Preparation method of human collagen and application thereof in anti-aging

Abstract

The application provides a preparation method of human collagen and application of the human collagen in anti-aging, and the amino acid sequence of the human collagen is shown as SEQ ID NO:1; the application is based on a natural collagen structure, provides a fusion sequence structure, and adjusts the proportion of individual amino acids; the collagen can promote skin cell growth and hyaluronic acid secretion, counteracts skin aging caused by ultraviolet radiation, inhibits expression of inflammatory factors, and counteracts oxidative stress.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology research and development, and specifically provides a preparation method of human collagen and its application in anti-aging. BACKGROUND

[0002] Skin is the largest organ of the body exposed to the external environment, and is affected by both intrinsic and extrinsic factors during the aging process. The characteristics of skin aging are dehydration, loss of skin elasticity, and appearance of wrinkles (see Lee D.E., Huh C.S., Ra J., et al. Clinical Evidence of Effects of Lactobacillus plantarum HY7714 on Skin Aging: A Randomized, Double Blind, Placebo-Controlled Study. J. Microbiol. Biotechnol. 2015; 25: 2160-2168.). With the improvement of living standards, people's demand for skin health management and beauty is getting higher and higher, and with many countries entering an aging society, the social and psychological impact of skin aging is gradually increasing, so skin aging has attracted considerable attention, and effective intervention measures need to be implemented to delay the skin aging process and maintain and repair the normal physiological function of the skin.

[0003] Collagen is the major protein found in skin, bone, cartilage, ligaments, tendons, and connective tissue, with humans producing at least 28 types of collagen, which are divided into fibril-forming collagens (e.g., types I, II, III, V, and XI) and non-fibrillar collagens (see Mukae S., Ogura Y., Hara Y. Characterization of the collagen network of human cheek skin using ultrasonic microscopy. Ultrasonics. 2024; 139: 107299). Collagen is characterized by a triple helix structure formed by a glycine every third residue repeat, i.e., a repeating Gly-X-Y amino acid triplet, with proline and hydroxyproline in the X, Y residues being relatively large (see Shoulders M.D., Raines R.T. Collagen structure and stability. Annu. Rev. Biochem. 2009; 78: 929-958). Collagen is the most common component of the extracellular matrix, providing mechanical support and guiding tissue development (see Frantz C., Stewart K.M., Weaver V.M. The extracellular matrix at a glance. J. Cell Sci. 2010; 123: 4195-4200).

[0004] Collagen I is the main ECM component in human skin and is widely used in dermatology and cosmetic applications due to its role in maintaining tissue biomechanics (see Akoa D.M., Helary C., Foda A., et al. Silicon impacts collagen remodelling and mineralization by human dental pulp stem cells in 3D pulp-like matrices. Dent. Mater. 2024;40: 1390-1399). It is formed as a heterotrimer of two a1(I) chains and one genetically distinct a2(I) chain, with a111(I) homotrimers also present in different tissues in the body, such as cancerous tissue, fibrosis, osteoarthritis and foetal (see Wong H.H., Seet S.H., Bascom C.C., et al. Tonic repression of collagen I by the bradykinin receptor 2 in skin fibroblasts. Matrix Biol. 2023;118: 110-128).Type I collagen can inhibit the accumulation of damaged mitochondria in cells treated with ultraviolet B (UVB), and pre-coating culture plates with type I collagen can reduce UVB-induced apoptosis of HaCaT cells (see Zhu J., Xu H.N., Lin T., et al. Silencing of cysteine and serine rich nuclear protein 1 inhibits apoptosis, senescence and collagen degradation in human-derived vaginal fibroblasts in response to oxidative stress or DNA damage. Exp. Cell Res. 2024;440:114139; Zhou X., Ye H., Wang X., et al. Ursolic acid inhibits human dermal fibroblasts hyperproliferation, migration, and collagen deposition induced by TGF-β via regulation the Smad2 / 3 pathway. Gene. 2023;867:147367).

