CN120699132A

CN120699132A - A recombinant humanized elastin with high biological activity and its preparation method and application

Info

Publication number: CN120699132A
Application number: CN202510682229.6A
Authority: CN
Inventors: 肖建喜; 黄文杰; 朱丽君; 孙秀霞; 石晶晶; 李飞
Original assignee: Guangzhou Advanced Regenerative Medicine Technology Co ltd
Current assignee: Guangzhou Advanced Regenerative Medicine Technology Co ltd
Priority date: 2025-05-26
Filing date: 2025-05-26
Publication date: 2025-09-26

Abstract

The invention provides recombinant humanized elastin with high bioactivity, a preparation method and application thereof, and belongs to the technical fields of genetic engineering and biological materials. The protein has a typical beta-helical hydrophobic domain and a lysine-rich hydrophilic domain, has good bionic structure and functional characteristics, can remarkably promote proliferation, adhesion and migration of human skin fibroblasts, has excellent antioxidant, anti-wrinkle and anti-aging activities, can be widely applied to the fields of skin repair dressings, injection implants, artificial skin, biomedical materials, functional skin care products and the like, and has good solubility, high stability and mature expression system, thereby being suitable for large-scale production.

Description

Recombinant humanized elastin with high bioactivity and preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering and biological materials, in particular to recombinant humanized elastin with high biological activity, and a preparation method and application thereof.

Background

Elastin (Elastin) is a key structural protein that is widely found in mammalian connective tissue that is elastic and deformable, such as skin, blood vessels, lungs, ligaments, etc., and has a primary function of imparting reversible elastic stretch properties to the tissue. Elastin is formed by tropoelastin (Tropoelastin) under the action of enzymatic crosslinking, and has a modularized structure formed by characteristic hydrophobic domains and hydrophilic domains alternately, wherein the hydrophobic region is usually rich in VPGXG repetitive sequences (such as VGVAPG, VAPGVG and the like), and the hydrophilic region is rich in positively charged amino acid residues such as lysine and the like, so that an active site is provided for molecular crosslinking. The structure endows elastin with good mechanical property, biocompatibility and thermoreversible phase change behavior, and is a core foundation for constructing biological material brackets, medical dressings and artificial tissue materials.

Currently, elastin shows wide application prospects in the fields of tissue engineering, skin injury repair, medical cosmetology, artificial skin, artificial blood vessels, functional skin care products, high-end medical injection materials and the like. However, the elastin from natural sources is mainly obtained by extraction from animal tissues (such as bovine aorta or pig ligament), and has the problems of complex extraction process, low yield, poor batch-to-batch stability, insufficient purity, destroyed biological activity, potential pathogen pollution and immunogenicity risks, and the like, which severely limit the application of the elastin in clinical and industrialized processes.

In recent years, with the development of genetic engineering and synthetic biology, the preparation of elastin by recombinant methods is becoming a research hotspot. The method can realize the manual optimization and functional modularized design of the protein structure, and has the advantages of controllable raw material source, uniform structure, large-scale production and the like. Patent CN102241747a and CN202110357328.9 report elastin-like molecules constructed with simplified short peptide repeat units (e.g. (GVGVAP) _n), but most of the above construction strategies only retain part of the hydrophobic domain of elastin, lack of complete hydrophilic region cross-linking sites and human sequences, resulting in significant deficiencies in their biological activity, and difficulty in meeting the functional requirements of biological materials. Meanwhile, the constructed expression strain has low yield, high protein purification difficulty, poor water solubility and stability, and difficulty in meeting the requirement of industrial mass production.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a recombinant humanized elastin with high bioactivity and excellent mechanical properties, and a preparation method and application thereof.

In order to achieve the above purpose, the specific technical scheme of the invention is as follows:

In a first aspect, the present invention provides a recombinant humanized elastin with high biological activity, the amino acid sequence of the recombinant humanized elastin comprises an amino acid sequence shown as SEQ ID NO. 1, or an amino acid sequence with the amino acid sequence shown as SEQ ID NO. 1 being substituted, deleted and/or added with one or more amino acids and having the same function as a protein composed of the amino acid sequence shown as SEQ ID NO. 1.

