Recombinant collagen with anti-aging and anti-oxidation effects and compound liquid thereof

Abstract

The invention discloses recombinant collagen with anti-aging and anti-oxidation effects and a compound solution of the recombinant collagen. The amino acid sequence of the recombinant human VII type collagen is as shown in SEQ ID No: 1. Compared with commercially available products, the recombinant human VII type collagen provided by the invention has better anti-aging and anti-oxidation effects in vivo or on body surface, and is low in cytotoxicity, high in safety and beneficial to industrial popularization, so that more users can benefit from the recombinant human VII type collagen.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a new recombinant human type VII collagen with anti-aging and anti-oxidation biological activities and a compound solution thereof. Background Art

[0002] Skin belongs to connective tissue, which consists of epidermis, dermis and subcutaneous tissue from outside to inside. The dermis is a mesh structure composed of collagen, elastin, hyaluronic acid and microvessels, among which collagen and elastin play a supporting role, making it have good firmness and elasticity. Sodium hyaluronate can lock the moisture of the skin as a moisturizing factor. According to scientific research, the speed of sodium hyaluronate and collagen production in the body slows down from the age of 20, and the elasticity of elastin that allows the skin to bounce back quickly decreases, causing these substances in the skin to be lost year by year. By the age of 40, the content is reduced to half, the skin tissue shrinks, gaps are formed, and the skin shows aging phenomena such as sagging, wrinkles, dryness, roughness, and large pores.

[0003] However, changes in the skin are not just as simple as aging. As society puts more and more pressure on people, people often lack time to pay attention to nutrition intake through diet, which leads to the lack of nutrients in the human body. The lack of these nutrients will be directly reflected on the facial skin, resulting in dry, dull and aging facial skin. The existing skin care nutrient solution focuses more on temporary protection and cannot supplement human nutrition to avoid skin problems caused by nutrient deficiency. Therefore, a collagen compound solution with skin repair and anti-aging effects is urgently needed to solve the above problems. Summary of the invention

[0004] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a new recombinant human type VII collagen and its compound solution with good anti-aging and antioxidant biological activities.

[0005] Specifically, the present invention includes:

[0006] 1. A recombinant human type VII collagen, the amino acid sequence of which is shown in SEQ ID No: 1.

[0007] SEQ ID No: 1

[0008] YGPTGLEKREAGFPPGPAKGPGSREGPPGPTGLEKREAGFPGPLGEAGEKGERGPPGPGGLAELSSRILSYMSSPGPLGELPGVAAGEKGERGSGISIGLPGPPGPPGLPGTSYEEGHGDPGPPGAPLLSLLRGSEFRIVGPPGPPGGIPGNVWSGGHGDPGPPGAPVQP PGPPGPPGPVTTITGETFGLAGPAGPQGPSGLGPEGPPPGVAGRPGAKGLEKREAGEGPEPAGSRGGRAGRRGMAEKGPAGERGMGGLEKRAPGAKAEGPPGPPEGKPGLQGPRGPPGPVGGLAGPAGPQGPSGLKTGPPGSISSSGQEIQQYISEYMQSDSIRSYLSGD.

[0009] 2. A nucleic acid encoding the recombinant human type VII collagen described in item 1.

[0010] 3. An expression vector comprising the nucleic acid according to item 2. Preferably, the expression vector is pGAPZaA.

[0011] 4. A host cell into which the expression vector described in item 3 is introduced. The host cell may be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells, preferably Escherichia coli or Pichia pastoris.

[0012] 5. A method for producing the recombinant human type VII collagen described in item 1, comprising: culturing the host cell described in item 4 to express the recombinant human type VII collagen, and collecting the cell culture. The expression can be constitutive expression, induced expression, or a combination of the two. The inducer for inducing expression can be an inducer such as IPTG, β-galactoside, methanol, ethanol, etc.

[0013] 6. The method according to item 5, further comprising the step of separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation. Preferably, the combination of chromatography and membrane separation is used, and more preferably, the combination of ion exchange chromatography and membrane separation is used.

[0014] 7. A collagen compound solution comprising the recombinant human type VII collagen described in item 1 and hydroxyapatite hollow microspheres.

[0015] 8. The collagen compound solution according to item 7 is prepared by the following method:

[0016] (1) Preparation of hydroxyapatite hollow microspheres: using equal amounts of calcium carbonate and disodium hydrogen phosphate to prepare hydroxyapatite hollow microspheres under normal pressure;

[0017] (2) Preparation of stock solution A: Weigh the recombinant human type VII collagen, vitamin E, antioxidants, and fibroblasts described in item 1 according to the amount and place them in a reaction container for mixing. Add deionized water in small amounts and in multiple times. Heat the reaction container to 30-45° C. After the above raw materials and deionized water are completely mixed, stock solution A is prepared. Stop heating the reaction container and allow stock solution A to cool naturally.

