Recombinant VII type collagen with firming and anti-wrinkle effects

Abstract

The invention discloses a recombinant VII type collagen as well as a preparation method and application thereof. The recombinant VII type collagen is obtained by repeating a short amino acid sequence GLPGAKGEKGAP for a plurality of times. The secretion yield of the recombinant VII type collagen in a commercial pichia pastoris expression system can be up to about 17.3 g / L, the extracellular secreted protein is basically not degraded in the fermentation process, the purity can reach 90% or above through simple ultrafiltration and decoloration, and industrial mass production can be achieved; and the recombinant VII type collagen has better biological activities of promoting autophagy, improving an endoplasmic reticulum stress state, promoting collagen secretion and inhibiting matrix metalloproteinase from degrading collagen, and shows good tightening and anti-wrinkle effects.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology, and particularly relates to a new recombinant type VII collagen with firming and anti-wrinkle effects, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen is a biological macromolecule that widely exists in the connective tissues of animals, such as skin, bone, tendon, cartilage and other parts. It plays an important role in the human body. It not only has good structural support performance, but also has various physiological functions such as moisturizing, promoting wound healing, regulating cell growth and differentiation, embryonic development, tissue repair and regeneration.

[0003] Recombinant collagen is prepared by using recombinant DNA technology to introduce the gene encoding collagen into a suitable host cell (such as bacteria, yeast, mammalian cells, etc.), so that the host cell expresses collagen, and then, through processes such as separation and purification, high-purity recombinant collagen is obtained. Recombinant collagen has the following advantages: 1) High safety. Compared with traditional animal-derived collagen, recombinant collagen avoids the risks of virus infection and immune rejection that may be brought by animal-derived materials. After strict quality control and testing, the safety of recombinant collagen is more guaranteed. 2) Stable quality. Genetic engineering technology can precisely control the structure and composition of collagen, and is not affected by animal individual differences and sources. The quality of recombinant collagen is more stable and its performance is more reliable. 3) Strong customizability. According to different needs, the structure and function of collagen can be modified and optimized by genetic engineering technology to produce recombinant collagen products with specific properties. With the continuous increase of people's demand for health and beauty, the demand for collagen will also continue to grow. As a safe, efficient and customizable collagen product, recombinant collagen has broad market prospects and development potential in the fields of medicine, beauty skin care and food.

[0004] Type VII anchored collagen is mainly present in the basement membrane zone of the skin, especially at the junction of the epidermis and the dermis. It acts like a "rivet" to tightly connect the epidermis and the dermis together, ensuring the stability of the skin structure. This connection is crucial for maintaining the integrity of the skin. If there is a problem with type VII anchored collagen, it may lead to the separation of the epidermis and the dermis, resulting in symptoms such as skin relaxation and blisters. Type VII anchored collagen, together with other collagens, provides elasticity and toughness to the skin. It can withstand a certain amount of external stretching force, enabling the skin to return to its original state after being subjected to external forces. With the increase of age, the synthesis of type VII anchored collagen decreases, and the connection function of the skin weakens, making it prone to aging phenomena such as skin relaxation and wrinkles. Some beauty methods, such as laser treatment and radiofrequency treatment, can stimulate the synthesis of skin collagen, including type VII anchored collagen, thereby improving the elasticity and firmness of the skin.

[0005] In addition, ultraviolet rays can induce autophagy, which is a catabolic process that can remove unwanted or damaged proteins, lipids, and organelles. Ultraviolet irradiation of dermal fibroblasts in the skin can induce the formation of autophagosomes, which can further protect cells to a certain extent. However, excessive ultraviolet exposure can lead to a decrease in autophagy levels, which in turn leads to increased endoplasmic reticulum stress and thus skin aging. Therefore, activating autophagy and improving the endoplasmic reticulum can be important strategies for combating skin aging. Summary of the Invention