Further, the recombinant humanized elastin is obtained after treatment of the precursor elastin with a protease.

Further, the amino acid sequence of the precursor elastin comprises an amino acid sequence shown as SEQ ID NO. 2, or an amino acid sequence with the same function as a protein consisting of the amino acid sequence shown as SEQ ID NO. 2, wherein one or more amino acids are substituted, deleted and/or added to the amino acid sequence shown as SEQ ID NO. 2.

Further, the protease comprises thrombin, pepsin, trypsin or bromelain.

In a second aspect, the present invention provides a method for preparing said recombinant humanized elastin, comprising the steps of:

(1) Synthesizing a gene sequence encoding a precursor elastin of the recombinant humanized elastin;

(2) Connecting the gene sequence with a vector, and transforming genetically engineered bacteria to construct recombinant genetically engineered bacteria;

(3) Expressing the constructed recombinant genetically engineered bacteria, collecting thalli, crushing to obtain supernatant, and purifying to obtain precursor elastin;

(4) Adding protease to treat the obtained precursor elastin, and purifying to obtain recombinant humanized elastin.

In a third aspect, the present invention provides a gene encoding a precursor elastin of the recombinant humanized elastin, the gene sequence encoding the precursor elastin comprising a gene sequence as shown in SEQ ID NO. 3, or a gene sequence as shown in SEQ ID NO. 3 substituted, deleted and/or added with one or several nucleotides and expressing the same protein as the gene sequence as shown in SEQ ID NO. 3.

In a fourth aspect, the present invention provides a recombinant vector or recombinant genetically engineered bacterium carrying the gene.

Further, the recombinant vector comprises pCold or pET.

Further, the recombinant genetically engineered bacteria include escherichia coli.

In a fifth aspect, the invention provides the use of said recombinant humanized elastin in skin care products, dressings, implants, artificial skin, vascular prostheses, medical devices, biomaterials, functional foods.

Compared with the prior art, the invention has the following advantages:

1. the invention provides a high-bioactivity recombinant humanized elastin, wherein 100% of the amino acid sequence of the elastin is derived from human elastin, has good tissue compatibility and is suitable for clinical application;

2. compared with elastin extracted from animal tissues, the elastin provided by the invention is obtained in a recombinant expression mode, so that the pollution risk of viruses, mycoplasma and animal-derived pathogens is avoided, the immunogenicity is obviously reduced, the biosafety and the consistency between batches of the product are improved, and the strict requirements of the medical and cosmetic fields on raw material sources are met;

3. The elastin molecule provided by the invention has a hydrophobic beta-helical structural domain and a hydrophilic structural domain rich in lysine, so that the structure and the function of the human elastin are well simulated, and a structural basis is provided for constructing an elastic fiber network and a functional hydrogel material;

4. The elastin provided by the invention has remarkable proliferation promoting, adhesion promoting and migration promoting effects on human skin fibroblast (HFF-1) and good cell activity;

5. The elastin provided by the invention has excellent antioxidant and anti-wrinkle properties, good solubility, strong compatibility and high thermal stability, and is suitable for being used as an active ingredient in a functional skin care product formula;

6. in a D-galactose-induced skin aging animal model, the elastin provided by the invention remarkably improves the thickness and density of skin, increases the moisture content of stratum corneum, reduces the loss of percutaneous moisture (TEWL), restores the skin elasticity, and has good anti-aging and skin barrier repairing effects;

7. In zebra fish experiments, the elastin provided by the invention has obvious anti-wrinkle effect and obvious effect of inhibiting active oxygen generation;

8. The invention constructs the engineering strain suitable for the elastin expression, the expression system is stable, the expression level is high, and the large-scale production feasibility is realized;

9. The recombinant humanized elastin with high bioactivity has wide application prospect in the fields of skin repair dressing, implant, artificial skin, biological material, medical instrument, cosmetics and the like.

