A recombinant humanized type 17 collagen peptide and its freeze-dried powder and anti-aging application

Abstract

The present invention discloses a recombinant humanized type 17 collagen peptide and its freeze-dried powder and anti-aging application. Its amino acid sequence is shown in SEQ NO.1, and its nucleic acid sequence is shown in SEQ NO.2. The freeze-dried powder is made of the following components in parts by weight: 0.02-0.2 parts of recombinant humanized type 17 collagen peptide, 0.05-0.5 parts of mannitol, 0.01-0.1 parts of trehalose, 0.01-0.1 parts of hyaluronic acid, and 70-95 parts of purified water. The freeze-dried powder prepared by the recombinant humanized type 17 collagen peptide provided by the present invention has a good anti-aging effect, especially in promoting cell adhesion, promoting cell migration, reducing cell damage or improving eye wrinkles. The freeze-dried powder prepared by the present invention has a beautiful and smooth appearance, no cracking, no atrophy, no wall detachment, no roughness, no stratification, and a long shelf life; at the same time, no preservatives are added, and the safety is relatively high.

Description

Technical Field

[0001] The present invention relates to the technical field of recombinant polypeptides, in particular to recombinant humanized type 17 collagen peptide freeze-dried powder and a preparation method thereof. Background Art

[0002] Skin is an important organ of human beings and a strong barrier against the invasion of external hazards. It is directly exposed to the external environment and is stimulated by harmful factors such as air pollution, oxidation, ultraviolet radiation, etc., which cause the skin to be damaged and gradually aged. In addition, the skin will undergo many natural aging phenomena as it ages, such as reduced skin elasticity, increased fine lines, and decreased skin barrier function. With the aging of the population and the improvement of living standards, people's constant pursuit of aesthetics and health has led to a growing demand for anti-aging products.

[0003] Type 17 collagen (Collagen XVII) is a transmembrane collagen that is mainly expressed in the epidermal basement membrane region. This collagen plays an important role in the adhesion and differentiation of skin cells and the structural integrity of the skin. In terms of anti-aging, type 17 collagen is closely related to maintaining the youthful state of the skin. Scientific research has found that type 17 collagen is one of the key components of epidermal stem cells and is involved in regulating the activity of these stem cells. Epidermal stem cells are the source of skin regeneration and repair and are essential for maintaining the youthful appearance of the skin. With age, stem cell function declines and the content of type 17 collagen decreases, which reduces the skin's ability to repair, gradually leading to the characteristics of skin aging, such as wrinkle formation, reduced elasticity and reduced water retention.

[0004] Among the key components of the extracellular matrix (ECM), there is a high-molecular-weight multifunctional adhesion protein that exists on the basement membrane and plays a vital role in maintaining cell structure and function. This protein acts as an adhesion point in the extracellular matrix, helping cells to stably attach, proliferate, migrate and differentiate, etc. These functions are very important for the integrity and repair of the skin. As we age, the structure and function of this protein may be affected, resulting in the skin's inability to effectively repair and renew itself. The loss of this adhesion protein in the skin is associated with skin aging, such as loss of skin elasticity, fine lines and wrinkles, and reduced skin barrier function. This is because adhesion proteins are essential for maintaining the stability and structure of the skin.

