A recombinant type III collagen Yves-Col3 with anti-aging efficacy, its preparation method and application

Abstract

The present invention discloses a recombinant type III collagen Yves-Col3 with anti-aging efficacy, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2. The encoding gene, expression vector, host bacterium, preparation method and application thereof are also disclosed. The recombinant type III collagen Yves-Col3 of the present invention can be expressed by prokaryotic fermentation, has no cytotoxicity, high thermal stability, can significantly scavenge hydroxyl radicals and DPPH radicals, promote cell proliferation, has significant anti-aging efficacy, and can be compounded with other proteins to become raw materials for tissue engineering products, cosmetics, health products or drugs.

Description

Technical Field

[0001] The present invention relates to a recombinant type III collagen Yves-Col3 with anti-aging efficacy, its preparation method and application, belonging to the technical field of protein expression. Background Art

[0002] Collagen is a high-level protein, the molecule of which is composed of various amino acids, including proline, glycine, etc., and its unique biological activity is built on the three-stranded superhelical three-dimensional structure. As the most abundant and widely distributed protein in the human body, collagen constructs a tough support structure in the skin, bones and connective tissues, and is closely related to key processes such as maintaining the youthful state of the skin, wound healing and cell regeneration. Adult skin is mainly rich in type I collagen, while infant skin is mainly type III collagen, which endows the skin with delicacy and elasticity. However, with the increase of age, the ability of the human body to synthesize type III collagen decreases, and the loss of collagen accelerates, thus triggering signs of skin aging.

[0003] Aging is a natural and inevitable physiological process in living organisms. Aging is usually accompanied by a chronic, low-grade, systemic inflammation, involving the activation of the inflammatory network and the release of related cytokines, including key pro-inflammatory mediators such as TNF-α, IL-6 and IL-1β, and this process is an important driving force for inducing aging and aging-related diseases. As a complex physiological mechanism, there are currently free radical theory, damage theory, genetic theory, immune theory, telomere theory, etc. The free radical theory of aging proposed by Denham Harman in 1956 holds that the degenerative changes during the aging process are caused by the harmful effects of free radicals generated during the normal metabolism of cells. The free radicals mentioned here are mainly oxygen free radicals, such as superoxide anion free radicals, lipid oxygen free radicals, hydroxyl free radicals, nitrogen dioxide and nitric oxide free radicals. And maintaining an appropriate level of antioxidants and free radical scavengers in the body can extend lifespan and delay aging.

[0004] More and more studies show that collagen may have anti-aging, anti-inflammatory and collagen-promoting regeneration effects. However, this requires in-depth analysis of the collagen sequence to obtain an appropriate sequence to effectively exert the corresponding effects, resulting in no suitable collagen being widely used at present. Summary of the Invention

[0005] The object of the present invention is to provide a recombinant type III collagen Yves-Col3 with anti-aging efficacy.

[0006] The technical solution adopted by the present invention:

[0007] A recombinant type III collagen Yves-Col3 with anti-aging effect, whose amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] The coding gene of the aforementioned recombinant type III collagen Yves-Col3.

[0009] Preferably, its nucleotide sequence is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0010] The expression vector of the aforementioned recombinant type III collagen Yves-Col3.

[0011] The host bacterium of the aforementioned recombinant type III collagen Yves-Col3.

[0012] The preparation method of the aforementioned recombinant type III collagen Yves-Col3, the steps of which include:

[0013] (1) Construct the aforementioned expression vector and transform it into an expression host bacterium;

[0014] (2) Culture the expression host bacterium to induce the expression of recombinant protein;

[0015] (3) Purify the expression product to obtain the aforementioned recombinant type III collagen Yves-Col3.

[0016] The application of the aforementioned recombinant type III collagen Yves-Col3 in promoting cell proliferation in vitro.

[0017] Preferably, the cell is a human fibroblast.

[0018] The application of the aforementioned recombinant type III collagen Yves-Col3 in scavenging free radicals or superoxide anions in vitro.