Drawings

FIG. 1 is a SDS-PAGE identification of recombinant humanized elastin of the invention;

FIG. 2 is a graph showing the results of reversible phase changes of the recombinant humanized elastin of the present invention;

FIG. 3 is a graph showing the results of cell experiments on recombinant humanized elastin of the present invention;

FIG. 4 is a graph showing the results of the Combo system characterization of the recombinant humanized elastin of the present invention;

FIG. 5 is a chart of staining of skin tissue sections with anti-aging characteristics of recombinant humanized elastin of the present invention;

FIG. 6 is a graph showing the results of an antioxidant stress test of the recombinant humanized elastin of the present invention;

FIG. 7 is a graph showing the results of anti-wrinkle and active oxygen scavenging experiments performed on zebra fish of the recombinant humanized elastin of the present invention.

Detailed Description

The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.

The invention provides a recombinant humanized elastin with high biological activity, wherein the amino acid sequence of the recombinant humanized elastin comprises an amino acid sequence shown as SEQ ID NO. 1, or an amino acid sequence with the same function as a protein formed by the amino acid sequence shown as SEQ ID NO. 1 by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1.

The recombinant humanized elastin is obtained by treating precursor elastin with protease.

The amino acid sequence of the precursor elastin comprises an amino acid sequence shown as SEQ ID NO. 2, or an amino acid sequence which is formed by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown as SEQ ID NO. 2 and has the same function as the protein consisting of the amino acid sequence shown as SEQ ID NO. 2.

In some examples, the protease comprises thrombin, pepsin, trypsin, or bromelain.

In the following examples, unless otherwise specified, the methods used are conventional and the reagents used are conventional and commercially available.

EXAMPLE 1 recombinant humanized elastin with high biological Activity

The amino acid sequence of the recombinant humanized Elastin C of the embodiment is shown as SEQ ID NO.1, and the protein is obtained by treating precursor Elastin with protease, and the preparation process is as follows:

(1) Determining the amino acid sequence of precursor Elastin of recombinant humanized Elastin C as shown in SEQ ID NO. 2;

(2) Constructing an expression strain of precursor elastin;

synthesizing a gene sequence for encoding precursor elastin as shown in SEQ ID NO. 3, constructing a plasmid introduced with the nucleic acid, and confirming successful synthesis of the plasmid through DNA sequencing, converting the plasmid into an escherichia coli BL21-DE3 strain to obtain a precursor elastin expression strain, adding glycerol into the strain which is successfully converted, and storing the strain in a refrigerator at-80 ℃;

(3) Preparation and purification of recombinant humanized elastin

Adding 200 μl of seed strain into 200 mL LB liquid medium containing antibiotics, performing proliferation culture on the strain overnight in a 37 ℃ constant temperature shaking table, transferring the strain to 1L LB liquid medium containing antibiotics according to 2% of inoculation amount, continuing amplification culture in the 37 ℃ constant temperature shaking table, adjusting the shaking table temperature to 25 ℃ when OD ₆₀₀ value reaches 0.8-1.0, adding 1mM IPTG final concentration for inducing expression, culturing at constant temperature overnight, centrifuging the bacterial liquid in a low temperature centrifuge, centrifuging at 3200 rpm,4 ℃ and 30min, and collecting bacterial cells.

The cells were resuspended in a buffer solution containing 20 mM sodium phosphate, 0.5M sodium chloride, and 20 mM imidazole at pH 7.4, the cells were disrupted by an ultrasonic disrupter, the disrupted suspension was centrifuged again, the supernatant was collected, and the collected supernatant was purified by a nickel ion affinity column to give a precursor elastin. Adding thrombin with the final concentration of 1-2U/mL to treat precursor Elastin, purifying to remove enzyme-cleaved impurities, and freeze-drying to obtain recombinant humanized Elastin-C with high biological activity.