[0006] The object of the present invention is to provide a recombinant type VII collagen with high secretion yield in a commercial expression system and good firming and anti-wrinkle effects, its preparation method and application. The present invention provides a new recombinant type VII collagen, which is obtained by repeating the short amino acid sequence GLPGAKGEKGAP multiple times. The secretion yield of this recombinant type VII collagen in the commercial Pichia pastoris expression system can be as high as about 17.3 g / L, and the extracellularly secreted protein is basically not degraded during the fermentation process. The purity can reach more than 90% through simple ultrafiltration and decolorization, and industrial mass production can be achieved; moreover, this recombinant type VII collagen has better biological activities of promoting autophagy, improving the endoplasmic reticulum stress state, promoting collagen secretion, and inhibiting matrix metalloproteinase from degrading collagen, showing good firming and anti-wrinkle effects, which are superior to the said dodecapeptide and also superior to the recombinant type VII collagen obtained by repeating other dodecapeptides multiple times.

[0007] The present invention includes:

[0008] 1. A polypeptide with the amino acid sequence shown in SEQ ID No.: 2.

[0009] SEQ ID No.: 2

[0010] GLPGAKGEKGAP

[0011] 2. A recombinant type VII collagen, which is obtained by repeating the short amino acid sequence GLPGAKGEKGAP, wherein the number of repetitions is 2 to 100 times.

[0012] 3. The recombinant type VII collagen according to item 2, wherein the number of repetitions is 5 to 40 times.

[0013] 4. The recombinant type VII collagen according to item 2, wherein the number of repetitions is 30 times. The amino acid sequence of this recombinant type VII collagen is shown in SEQ ID No.: 1.

[0014] SEQ ID No.: 1

[0015] GLPGAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPG

[0016] LPGAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLP

[0017] GAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPG

[0018] AKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGA

[0019] KGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAK

[0020] GEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAKG

[0021] EKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAKGEKGAPGLPGAKGE

[0022] KGAPGLPGAKGEKGAP

[0023] 5. The nucleic acid of recombinant type VII collagen as described in coding item 2.

[0024] 6. An expression vector comprising the nucleic acid as described in item 5.

[0025] 7. A host cell comprising the expression vector as described in item 6. The host cell can be a prokaryotic cell or a eukaryotic cell, including: bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, etc., eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, plant cells, etc., preferably Escherichia coli and Pichia pastoris, more preferably Pichia pastoris.

[0026] 8. A method for producing the recombinant type VII collagen as described in item 2, which comprises the step of culturing the host cell as described in item 7 and expressing the recombinant type VII collagen.

[0027] 9. Use of the recombinant type VII collagen as described in item 2 in the preparation of cosmetics, drugs or medical devices for firming and anti-wrinkle, as a firming and anti-wrinkle active ingredient.

[0028] 10. The use as described in item 9, wherein the cosmetics, drugs or medical devices are formulated in a form suitable for application to the skin.

[0029] 11. Use of the recombinant type VII collagen described in item 2 in the preparation of cosmetics, drugs or medical devices for promoting autophagy, alleviating endoplasmic reticulum stress, promoting collagen secretion, and / or inhibiting matrix metalloproteinase from degrading collagen, as an active ingredient.

[0030] 12. The use described in item 11, wherein the cosmetics, drugs or medical devices are made into forms suitable for application to the skin. Brief Description of the Drawings

[0031] Figure 1 It is an electrophoresis diagram showing the purification result of recombinant type VII collagen.

[0032] Figure 2 It is an immunoblot diagram of Beclin 1, p62 and LC3II proteins.

[0033] Figure 3 It is a fluorescence staining diagram of endoplasmic reticulum green fluorescence probe and calcium ion fluorescence probe for human skin fibroblasts.

[0034] Figure 4 It is a diagram showing the expression of COL1A1 detected by q-PCR.

[0035] Figure 5 It is a diagram showing the expression of COL3A1 detected by q-PCR.

[0036] Figure 6 It is a diagram showing the expression of COL7A1 detected by q-PCR.

[0037] Figure 7 It is a diagram showing the expression of MMP1 detected by q-PCR.

[0038] Figure 8 It is a diagram showing the expression of MMP3 detected by q-PCR.