[0019] The application of the aforementioned recombinant type III collagen Yves-Col3 in the preparation of anti-inflammatory agents.

[0020] The application of the aforementioned recombinant type III collagen Yves-Col3 in promoting the in vitro synthesis of type III collagen.

[0021] The beneficial effects of the present invention:

[0022] The recombinant type III collagen Yves-Col3 of the present invention can be expressed by prokaryotic fermentation, has no cytotoxicity, high thermal stability, can significantly scavenge hydroxyl radicals and DPPH radicals, promote cell proliferation, has a significant anti-aging effect, and can be compounded with other proteins to become raw materials for tissue engineering products, cosmetics, health products or drugs. Description of the Drawings

[0023] Figure 1 Expression vector map of recombinant type III collagen Yves - Col3 of the present invention.

[0024] Figure 2 Purification SDS - PAGE map of recombinant type III collagen Yves - Col3 of the present invention. Lane 1: Supernatant after heating Yves - Col3 - A3 at 70°C for 1 h. Lane 2: After purification of Yves - Col3 - A3 by ion exchange column. Lane 3: Supernatant after heating Yves - Col3 - A6 at 70°C for 1 h. Lane 2: After purification of Yves - Col3 - A6 by ion exchange column.

[0025] Figure 3 Cell proliferation experiment of recombinant type III collagen Yves - Col3 of the present invention.

[0026] Figure 4 Experiment on scavenging hydroxyl radicals and DPPH radicals of recombinant type III collagen Yves - Col3 of the present invention.

[0027] Figure 5 Anti - inflammatory efficacy detection experiment of recombinant type III collagen Yves - Col3 of the present invention.

[0028] Figure 6 Experiment on promoting collagen synthesis in fibroblasts of recombinant type III collagen Yves - Col3 of the present invention. Detailed implementation manners

[0029] The present invention is further illustrated by the following examples, but it is not intended to limit the present invention. The specific materials used in the implementation schemes of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention. Materials with the same or similar types, models, qualities, properties or functions as the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0030] Example 1: Preparation of recombinant type III collagen Yves - Col3 series plasmids

[0031] (1) Gene design and synthesis

[0032] In the present invention, an expression vector of recombinant type III collagen Yves-Col3 is constructed, wherein the recombinant type III collagen Yves-Col3 is respectively Yves-Col3-A3 and Yves-Col3-A6, whose corresponding amino acid sequences are respectively shown in SEQ ID No.1 and SEQ ID No.2, and whose nucleotide sequences are respectively shown in SEQ ID No.3 and SEQ ID No.4. The nucleotide sequences SEQ ID No.3 and SEQ ID No.4 are entrusted to Beijing Liuhe BGI Genomics Technology Co., Ltd. for synthesis.

[0033] (2) Construction of recombinant expression vector

[0034] The above-synthesized gene fragment was used as a template for PCR amplification, and the target gene fragment and the pET30a expression vector backbone were connected by Gibson-plasmid connection to obtain the corresponding plasmid.

[0035] Primer design:

[0036]

[0037] PCR amplification system:

[0038] System components Component volume Primer F 2.5 μL Primer R 2.5 μL Q5 High-Fidelity 2x Master Mix 2 μL The coding gene synthesized by BGI 25 μL <![CDATA[ddH2O]]> To 50 μL

[0039] After the PCR system was prepared, it was mixed and centrifuged. The PCR amplification conditions were as follows: the first stage was pre-denaturation at 98°C for 30s; the second stage was denaturation at 98°C for 10s, annealing at 50-72°C for 30s, extension at 72°C for 30s / kb, and 32 cycles; the third stage was final extension at 72°C for 2min. The above vector or gene fragment was recovered using a universal DNA purification kit (Tiangen Biochemical Technology Co., Ltd.) and the operation steps in the product manual were followed.

