Oral recombinant human elastin capable of repairing skin subacute aging injury and application of oral recombinant human elastin

Abstract

The invention provides oral recombinant human elastin capable of repairing skin subacute aging injury and application of the oral recombinant human elastin, and belongs to the technical field of biotechnology and functional food. The oral recombinant human elastin capable of repairing skin subacute aging injury is prepared in a gene recombination expression mode. The recombinant human elastin has both a beta-spiral hydrophobic structure domain and a lysine-rich hydrophilic region, and shows excellent biological safety and biological activity; after the traditional Chinese medicine composition is prepared into an oral preparation, skin wrinkles can be remarkably reduced, the skin water content and dermis density can be improved, transdermal water loss can be reduced, the hydroxyproline level can be increased, collagen generation can be promoted, elasticity can be recovered, and skin oxidative stress reaction can be effectively inhibited. The method solves the problems that the natural elastin source is limited and the biological safety is insufficient, and has a wide industrialization prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and functional food technology, in particular to a recombinant human elastin for repairing subacute aging damage of skin and its application. BACKGROUND

[0002] Skin aging is the result of natural physiological degenerative changes of the body and the combined effects of external environmental factors such as ultraviolet radiation, air pollution and unhealthy lifestyle. Its main pathological features include the gradual decrease of collagen and elastic fiber content in the dermis, the decline of the synthesis and repair function of fibroblasts, the destruction of the extracellular matrix (ECM) network structure, the impairment of skin barrier function and the significant increase of oxidative stress level. The accumulation of reactive oxygen species (ROS) can accelerate the degradation of collagen and elastin mediated by matrix metalloproteinases (MMPs), forming a vicious cycle, which ultimately leads to decreased skin elasticity, relaxation and wrinkle formation, seriously affecting the appearance and quality of life of individuals.

[0003] Currently, the intervention techniques for skin aging mainly include two categories: topical repair preparations and oral functional factors. Commercially available topical repair preparations such as vitamin E, hyaluronic acid and peptide small molecules can quickly improve the hydration and barrier function of the epidermis and superficial dermis through topical application, but due to the barrier limitation of the stratum corneum, their deep penetration ability is insufficient, making it difficult to effectively rebuild the dermal ECM, and the drug delivery persistence is poor, requiring frequent use, which cannot achieve deep repair and long-term effect.

[0004] Oral functional factors such as nicotinamide, mononucleotides, resveratrol and collagen peptides can regulate multiple aging-related pathways through systemic circulation, with the advantages of systemic effect and dose controllability, but they generally have low bioavailability, unstable tissue distribution, inconsistent control of raw material source and quality, insufficient long-term safety evidence, and some active ingredients are easily degraded or cause discomfort in the gastrointestinal tract, which limits their clinical transformation value.

[0005] Elastin, as a key component to maintain the structural integrity of dermal ECM and the recovery ability of skin elasticity, its degradation or absence is recognized as a core marker of skin aging; however, the existing natural elastin mainly depends on animal tissue extraction, which is scarce and difficult to produce on a large scale, and is easily contaminated by animal-derived pathogens such as viruses and mycoplasma, with poor batch consistency and high immunogenicity, which is difficult to meet the stringent requirements of high-purity and high-safety raw materials in the fields of medical cosmetology and functional health products. SUMMARY

[0006] In view of the above deficiencies of the prior art, the present application provides a recombinant human elastin for oral repair of subacute aging damage of skin and an application thereof, which is obtained by gene recombination expression and has excellent biological safety and biological activity, and after being prepared into an oral preparation, can significantly reduce skin wrinkles, improve skin water content and skin density, and effectively inhibit skin oxidative stress reaction; the oral preparation is suitable for the fields of health products, functional foods and medical cosmetic preparations, and has broad market application prospects and industrialization potential.