[0039] Figure 9 It is a diagram showing the efficacy of recombinant type VII collagen in improving the skin condition of animals in the photoaging model. Detailed Description of the Invention

[0040] In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0041] Example 1 Screening of Active Polypeptides

[0042] GL Biochem chemically synthesized polypeptides with 12 amino acid residues covering the entire human type VII collagen, and finally synthesized 30 short amino acid sequences, as shown in Tables 1, 2, and 3 below.

[0043] 1-1 Screening for autophagy activation activity

[0044] 1) Seed cells into a 6-well plate at an inoculation density of 2×10 5 cells / well and incubate overnight in an incubator (37°C, 5% CO2).

[0045] 2) UVB stimulation: The negative control and the experimental group were irradiated with 100 mJ / cm 2 of UVB for modeling.

[0046] 3) Sample treatment: After the irradiation modeling is completed, prepare each short amino acid sequence with a concentration of 1 mg / mL respectively. Add 2 mL of the test substance working solution to the experimental group, and add an equal volume of cell culture medium to the blank control group and the negative control group respectively.

[0047] 4) Extract total protein and quantify protein: After 24 h, discard the culture medium, gently wash the cells 2-3 times with pre-cooled phosphate buffer (PBS) to remove components such as culture medium and serum. Add an appropriate amount of protein lysate to lyse the cells, collect the cells into a centrifuge tube using a cell scraper, and lyse on ice for 30 min. Centrifuge the lysed sample at 12,000 rpm for 20 min in a low-temperature centrifuge. Use a BCA kit to measure the protein content, adjust to the same protein concentration according to the BCA detection results, add loading buffer and boil in a boiling water bath, and store at -80°C in the refrigerator for later use.

[0048] 5) WB detection of key proteins in the autophagy signaling pathway: Separate proteins with different molecular weights through SDS-PAGE gel, then transfer the target protein to a carrier (PVDF) membrane. Using the principle of specific binding of antigen and antibody, bind the target protein with a specific primary antibody, bind the primary antibody with an HRP-labeled secondary antibody, and then develop color with ECL luminescent solution to detect the expression level of LC3II protein in human skin fibroblasts.

[0049] The experimental results are shown in Table 1. Among these 30 dodecapeptides, the 28th dodecapeptide GLPGAKGEKGAP had the best effect on enhancing the expression of LC3II protein in HSF cells after UVB damage. The enhancement rate compared to the model group was 48.6%, significantly higher than that of other dodecapeptides, indicating that we screened out a short amino acid sequence from human type VII collagen with significant autophagy activation activity.

[0050] Table 1 Enhancement rates of different dodecapeptides on the expression of LC3II protein in HSF cells after UVB damage

[0051]

[0052]

[0053] 1-2 Screening for Activity of Promoting Collagen Synthesis

[0054] 1) Seed cells into a 6-well plate at an inoculation density of 2×10 5 cells / well and incubate overnight in an incubator (37°C, 5% CO2).

[0055] 2) UVB stimulation: The negative control and the experimental group were irradiated with 100 mJ / cm 2 UVB for modeling.

[0056] 3) Sample treatment: After the irradiation modeling is completed, prepare short amino acid sequences with a concentration of 1 mg / mL respectively. Add 2 mL of the test substance working solution to the experimental group, and add an equal volume of cell culture medium to the blank control group and the negative control group respectively.

[0057] 4) Wash twice with PBS buffer, add 1 mL of Trizol on ice, lyse for 5 - 10 min, aspirate into a non-enzymatic EP tube, incubate in a water bath at 25°C for 5 - 10 min, add 200 μL of chloroform, mix well, centrifuge at 12,000 g for 15 min at 4°C, and collect 300 - 400 μL of the upper aqueous phase into a new EP tube. Add 500 μL of isopropanol, shake well, centrifuge at 12,000 g for 10 min at 4°C, pour off the supernatant directly without aspiration. Prepare 75% ethanol with enzyme-free water, add 1 mL to the sample, mix well, centrifuge at 7,000 g for 5 min at 4°C, discard the supernatant and air-dry, and dissolve the precipitate with enzyme-free water. Synthesize cDNA using a reverse transcription kit, measure the RNA concentration with a ultra-micro spectrophotometer, and a sample with a concentration greater than 100 ng / μl and an A260 / A280 value greater than 1.8 is a qualified sample. Detect COL3A1 (type III collagen) by fluorescence quantitative PCR.