[0040] Gibson Connection:

[0041] System components Component volume Gibson Assembly Master Mix(2X) 5 μL Ligation fragment 0.2–1 pmols*X μL <![CDATA[dd H2O]]> (5-X) μL Total system 10 μL

[0042] After mixing the above components on ice, place in a 37°C heat bath for 60 min. The obtained ligation product is stored on ice or at -20°C for subsequent competent cell transformation.

[0043] The ligation product was transformed into the host bacteria E. coli DH5α by the heat shock method, spread on LB culture resistance plates, and incubated at 37°C overnight. Positive clones were randomly picked and inoculated into LB liquid culture medium, and cultured at 37°C, 220rpm overnight. The plasmid was extracted using a plasmid rapid extraction kit, and the successfully constructed plasmids were named pET30a-C3-A3 and pET30a-C3-A6, respectively.

[0044] (3) Construction of Engineered Bacteria

[0045] The above-obtained recombinant expression plasmid was transferred into Escherichia coli competent cell BL21(DE3) by heat shock method, and positive Escherichia coli genetic engineering bacteria were screened. The specific process was as follows: ① 4 μL of the recombinant expression plasmid was added to 100 μL of Escherichia coli competent cell BL21(DE3), and the mixture was left standing on ice for 30 min; ② The mixture was heat shocked in a 42 °C water bath for 90 s, and then quickly placed on ice and left standing for 2 min; ③ 700 μL of antibiotic-free LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) was added to the mixture, and the mixture was cultured at 37 °C and 220 rpm for 1 h; ④ 200 μL of the bacterial solution was evenly spread on an LB solid medium plate containing ampicillin (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 100 μg / mL ampicillin); ⑤ The plate was inverted and cultured in a 37 °C incubator for about 16 h. When clearly visible colonies grew out, the corresponding DE3-pET30a-C3-A3 and DE3-pET30a-C3-A6 engineered bacteria were obtained.

[0046] Example 2: Induced Expression of Engineered Bacteria

[0047] The single colonies on the above plate were placed in an LB liquid medium containing ampicillin and cultured at 37 °C and 220 rpm for 10 h. This was the primary seed solution. It was inoculated into a new LB medium at an inoculation amount of 1%, cultured overnight at 37 °C, inoculated into a new LB medium at an inoculation amount of 5%, cultured at 37 °C for 2 h, IPTG with a final concentration of 0.5 mM was added, and induced expression was carried out at 18 °C for 16 h. The bacterial cells were collected by centrifugation at 4000 g and 4 °C for 20 min.

[0048] The bacterial cells were resuspended in lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 500 mM NaCl, pH 8.5), 100X protease inhibitor PMSF was added, the bacterial cells were dispersed using a high-shear dispersion emulsifier, and a high-pressure homogenizer was used at 800 - 1000 bar for homogenization for 15 - 30 min. After cell disruption, 1 mL of the disrupted solution was centrifuged at 4 °C and 12,000 rpm for 2 min, 80 μL of the supernatant was taken, the precipitate was resuspended in 200 μL of lysis buffer, 120 μL of the resuspended solution was discarded, 20 μL of 5X protein loading buffer was added to both the supernatant and the precipitate, and after mixing, it was heated in a boiling water bath for 10 min for SDS-PAGE to confirm protein expression.

[0049] Example 3: Purification of Expression Products

[0050] The supernatant of the broken liquid obtained in Example 2 was heated in a water bath at 70 °C for 1 h to remove most of the miscellaneous proteins. After the heating of the protein solution was completed, the temperature was lowered to room temperature, and it was centrifuged at 4 °C and 12,000 rpm for 20 min. The supernatant was taken and the precipitate was discarded. Among them, 80 μL of the supernatant was added with 20 μL of 5X protein loading buffer, and after mixing, it was heated in a boiling water bath for 10 min for SDS-PAGE, and the gel image was analyzed by ImageJ software. The protein yield and purity are shown in the following table:

[0051]

[0052]