[0007] To achieve the above-mentioned purpose, the specific technical solutions of the present application are as follows:

[0008] In the first aspect, the present application provides a recombinant human elastin for oral repair of subacute aging damage of skin, and the amino acid sequence of the recombinant human elastin is:

[0009] (a) the amino acid sequence shown in SEQ ID NO. 1; or

[0010] (b) an amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids based on the sequence shown in SEQ ID NO. 1, and having substantially the same function as the protein shown in SEQ ID NO. 1.

[0011] Further, the recombinant human elastin is obtained by treating a precursor elastin with a protease.

[0012] Further, the amino acid sequence of the precursor elastin is:

[0013] (a) the amino acid sequence shown in SEQ ID NO. 2; or

[0014] (b) an amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids based on the sequence shown in SEQ ID NO. 2, and having substantially the same function as the protein shown in SEQ ID NO. 2.

[0015] In the second aspect, the present application provides a gene encoding the precursor elastin, and the nucleotide sequence of the gene encoding the precursor elastin is:

[0016] (a) the nucleotide sequence shown in SEQ ID NO. 3; or

[0017] (b) a nucleotide sequence obtained by substitution, deletion and / or addition of one or more nucleotides based on the nucleotide sequence shown in SEQ ID NO. 3, and expressing the same protein as the nucleotide sequence shown in SEQ ID NO. 2.

[0018] In a third aspect, the present application provides a preparation method of the recombinant human elastin for repairing subacute aging damage of skin, comprising the following steps:

[0019] (1) synthesizing a gene sequence SEQ ID NO. 3 encoding a precursor elastin of the recombinant human elastin;

[0020] (2) connecting the gene sequence with a vector, transforming a genetic engineering bacterium, and constructing a recombinant genetic engineering bacterium;

[0021] (3) expressing the constructed recombinant genetic engineering bacterium, collecting bacterial bodies, crushing to obtain supernatant, and purifying to obtain the precursor elastin;

[0022] (4) adding a protease to treat the obtained precursor elastin, and purifying to obtain the recombinant human elastin.

[0023] Further, the protease includes thrombin, pepsin, trypsin or bromelain.

[0024] In a fourth aspect, the present application provides a recombinant vector or a recombinant genetic engineering bacterium carrying the precursor protein gene of the recombinant human elastin.

[0025] Further, the recombinant vector includes pCold or pET, and the recombinant engineering bacterium includes Escherichia coli.

[0026] In a fifth aspect, the present application provides an application of the recombinant human elastin in preparing an oral product for repairing subacute aging of skin.

[0027] In a sixth aspect, the present application provides an oral product for repairing subacute aging of skin, wherein an active ingredient of the oral product includes the recombinant human elastin.

[0028] Further, in a mouse experiment, the recombinant human elastin is used in an amount of 10-40 mg / kg per day.

[0029] Compared with the prior art, the present application has the following advantages:

[0030] (1) The recombinant human elastin provided by the present application has both a β-helix hydrophobic domain and a lysine-rich hydrophilic region, and shows good biological safety and biological activity.

[0031] (2) Based on the oral liquid of the recombinant human elastin, in a mouse subacute aging model, wrinkles can be significantly reduced, skin water content and dermal density can be increased, transdermal water loss can be reduced, hydroxyproline level can be increased, collagen production can be promoted, and elasticity can be restored; meanwhile, the oral liquid can significantly improve SOD, CAT and GSH activities and reduce MDA content, and has no adverse effect on mouse body weight, spleen and thymus index.

[0032] (3) Compared with the commercially available elastin peptides, the recombinant human elastin of the present application shows more significant ability to repair and improve aging skin.