[0005] Type III collagen exists in the endometrium, which is composed of the main part of the extracellular matrix, has the ability to affect cell proliferation, adhesion and migration, and can bind to integrin. As the second abundant collagen protein, type III collagen is widely distributed in various connective tissues such as the vascular system and internal organs (see Yang L., Wu H., Lu L., et al. A tailored extracellular matrix (ECM)-Mimetic coating for cardiovascular stents by stepwise assembly of hyaluronic acid and recombinant human type III collagen. Biomaterials. 2021;276:121055). Studies have found that collagen III and collagen I both play an important role in the activation of fibroblasts, and they are both the main components of tissue regeneration and wound healing (see Jia Y., Han Y., Zhang Y., et al. Multifunctional type III recombinant human collagen incorporated sodium alginate hydrogel with sustained release of extracellular vehicles for wound healing multimodal therapy in diabetic mice. Regenerative Ther. 2024;27:329-341); type III and type I collagen bind to collagen receptors and play multiple biological roles, such as supporting functions in cell migration, differentiation and adhesion, and M2 macrophage polarization, and promoting ECM, platelet aggregation and angiogenesis (see Li H., You S., Yang X., et al. Injectable recombinant human collagen-derived material with high cell adhesion activity limits adverse remodelling and improves pelvic floor function in pelvic floor dysfunction rats. Biomater. Adv. 2022;134:112715).Yang et al. used recombinant type III humanized collagen to regulate the structure and composition of rat skin tissue and inflammatory infiltration through the interleukin 6 / matrix metalloproteinase 1 signaling pathway, and improved the skin photoaging caused by ultraviolet irradiation in rats (see Yang Lixia, Diao Liqin, Li Hua, et al. Recombinant type III humanized collagen improves the regulatory mechanism of rat photoaging skin. Chinese Tissue Engineering Research, 2025; 30 (08) : 1988-2000).

[0006] Collagen derived from animal tissue has the risk of allergic reaction, immunogenicity and pathogen transmission (see Galdyszynska M., Zwolinski R., Piera L., et al. Stiff substrates inhibit collagen accumulation via integrin α2β1, FAK, Src kinases in human atrial fibroblast and myofibroblast cultures derived from patients with aortal stenosis. Biomed. Pharmacother. 2023; 159: 114289). In contrast, recombinant human collagen has the advantages of high purity, non-animal origin and elimination of the risk of viral or prion contamination (see Munisso M.C., Saito S., Tsuge I., et al. Three-dimensional analysis of load-dependent changes in the orientation of dermal collagen fibers in human skin: A pilot study. J. Mechanic. Behav. Biomed. Mater. 2023; 138: 105585). Therefore, in the present application, recombinant expression technology is used to produce target proteins in order to improve the safety and effectiveness of collagen use.

[0007] Based on the characteristics of natural human collagen, the present application respectively intercepts part of the active sequence in type I and type III collagen to form a fusion protein, and obtains the current protein through recombinant expression, which can effectively resist skin aging and maintain normal physiological function of the skin. SUMMARY

[0008] In view of the deficiencies of the prior art, the first aspect of the present application provides a human collagen, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] Further, the nucleotide sequence encoding the human collagen is shown as SEQ ID NO: 2.

[0010] The second aspect of the present application provides a preparation method of the human collagen, comprising the following steps: introducing a target gene encoding the human collagen into an expression vector, transferring the expression vector into an E. coli host cell, fermentation culture and separation and purification, and obtaining the human collagen.

[0011] Further, the expression vector is pET21a.

[0012] Further, the fermentation culture comprises inoculation at an inoculation amount of 10%, 37℃, 220 r / min incubation on a shaker, addition of IPTG at a final concentration of 0.4 mmol / L to induce protein expression when OD600 is 0.6-0.8, and induction of expression under the condition of 16℃, 220 r / min for 12-16 h.

[0013] Further, the separation and purification comprises ultrasonic disruption of bacterial cells at a power of 200-500 W, ultrasonic disruption for 3 s, interval of 3 s, and ultrasonic disruption for 15-30 min.

[0014] The third aspect of the present application provides an anti-skin aging composition comprising the human collagen.

[0015] The fourth aspect of the present application provides application of the human collagen and the anti-skin aging composition in preparation of an anti-skin aging product.

[0016] Further, the skin aging is ultraviolet-induced skin aging.

[0017] Further, the product is a pharmaceutical product or a cosmetic product.

[0018] Beneficial effects

[0019] The present application provides a preparation method of a human collagen and application thereof in anti-aging, specifically as follows:

[0020] 1. Based on the structure of natural collagen, a fusion sequence structure is provided, and the proportion of individual amino acids is adjusted.

[0021] 2. The collagen can promote skin cell growth and hyaluronic acid secretion.

[0022] 3. The collagen can resist skin aging caused by ultraviolet radiation, inhibit expression of inflammatory factors, and resist oxidative stress. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 : Collagen promotes skin cell proliferation;

[0024] Figure 2 : Collagen promotes skin cell hyaluronic acid secretion;

[0025] Figure 3 : IL-6 expression level;

[0026] Figure 4 : TGF-β expression level;

[0027] Figure 5 : MMP3 expression level. DETAILED DESCRIPTION

[0028] The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents, biological materials, and detection kits described are all commercially available unless otherwise specified.