[0005] However, collagen and adhesion proteins extracted directly from human or animal tissues are expensive and pose risks of immunogenicity and pathogen transmission. In recent years, the rapid development of recombinant technology has provided a feasible technical approach for the preparation of humanized type 17 collagen and adhesion protein fusion proteins, which can produce high-purity, low-immunogenicity proteins on a large scale in microbial or mammalian cell expression systems. In addition, fusion proteins combine the structural and functional advantages of both proteins in the skin and reduce the complexity of protein production and improve the consistency of ingredients through fusion expression. Active protein lyophilized powder preparations provide the cosmetics industry with highly stable, high-purity active ingredients and significantly extend the shelf life of the products. Lyophilization technology allows active proteins to exist in high concentrations and remain inactive before use to prevent premature degradation of ingredients. Through lyophilized powder preparations, cosmetics can achieve immediate activity, reduce the need for preservatives, and are easy to use and carry. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a recombinant humanized type 17 collagen peptide and a lyophilized powder thereof. The recombinant humanized type 17 collagen peptide provided by the present invention has a significant anti-aging effect, and the lyophilized powder prepared by using the recombinant humanized type 17 collagen peptide has a good anti-aging effect, and the frozen appearance is beautiful and smooth, without cracking, shrinkage, wall detachment, roughness, and stratification, and the shelf life is long; at the same time, no preservatives are added, and the safety is relatively high.

[0007] In order to achieve the purpose of the present invention, the present invention adopts the following technical solution:

[0008] A recombinant humanized type 17 collagen peptide, the amino acid sequence of which is shown in SEQ NO.1.

[0009] The nucleic acid sequence encoding the above-mentioned recombinant humanized type 17 collagen peptide is shown in SEQ NO.2.

[0010] A recombinant humanized type 17 collagen peptide freeze-dried powder is prepared from the following components in parts by weight:

[0011] 0.02-0.2 parts of recombinant humanized type 17 collagen peptide, 0.05-0.5 parts of mannitol, 0.01-0.1 parts of trehalose, 0.01-0.1 parts of hyaluronic acid, and 70-95 parts of purified water.

[0012] The method for preparing the above-mentioned recombinant humanized type 17 collagen peptide freeze-dried powder comprises the following steps:

[0013] (1) Take 0.02-0.2 parts of recombinant humanized type 17 collagen peptide and 30-35 parts of purified water, mix and dissolve, and stir evenly until completely transparent to obtain phase A;

[0014] (2) Take 0.01-0.1 parts of trehalose, 0.05-0.5 parts of mannitol and 30-45 parts of purified water, mix and dissolve them and stir them evenly until they are completely transparent to obtain a phase B solution;

[0015] (3) Mix phases A and B and stir for 8-10 minutes until they are homogeneous and transparent to obtain a mixed solution of phases AB;

[0016] (4) Add 10-15 parts of purified water to the AB phase mixture, filter the material with a sterilized 0.22μ filter, and vacuum freeze-dry to obtain recombinant humanized type 17 collagen peptide freeze-dried powder.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The freeze-dried powder prepared from the recombinant humanized type 17 collagen peptide provided by the present invention has a good anti-aging effect, especially in promoting cell adhesion, promoting cell migration, reducing cell damage or improving eye wrinkles. The freeze-dried powder prepared by the present invention has a beautiful and smooth appearance, no cracking, no shrinkage, no wall detachment, no roughness, no stratification, and a long shelf life; at the same time, no preservatives are added, and the safety is relatively high. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the recombinant expression plasmid pET28a-ABLG of the present invention.

[0020] Figure 2 This is a diagram showing the results of SDS-PAGE detection of the target protein expression of the present invention.

[0021] Figure 3 This is a diagram showing the SDS-PAGE detection results of the target protein purification product of the present invention.

[0022] Figure 4 This is a diagram showing the Western Blot detection results of the purified product of the target protein of the present invention.

[0023] Figure 5 This is the appearance of the recombinant humanized type 17 collagen peptide lyophilized powder of the present invention.

[0024] Figure 6 This is a diagram showing the cell adhesion promoting effect of the recombinant humanized type 17 collagen peptide freeze-dried powder of the present invention.

[0025] Figure 7 This is a diagram showing the effect of the recombinant humanized type 17 collagen peptide freeze-dried powder of the present invention on promoting cell migration.

[0026] Figure 8 This is a diagram showing the effect of the recombinant humanized type 17 collagen peptide freeze-dried powder of the present invention on reducing cell damage.