[0033] (4) The present application overcomes the defects of limited source and insufficient biological safety of natural elastin, and provides an innovative oral active protein product with high purity, high consistency and systematic repair of subacute aging damage of skin, which is suitable for health care products, functional foods and medical cosmetology fields, and has broad industrialization prospects. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 SDS-PAGE identification chart of the recombinant human elastin Elastin F of the present application;

[0035] Figure 2 Qualitative evaluation of oral recombinant human elastin in repairing the apparent state of aging skin by DermaLab®-Combo;

[0036] Figure 3 Qualitative evaluation of oral recombinant human elastin in repairing the density of aging skin by DermaLab®-Combo;

[0037] Figure 4 Quantitative evaluation of oral recombinant human elastin in repairing the skin density, trans-epidermal water loss, skin water content and skin rebound time of aging skin by DermaLab®-Combo; wherein, Figure 4 a is the dermal layer density detection result of the skin; Figure 4 b is the trans-epidermal water loss detection result; Figure 4 c is the skin water content detection result; Figure 4 d is the skin rebound time detection result;

[0038] Figure 5 Evaluation of the repair effect of oral recombinant human elastin on aging skin by tissue staining;

[0039] Figure 6 Quantitative evaluation of the repair effect of oral recombinant human elastin on aging skin tissue; wherein, Figure 6 a is the hydroxyproline content detection result in the skin; Figure 6 b is the collagen fiber content detection result in the skin; Figure 6 c is the elastin fiber content detection result in the skin.

[0040] Figure 7 Quantitative evaluation of the antioxidant capacity of oral recombinant human elastin; wherein, Figure 7 a is the malondialdehyde content detection result; Figure 7b is the result of superoxide dismutase activity detection; Figure 7 c is the result of catalase activity detection; Figure 7 d is the result of glutathione content detection. DETAILED DESCRIPTION

[0041] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0042] The present application provides a recombinant human elastin for repairing subacute aging damage of skin orally, and the amino acid sequence of the recombinant human elastin is:

[0043] (a) the amino acid sequence shown in SEQ ID NO. 1; or

[0044] (b) an amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids based on the sequence shown in SEQ ID NO. 1, and having substantially the same function as the protein shown in SEQ ID NO. 1.

[0045] The recombinant human elastin is obtained by treating a precursor elastin with a protease.

[0046] The amino acid sequence of the precursor elastin is:

[0047] (a) the amino acid sequence shown in SEQ ID NO. 2; or

[0048] (b) an amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids based on the sequence shown in SEQ ID NO. 2, and having substantially the same function as the protein shown in SEQ ID NO. 2.

[0049] The nucleotide sequence of the gene encoding the precursor elastin is:

[0050] (a) the nucleotide sequence shown in SEQ ID NO. 3; or

[0051] (b) a nucleotide sequence obtained by substitution, deletion and / or addition of one or more nucleotides based on the nucleotide sequence shown in SEQ ID NO. 3, and expressing the same protein as the nucleotide sequence shown in SEQ ID NO. 2.

[0052] In the following specific embodiments, if not specifically stated, the methods used are conventional methods, and the reagents used are conventional reagents, which can be obtained from commercial channels.

[0053] Preparation of recombinant human elastin F

[0054] The amino acid sequence of the recombinant human elastin F of this embodiment is shown as SEQ ID NO. 1, which is obtained by treating the precursor elastin with a protease, and the preparation process is as follows:

[0055] (1) The amino acid sequence of the precursor elastin of the recombinant human elastin F is shown as SEQ ID NO. 2;

[0056] (2) Construction of the expression strain of the precursor elastin

[0057] The gene sequence encoding the precursor elastin is shown as SEQ ID NO. 3, a plasmid into which the above nucleic acid is introduced is constructed, and the successful synthesis of the plasmid is confirmed by DNA sequencing; the plasmid is transformed into an E. coli BL21-DE3 strain to obtain the precursor elastin expression strain, and the transformed strain is added with glycerol and stored in a refrigerator at -80 ℃;

[0058] (3) Preparation and purification of the recombinant human elastin

[0059] After 200 μL of the seed strain is added to 200 mL of LB liquid medium containing an antibiotic and proliferated at 37 ℃ overnight, it is transferred to 1 L of LB liquid medium containing an antibiotic at a 2% inoculation amount, and the culture is continued to expand at 37 ℃; when the OD value reaches 0.8-1.0, the temperature of the shaker is adjusted to 25 ℃, and 1 mM IPTG is added to induce expression, and the culture is continued overnight at a constant temperature; the bacterial solution is centrifuged in a low-temperature centrifuge at 3200 rpm and 4 ℃ for 30 min, and the bacterial body is collected. 600