[0029] Example 1 Preparation of human-derived collagen

[0030] 1.1 Design of human-derived collagen

[0031] Type I and type III collagen are the most important collagen subtypes, not only the main components of the extracellular matrix, but also exhibit protective effects on the skin in various in vivo and in vitro experiments, and are important active ingredients of various skin care products. However, the molecular weight of natural human-derived type I and type III collagen is too large, which is not conducive to preparation, storage and use. Therefore, based on the sequence structure of natural type I and type III collagen, different parts are intercepted to construct fusion proteins in order to overcome the above defects. At the same time, it has been reported that serine and aspartic acid are important factors for skin moisturizing, which can maintain skin luster and improve skin physiological function. Therefore, the amino acids in part of the triple helix structure are replaced, and the proportion of serine and aspartic acid is increased. The amino acid sequence of the human-derived collagen is shown in SEQ ID NO: 1. In order to facilitate the preparation of the human-derived collagen using the E. coli system, the codon is optimized, and the nucleotide sequence encoding the human-derived collagen is shown in SEQ ID NO: 2.

[0032] 1.2 Preparation of human-derived collagen

[0033] The target nucleic acid sequence was amplified by PCR method, and Nde I and Xho I enzyme cutting sites were introduced into the upstream and downstream primers, respectively. The PCR solution system: PCR buffer 4.8 μL, ddH2O 4.8 μL, single colony template, upstream primer 0.2 μL, downstream primer 0.2 μL, total system 10 μL. The PCR program is: preheating 94 ℃, 10 min; denaturation 94 ℃, 30 s, annealing 60 ℃, 30 s, extension 72 ℃, 1 min, a total of 30 cycles; recombination 72 ℃, 10 min, 4 ℃ incubation. The plasmid pET21a and the above-mentioned amplified nucleic acid fragment were digested with Nde I and Xho I endonuclease, and then the T4 ligase was used for ligation reaction, and the plasmid pET21a-mucol carrying the target nucleic acid fragment was obtained. Sequencing identification showed that the target fragment sequence carried by the plasmid was correct.

[0034] The target plasmid pET21a-mucol was introduced into the competent cell BL21 (DE3) by electroporation method, and then inoculated in LB solid medium containing ampicillin, cultured at 37 ℃ overnight, and positive clones were selected. The positive clone strain was inoculated in LB liquid medium containing ampicillin, cultured at 37 ℃, 220 r / min in a shaker overnight, and the activated seed liquid was obtained; the seed liquid was inoculated in a shake flask containing 200 mL of LB liquid medium at a 10% inoculation amount, and cultured at 37 ℃, 220 r / min in a shaker, and when OD600 was 0.6-0.8, the final concentration of 0.4 mmol / L IPTG was added to induce protein expression, and the expression was induced at 16 ℃, 220 r / min for 12-16 h.

[0035] The above fermentation liquid was centrifuged at 4 ℃, 6000 r / min for 10 min, the bacterial cells were collected and washed with sterile PBS. The resuspended bacterial liquid was placed in an ice bath condition, and ultrasonic was performed at 250W power, 3s interval, 20min; 10 000 r / min centrifugation for 30 min, and the supernatant was collected. The AKTA protein purification system (purchased from General Electric Company, USA) was used to purify the target protein, and the protein molecular weight was correct by SDS-PAGE electrophoresis detection.

[0036] Example 2 Human-derived collagen improves the physiological function of skin cells

[0037] 2.1 Promote skin cell proliferation

[0038] HaCaT cells (preserved in the laboratory) were inoculated in 96-well plates at a density of 1 x 104 per well, 3 replicates per group, and incubated in a carbon dioxide incubator (temperature 37°C, CO2volume fraction 5%) overnight. Recombinant collagen was added at a final concentration of 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively, and an equal volume of DMEM medium was used as a blank control well, and incubated at 37°C for 48 h. The absorbance at 450 nm of each well was determined using a CCK-8 kit, and the cell viability (%) was calculated as the ratio of the OD450 of the sample group to that of the control group. The results are shown in Figure 1 The collagen provided by the present application can effectively promote the proliferation of skin cells, and exhibits a certain dose-dependent effect.

[0039] 2.2 Promotion of hyaluronic acid secretion by skin cells

[0040] 5 x 10 4 4HaCaT cells were inoculated in 6-well plates, 3 replicates per group, and incubated in a carbon dioxide incubator (temperature 37°C, CO2volume fraction 5%) overnight. When the cell growth area reached about 50%, recombinant collagen was added at a final concentration of 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively, and an equal volume of DMEM medium was used as a blank control well, and incubated at 37°C for 48 h. The cell supernatant was collected and centrifuged at 3000 rpm for 10 min, and the supernatant was taken and used to detect the level of hyaluronic acid (HA) using an ELISA kit (purchased from Jingmei Biotechnology Co., Ltd.). The results are shown in Figure 2 After the addition of recombinant collagen, the HA secretion capacity of HaCaT cells was improved, and the medium and high dose groups showed significant differences, indicating that the recombinant collagen can effectively regulate the physiological function of skin cells.

