO.1):

[0032] MGSSSGSSSGPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGSSSGPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPL GSSSGPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGSSSGPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGSSSG.

[0033] The corresponding nucleotide sequence (SEQ ID NO.2) is:

[0034] ATGGGAAGCAGCTCAGGCTCAAGTAGCGGTCCGGGCCCGCCGGGTAGCAATGGCAACCCGGGCCCGCCGGGCCCGAGTGGTAGCCCGGGCAAAGATGGTCCTCCGGGACCGGCGGGCAATACCGGCGCGCCGGGTAGCCCGGGCGTGTCTGGCCCGAAAGGTGATGCGGGTCAGCCGGGGGAAAAAGGCAGCCCGGGCGCACAGGGCCCGCCTGGAGCGCCGGGCCCTCTGGGCAGCAGCTCTGGCCCGGGCCCGCCGGGTTCGAATGGCAACCCGGGCCCACCGGGCCCGAGCGGCAGCCCGGGCAAAGATGGCCCGCCGGGCCCGGCGGGTAATACCGGCGCTCCGGGTAGCCCGGGTGTATCGGGCCCGAAAGGCGACGCCGGCCAGCCGGGCGAAAAAGGCTCACCGGGCGCACAGGGTCCGCCGGGCGCCCCGGGCCCGCTGGGCAGCAGCAGCGGCCCAGGCCCGCCGGGTAGCAACGGCAATCCGGGTCCGCCGGGTCCGAGCGGCAGCCCTGGCAAAGATGGCCCGCCGGGCCCGGCCGGCAACACTGGTGCGCCGGGCAGCCCCGGAGTGAGCGGCCCGAAAGGTGATGCCGGTCAGCCGGGCGAAAAGGGCTCACCGGGTGCGCAGGGTCCGCCGGGTGCCCCGGGTCCGCTGGGCTCATCAAGCGGACCGGGCCCGCCGGGCAGCAATGGTAATCCGGGCCCGCCGGGCCCGAGCGGCAGTCCGGGCAAAGATGGCCCGCCGGGCCCGGCGGGCAATACGGGCGCCCCGGGCAGCCCGGGTGTGAGCGGTCCGAAAGGCGATGCCGGCCAGCCGGGCGAAAAAGGCAGCCCGGGCGCGCAGGGTCCGCCGGGCGCGCCGGGCCCGCTGGGTAGCAGCAGCGGCTGA。

[0035] Insert the nucleotide sequence corresponding to the above amino acid sequence between NcoI and EcoRI of the pET28a vector and name it COL01_pET28a. Take 0.5uL of the successfully constructed plasmid and transform it into competent cells (e.g., BL21, Rosetta and other expression strains) in a biosafety cabinet. Put the 1.5mL centrifuge tube containing competent cells and plasmids in an ice bath for 5 minutes, heat shock at 42℃, 45S, and continue to ice bath for 10 minutes. After the ice bath, add 900uL of LB liquid culture medium (10g / L, yeast extract powder 5g / L, NaCl 10g / L) to the 1.5mL centrifuge tube in the biosafety cabinet, put it in a shaker at 37℃, 100rpm, 2h, and after 2h, centrifuge at 900Xg at room temperature for 3min. Discard the supernatant in the biosafety cabinet, retain 100uL of supernatant, and gently pipette to mix. Use a spreader to evenly spread kanamycin with a final concentration of 50ug / mL on a solid plate medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, Agar 15g / L to 100mL, sterilized at high temperature). Take 20uL of the mixed product to spread on the plate, and place it in a 37℃ incubator for overnight culture.

[0036] 2. Pick a single bacteria

[0037] Pick 3-5 single colonies from the overnight cultured solid plate medium and place them into 50 mL of liquid culture medium as seed strains for scale-up culture.

[0038] 3. Protein Expression

[0039] COL01_pET28a was expressed on a large scale using different IPTG concentrations, different temperatures, and different culture media.