[0060] The bacterial body is resuspended in a buffer solution containing 20 mM sodium phosphate, 0.5 M sodium chloride, and 20 mM imidazole at pH 7.4, the bacterial body is broken using an ultrasonic crusher, the broken suspension is centrifuged again, the supernatant is collected, and the collected supernatant is purified by a nickel ion affinity chromatography column to obtain the precursor elastin. The precursor elastin is treated with thrombin at a final concentration of 2-4 U / mL, the enzyme-cut impurities are removed, and the high-biological-activity recombinant human elastin is obtained by freeze-drying.

[0061] The recombinant human elastin F prepared in Example 1 is identified by SDS-PAGE, and the results are shown in Figure 1 The recombinant human elastin F prepared in this embodiment is a single band, indicating that the recombinant human elastin prepared in the present application has high purity.​

[0062] Example 2 Evaluation of the ability of recombinant human elastin to repair aging skin

[0063] 1. Modeling of subacute aging mice

[0064] Adult female Kunming mice were purchased from the Experimental Animal Center of Lanzhou University. All animals were fed with standard laboratory diet. A total of 108 mice were used to establish a D-galactose-induced subacute aging model. The mice were randomly divided into 6 groups: normal group (Normal), model group (Model), commercial product group (CP, elastin peptide, 40 mg / kg), Elastin-10 group (recombinant human elastin Elastin F, 10 mg / kg), Elastin-20 group (recombinant human elastin Elastin F, 20 mg / kg), and Elastin-40 group (recombinant human elastin Elastin F, 40 mg / kg). Except for the normal group, mice were subcutaneously injected with 500 mg / kg of D-galactose daily for 8 weeks to establish an aging model. After two weeks of subcutaneous injection, the normal and model groups of mice were orally administered with 0.4 mL of normal saline daily. The commercial product group, Elastin-10 group, Elastin-20 group, and Elastin-40 group were respectively administered with commercial elastin peptide CP and recombinant human elastin Elastin-10, Elastin-20, and Elastin-40 by gavage. The body weight of mice was recorded at 2, 4, and 8 weeks to monitor their health status. During the entire experimental period, mice could freely obtain food and water.

[0065] 2. Toxicological effects of oral administration of recombinant human elastin

[0066] The body weight of mice in each group at 2, 4, and 8 weeks was respectively recorded by weighing to evaluate whether the recombinant human elastin of the present application had a significant adverse effect on the growth of mice. The experimental results are shown in Table 1. The experimental results showed that the body weight of mice in the normal group, model group, commercial product group, Elastin-10 group, Elastin-20 group, and Elastin-40 group all showed a steady increasing trend during the entire experimental period. Statistical analysis showed that the difference in body weight between each group at the same time point did not reach statistical significance. This result indicated that the recombinant human elastin of the present application did not have a significant adverse effect on the growth of mice.

[0067] At the 8th week, the spleen and thymus of each group of mice were weighed to comprehensively evaluate the possible toxicological effects of oral elastin on mice, and the experimental results are shown in Table 1. The spleen and thymus, as key immune organs, play an important role in the immune system of the body. The spleen is rich in lymphocytes and macrophages, and plays a regulatory role in the immune response of the body; while the thymus is an important place for T cell differentiation and maturation, and is essential for maintaining the balance of the immune system. The experimental results show that the average values of the spleen index (SI) and thymus index (TI) of the normal group are 2.92±0.83 mg / g and 3.28±0.71 mg / g, respectively, and those of the model group, the commercial product group, the Elastin-10 group, the Elastin-20 group and the Elastin-40 group are 3.48±0.35 mg / g and 3.32±0.57 mg / g, 3.83±0.74 mg / g and 3.08±0.48 mg / g, 3.27±0.44 mg / g and 2.38±0.09 mg / g, 3.35±0.21 mg / g and 3.42±0.97 mg / g, 4.69±0.64 mg / g and 2.74±0.62 mg / g, respectively. The SI and TI of each group of mice compared with the normal group did not show significant difference. This result shows that the recombinant human elastin of the application does not cause any damage to the immune organs of mice, i.e. the recombinant human elastin of the application does not produce obvious toxicological effects on mice, and has good biological safety.