[0053] It can be seen from the above table that Yves-Col3-A3 and Yves-Col3-A6 have high yields and good thermal stabilities. The supernatant protein solutions of Yves-Col3-A3 and Yves-Col3-A6 were enriched and purified by cation exchange resin. The purified Yves-Col3-A3 and Yves-Col3-A6 proteins were collected. 80 μL of the supernatant was added with 20 μL of 5X protein loading buffer, and after mixing, it was heated in a boiling water bath for 10 min for SDS-PAGE, and the gel image was analyzed by ImageJ software. The protein yield and purity are shown in the following table:

[0054] Protein Yield mg / L Purity % Yves-Col3-A3 350 99 Yves-Col3-A6 380 99

[0055] The purification results of Yves-Col3-A3 and Yves-Col3-A6 are as Figure 2 shown. The purified protein band is single and the purity is high.

[0056] Example 4: Biological Activity Detection of Recombinant Humanized Collagen Type III Yves-Col3

[0057] (1) Cell Proliferation Experiment of Recombinant Collagen Type III Yves-Col3

[0058] The MTT cell proliferation detection kit was used to detect the proliferation effects of the recombinant collagen type III Yves-Col3-A3 and Yves-Col3-A6 prepared in Example 3 on human foreskin fibroblasts HFF-1. The specific detection steps can be routinely adjusted according to the kit instructions. Among them, the control group was DMEM complete medium, and the experimental groups 1-3 were DMEM complete medium containing 1, 10, and 100 ng / mL Yves-Col3-A3 or Yves-Col3-A6 respectively, and the positive control group was DMEM complete medium containing 100 ng / mL TGF-β1. The cell dosage in each group was 1×10 4 cells.

[0059] The detection results are asFigure 3 As shown in Figure 3 , 10 ng / mL of recombinant type III collagen Yves-Col3-A3 and Yves-Col3-A6 can effectively promote the proliferation of fibroblasts, and there is a dose effect. The greater the dose, the more significant the promotion of fibroblast growth. It is confirmed that the recombinant type III collagen Yves-Col3-A3 and Yves-Col3-A6 of the present invention have the effect of promoting fibroblast growth.

[0060] (2) Experiments on the scavenging of hydroxyl radicals, DPPH radicals and superoxide anions by recombinant type III collagen Yves-Col3

[0061] Determination of DPPH radical scavenging ability: Take test tubes, add 2 mL of the sample solution, and then add 2.5 mL of 0.1 mmol / L DPPH solution to each test tube. After shaking well, let it stand in the dark at room temperature for 30 min, centrifuge, and take the supernatant to measure the absorbance at 517 nm. The calculation formula for its scavenging rate is as follows:

[0062] Scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%

[0063] In the formula, A0 is the absorbance of distilled water instead of the sample solution; A1 is the absorbance after reacting with the sample solution; A2 is the absorbance of distilled water instead of the DPPH solution.

[0064] Determination of hydroxyl radical scavenging ability: Take test tubes, first add 2 mL of 8 mmol / L ferrous sulfate solution respectively, then add 2 mL of 8 mmol / L salicylic acid-ethanol solution, then add 2 mL of the sample solution, and finally add 1 mL of hydrogen peroxide with a volume fraction of 0.03% to start the reaction. After shaking well, let it stand at room temperature for 20 min, centrifuge, and take the supernatant to measure the absorbance at 510 nm. The calculation formula for its scavenging rate is as follows:

[0065] Scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%

[0066] In the formula, A0 is the absorbance of distilled water instead of the sample solution; A1 is the absorbance after reacting with the sample solution; A2 is the absorbance of distilled water instead of the salicylic acid-ethanol solution.

[0067] Determination of the scavenging ability against superoxide anions: Take test tubes and add 4.5 mL of Tris-HCl buffer solution with pH = 8.2 respectively, and react in a water bath at 30 °C for 10 min. Then add 0.5 mL of each sample solution, add 1 mL of 45 mmol / L pyrogallol solution, mix well, and then react with stirring in a water bath at 30 °C for 5 min. Immediately after the water bath ends, terminate the reaction with 6 mol / L concentrated hydrochloric acid, and measure the absorbance value of the supernatant at 325 nm. The calculation formula for the scavenging rate is as follows:

[0068] Scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%

[0069] In the formula, A0 is the absorbance of distilled water instead of the sample solution; A1 is the absorbance after reacting with the sample solution; A2 is the absorbance of distilled water instead of the pyrogallol solution.