[0027] Fig. 9 This is a graph evaluating the effect of the recombinant humanized type 17 collagen peptide freeze-dried powder of the present invention on improving wrinkles at the corners of the eyes. DETAILED DESCRIPTION

[0028] Example 1: Construction and expression of recombinant humanized type 17 collagen polypeptide containing anti-aging functional fragments

[0029] (1) Optimization of recombinant human collagen polypeptide sequence: The DNA sequence of pET28a-ABLG was modified, and a hexahistidine tag was added to the C-terminus to facilitate immunological antibody detection and specific affinity chromatography purification. The optimized pET28a-ABLG contains a total of 757 amino acids, and its sequence is shown in ABLG (Protein), and the amino acid sequence is shown in SEQ NO.1. Its gene coding sequence is shown in ABLG (Gene), and the nucleic acid sequence is shown in SEQ NO.2.

[0030] (2) Gene synthesis and construction of recombinant expression plasmid: Shanghai Jierui Biotechnology Co., Ltd. was commissioned to synthesize the INTRA gene sequence. pET-28a was used as the vector plasmid. BamH I and Not1 restriction sites were selected. The gene synthesis sequence was ligated into the pET-28a vector using T4 DNA ligase (TaKaRa, product number 2011A) (refer to the product manual for the method). The ligation product was transformed into Escherichia coli DH5α competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.). The recombinant expression plasmid map is shown in Figure 2. Figure 1 Positive clones were screened on LB resistance plates containing kanamycin (50 μg / mL) and colony PCR amplification and sequencing verification were performed (Shenzhen BGI Technology Co., Ltd.).

[0031] (3) Preparation and expression of recombinant engineering bacteria: The verified recombinant vector plasmid was transformed into BL21 (DE3) competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.), and the expression bacteria were successfully obtained. SDS-PAGE expression identification was performed. The results are as follows Figure 2 As shown, the apparent molecular weight is approximately 95 kDa.

[0032] (5) Purification of peptides

[0033] Since the recombinant collagen polypeptide contains a His tag, the recombinant fusion protein expressed by the present invention can be separated and purified by Ni column affinity chromatography (Ni Sepharose 6Fast Flow) and molecular sieve (Sephadex G-25) to obtain pure protein. The purified product is identified by SDS-PAGE and Western Blot analysis. Figure 3 and Figure 4 shown.

[0034] Example 2: Preparation of recombinant humanized type 17 collagen peptide lyophilized powder:

[0035] (1) Pre-prepare phase A: add 0.05 parts by weight of the recombinant humanized type 17 collagen peptide prepared in Example 1 to 30 parts by weight of purified water, and stir to dissolve evenly until completely transparent.

[0036] (2) Pre-prepare phase B: add 0.2 parts by weight of mannitol and 0.05 parts by weight of trehalose into 35 parts by weight of purified water, and stir to dissolve evenly until completely transparent.

[0037] (3) Mix phases A and B and stir for 9 minutes until they become homogeneous and transparent.

[0038] (4) Add 10 parts by weight of purified water, filter the material with a sterilized 0.22μ filter, and vacuum freeze-dry to obtain recombinant humanized type 17 collagen peptide lyophilized powder. The appearance of the recombinant humanized type 17 collagen peptide lyophilized powder is as follows: Figure 5 As shown. Figure 5 It can be seen that the freeze-dried powder has a beautiful and smooth appearance, without cracking, shrinkage, wall separation, roughness or stratification.

[0039] Example 3: Experiment on promoting cell adhesion by recombinant humanized type 17 collagen peptide lyophilized powder

[0040] (1) The recombinant humanized type 17 collagen peptide and the negative control group solution were coated onto a 96-well plate. Three replicates were set for each concentration, 100 μL per well, and the plate was sealed and placed at 4°C for coating overnight.

[0041] (2) The test cells were cultured in complete culture medium at 37°C and 5% CO2, and the cell concentration was controlled to be 1.0×10 per mL. 5 -5.0×10 6 The cells were used for biological activity assay 24-36 hours after passage.