[0040] The recombinant humanized type III collagen Escherichia coli was inoculated into LB / TB / TRB medium in a shake flask at an inoculum size of 0.2% (the LB medium is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; the TRB medium is: 8 g / L tryptone, 16 g / L yeast extract, 10 g / L NaCl, 4 g / L glycerol, 0.4 g / L disodium hydrogen phosphate dodecahydrate, and 0.62 g / L potassium dihydrogen phosphate; the TB medium is: 8 g / L tryptone, 16 g / L yeast extract, 10 g / L NaCl, 7.5 g / L glycerol, 0.4 g / L disodium hydrogen phosphate dodecahydrate, 0.34 g / L sodium dihydrogen phosphate, 9 g / L potassium sulfate, 7.5 g / L magnesium sulfate heptahydrate, 3 g / L potassium hydroxide, 15 g / L phosphoric acid, and 0.42 g / L zinc sulfate). Put it in a shaker at 16℃ / 25℃ / 37℃, 200rpm for shaking. When OD: 0.6-0.8, add 0.5mM and 1mM IPTG to induce expression. After 24h, collect the bacterial solution by centrifugation at 10000Xg for 20min. Take 20uL of bacterial solution and centrifuge it at 4℃, 13000rpm, 5min. After centrifugation, discard the supernatant, add 20uL of protein loading buffer, blow and mix, use a constant temperature mixer (heating type) at 98℃, boil for 10min, take 5uL of sample and load it, and perform SDS-PAGE to observe whether it is expressed. This step is the premise of obtaining the target protein to ensure that there is enough starting material for subsequent purification operations.

[0041] 4. Protein Crushing

[0042] The protein that has been successfully expressed is suspended in pre-cooled disruption buffer at a ratio of 1:10 between strain and disruption buffer, and a protease inhibitor with a final concentration of 1mM is added. After inversion and mixing, ultrasonic disruption is performed. The ultrasonic disruption conditions are: 250W, on for 1s and off for 3s, 7min / time, twice in total, and the whole process is performed on ice to ensure that the cells are completely disrupted. If the cells are not sufficiently disrupted, the recovery rate of the target protein will be reduced; however, excessive lysis may denature the protein or release too many impurities, which will increase the difficulty of subsequent purification. Samples of the cells that have been disrupted are taken and SDS-PAGE is performed to detect whether they are soluble proteins.

[0043] 5. Protein Purification

[0044] The completely broken soluble protein is subjected to high-speed centrifugation at 4°C, 13,000 rpm, 30 min. After the centrifugation, the supernatant is filtered to remove impurities that have not been completely centrifuged after high-speed centrifugation. The broken protein is purified using an AKTA protein purifier, and a new DEAE column (ion exchange column) is prepared. Note: The whole process is operated at low temperature.

[0045] (1) Column equilibration: Use 5 column volumes of equilibration buffer (Buffer A: 25 mM Tris, 10% glycerol pH: 8.0) at a flow rate of 1.5 mL / min to balance the column and allow the environment inside the column to reach a stable state for subsequent sample loading and separation.

[0046] (2) Protein loading: Load the filtered protein at the same flow rate for two cycles to ensure that the protein is completely adsorbed on the column.

[0047] (3) Elution: The eluent is prepared by adding different concentrations of NaCl to Buffer A for protein elution. The concentration of NaCl in the eluent is 0M / 0.05mM / 0.1M / 0.3M / 0.5M / 1M, and the pH is 8.0. As the concentration of NaCl increases, the electrostatic interaction between the biomacromolecules and the DEAE ion exchange column will gradually weaken, thereby eluting the biomacromolecules. The elution volume for each concentration is 12 column volumes. The elution volume is selected according to the protein peak to be eluted. The eluent containing the target protein is dialyzed. A suitable dialysis bag needs to be selected. The dialysis bag is a semipermeable membrane. Proteins as biomacromolecules cannot pass through the dialysis bag, while small molecules can freely pass through the semipermeable membrane and exchange liquid with the surrounding dialysis solution to dilute the NaCl concentration in the protein solution. The NaCl concentration is kept consistent with the buffer solution through multiple dialysis. The protein eluate that has completed dialysis is taken out for centrifugation and filtration to remove possible particulate impurities and avoid clogging the chromatography column. If the sample concentration is too high, it can be diluted appropriately.