[0068] Table 1: Statistics of body weight, spleen index and thymus index of mice during the experiment

[0069]

[0070] 3. Qualitative evaluation of oral recombinant human elastin in repairing the apparent state of aging skin by DermaLab®-Combo

[0071] The dermoscope of DermaLab®-Combo was used to evaluate the ability of recombinant human elastin to repair aging skin (the experimental results are shown in Figure 2). The experimental results show that the back skin of the normal group of mice remains smooth and delicate during the experiment. At the 2nd week, fine wrinkles appear in the experimental groups except the normal group. At the 4th week, the skin wrinkles of the model group and the CP group are deepened, and the wrinkles of the Elastin-10 group, the Elastin-20 group and the Elastin-40 group are reduced compared with the 2nd week. At the 8th week, the skin wrinkles of the model group are further deepened, and a small amount of erythema is generated. The CP group has no obvious improvement effect, and the skin of the mice in the Elastin-10 group, the Elastin-20 group and the Elastin-40 group is smooth and delicate. The above results show that the recombinant human elastin of the application can repair the apparent state of aging skin, which is specifically manifested in reducing skin wrinkles and improving skin roughness.

[0072] 4. Qualitative evaluation of oral recombinant human elastin in repairing the density of aging skin by DermaLab®-Combo

[0073] The dermal density and epidermal thickness of the mice at the 2nd, 4th and 8th weeks are detected by using the Combo high-frequency ultrasound probe to evaluate the ability of oral elastin in repairing aging skin (the experimental results are shown in Figure 3 ). The color of the ultrasound image is related to the density and thickness of the skin, and the relationship is black < green < blue < red < yellow < white. The experimental results show that at the 2nd week, the skin density of the normal group of mice is the highest, and the epidermal thickness is uniform, while the experimental groups injected with galactose subcutaneously all show the phenomenon of thin epidermis and decreased density. At the 4th week, the epidermis of the model group and the CP group of mice is thinner, and the dermal density is decreased. After oral administration of the recombinant human elastin of the application, the decrease of dermal density caused by aging can be obviously reduced, and the effect of 20 mg / kg and 40 mg / kg is better. After 8 weeks, the dermal density of the model group of mice is very low. Compared with oral administration of commercial elastin, the dermal density of the mice is significantly improved after oral administration of the recombinant human elastin of the application.

[0074] 5. Quantitative evaluation of skin density, trans-epidermal water loss (TEWL), moisture and elasticity

[0075] The dermal layer density of the skin is measured by using the Combo high-frequency ultrasound probe, and the experimental results are shown in Figure 4 a. The higher the detected skin density, the better the skin state. Figure 4aIt is shown that the skin density of the mice in other experimental groups is obviously lower than that of the normal group (62.55±4.71) at the 2nd week, and the skin density of the mice in the model group decreases with aging, and the skin density decreases to 29.34±2.57 at the 8th week, and the skin density of the mice in the CP group gradually decreases. At the 8th week, the skin density of the mice in the Elastin-20 group and the Elastin-40 group has no significant difference (P>0.05) with that of the normal group. The results show that after oral administration of the recombinant human elastin, the decrease in skin density caused by aging can be obviously reduced, and the effect of 20 mg / kg and 40 mg / kg is better.