[0042] (3) The adherent cells were fixed and stained with 1% crystal violet methanol solution for 20 min, washed twice with ultrapure water, observed under a microscope and photographed for analysis.

[0043] like Figure 6 As shown, the results showed that the recombinant humanized type 17 collagen peptide had good activity in promoting cell migration and adhesion.

[0044] Example 4: Experiment on promoting cell migration with recombinant humanized type 17 collagen peptide lyophilized powder

[0045] (1) Before inoculating cells on the culture plate, use a Maker pen to draw reference lines on the back of the 12-well plate using a ruler. Draw reference lines across the center of each well, with at least two lines crossing each well.

[0046] (2) NIH3T3 cells in the logarithmic phase (purchased from Thermo Fisher Scientific) were trypsinized and resuspended in 10% FBS culture medium (purchased from Gibico). The cells were inoculated into a 12-well plate at a concentration of 100,000 cells / mL and 1 mL / well.

[0047] (3) After the cells have grown to 90% on the second day, discard the supernatant in the 12-well plate, leaving 100-200 μL of culture medium. Use a 200 μL pipette tip to measure the ruler and scratch as perpendicularly as possible to the reference line on the back. Wash the cells 1-2 times with PBS to remove the detached cells. If there are no floating cells under the microscope, the cells are clean.

[0048] (4) Aspirate the cell culture medium and rinse the plate three times with PBS to remove the cell debris produced by the scratches.

[0049] (5) Add the prepared samples and standards, 900uL / well, and mark them. Place in a 37°C, 5% CO2 incubator for incubation; select positions for photography at 0, 24, and 48 hours (each group of photography must be at the same position at different time points). The photography time can be adjusted according to actual conditions.

[0050] (6) Based on the collected pictures, the scratch area was analyzed using image J software, and the data was sorted to obtain the experimental results. Calculation of wound healing percentage: Based on the initial scratch area, the ratio of the area that healed at a certain time (initial area - area at a certain time point) to the initial area can be used to obtain the percentage of wound healing. That is, wound healing rate % = (scratch area before drug administration - scratch area of ​​the drug administration group at a certain time point) / scratch area before drug administration × 100%.

[0051] like Figure 7 As shown, the results showed that the recombinant humanized type 17 collagen peptide had good activity in promoting cell migration.

[0052] Example 5: Comet assay of recombinant humanized type 17 collagen peptide lyophilized powder

[0053] (1) Isolation and preparation of single cell suspension: Digest the cells with trypsin and place them in a centrifuge tube. Count the cells and adjust the cell density to 10 per ml using PBS. 5 -10 6 indivual.

[0054] (2) Gel preparation: Add the first layer of 0.5% normal melting point agarose to the preheated slide, then quickly cover with a clean coverslip and place at 4°C for 10 minutes to solidify. Prepare a mixed solution by mixing 10 μL of PBS containing 10,000 cells and 75 μL of 10.5% low melting point agarose at 37°C, then gently remove the coverslip, drop the low melting point agarose containing cells onto the first layer of gel, immediately cover with a clean coverslip, and place at 4°C for 10 minutes to solidify. The preparation of the third layer of gel is the same as the first layer, and then cover with a coverslip again.

[0055] (3) Lysis: Remove the coverslip and immerse the slide in the newly prepared RIPA cell lysis buffer for at least 2.5 hours (try to make the lysis time of each slide the same).

[0056] (4) Unwinding and electrophoresis: After cell lysis, remove the slide, rinse twice with PBS, and then place the slide in a horizontal electrophoresis tank. Pour 1X TBE electrophoresis buffer to cover the gel surface by 0.25 cm, perform alkaline unwinding for 20 minutes, and then electrophoresed in a 4°C refrigerator for 25-30 minutes, with the voltage set to 25V.