[0048] 6. Secondary purification with molecular sieve: Open the AKTA protein purifier and assemble the molecular sieve. Note: The whole process should be operated at low temperature.

[0049] (1) Column equilibration: Use Buffer A at a flow rate of 1.5 mL / min to equilibrate the column and allow the environment inside the column to reach a stable state for subsequent sample loading and separation.

[0050] (2) Protein loading: The dialyzed target protein was subjected to low-temperature high-speed centrifugation at 4°C, 12,000 rpm, for 30 min, and then filtered. The sample was loaded repeatedly for two cycles.

[0051] (3) Elution: Use eluent (add different concentrations of NaCl to Buffer A for protein elution, the concentration of NaCl in the eluent is 0 M / 0.05 mM / 0.1 M / 0.3 M / 0.5 M / 1 M, pH is 8.0) to elute the protein, collect samples according to the protein peak, take samples of different gradients for gel running, and observe the purity and concentration of the target protein.

[0052] (4) The secondary purified protein was dialyzed to remove the high concentration of salt. After the dialysis was completed, the target protein was concentrated and the protein concentration was detected by UV spectrophotometer. After running the gel, the actual concentration was determined to be 20 mg / mL.

[0053] Example 2

[0054] This example provides a preparation process of recombinant humanized type III collagen gel, which is as follows:

[0055] Dissolve Carbomer: Slowly add the weighed 0.8% Carbomer into 200 mL of purified water, stirring while adding. Carbomer tends to agglomerate in water, so start with low-speed stirring of 50-100 rpm / min. As the Carbomer gradually disperses until it is completely dissolved, a uniform and transparent solution is formed. This may take a long time, generally 30 minutes to several hours, depending on the specific situation.

[0056] Similarly, add the 0.15% hydroxyethyl cellulose to 150mL purified water, and stir in a similar manner to the dissolution process of carbomer, stirring at a low speed first and then appropriately increasing the speed until the hydroxyethyl cellulose is completely dissolved to obtain a uniform solution. Slowly pour the dissolved carbomer solution and hydroxyethyl cellulose solution into the same container, stir while pouring, and keep the stirring speed at 300-500rpm / min to fully mix the two solutions to form a mixed thickener solution. Slowly add the recombinant type III humanized collagen solution prepared above to the mixed thickener solution according to the required proportion, continue stirring, and keep the stirring speed at 300-500rpm / min to ensure that the collagen is evenly distributed in the mixed solution. Add different proportions of trehalose, glycerol, hexylene glycol, β-glucan and other ingredients in sequence, and stir each ingredient thoroughly, and keep the stirring speed at 300-500rpm / min to make the ingredients evenly mixed in the solution. According to the total amount of the final gel required, calculate the amount of purified water that needs to be added, adjust the pH, and finally fix the volume. In order to ensure the accuracy of the test, comparative example 1 was set up, except that COL01 was not added and the missing volume was supplemented with pure water, and the rest of the operations were the same.

[0057] Example 3

[0058] COL01 (prepared in Example 1, concentration is 20 mg / mL): 1% w / v, trehalose: 1% w / v, carbomer 0.8% w / v, hydroxyethyl cellulose: 0.15% w / v, glycerol: 6% w / v, hexylene glycol: 0.5% w / v, β-glucan: 0.1% w / v, triethanolamine: 0.08% w / v, purified water: balance, the operation is the same as in Example 2.

[0059] Example 4

[0060] COL01 (same as Example 3): 1% w / v, trehalose: 1.5% w / v, carbomer: 0.8% w / v, hydroxyethyl cellulose: 0.15% w / v, glycerol: 6% w / v, hexylene glycol: 0.8% w / v, β-glucan: 0.1% w / v, triethanolamine: 0.08% w / v, purified water: balance, the operation is the same as Example 2.

[0061] Example 5

[0062] COL01 (same as Example 3): 1% w / v, trehalose: 1% w / v, carbomer: 0.8% w / v, hydroxyethyl cellulose: 0.15% w / v, glycerol: 4% w / v, hexylene glycol: 0.5% w / v, β-glucan: 0.5% w / v (to increase anti-inflammatory effect), triethanolamine: 0.08% w / v, purified water: balance, the operation is the same as Example 2.