[0058] The experimental results are shown in Table 2. Among these 30 dodecapeptides, the 28th dodecapeptide GLPGAKGEKGAP has the best promoting effect on the expression of COL3A1 in HSF cells after UVB damage. The promotion rate is 132.1% compared with the model group, which is significantly higher than other dodecapeptides. This indicates that GLPGAKGEKGAP is also a short amino acid sequence from human type VII collagen with good activity of promoting collagen synthesis.

[0059] 1-2 Promoting Effects of Different Dodecapeptides on the Synthesis of Type III Collagen

[0060]

[0061] 1 - 3 Screening for Inhibiting Matrix Metalloproteinase Activity

[0062] 1) Seed cells into a 6 - well plate at an inoculation density of 2×10 5 cells / well and incubate overnight in an incubator (37°C, 5% CO2).

[0063] 2) UVB Stimulation: The negative control and the experimental group were irradiated with 100 mJ / cm 2 of UVB for modeling.

[0064] 3) Sample Treatment: After the irradiation modeling, prepare each short amino acid sequence with a concentration of 1 mg / mL. Add 2 mL of the test substance working solution to the experimental group, and add an equal volume of cell culture medium to the blank control group and the negative control group respectively.

[0065] 4) Wash twice with PBS buffer. Add 1 mL of Trizol on ice, lyse for 5 - 10 min, inhale into a non - enzymatic EP tube, incubate in a water bath at 25°C for 5 - 10 min, add 200 μL of chloroform, mix well, centrifuge at 4°C, 12000 g for 15 min, and collect 300 - 400 μL of the upper aqueous phase into a new EP tube. Add 500 μL of isopropanol, shake well, centrifuge at 4°C, 12000 g for 10 min, pour off the supernatant directly without aspiration. Prepare 75% ethanol with non - enzymatic water, add 1 mL and mix with the sample, centrifuge at 4°C, 7000 g for 5 min, discard the supernatant and air - dry, and dissolve the precipitate with non - enzymatic water. Synthesize cDNA using a reverse transcription kit, measure the RNA concentration with a ultra - micro spectrophotometer. Samples with a concentration greater than 100 ng / μl and an A260 / A280 value greater than 1.8 are qualified. Detect the gene expression level of matrix metalloproteinase 1 (MMP - 1) by fluorescence quantitative PCR.

[0066] The experimental results are shown in Table 3. Among these 30 dodecapeptides, the 28th dodecapeptide GLPGAKGEKGAP has the best inhibitory effect on the gene expression of matrix metalloproteinase (MMP - 1) in HSF cells after UVB damage. The inhibition rate is 73.4% compared with the model group, which is significantly higher than that of other dodecapeptides. This dodecapeptide GLPGAKGEKGAP is also a short amino acid sequence from human type VII collagen with good activity in inhibiting the degradation of collagen by matrix metalloproteinase.

[0067] Table 3 Inhibitory Effects of Different Dodecapeptides on MMP - 1

[0068]

[0069]

[0070] Example 2 Preparation of Recombinant Type VII Collagen in Yeast Expression System

[0071] I. Experimental methods

[0072] 1. Construction of expression vector

[0073] (1) Preparation of shuttle plasmid

[0074] The amino acid sequence of recombinant type VII collagen was obtained by repeating the short amino acid sequence GLPGAKGEKGAP 30 times, as shown in SEQ ID No.1. Its codons were optimized for the yeast expression system to obtain the gene sequence of recombinant type VII collagen. The obtained target gene sequence was entrusted to Tsingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was ligated into the pPicZαA plasmid to obtain the pPicZαA-I-α1 plasmid.