[0018] (3) Preparation of stock solution B: Weigh sodium chloride diluent, lidocaine hydrochloride injection and Panax notoginseng extract according to the amount and put them into a reaction container for mixing, add deionized water in small amounts and multiple times, heat the reaction container to 40-60° C., and after the above raw materials and deionized water are completely mixed, stock solution B is prepared, stop heating the reaction container, and allow stock solution B to cool naturally;

[0019] (4) Mixing stock solution A and stock solution B, pouring stock solution A and stock solution B into an emulsifying pot under a sterile environment at 25 to 35° C., stirring evenly, and then adding an emulsifier to obtain a sufficient mixture of stock solution A and stock solution B;

[0020] (5) After the mixture is allowed to stand for 1 to 2 hours, a pH adjuster is added to adjust the pH value of the mixture to 7 to 8, and the mixture is stirred for 15 to 20 minutes to prepare a nutrient solution;

[0021] (6) Put the above-mentioned hydroxyapatite hollow microspheres and nutrient solution into a mixing container, heat the mixing container to 30-45° C., stir the hydroxyapatite hollow microspheres and nutrient solution for 20-40 minutes to obtain a composite solution, and cool it naturally.

[0022] 9. A product comprising the recombinant human type VII collagen described in item 1, wherein the product is a medicine, a cosmetic or a medical device.

[0023] 10. Use of the recombinant human type VII collagen described in item 1 in the preparation of medicines, cosmetics or medical devices. Preferably, the medicines, cosmetics or medical devices are used for anti-aging and anti-oxidation.

[0024] Effects of the Invention

[0025] The anti-aging and antioxidant activities of recombinant human type VII collagen are significantly better than those of full-length human type VII collagen and commercially available anti-aging and antioxidant collagen products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A diagram showing the results of SDS-PAGE electrophoresis of proteins.

[0027] Figure 2 UVB photoaging model, β-galactose aging cell staining after various sample interventions. Among them, the aged cells appear green under the β-galactose kit.

[0028] Figure 3 The distribution of hydrogen peroxide-oxidized cells. The oxidized cells show green fluorescence under the ROS kit.

[0029] Figure 4 MDA malondialdehyde content determination diagram. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below through specific embodiments. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute limitations on the scope of the present invention.

[0031] Example 1: Preparation of recombinant human type VII collagen in large intestine expression system

[0032] 1. Construction of pGAPZaA-HLC expression strain

[0033] The amino acid sequence of SEQ ID No: 1 was optimized according to the codon preference of Escherichia coli, synthesized by whole gene synthesis, and directly connected to the pGAPZaA plasmid to obtain the pGAPZaA plasmid. The pGAPZaA was transferred into DE3 competent cells by heat shock, coated on a resistance plate, and a single colony on the plate was picked as the expression strain.

[0034] 2. Expression of Target Protein

[0035] (1) First, the engineered bacteria were inoculated into 50 ml YPD liquid medium for cultivation at 30°C. When the OD600 reached 4-8, it could be used as fermentation seeds.

[0036] (2) The fermentation tank parameters were set to a temperature of 30°C, a rotation speed of 200-700 rpm for 2 days, a ventilation volume of 4 L / min (automatically adjusted by the fermentation tank), dissolved oxygen maintained above 30%, and ammonia water was used to adjust the pH to 5.0.

[0037] (3) The fermentation seed liquid is pumped into a sterile fermentation tank and fermentation culture is started. When the carbon source is exhausted, feed medium is added and fermentation is terminated when the wet weight of the bacteria reaches 220 g / L.

[0038] (4) A 10% (g wet bacteria / mL PBS) bacterial suspension was prepared with a PBS buffer solution at pH 6.0, and the suspension was homogenized at 800 bar for 3 min. The suspension was centrifuged at 9000 rpm for 10 min, and the supernatant was collected as the crude protein solution.

[0039] 3. Isolation and purification of target protein

[0040] (1) Add NaCl with a final concentration of 60% to the crude protein supernatant collected by centrifugation, stir to dissolve, let stand at room temperature for 2 hours, centrifuge at 9000 rpm for 10 minutes, and collect the supernatant. The collected supernatant is concentrated and desalted by a 30KD ultrafiltration membrane.

[0041] (2) The pH of the concentrated and desalted protein supernatant was adjusted to 6.5 with phosphoric acid, loaded onto a cation exchange resin, and the target protein was obtained after elution with 0.5 M NaCl;

[0042] (3) The collected target protein solution was desalted by a 30KD ultrafiltration membrane, then placed in a -20°C refrigerator for pre-freezing for 4 hours, and then transferred to a vacuum freeze dryer for freeze-drying. The freeze-dried protein was collected after 48 hours.