[0057] (5) Staining: Take out the slides, absorb the electrophoresis buffer with filter paper, and then neutralize with Tris-HCl (pH 7.5) for 15 minutes. Then, add 50 microliters of 30ug / mL ethidium bromide (EB) solution on each slide and keep it away from light. Cover with a coverslip and stain for 20 minutes away from light.

[0058] (6) Nuclear image analysis under a microscope: Place the EB-stained sample under a fluorescence microscope for observation as soon as possible, and take an image of the electrophoresis pattern.

[0059] like Figure 8 As shown, the results indicate that recombinant humanized type 17 collagen peptide can reduce the cell damage effect.

[0060] Example 6: Experimental evaluation of the efficacy of recombinant humanized type 17 collagen peptide freeze-dried powder in improving eye wrinkles

[0061] (1) Before the experiment, volunteers were required to clean their faces with a Uni-brand cleanser and sit quietly in a human efficacy evaluation room (temperature: 20℃~24℃, humidity: 40%~60%) for 30 minutes. During this period, they were not allowed to drink water or beverages. Their faces were allowed to dry naturally without wiping with facial tissues. The test area for volunteers was the crow's feet at the left and right corners of the eyes. The test area was at least 3cm×3cm.

[0062] (2) After cleaning the face, pat the face with toner. After it is dry, dissolve the recombinant humanized type 17 collagen peptide freeze-dried powder and apply it to the skin around the eyes. Massage thoroughly until it is completely absorbed, and then apply facial cream (use your own toner and facial cream. Only moisturizing skin care products can be used. Other functional skin care products cannot be used). Use once a day in the morning and evening for 8 consecutive weeks.

[0063] (3) The face was photographed using VISIA facial image analyzer, the corners of the eyes were photographed using PRIMOS-CR, and the wrinkle target sites were selected using random software to analyze wrinkle depth and length, etc. The test samples were used at home for 8 consecutive weeks, and the same indicators were tested after 2, 4, 6, and 8 weeks of use. The test of the same volunteer was completed by the same measurement personnel.

[0064] like Fig. 9 As shown, the results indicate that the recombinant humanized type 17 collagen peptide freeze-dried powder product has a significant effect in improving eye wrinkles.

Claims

1. A recombinant humanized type 17 collagen peptide, the amino acid sequence of which is shown in SEQ NO.

1.

2. A gene encoding the recombinant humanized type 17 collagen peptide according to claim 1, wherein the nucleic acid sequence thereof is shown in SEQ NO.

2.

3. A recombinant humanized type 17 collagen peptide freeze-dried powder, characterized in that Made from the following components in parts by weight: 0.02-0.2 parts of the recombinant humanized type 17 collagen peptide according to claim 1, 0.05-0.5 parts of mannitol, 0.01-0.1 parts of trehalose, 0.01-0.1 parts of hyaluronic acid, and 70-95 parts of purified water.

4. A method for preparing the recombinant humanized type 17 collagen peptide freeze-dried powder as claimed in claim 3, characterized in that The steps include: (1) Take 0.02-0.2 parts of recombinant humanized type 17 collagen peptide and 30-35 parts of purified water, mix and dissolve, and stir evenly until completely transparent to obtain phase A; (2) Take 0.01-0.1 parts of trehalose, 0.05-0.5 parts of mannitol and 30-45 parts of purified water, mix and dissolve, and stir evenly until completely transparent to obtain phase B solution; (3) Mix phases A and B and stir for 8-10 minutes until they are homogeneous and transparent to obtain a mixed solution of phases AB; (4) Add 10-15 parts of purified water to the AB phase mixture, filter the material with a sterilized 0.22μ filter, and vacuum freeze-dry to obtain recombinant humanized type 17 collagen peptide freeze-dried powder.

5. Use of the recombinant humanized type 17 collagen peptide freeze-dried powder according to claim 3 in the preparation of anti-aging cosmetics.

6. The use according to claim 5, characterized in that The anti-aging effect refers to promoting cell adhesion, promoting cell migration, reducing cell damage or improving wrinkles at the corners of the eyes.

Images