[0075] (2) Preparation of yeast expression strain

[0076] pPicZαA-I-α1 was linearized with Pme I and then transformed into the competent cells of Pichia pastoris X-33. Using bleomycin resistance as the screening marker, transformants were screened to obtain the yeast expression strain.

[0077] 2. Induced expression of target protein

[0078] (1) Pick a single colony of the constructed yeast expression strain and add it to 10 ml of YPD liquid medium (1% yeast extract, 2% peptone, and 2% glucose). Incubate at 30 °C and 250 rpm for 48 h for activation;

[0079] (2) Transfer with an inoculation amount of 2% to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium). Incubate at 30 °C and 250 rpm for 24 h as the seed for the fermenter;

[0080] (3) Prepare 7 L of BSM medium and add it to a 10 L fermenter. Sterilize at 121 °C for 20 min. After cooling to 30 °C, adjust the pH to 5.5. Add the seed prepared in (2) to the fermenter in the form of flame inoculation for fermentation culture;

[0081] (4) When the OD 600 reaches 65, start supplementing 50% glycerol until the OD 600 reaches about 115, stop supplementing. Wait for the dissolved oxygen to rebound to 100%, and start feeding methanol for induction;

[0082] (5) During the induction process, control the dissolved oxygen not less than 30% and the pH around 5.5. Induce for 45 h to end the fermentation. Take the culture solution and centrifuge at 12000 rpm for 5 min. Take the supernatant and detect the protein yield by the BCA method.

[0083] 3. Collagen Purification

[0084] The collected supernatant was passed through a 10KD ultrafiltration membrane, then passed through a hollow fiber column and freeze-dried to obtain recombinant type VII collagen. 10 mg of the freeze-dried collagen was taken and added to 10 mL of ultrapure water until completely dissolved to prepare a recombinant collagen solution. 10 μL of the recombinant collagen solution was taken and the protein purity was detected by SDS-PAGE method.

[0085] II. Results

[0086] The BCA detection result of the yield of recombinant type VII collagen in the fermenter prepared by the above method was as high as 17.3 g / L. The detection result of electrophoresis by SDS-PAGE method showed that the protein purity after ultrafiltration through the hollow fiber column could reach more than 90%. The protein electrophoresis result was as Figure 1 shown.

[0087] Example 3 Recombinant Type VII Collagen Activates Autophagy Signaling Pathway

[0088] 1) Cells were inoculated into 6-well plates at an inoculation density of 2×10 5 cells / well and incubated overnight in an incubator (37 °C, 5% CO2).

[0089] 2) UVB stimulation: The negative control and the experimental group were irradiated with 100 mJ / cm 2 of UVB for modeling.

[0090] 3) Sample treatment: After the irradiation modeling was completed, grouping and drug administration were carried out. 2 ml of the test substance working solution was added to the experimental group respectively, and the same volume of cell culture medium was added to the blank control group and the negative control group respectively.

[0091] 4) Total protein extraction and protein quantification: After 24 h, the culture medium was discarded. The cells were gently washed 2-3 times with pre-cooled phosphate buffer (PBS) to remove components such as the culture medium and serum. An appropriate amount of protein lysate was added to lyse the cells, and a cell scraper was used to collect the cells into a centrifuge tube and lysed on ice for 30 min. The lysed sample was centrifuged at 12,000 rpm for 20 min in a low-temperature centrifuge. The protein content was measured using a BCA kit, adjusted to the same protein concentration according to the BCA detection result, loading buffer was added and boiled in a boiling water bath, and stored in a -80 °C refrigerator for later use.

[0092] 5) WB detection of key proteins in the autophagy signaling pathway: Proteins with different molecular weights were separated by SDS-PAGE gel, and then the target proteins were transferred to a carrier (PVDF) membrane. Using the principle of specific binding of antigen and antibody, specific primary antibodies were used to bind to the target proteins, and secondary antibodies labeled with HRP were used to bind to the primary antibodies. Then, ECL luminescent solution was added for color development to detect the expression levels of Beclin 1, p62, and LC3II proteins in human skin fibroblasts.