[0076] The trans-epidermal water loss of the skin is measured by using the TEWL probe of Combo, and the experimental results are shown in Table 2. Figure 4 bThe higher the detected TEWL value is, the worse the skin state is. Figure 4 bIt is shown that the skin TEWL value of the mice in the model group gradually increases after injection of D-galactose, and the skin TEWL value is 20.53±4.47 g / m 2 / h at the 2nd week and 30.88±3.87 g / m 2 / h at the 8th week. The CP group has a certain improvement effect within 8 weeks. After oral administration of the recombinant human elastin, the increase in the skin TEWL value caused by aging can be obviously reduced, and the effect of 20 mg / kg and 40 mg / kg is better, and at the 8th week, the skin TEWL value of the mice has no significant difference with that of the normal group.

[0077] The skin moisture level is measured by using the moisture probe of Combo, and the experimental results are shown in Table 3. Figure 4 c. Figure 4 cIt is shown that the skin moisture of the mice in the normal group is maintained at a high level (296.33±21.75 μS), and the skin moisture content of the mice in the model group gradually decreases, and the skin moisture content is 112.59±23.31 μS at the 8th week. The CP group has a certain improvement effect within 8 weeks. After oral administration of the recombinant human elastin, the decrease in the skin moisture caused by aging can be obviously reduced, and the effect of 20 mg / kg and 40 mg / kg is better, and at the 8th week, the skin moisture of the mice has no significant difference with that of the normal group.

[0078] The skin rebound time is measured by using the elasticity probe of Combo, and the experimental results are shown in Table 4. Figure 4 d. Figure 4 dIt is shown that the skin rebound time of the mice in the normal group is short (82.33±10.58 mS), and the skin rebound time of the mice in the model group gradually increases, and the improvement effect of the CP group is not obvious within 8 weeks. After oral administration of the recombinant human elastin, the decrease in the skin elasticity caused by aging can be obviously reduced, and the effect of 20 mg / kg and 40 mg / kg is better, and at the 8th week, the skin rebound time of the mice has no significant difference with that of the normal group.

[0079] 6. Organizational Analysis

[0080] H&E staining was used to investigate at the tissue level whether oral administration of the recombinant human elastin of this invention could improve the condition of aging skin. The experimental results are shown below. Figure 5 The figure shows that the normal group mice had regular epidermal structure, uniform thickness, tightly packed epidermal cells, normal morphology, and a clear boundary between the epidermis and dermis. The model group mice had irregular epidermis, uneven thickness, localized keratinization, and thinner dermis. Compared with the model group, oral administration of the recombinant human elastin of this invention improved skin tissue morphology to some extent, resulting in a more regular epidermal morphology and increased dermal thickness. Specifically, the Elastin-40 group showed intact skin structure, smooth epidermis, and uniform thickness.

[0081] Masson staining was used to study the effect of orally administered elastin on collagen regeneration in mouse skin at the tissue level. The experimental results are shown below. Figure 5 The figure shows that collagen fibers in the dermis of normal mice are arranged in a cross-hatched pattern, representing good elasticity and mechanical properties. After aging, collagen fibers break down and become disordered. However, after oral administration of the recombinant human elastin of this invention, collagen fibers remodel and regain their orderly arrangement. The results of quantifying mouse skin collagen fibers using ImageJ are shown in the figure. Figure 6 b. The results showed that oral administration of the recombinant human elastin of the present invention could improve the breakage of collagen fibers caused by aging and increase collagen deposition. The collagen deposition in the Elastin-40 group was 30.16±1.49%, which was close to the collagen fiber content of 31.04±2.44% in the normal group.

[0082] In addition, the hydroxyproline (HYP) content in skin tissue was analyzed, and the experimental results are shown in […]. Figure 6 Using the normal group as 100% HYP content, the results showed that the HYP content in the back skin of aged mice significantly decreased to 54.00±2.26%. The HYP content in the commercially available product group was 57.27±1.24%, showing limited improvement; while in the oral recombinant human elastin group, the HYP content in the Elastin-10 group, Elastin-20 group, and Elastin-40 group increased to 81.63±1.41%, 84.58±1.99%, and 96.69±1.55%, respectively, with the Elastin-40 group approaching the level of the normal group.