[0070] The results are as Figure 4 shown. Both recombinant type III collagen Yves-Col3-A3 and Yves-Col3-A6 have significant effects on scavenging free radicals. Among them, the protein Yves-Col3-A6 has a better effect and has good anti-aging efficacy.

[0071] (3) Anti-inflammatory efficacy experiment of recombinant type III collagen Yves-Col3

[0072] Experimental principle: During the skin inflammation process, many inflammatory factors will be secreted, such as tumor necrosis factor-α (TNF-α). When the inflammatory effect slows down, the inflammatory factors will also decrease accordingly. In this experiment, UVB (irradiation dose is 300 mJ / cm 2 , irradiation time is 228 s) is used to stimulate human keratinocytes to construct an inflammation model, so that the cells secrete inflammatory factors. Then, the cells are treated with recombinant type III collagen Yves-Col3, and the expression of TNF-α in the supernatant is quantitatively detected by ELISA to investigate whether recombinant type III collagen Yves-Col3 has an inhibitory effect on the release of TNF-α, so as to achieve the anti-inflammatory effect.

[0073] The basic principle of ELISA is to utilize the specific reaction principle of antigen-antibody. The solid-phase immobilization of antigens or antibodies and the enzyme labeling of antigens or antibodies are carried out. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product, and the amount of the product is directly related to the amount of TNF-α in the supernatant. The ELISA experiment can convert chemical signals into electrical signals, and quantitatively analyze TNF-α by detecting the OD value with an enzyme-labeled instrument.

[0074] Dexamethasone can inhibit the release of cytokines, thereby reducing the inflammatory response and immune response. The mechanism of action of dexamethasone mainly exerts its anti-inflammatory, anti-allergic and immunosuppressive effects by affecting the intracellular signal transduction pathway. It can inhibit the synthesis of inflammatory mediators, inhibit the migration and infiltration of inflammatory cells, inhibit the activation and proliferation of immune cells, inhibit the release of cytokines, etc. It was used as a reference control in this experiment.

[0075] Experimental procedure: The experimental design is shown in the following table. The ELISA kit in the table is a kit for measuring Human TNF-α.

[0076] Anti-inflammatory experimental design

[0077]

[0078]

[0079] Experimental method: Digest the cells in the logarithmic growth phase and inoculate them into 6-well plates at a certain seeding density, and incubate overnight in a 37°C, 5% CO2 incubator; when the cell confluence rate in the 6-well plates reaches 50%, perform the treatment. The blank group and the stimulation group do not need to be treated. The reference control group is treated with 0.01% dexamethasone, and the experimental group is treated with 0.1% Yves-Col3-A3 or Yves-Col3-A6. Each group has 3 replicate wells. After treatment, continue to culture in a 37°C, 5% CO2 incubator for 24 h. Except for the blank group, other groups are irradiated with UVB according to the experimental requirements (irradiation dose is 300 mJ / cm 2 , irradiation time is 228 s), and then the experimental group and the control group are both changed to normal medium, and the supernatant is collected after continuing to culture in a 37°C, 5% CO2 incubator for 24 h. The ELISA detection kit is used to detect and analyze the secretion of TNF-a.