[0043] The results of SDS-PAGE electrophoresis of the recombinant human type VII collagen are as follows: Figure 1 shown.

[0044] Example 2: Preparation of recombinant human type VII collagen complex solution

[0045] Hydroxyapatite hollow microspheres were prepared by 3% calcium carbonate and 3% disodium hydrogen phosphate under normal pressure. 22% recombinant human type VII collagen prepared in Example 1, 6% vitamin E, 2% antioxidant, and 7% fibroblasts were weighed and put into a reaction container for mixing, and deionized water was added in small amounts and multiple times. The temperature of the reaction container was heated to 40° C. After the raw materials and deionized water were completely fused, a stock solution A was prepared, and the heating of the reaction container was stopped to allow the stock solution A to cool naturally.

[0046] Weigh 4% sodium chloride dilution, 8% lidocaine hydrochloride injection and 10% Panax notoginseng extract into a reaction container and mix them. Add deionized water in small amounts and multiple times. Heat the reaction container to 55°C. After the raw materials and deionized water are completely blended, prepare stock solution B. Stop heating the reaction container and allow stock solution B to cool naturally.

[0047] Pour stock solution A and stock solution B into an emulsifying pot under a sterile environment at 32°C, stir evenly, and then add an emulsifier to obtain a sufficient mixture of stock solution A and stock solution B. After the nutrient solution is allowed to stand for 1 hour, a pH adjuster is added to adjust the pH value of the mixture to 7.5, and stir for 20 minutes to obtain a nutrient solution. Put the hydroxyapatite hollow microspheres and the nutrient solution into a mixing container, heat the mixing container to 40°C, stir the hydroxyapatite hollow microspheres and the nutrient solution for 50 minutes, obtain a composite solution, and let it cool naturally.

[0048] Example 3: Characterization of the anti-photoaging performance of different anti-aging products

[0049] 1. Inoculate HaCat (human immortalized epidermal cells) cells 1 mL per well in a 24-well plate (8×10 4 cells / well) and incubated at 37°C, 5% CO2 for 24 h.

[0050] 2. Modeling: After the incubation, the culture medium was removed, the cells were washed once or twice with PBS, and then the modeling process was performed; the model group and the drug-treated group were replaced with 1 mL of PBS per well and irradiated with UVB for 10 5 mJ / cm 2 At the same time, the blank control group was replaced with 1 mL of PBS per well and placed in the dark.

[0051] 3. Administration: After UVB irradiation, the PBS in the model group and the blank control group was removed and replaced with 1 mL of fresh basal medium per well. The sample group was replaced with fresh basal medium containing Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3, and incubated at 37° C., 5% CO2 for 24 h.

[0052] The drug administration group was given the following samples prepared with fresh basal medium:

[0053] Example Sample 1: an aqueous solution of recombinant human type VII collagen prepared in Example 1, with a protein concentration of 0.5 mg / ml;

[0054] Example Sample 2: The recombinant human type VII collagen solution prepared in Example 2, with a protein concentration of 0.5 mg / ml;

[0055] Comparative Example Sample 1: Aqueous solution of recombinant human type VII collagen (Shanghai Hewu Biotechnology Co., Ltd.), protein concentration 0.5 mg / ml;

[0056] Comparative Example Sample 2: Zhende Anshida Medical Recombinant Collagen Cold Compress Extract (Hunan Xinxiekang Medical Instrument Co., Ltd.), protein concentration 0.5 mg / ml;

[0057] Comparative Example Sample 3: Medical recombinant humanized collagen anti-wrinkle, firming and anti-aging essence (Guangzhou Suxiuchen Biotechnology Co., Ltd.), protein concentration 0.5 mg / ml.

[0058] 4.β-galactose detection kit, after staining and washing three times, the degree of cell senescence is detected.

[0059] Test results see Figure 2

[0060] 5. Use the above samples to test in the same way as above, and set up a blank control, in which no anti-aging products are placed.

[0061] 6. From Figure 2 It can be seen that the samples of comparative examples 1, 2, and 3 have partial anti-aging properties, and the green spots stained by β-galactose are slightly reduced. The samples of Examples 1 and 2 of the present invention have better anti-aging properties than the samples of comparative examples 1-3, and the green spots stained by β-galactose are significantly reduced. The samples of Examples 1 and 2 have better and higher anti-aging effects.

[0062] Example 4 Characterization of the antioxidant properties of different anti-aging products

[0063] 1. Inoculate HaCat (human immortalized epidermal cells) cells 1 mL per well in a 24-well plate (8×10 4 cells / well) and incubated at 37°C, 5% CO2 for 24 h.