[0083] The effects of orally administered elastin on the regeneration of elastic fibers in mouse skin were studied at the tissue level using EVG staining. The experimental results are shown below. Figure 5The figure shows that the elastic fibers in the dermis of the normal group of mice are complete, and aging causes the elastic fibers to break and decrease. Oral administration of the recombinant human-derived elastin of the application can resist aging and increase the elastic fibers in the dermis of mice. The elastic fibers in the skin of mice were quantified using image J, and the experimental results are shown in Table 1 Figure 6 c, the normal group was 10.30 ± 0.66%, the model group was 5.94 ± 0.26%, the CP group was 6.09 ± 0.28%, the Elastin-10 group was 8.17 ± 0.51%, the Elastin-20 group was 8.95 ± 0.57%, and the Elastin-40 group was 11.10 ± 0.60%.

[0084] 7. In vivo antioxidant activity analysis of oral administration of recombinant human-derived elastin

[0085] MDA (malondialdehyde) content detection kit, SOD (superoxide dismutase) activity detection kit, CAT (catalase) activity detection kit, and GSH (glutathione) activity detection kit were used to further evaluate the in vivo antioxidant activity of oral administration of elastin, and the experimental results are shown in Table 2 Figure 7 .

[0086] MDA is a lipid oxidation product, and an increase in its content is usually associated with oxidative stress and cell damage. Aging can cause an increase in the content of MDA in mice. Figure 7 a shows that after 8 weeks, the content of MDA in the serum of the model group of mice reached 3.76 ± 0.22 nmol / mL, and oral administration of commercially available elastin peptides had no obvious improvement effect, the Elastin-10 group was 2.56 ± 0.06 nmol / mL, the Elastin-20 group was 2.38 ± 0.10 nmol / mL, and the Elastin-40 group was 2.12 ± 0.05 nmol / mL. The results show that the recombinant human-derived elastin of the application can significantly reduce the content of MDA, and the Elastin-40 group has the best effect.

[0087] SOD plays a key antioxidant role in the skin, and it can help scavenge free radicals to reduce oxidative stress, thereby protecting skin cells from oxidative damage. Aging can cause a decrease in the activity of SOD in the serum. Figure 7b shows that the normal group is 79.91±1.56 U / g, the model group is 42.00±1.45 U / g, the CP group is 43.10±1.17 U / g, the Elastin-10 group is 60.66±1.24 U / g, the Elastin-20 group is 75.63±1.17 U / g, and the Elastin-40 group is 76.28±1.92 U / g. The results show that the recombinant human elastin can significantly improve the SOD activity, and the Elastin-20 and Elastin-40 groups are the best.

[0088] Aging can lead to a decrease in CAT activity. Figure 7 c shows that the normal group is 40.54±1.09 U / mL, the model group is 23.62±1.82 U / mL, the CP group is 26.13±1.15 U / mL, the Elastin-10 group is 31.05±1.86 U / mL, the Elastin-20 group is 39.60±1.73 U / mL, and the Elastin-40 group is 39.90±1.23 U / mL. The results show that the recombinant human elastin can significantly improve the CAT activity, and the Elastin-20 and Elastin-40 groups are the best.

[0089] Aging can also lead to a decrease in GSH content. Figure 7 d shows that the normal group is 68.61±2.78 μg / mL, the model group is 34.39±2.07 μg / mL, the CP group is 50.67±0.84 μg / mL, the Elastin-10 group is 56.70±1.09 μg / mL, the Elastin-20 group is 59.26±1.47 μg / mL, and the Elastin-40 group is 63.47±1.39 μg / mL. The results show that the recombinant human elastin can significantly improve the GSH content, and the Elastin-40 group is the best, and has no significant difference with the normal group.

[0090] The above results show that oral administration of the recombinant human elastin can significantly reduce the oxidative stress level in the mouse body to prevent skin aging.