[0093] The experimental results showed that after UVB stimulation of human skin fibroblasts HSF, compared with the NC group and the BC group, the expression levels of Beclin 1 and LC3II were significantly down-regulated, and the expression level of p62 was down-regulated ( Figure 2 ), and the model was successfully established. Based on UVB stimulation of human skin fibroblasts, when the working concentration of the sample recombinant type VII collagen was 0.25 mg / mL and 1 mg / mL, it could significantly increase the expression levels of Beclin 1 and LC3II and inhibit the expression of p62, improve the autophagy level, and the effect of the same concentration of recombinant type VII collagen in enhancing the expression level of LC3II was significantly higher than that of the dodecapeptide.

[0094] Example 4 Recombinant type VII collagen alleviates endoplasmic reticulum stress

[0095] 1) Cells were inoculated into 6-well plates at an inoculation density of 2×10 5 cells / well and incubated overnight in an incubator (37 °C, 5% CO2).

[0096] 2) UVB stimulation: The negative control and the experimental group were irradiated with 100 mJ / cm 2 of UVB for modeling.

[0097] 3) Sample treatment: After the irradiation modeling was completed, grouping and drug administration were carried out. 2 ml of the test substance working solution was added to the experimental group, and an equal volume of cell culture medium was added to the blank control group and the negative control group respectively.

[0098] 4) Fluorescent probe detection of the endoplasmic reticulum stress status:

[0099] ER-Tracker Green was added to the diluent at a ratio of 1:1000 and mixed evenly to obtain the ER-Tracker Green working solution. The working solution was preheated at 37 °C for 10 minutes in advance. The cell culture medium was removed, and the cells were washed 2 times with HBSS solution. The preheated working solution was added, and the cells were incubated in an incubator (37 °C, 5% CO2) for 30 minutes. The working solution was removed, and the cells were washed 1-2 times with cell culture medium. Subsequently, fluorescence microscopy was used for observation.

[0100] Prepare a calcium ion probe staining solution containing 5 μM Fura-2AM. Remove the cell culture medium, wash the cells twice with PBS, add the staining solution, and incubate in an incubator (37 °C, 5% CO2) for 1 hour to complete the loading of the fluorescent probe. Observe using a fluorescence microscope.

[0101] The fluorescence results showed that after the cells irradiated with UVB were stained with the endoplasmic reticulum green fluorescent probe and the calcium ion fluorescent probe, the fluorescence intensity was significantly up-regulated ( Figure 3 ), indicating that the endoplasmic reticulum stress model was successfully constructed. After intervention with recombinant type VII collagen at 0.25, 0.5, and 1 mg / mL, the fluorescence intensities of the endoplasmic reticulum green fluorescent probe and the calcium ion fluorescent probe staining were significantly lower than those in the model group, and with the increase in the concentration of recombinant type VII collagen, the fluorescence intensity gradually decreased, showing a good concentration-dependent relationship. The fluorescence results proved that recombinant type VII collagen could improve the endoplasmic reticulum stress state after UVB irradiation and could improve skin aging caused by endoplasmic reticulum stress.

[0102] Example 5 Recombinant type VII collagen promotes collagen secretion and inhibits collagen degradation

[0103] Six-week-old male Kunming mice were provided by the Experimental Animal Center of the School of Medicine, Xi'an Jiaotong University. The back skin of the mice in the model group and the drug administration group (n = 8) was exposed to UVB radiation of 300 mJ / cm 2 . After 8 weeks of UVB radiation three times a week, recombinant type VII collagen at 0.25 mg / mL (low-dose sample group) and 1 mg / mL (high-dose sample group) was topically applied for 4 weeks, and the normal group was topically applied with the same volume of solvent (normal saline) for 4 weeks.

