A recombinant humanized type III collagen gel, its preparation method and application

Abstract

The present invention discloses a recombinant humanized type III collagen gel and its preparation method and application, belonging to the field of biotechnology. 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 balance 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 as shown in SEQ ID NO.1. The recombinant humanized collagen gel prepared by the present invention has significant moisturizing, isolating and repairing effects.

Description

Technical Field

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

[0002] As a novel biomaterial, recombinant humanized type III collagen gel shows broad application prospects in the field of medical skincare due to its unique biological characteristics and advantages. The recombinant humanized type III collagen gel can supplement collagen in the skin, increase skin elasticity and firmness, reduce the generation of wrinkles, and can also be used for skin repair of sensitive skin and after laser beauty treatment. It has an obvious effect on improving skin moisture function, can significantly enhance the effectiveness of moisturizing, and has a high anti-wrinkle effect and skin repair effect, etc., and has the value of popularization.

[0003] Currently, in the preparation of recombinant humanized type III collagen gel, there are mainly the following two common methods:

[0004] (1) Genetic engineering technology: By optimizing and modifying the human collagen gene, introducing it into a suitable expression system for expression, and then obtaining recombinant collagen through purification and other steps. Then it is mixed with a gelling agent, etc. to prepare a gel.

[0005] (2) Formulation optimization technology: Adding various components with good biocompatibility with recombinant collagen to the gel formulation to enhance its performance.

[0006] Problems existing in the prior art:

[0007] (1) Quality control problems: Fluctuations in raw material quality may lead to unstable product performance. Each link in the production process requires precise control and monitoring to ensure product quality and consistency. For example, improper control of fermentation process conditions may affect the expression level and quality of collagen, and improper operation in the purification process may introduce impurities.

[0008] (2) Cost problems: The production process is complex, requiring advanced technologies and equipment as well as high-quality raw materials, resulting in high production costs and restricting its wide 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 balance 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 as shown in SEQ ID NO.1.

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

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

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

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

[0014] Preferably, the recombinant humanized type III collagen gel is prepared from the following components: 1.5% w / v of recombinant humanized type III collagen solution, 1% w / v of trehalose, 0.8% w / v of carbomer, 0.15% w / v of hydroxyethyl cellulose, 2% w / v of glycerol, 0.5% w / v of hexylene glycol, 0.1% w / v of β-glucan, 0.08% w / v of triethanolamine, and the balance of 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, and the preparation method includes 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) Pour the dissolved carbomer solution and hydroxyethyl cellulose solution into the same container, mix them thoroughly to form a mixed thickener solution;

[0019] (4) Add the recombinant type III humanized collagen solution to the above-mentioned mixed thickener solution in the required proportion, continue to stir, and then sequentially add trehalose, glycerol, hexylene glycol, β-glucan, and triethanolamine;

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

[0021] The third object of the present invention is to provide the application 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] Based on recombinant humanized collagen, the present invention adds trehalose, which can form a protective film on the cell surface, lock in moisture, prevent skin moisture loss, keep the skin in a moist state, and help improve the skin's moisturizing ability. Carbomer helps to form a uniform film on the skin surface, which can play a certain role in isolation, moisturization, etc., and enhance the protective effect on the skin. Hydroxyethyl cellulose helps to stabilize the product formulation system and prevent unstable phenomena such as stratification and precipitation due to the interaction between different components. β-glucan can also promote the repair and regeneration of skin cells and is helpful for the recovery of damaged skin. Description of the Drawings

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

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

[0027] Figure 3 It is the protein concentration diagram of recombinant humanized type III collagen (COL01) in Example 1.

[0028] Figure 4 It is the recombinant type III humanized collagen gel prepared in Examples 3-7 and Comparative Example 1. Detailed implementation mode

[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] MGSSSGSSSGPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGSSSGPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGSSSGPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGSSSGPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGSSSG.