[0091] In summary, the present application provides a recombinant human elastin and an oral preparation thereof, which can repair skin subacute aging damage orally, and the recombinant human elastin has high purity, high consistency and excellent biological safety; after being prepared into an oral liquid, the recombinant human elastin can significantly reduce the number of wrinkles, increase the skin water content and dermal layer density, reduce the transdermal water loss rate, promote the regeneration of collagen, and accelerate the recovery of skin elasticity in a D-galactose-induced mouse subacute skin aging model; meanwhile, the preparation can significantly improve the activities of superoxide dismutase, catalase and glutathione, and reduce the content of malondialdehyde, and has no adverse effects on the body weight, spleen and thymus indices of mice; compared with existing animal-derived elastin or elastin peptide preparations, the present application overcomes the defects of limited raw material sources and insufficient biological safety, has a better anti-aging repair effect, is suitable for the fields of health products, functional foods and medical cosmetic preparations, and has a broad industrialization prospect.

[0092] The above detailed description of the embodiments of the present application, but the present application is not limited to the specific details of the above-described embodiments. Within the scope of the claims and technical concepts of the present application, the technical solutions of the present application can be modified and changed in many ways, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A recombinant human-derived elastin for orally repairing subacute aging damage of skin, characterized in that, The amino acid sequence of the recombinant human-derived elastin is: (a) the amino acid sequence shown in SEQ ID NO. 1; or (b) an amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids based on the sequence shown in SEQ ID NO. 1, and having substantially the same function as the protein shown in SEQ ID NO.

1.

2. The recombinant human-derived elastin for orally repairing sub-acute skin aging damage according to claim 1, characterized in that, The recombinant human-derived elastin is obtained by treating a precursor elastin, and the amino acid sequence of the precursor elastin is: (a) the amino acid sequence shown in SEQ ID NO. 2; or (b) an amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids based on the sequence shown in SEQ ID NO. 2, and having substantially the same function as the protein shown in SEQ ID NO.

2.

3. The recombinant human elastin of claim 2, wherein the recombinant human elastin is orally administrable to repair sub-acute skin aging damage. The gene sequence encoding the precursor elastin is: (a) the nucleotide sequence shown in SEQ ID NO. 3; or (b) a nucleotide sequence obtained by substitution, deletion and / or addition of one or more nucleotides based on the sequence shown in SEQ ID NO. 3, and expressing the same protein as the nucleotide sequence shown in SEQ ID NO.

2.

4. The method of producing the recombinant human elastin according to any one of claims 1 to 3, wherein, The method comprises the following steps: (1) synthesizing the gene sequence SEQ ID NO. 3 encoding the precursor elastin of the recombinant human-derived elastin; (2) connecting the gene sequence with a vector, transforming a genetically engineered bacterium, and constructing a recombinant genetically engineered bacterium; (3) expressing the constructed recombinant genetically engineered bacterium, collecting the bacterium, breaking it to obtain supernatant, and purifying to obtain the precursor elastin; (4) adding a protease to treat the obtained precursor elastin, and purifying to obtain the recombinant human-derived elastin.

5. The method for preparing recombinant human elastin according to claim 4, characterized in that, The protease includes thrombin, pepsin, trypsin or bromelain.

6. A recombinant vector or a recombinant genetically engineered bacterium carrying the gene of the precursor protein of the recombinant human-derived elastin according to any one of claims 1-3.

7. The recombinant vector or the recombinant genetically engineered bacteria according to claim 6, characterized in that, The recombinant vector includes pCold or pET, and the recombinant genetically engineered bacterium includes Escherichia coli.

8. Use of the recombinant human-derived elastin according to any one of claims 1-3 in the preparation of an oral product for repairing subacute aging of skin.

9. An oral product for repairing subacute aging of the skin, characterized in that, The active ingredient of the oral product includes the recombinant human-derived elastin according to any one of claims 1-3.

10. An oral product for repairing subacute aging of the skin according to claim 9, characterized in that, The amount of the recombinant human-derived elastin is 10-40 mg / kg per day.

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