[0064] 2. Modeling: After the incubation, the culture medium was removed, the cells were washed once or twice with PBS, and then the modeling was performed; the model group and the drug-treated group were replaced with 1 mL of 200uM hydrogen peroxide prepared in serum-free culture medium per well for 1 hour, then replaced with serum culture medium after 3 hours of recovery, and then replaced with normal culture medium. At the same time, the blank control group was replaced with normal culture medium.

[0065] 3. Administration: After completion, the blank control group was removed and replaced with 1 mL of fresh basal medium per well, and the sample group was replaced with fresh basal medium containing Example 2, and incubated at 37°C, 5% CO2 for 24 hours. The samples of the administration group were the same as those of Example 3 above.

[0066] 4. After the cells are treated with the reactive oxygen species detection kit and excited with blue light, the oxidized cells show green fluorescence under the microscope. The detection results are shown in Figure 3 .

[0067] 5. Extract cell supernatant and measure MDA content using ELISA kit. The test results are shown in Figure 4 .

[0068] 6. Use the above samples to test in the same way as above, and set up a blank control, in which no antioxidant product is placed.

[0069] 7. From Figure 3 It can be seen that the samples of comparative examples 1, 2, and 3 all have partial antioxidant properties, and the green fluorescence of fluorescent dyeing is slightly reduced. The samples of embodiments 1 and 2 have better antioxidant properties than comparative examples 1-3, and the green fluorescence of fluorescent dyeing is significantly reduced. The samples of embodiments 1 and 2 have higher antioxidant efficacy.

[0070] 8. From Figure 4 It can be seen that the samples of Comparative Examples 1, 2, and 3 all have partial antioxidant properties, and the MDA malondialdehyde is slightly reduced. The protein compound liquid in the samples of Examples 1 and 2 has better antioxidant properties than that of Comparative Examples 1-3, and the MDA malondialdehyde is significantly reduced. The samples of Examples 1 and 2 of the present invention all have better antioxidant properties.

Claims

1. A recombinant human type VII collagen, the amino acid sequence of which is shown in SEQ ID No:

1.

2. A nucleic acid encoding the recombinant human type VII collagen according to claim 1.

3. An expression vector comprising the nucleic acid of claim 2; preferably, the expression vector is pGAPZaA.

4. A host cell into which the expression vector according to claim 3 has been introduced; the host cell is preferably Escherichia coli or Pichia pastoris.

5. A method for producing the recombinant human type VII collagen according to claim 1, comprising: The step of culturing the host cell according to claim 4 to express the recombinant human type VII collagen, and collecting the cell culture.

6. The method according to claim 5, further comprising the step of separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation.

7. A collagen compound solution comprising the recombinant human type VII collagen according to claim 1 and hydroxyapatite hollow microspheres.

8. The collagen compound solution according to claim 7 is prepared by the following method: (1) Preparation of hydroxyapatite hollow microspheres: using equal amounts of calcium carbonate and disodium hydrogen phosphate to prepare hydroxyapatite hollow microspheres under normal pressure; (2) Preparing stock solution A: Weigh the recombinant human type VII collagen, vitamin E, antioxidants, and fibroblasts described in claim 1 according to the amount, put them into a reaction container and mix them, add deionized water in small amounts and multiple times, heat the reaction container to 30-45° C., and prepare stock solution A after the above raw materials and deionized water are completely mixed. Stop heating the reaction container and allow the stock solution A to cool naturally; (3) Preparation of stock solution B: Weigh sodium chloride diluent, lidocaine hydrochloride injection and Panax notoginseng extract according to the amount and put them into a reaction container for mixing, add deionized water in small amounts and multiple times, heat the reaction container to 40-60° C., and after the above raw materials and deionized water are completely mixed, stock solution B is prepared, stop heating the reaction container, and allow stock solution B to cool naturally; (4) Mixing stock solution A and stock solution B, pouring stock solution A and stock solution B into an emulsifying pot under a sterile environment at 25 to 35° C., stirring evenly, and then adding an emulsifier to obtain a sufficient mixture of stock solution A and stock solution B; (5) After the mixture is allowed to stand for 1 to 2 hours, a pH adjuster is added to adjust the pH value of the mixture to 7 to 8, and the mixture is stirred for 15 to 20 minutes to prepare a nutrient solution; (6) Put the above-mentioned hydroxyapatite hollow microspheres and nutrient solution into a mixing container, heat the mixing container to 30-45° C., stir the hydroxyapatite hollow microspheres and nutrient solution for 20-40 minutes to obtain a composite solution, and cool it naturally.

9. A product comprising the recombinant human type VII collagen according to claim 1, wherein the product is a medicine, a cosmetic or a medical device.

10. Use of the recombinant human type VII collagen according to claim 1 in the preparation of medicines, cosmetics or medical devices; preferably, the medicines, cosmetics or medical devices are used for anti-aging and anti-oxidation.

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