[0041] Example 3 Human-derived collagen against ultraviolet-induced skin aging

[0042] 3.1 Modeling and drug administration

[0043] Thirty female nude mice weighing 20±2 g were selected, and after adaptive feeding for 3-7 days, they were randomly divided into 3 groups, namely the blank group, the model group, and the collagen group. The blank group was not treated with UVB irradiation, and the model group and the collagen group were irradiated with an ultraviolet crosslinking instrument (UVB) according to the ultraviolet radiation gradient of 300 mJ / cm 2 2. The collagen group was injected with 100 μg / kg of recombinant collagen at multiple points on the modeled skin, and the model group was injected with an equal volume of normal saline, once every 2 days, for a total of 3 weeks.

[0044] 3.2 Inflammatory factor detection

[0045] After 3 weeks of experiment, the mice were sacrificed and the skin tissues on the back of the mice were taken for testing. 0.1 g of skin tissue was cut and washed with 4°C normal saline, wiped dry with absorbent paper, cut into pieces, and added with protein lysate mixed with protease inhibitor (PMSF) at 4°C, homogenized with a tissue homogenizer (90 s each time, repeated 3 times), and centrifuged at 3000 r / min for 15 min to obtain the supernatant; centrifuged at 1000 rpm for 5 min to remove the residue. The contents of IL-6, TGF-β and MMP3 in the supernatant were detected by using an ELISA kit (purchased from R&D Systems Company).

[0046] IL-6 is an important inflammatory factor. After ultraviolet radiation, keratinocytes and fibroblasts secrete a large amount of IL-6, which can amplify the inflammatory response and induce the expression of other inflammatory factors such as TNF-α and IL-1β, thereby aggravating skin inflammation, such as Figure 3 As shown in the figure, the collagen in the application can effectively inhibit IL-6 and relieve inflammatory response; TGF-β is a major fibrosis-promoting factor. Ultraviolet radiation usually leads to an increase in TGF-β expression, resulting in a decrease in collagen synthesis and accelerating skin aging, such as Figure 4 As shown in the figure, the collagen in the application can reduce the expression amount of TGF-β after radiation and prevent excessive fibrosis of skin tissue; MMP3 can degrade various matrix components (such as collagen, laminin and proteoglycan), directly destroy the dermal structure and promote wrinkle formation, such as Figure 5 As shown in the figure, the collagen in the application can inhibit the expression of MMP3 and maintain the normal physiological function of the skin.

[0047] 3.3 Antioxidant capacity detection

[0048] The supernatant of the mouse tissue homogenate (preparation method same as section 3.2) was taken and SOD and GSH-Px detection kits (purchased from Wuhan Solabio Technology Co., Ltd.) were used to detect the antioxidant enzyme activity in the skin tissue of the mice. As shown in Table 1, the expression levels of SOD and GSH-Px significantly decreased after modeling, and the oxidative stress level increased; after treatment with the recombinant collagen protein of the application, the expression ability of SOD and GSH-Px recovered, and the oxidative stress was inhibited.

[0049] ;

[0050] The above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A human-derived collagen, characterized in that, The amino acid sequence of the human collagen is shown in SEQ ID NO:

1.

2. The method for preparing human collagen according to claim 1, comprising the following steps: introducing the target gene encoding the human collagen into an expression vector, transferring the expression vector into Escherichia coli host cells, fermenting and culturing, and separating and purifying to obtain the human collagen.

3. The preparation method according to claim 2, characterized in that, The expression vector is pET21a.

4. The preparation method according to claim 2, characterized in that, The fermentation culture includes: inoculating at a 10% inoculum, culturing in a shaker at 37°C and 220 r / min, adding IPTG at a final concentration of 0.4 mmol / L to induce protein expression when the OD600 reaches 0.6-0.8, and inducing expression for 12-16 h at 16°C and 220 r / min.

5. The preparation method according to claim 2, characterized in that, The separation and purification process includes ultrasonic disruption of bacterial cells, using a power of 200-500W, ultrasonication for 3 seconds, with a 3-second interval, for 15-30 minutes.

6. An anti-skin aging composition comprising the human collagen of claim 1.

7. The use of the human collagen of claim 1 and the anti-skin aging composition of claim 6 in the preparation of anti-skin aging products.

8. The application as described in claim 7, characterized in that, Skin aging is caused by ultraviolet radiation.

9. The application as described in claim 7, characterized in that, The product in question is a cosmetic product.

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