[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 0.5 μL of the successfully constructed plasmid in a biosafety cabinet and transform it into competent cells (such as expression strains like BL21, Rosetta, etc.). Place the 1.5 mL centrifuge tube containing the competent cells and the plasmid in an ice bath for 5 min, then perform heat shock at 42 °C for 45 s, and continue to ice bath for 10 min. After the ice bath, add 900 μL of LB liquid medium (10 g / L, yeast extract 5 g / L, NaCl 10 g / L) to the 1.5 mL centrifuge tube in the biosafety cabinet, place it in a shaker at 37 °C, 100 rpm for 2 h. After 2 h, centrifuge at 900 Xg at room temperature for 3 min. Discard the supernatant in the biosafety cabinet, retain 100 μL of the supernatant, and gently pipette and mix well. Use a spreading rod to evenly spread kanamycin with a final concentration of 50 μg / mL on the solid plate medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, Agar 15 g / L, made up to 100 mL and autoclaved). Take 20 μL of the already mixed product for plating, and after plating, place it in an incubator at 37 °C and incubate overnight.

[0036] 2. Pick single colonies

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

[0038] 3. Protein expression

[0039] Perform scale-up expression of COL01_pET28a according to different concentrations of IPTG, different temperatures, and different media.

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

[0041] 4. Protein fragmentation

[0042] Suspend the successfully expressed protein with pre-cooled lysis buffer at a ratio of strain:lysis buffer of 1:10, add protease inhibitor with a final concentration of 1 mM, invert and mix well, then perform ultrasonic fragmentation. Use the ultrasonic fragmentation conditions: 250 W, on for 1 s and off for 3 s, 7 min / time, for a total of two times, and operate on ice throughout the process to ensure complete cell fragmentation. If the cell fragmentation is insufficient, it will lead to a decrease in the recovery rate of the target protein; but excessive lysis may cause protein denaturation or release too many impurities, which will increase the difficulty of subsequent purification. Take a sample of the already fragmented cells and perform SDS-PAGE to detect whether it is a soluble protein.

[0043] 5. Protein purification

[0044] Centrifuge the completely fragmented soluble protein at 4°C and 13000 rpm for 30 min. After centrifugation, take the supernatant for filtration to remove the impurities that still have not been completely centrifuged off after high-speed centrifugation. Use an AKTA protein purifier to purify the fragmented protein, and at the same time prepare a new DEAE column (ion exchange column). Note: Operate at low temperature throughout the process.

[0045] (1) Column equilibration: Equilibrate the column with 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, and bring the internal environment of the column to a stable state for subsequent sample loading and separation.

[0046] (2) Protein loading: Load the filtered protein at the same flow rate and perform two cycles of loading to ensure complete adsorption of the protein onto the column.

[0047] (3) Elution: The eluent is prepared by adding different concentrations of NaCl to Buffer A for protein elution. The concentrations of NaCl in the eluent are 0 M / 0.05 mM / 0.1 M / 0.3 M / 0.5 M / 1 M, and the pH is 8.0. As the NaCl concentration increases, the electrostatic interaction between biomacromolecules and the DEAE ion exchange column will be gradually weakened, thereby eluting the biomacromolecules. The elution volume for each concentration is 12 column volumes. Select the receiving volume of the protein according to the eluted protein peak. Dialyze the eluent containing the target protein. A suitable dialysis bag needs to be selected. The dialysis bag is a semipermeable membrane. As a biomacromolecule, the protein cannot pass through the dialysis bag, while small molecule substances can freely pass through the semipermeable membrane and exchange liquids with the surrounding dialysis solution to dilute the NaCl concentration in the protein solution. Through multiple dialysis, the NaCl concentration is made consistent with the buffer. Take out the dialyzed protein eluent, centrifuge and filter it to remove possible particulate impurities and avoid clogging the chromatography column. If the sample concentration is too high, appropriate dilution can be performed.

[0048] 6. Secondary purification by molecular sieve: Turn on the AKTA protein purifier and assemble the molecular sieve. Note: Operate at low temperature throughout the process.

[0049] (1) Column equilibration: Equilibrate the column with Buffer A at a flow rate of 1.5 mL / min, and bring the internal environment of the column to a stable state for subsequent sample loading and separation.

[0050] (2) Protein loading: Centrifuge the target protein that has completed dialysis at 4°C, 12000 rpm for 30 min at high speed, then filter it, and repeat the loading twice.