[0063] Example 6

[0064] COL01 (same as Example 3): 1.5% w / v, trehalose: 1% w / v, carbomer: 0.8% w / v, hydroxyethyl cellulose: 0.15% w / v, glycerol: 4% w / v, hexylene glycol: 0.8% w / v, β-glucan: 0.1% w / v, triethanolamine: 0.08% w / v, purified water: balance, the operation is the same as Example 2.

[0065] Example 7

[0066] COL01 (same as Example 3): 1.5% w / v, trehalose: 1% w / v, carbomer: 0.8% w / v, hydroxyethyl cellulose: 0.15% w / v, glycerol: 2% w / v, hexylene glycol: 0.5% w / v, β-glucan: 0.1% w / v, triethanolamine: 0.08% w / v, purified water: balance, the operation is the same as Example 2.

[0067] Comparative Example 1

[0068] Trehalose: 1% w / v, carbomer 0.8% w / v, hydroxyethylcellulose: 0.15% w / v, glycerol: 6% w / v, hexylene glycol: 0.5% w / v, β-glucan: 0.1% w / v, triethanolamine: 0.08% w / v, and purified water: the balance.

[0069] Figure 4 They are Example 3, Example 4, Example 5, Example 6, Example 7 and Comparative Example 1.

[0070] The pH detection test was performed on the recombinant type III humanized collagen gels of Examples 3-7 and the negative control of Comparative Example 1. The test results are shown in Table 1.

[0071] Table 1

[0072] standard 5.5≤PH≤7.5 Example 3 6.57 Example 4 6.42 Example 5 6.54 Example 6 6.54 Example 7 6.51 Comparative Example 1 6.48 in conclusion The above tests all meet the requirements

[0073] The viscosity test results of the recombinant type III humanized collagen gel of Examples 3-7 and the negative control of Comparative Example 1 are shown in Table 2.

[0074] Table 2

[0075]

[0076]

[0077] The performance of the recombinant type III humanized collagen gels of Examples 3-7 and Comparative Example 1 was verified.

[0078] 1. Skin sensitization test

[0079] Select 16 albino guinea pigs, regardless of gender. Prepare the recombinant type III humanized collagen gel of Examples 3-7 and the negative control of Comparative Example 1, and prepare the test solution as needed. Use 3 guinea pigs for each collagen gel sample, for a total of 15 guinea pigs, and use 1 guinea pig as a negative control. During the induction period, shave the inner part of the scapula on the back of each guinea pig, apply the patch soaked with the test solution to the shaved area, and remove it after 48 hours. During the stimulation period, shave the untested areas on the left and right ventral sides of the guinea pig, apply the patch soaked with the test solution to the shaved area, and remove it after 24 hours. Observe the erythema and edema reactions at the stimulated site of the guinea pig 24 hours and 48 hours after stimulation. The test results are shown in Table 4.

[0080] Table 3 Skin reaction grade standard

[0081]

[0082] Table 4 Irritation test results

[0083]

[0084]

[0085] 2. Moisture test

[0086] 30 healthy adult guinea pigs were selected as experimental animal models. The test samples were the recombinant type III humanized collagen gel of Examples 3-7, and the negative controls were comparative example 1 and 1 commercial control (Fuerjia medical recombinant type III humanized collagen patch, medical device registration certificate number Hei Xie Zhu Zhu 20222140048). The backs of adult guinea pigs were shaved to prepare the test area. Use a moisture tester to measure the baseline moisture content (H0) of the shaved area, and evenly apply a certain amount of sample to each test area to ensure that the entire area is covered. At 0.5h, 2h and 4h after application, use a moisture tester to measure the moisture content (H1) of each area, record the moisture content at each time point, and calculate the moisture change rate. Moisture change rate calculation formula: Moisture content change rate = (H1-H0) / H0 X100% The test results are shown in Table 5.