[0104] After the experiment started, the skin appearance changes were observed every day and photographed once a week for recording. Thereafter, every week

[0105] Fluorescent quantitative PCR was used to detect the expression of genes related to collagen synthesis and degradation: Mouse back tissue samples were taken out and placed in a homogenizer. Add 1 mL of Trizol on ice and lyse for 5 - 10 min. Aspirate into a sterile EP tube, incubate in a water bath at 25°C for 5 - 10 min, add 200 μL of chloroform, mix well, centrifuge at 4°C, 12000g for 15 min, and collect 300 - 400 μL of the upper aqueous phase (without aspirating the middle layer) into a new EP tube. Add 500 μL of isopropanol, shake well, centrifuge at 4°C, 12000g for 10 min, and directly pour out the supernatant without aspirating. Prepare 75% ethanol with enzyme-free water, add 1 mL to the sample, mix well, centrifuge at 4°C, 7000g for 5 min, discard the supernatant and air-dry, then dissolve the precipitate with enzyme-free water. Reverse transcribe to synthesize cDNA, and use a ultra-micro spectrophotometer to measure the RNA concentration. Samples with a concentration greater than 100 ng / μl and an A260 / A280 value greater than 1.8 are qualified samples. Fluorescent quantitative PCR was used to detect MMP1, MMP3, COL1A1, COL3A1, and COL7A1.

[0106] The experimental results showed that after UVB modeling, the expression levels of COL1A1, COL3A1, and COL7A1 were significantly decreased ( Figure 4 ), while MMP1 and MMP3 were significantly up-regulated ( Figure 5 ), indicating that the construction of the photoaging animal model was successful. After intervention with recombinant type VII collagen, after intervention with 0.25, 0.5, and 1 mg / mL of recombinant type VII collagen, the expression of COL1A1, COL3A1, and COL7A1 was significantly increased compared with the model group ( Figure 4 , 5 , 6), and the expression of MMP1 and MMP3 was significantly decreased ( Figure 7 , 8 ) and showed a concentration-dependent relationship.

[0107] Example 6 Verification of the firming and anti-wrinkle effect of recombinant type VII collagen

[0108] The foregoing Examples 3 - 5 respectively demonstrated that the recombinant type VII collagen prepared in Example 2 by repeating the short amino acid sequence GLPGAKGEKGAP 30 times has good biological activities of activating autophagy, alleviating endoplasmic reticulum stress, promoting collagen secretion, and inhibiting matrix metalloproteinase from degrading collagen. Based on these biological activities, it can be inferred that the recombinant type VII collagen has good firming and anti-wrinkle effects.

[0109] For verification, in the experiment of Example 5 above, the skin folds on the backs of mice were observed. The skin folds on the backs of mice in the model group increased significantly. With the intervention of different concentrations of recombinant type VII collagen, the skin folds were significantly improved and the skin elasticity was significantly enhanced ( Figure 9)。The above results indicate that recombinant type VII collagen can indeed improve skin wrinkles and increase skin elasticity, and it has good firming and anti-wrinkle effects.

Claims

1. A polypeptide having an amino acid sequence as shown in SEQ ID No.:

2.

2. A recombinant type VII collagen, which is obtained by repeating the short amino acid sequence GLPGAKGEKGAP, wherein, The number of repetitions is from 2 to 100 times.

3. The recombinant type VII collagen according to claim 2, wherein, The number of repetitions is from 5 to 40 times.

4. The recombinant type VII collagen according to claim 2, wherein, The number of repetitions is 30 times.

5. A nucleic acid encoding the recombinant type VII collagen according to claim 2.

6. An expression vector comprising the nucleic acid according to claim 5.

7. A host cell comprising the expression vector according to claim 6.

8. A method for producing the recombinant type VII collagen according to claim 2, which comprises culturing the host cell according to claim 7 and causing it to express the recombinant type VII collagen.

9. Use of the recombinant type VII collagen according to claim 2 in the preparation of a cosmetic, drug or medical device for firming and anti-wrinkle, as a firming and anti-wrinkle active ingredient.

10. The use according to claim 9, wherein, The cosmetic, drug or medical device is made in a form suitable for application to the skin.

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