[0080] The results are as Figure 5 shown. The secretion of the inflammatory factor TNF-a in the stimulation group increased significantly compared with the blank group, indicating that the UVB irradiation stimulation successfully established an inflammatory model; compared with the stimulation group, the reference control group (0.01% dexamethasone) could significantly reduce the release of TNF-a; compared with the stimulation group, the experimental group (0.1% Yves-Col3-A3 or Yves-Col3-A6) could significantly reduce the release of TNF-a, indicating that Yves-Col3-A3 and Yves-Col3-A6 have anti-inflammatory effects, and among them, Yves-Col3-A6 has a better effect. (4) Fibroblast collagen synthesis promotion experiment of recombinant type III collagen Yves-Col3

[0081] Experimental principle: The content of collagen is the key to plump skin. Aging skin often accompanies the loss of collagen. Therefore, in anti-aging products, it is necessary to promote collagen synthesis to achieve the effect of filling the skin. In this experiment, after incubating fibroblasts with collagen aqueous solutions at different concentrations and the positive control TGFβ1 for 24 hours, the content of type III collagen in cells under different treatment conditions was detected by ELISA experiment.

[0082] The basic principle of ELISA experiment is the specificity of antigen-antibody. After binding the antibody of type III collagen labeled with enzyme to the type III collagen in cells, the substrate of the enzyme reaction is added. The substrate is catalyzed by the enzyme to become a colored product. The amount of the colored product is directly related to the amount of type III collagen. By detecting the OD value of the color of the colored product, quantitative analysis of type III collagen is carried out.

[0083] Experimental steps: The experimental concentrations are set as shown in the following table. The medium in the table is FbGrowth medium; the ELISA kit is a human type III collagen (COL3) ELISA kit.

[0084] Experimental design for promoting collagen synthesis in fibroblasts

[0085]

[0086]

[0087] 1) Cell seeding: Collect cells in the logarithmic growth phase and seed them into a 96-well plate at a cell density of 1×10 4 cells / well and culture them in an incubator at 37°C and 5% CO2 for 24 h.

[0088] 2) Sample addition: When the cell confluence rate in the 96-well plate reaches 40% - 60%, sample addition is carried out. The blank control group is cells inoculated with the culture medium, the positive control group is cells inoculated with the culture medium containing TGFβ1, and the sample group is cells inoculated with the culture medium containing diluted samples at each concentration. After sample addition, place the 96-well plate in an incubator (37°C, 5% CO2) and culture for 24 h.

[0089] 3) ELISA detection: After the culture is completed, collect the cell culture medium into a 1.5 mL centrifuge tube and then operate according to the ELISA kit instructions.

[0090] Upregulation rate of type III collagen in the positive control group = (average value of the positive control group - average value of the blank control group) / average value of the blank control group × 100%

[0091] Upregulation rate of type III collagen in the experimental group = (average value of the experimental group - average value of the blank control group) / average value of the blank control group × 100%

[0092] The results are as follows Figure 6 shown, Yves-Col3-A3 and Yves-Col3-A6 can significantly up-regulate the content of type III collagen and have the ability to promote the synthesis of type III collagen.

Claims

1. A recombinant type III collagen Yves-Col3 with anti-aging effect, characterized in that Its amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The gene encoding the recombinant type III collagen Yves-Col3 according to claim 1.

3. The coding gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.3 or SEQ ID NO.

4. The expression vector of the recombinant type III collagen Yves-Col3 according to claim 1 .

5. The host bacteria of the recombinant type III collagen Yves-Col3 according to claim 1.

6. The method for preparing the recombinant type III collagen Yves-Col3 according to claim 1, characterized in that The steps include: (1) constructing the expression vector according to claim 4 and transforming it into an expression host bacterium; (2) Cultivating the expression host bacteria and inducing the expression of the recombinant protein; (3) Purify the expression product to obtain the recombinant type III collagen Yves-Col3 as claimed in claim 1.

7. Use of the recombinant type III collagen Yves-Col3 according to claim 1 in promoting cell proliferation in vitro.

8. The use according to claim 7, characterized in that The cells are human fibroblasts.

9. Use of the recombinant type III collagen Yves-Col3 according to claim 1 in scavenging free radicals or superoxide anions in vitro.

10. Use of the recombinant type III collagen Yves-Col3 according to claim 1 in the preparation of anti-inflammatory agents.

11. Use of the recombinant type III collagen Yves-Col3 according to claim 1 in promoting the in vitro synthesis of type III collagen.

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