[0087] Table 5

[0088] Time Example 3 Example 4 Example 5 Example 6 Example 7 Comparative Example 1 Product comparison 0.5H 24.3％ 26.9％ 23.4％ 29.5％ 26.2％ 19.3％ 32.7％ 2H 24.7％ 29.3％ 24.2％ 31.1％ 28.5％ 20.1％ 29.3％ 4H 23.1％ 26.9％ 22.8％ 30.4％ 27.8％ 18.5％ 24.6％

[0089] Conclusion: This animal test shows that the skin moisture change rate of the product control is 32.7% 0.5 hours after application, but the moisture content drops to 29.3% after 2 hours, and 24.6% remains after 4 hours. The overall moisturizing time is relatively short. Compared with implementation 6, the moisturizing effect is obvious in the early stage, but the durability is not as long as implementation 6. In comparison, the effect of embodiment 6 is the best.

[0090] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant humanized type III collagen gel, characterized in that: The recombinant humanized type III collagen gel is prepared from the following components: 0.5-2% w / v of recombinant humanized type III collagen solution, 0.5-2% w / v of trehalose, 0.5-1% w / v of carbomer, 0.1-0.2% w / v of hydroxyethyl cellulose, 1-7% w / v of glycerol, 0.4-0.9% w / v of hexylene glycol, 0.05-0.6% w / v of β-glucan, 0.07-0.09% w / v of triethanolamine, and the remainder of purified water; the concentration of the recombinant humanized type III collagen solution is 18-22 mg / mL, and the amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID NO.

1.

2. The recombinant humanized type III collagen gel according to claim 1, characterized in that: The recombinant humanized type III collagen gel is prepared from the following components: 1% w / v recombinant humanized type III collagen solution, 1% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 6% w / v glycerol, 0.5% w / v hexylene glycol, 0.1% w / v β-glucan, 0.08% w / v triethanolamine, and the balance purified water.

3. The recombinant humanized type III collagen gel according to claim 1, characterized in that: The recombinant humanized type III collagen gel is prepared from the following components: 1% w / v recombinant humanized type III collagen solution, 1.5% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 6% w / v glycerol, 0.8% w / v hexylene glycol, 0.1% w / v β-glucan, 0.08% w / v triethanolamine, and the balance purified water.

4. The recombinant humanized type III collagen gel according to claim 1, characterized in that: The recombinant humanized type III collagen gel is prepared from the following components: 1% w / v recombinant humanized type III collagen solution, 1% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 4% w / v glycerol, 0.5% w / v hexylene glycol, 0.5% w / v β-glucan, 0.08% w / v triethanolamine, and the balance purified water.

5. The recombinant humanized type III collagen gel according to claim 1, characterized in that: The recombinant humanized type III collagen gel is prepared from the following components: 1.5% w / v recombinant humanized type III collagen solution, 1% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 4% w / v glycerol, 0.8% w / v hexylene glycol, 0.1% w / v β-glucan, 0.08% w / v triethanolamine, and the balance purified water.

6. The recombinant humanized type III collagen gel according to claim 1, characterized in that: The recombinant humanized type III collagen gel is prepared from the following components: 1.5% w / v recombinant humanized type III collagen solution, 1% w / v trehalose, 0.8% w / v carbomer, 0.15% w / v hydroxyethyl cellulose, 2% w / v glycerol, 0.5% w / v hexylene glycol, 0.1% w / v β-glucan, 0.08% w / v triethanolamine, and the balance purified water.

7. A method for preparing the recombinant humanized type III collagen gel according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: (1) Add carbomer to purified water and stir until completely dissolved; (2) Add hydroxyethyl cellulose to purified water and stir until completely dissolved; (3) pouring the dissolved carbomer solution and hydroxyethyl cellulose solution into the same container and mixing them thoroughly to form a mixed thickener solution; (4) adding the recombinant type III humanized collagen solution to the mixed thickener solution in the required proportion, continuing to stir, and then adding trehalose, glycerol, hexylene glycol, β-glucan, and triethanolamine in sequence; (5) Add the remaining amount of purified water, adjust the pH, and finally make up to volume.

8. Use of the recombinant humanized type III collagen gel according to any one of claims 1 to 7 in the preparation of skin care products.

9. The use according to claim 8, characterized in that: The product is a facial mask.

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