[0051] (3) Elution: Use the eluent (prepared by adding different concentrations of NaCl to Buffer A for protein elution. The concentrations of NaCl in the eluent are 0 M / 0.05 mM / 0.1 M / 0.3 M / 0.5 M / 1 M, and the pH is 8.0) to elute the protein. Collect samples according to the protein peak, and take samples at different gradients for gel running detection to 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: the balance. The operation is the same as that in 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: the balance. The operation is the same as that in 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: the balance. The operation is the same as that in 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: the balance. The operation is the same as that in Example 2.

[0067] Comparative Example 1

[0068] 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: the balance.

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

[0070] pH detection tests were carried out on the recombinant type III humanized collagen gel 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 Conclusion All of the above tests 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 gel of Examples 3-7 and Comparative Example 1 was verified.

[0078] 1. Skin sensitization test

[0079] Sixteen albino guinea pigs were selected, regardless of gender. Prepare the test article solutions as needed for the recombinant type III humanized collagen gel of Examples 3-7 and the negative control of Comparative Example 1. Three guinea pigs were used for each collagen gel sample, a total of 15 guinea pigs were needed, and 1 guinea pig was used as the negative control. During the induction period, the hair on the inner part of the scapula on the back of each guinea pig was shaved, and the patch soaked with the test article solution was applied to the shaved area and removed after 48 hours. During the challenge period, the hair on the untested areas on the left and right ventral sides of the guinea pigs was shaved, and the patch soaked with the test article solution was applied to the shaved area and removed after 24 hours. The erythema and edema reactions at the challenge sites of the guinea pigs were observed 24 hours and 48 hours after the challenge. The test results are shown in Table 4.

[0080] Table 3 Skin reaction grade criteria

[0081]

[0082] Table 4 Irritation test results

[0083]

[0084]

[0085] 2. Moisture retention 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 point Example 3 Example 4 Example 5 Example 6 Example 7 Comparative Example 1 Product control 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: recombinant humanized type III collagen solution 0.5 - 2% w / v, trehalose 0.5 - 2% w / v, carbomer 0.5 - 1% w / v, hydroxyethyl cellulose 0.1 - 0.2% w / v, glycerol 1 - 7% w / v, hexylene glycol 0.4 - 0.9% w / v, β-glucan 0.05 - 0.6% w / v, triethanolamine 0.07 - 0.09% w / v, and the balance 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 as shown in SEQ ID NO.

1.

2. The recombinant humanized type III collagen gel according to claim 1, wherein The recombinant humanized type III collagen gel is prepared from the following components: recombinant humanized type III collagen solution 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, and the balance of purified water.

3. The recombinant humanized type III collagen gel according to claim 1, wherein The recombinant humanized type III collagen gel is prepared from the following components: recombinant humanized type III collagen solution 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, and the balance of purified water.

4. The recombinant humanized type III collagen gel according to claim 1, wherein The recombinant humanized type III collagen gel is prepared from the following components: recombinant humanized type III collagen solution 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, triethanolamine 0.08% w / v, and the balance of purified water.

5. The recombinant humanized type III collagen gel according to claim 1, wherein The recombinant humanized type III collagen gel is prepared from the following components: recombinant humanized type III collagen solution 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, and the balance of purified water.

6. The recombinant humanized type III collagen gel according to claim 1, wherein The recombinant humanized type III collagen gel is prepared from the following components: recombinant humanized type III collagen solution 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, and the balance of purified water.

7. A method for preparing the recombinant humanized type III collagen gel according to any one of claims 1-6, characterized in that, The preparation method includes 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) Pour the dissolved carbomer solution and hydroxyethyl cellulose solution into the same container and mix them thoroughly to form a mixed thickener solution; (4) Add the recombinant type III humanized collagen solution to the above-mentioned mixed thickener solution in the required proportion, continue to stir, and then add trehalose, glycerol, hexylene glycol, β-glucan, and triethanolamine in sequence; (5) Add the remaining purified water, adjust the pH, and finally make up the volume.

8. Use of the recombinant humanized type III collagen gel according to any one of claims 1-6 in the preparation of skin care products.

9. The application according to claim 8, wherein The product is a facial mask.

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