The recombinant humanized Elastin C with high bioactivity prepared in example 1 was subjected to SDS-PAGE for identification, and the results are shown in FIG. 1. FIG. 1 shows that the recombinant humanized Elastin-C prepared in this example is a single band, indicating that the recombinant humanized Elastin prepared in this invention has high purity.

EXAMPLE 2 reversible phase-change characterization of recombinant humanized elastin

Reversible phase transition is a property unique to elastin, and the reversible phase transition temperature of recombinant humanized elastin is determined by performing turbidity experiments with an ultraviolet-visible spectrophotometer equipped with a temperature control system. As shown in fig. 2, the absorbance of the 100 μ M ELASTIN C solution remained stable over the temperature range of 20 ℃ to 25 ℃. When the temperature exceeds 25 ℃, the solution starts to form a precipitate, the absorbance rises rapidly, and as the temperature rises to 45 ℃, the absorbance reaches a plateau, and the maximum value is 1.5. The different concentrations of Elastin C showed similar trend of change (fig. 2A). A linear relationship model was established with Elastin C reversible phase transition temperature as Y axis and its concentration logarithm (lgC _{Elastin C})) as X axis (FIG. 2B). In addition, the present study explored the effect of salt ion concentration on reversible phase change behavior of 100 μ M ELASTIN C solutions (FIGS. 2C, 2D). The Elastin C phase transition temperature shows a gradient decrease as the NaCl concentration increases from 0.25M to 1.5M. The result shows that the recombinant humanized elastin provided by the invention has the characteristic of reversible phase change.

EXAMPLE 3 cytobiological functional verification of recombinant humanized elastin

(1) Cytotoxicity test

100. Mu.L of human skin fibroblast HFF-1 with a cell density of 1x10 ⁵/mL was added to a 96-well plate, the culture solution in the 96-well plate was aspirated after incubation for 24 h and 24 h at 37℃with 5% CO ₂, the recombinant humanized Elastin C prepared in example 1 was prepared in a solution of 0, 5, 10, 50, 100, 500, 1000. Mu.g/mL in a 96-well plate with DMEM high sugar medium, and the control group was supplemented with DMEM high sugar medium alone and continued incubation for 24 h in an incubator at 37℃with 5% CO ₂. The cck-8 was used to examine whether the designed recombinant humanized elastin was toxic to human skin fibroblast HFF-1.

As shown in FIG. 3A, after the Elastin C with different concentrations is added, the cell survival rate is over 100 percent, which indicates that the recombinant humanized Elastin provided by the invention has no cytotoxicity and excellent biocompatibility.

(2) Cell proliferation assay

HFF-1 cells were seeded at a density of 5X 103 cells/well in 24 well plates and pre-incubated for 24 hours with complete DMEM medium containing 10% fetal bovine serum. DMEM medium containing 1.0 mg/mL of recombinant humanized Elastin C prepared in example 1 was added to 6 experimental wells in a 24-well plate, and wells without Elastin C were set as blank. After culturing at 37℃for 1, 3 and 5 days, cell proliferation was examined by CCK-8 method. The relative cell growth rates of the blank and Elastin C groups at different time points were calculated based on the cell growth status (set to 100% growth rate) on day 1 of the blank.

HFF-1 cells were inoculated at a density of 1X 10 ⁵/mL into a culture system containing 1.0: 1.0 mg/mL of the recombinant Elastin C prepared in example 1, and after culturing at 37℃for 1,3, 5 days, respectively, cell activity was assessed using a live/dead cell double-staining kit.

As shown in fig. 3B, the cell proliferation rate of the Elastin C group was slightly higher than 100% on day 1, the proliferation rate of the Elastin C group (182%) was significantly higher than that of the blank group (137%) (. P < 0.001) by day 3, and the proliferation rates of both the Elastin C group (223%) and the blank group (197%) were continuously increased on day 5, and the proliferation rate of the Elastin C group was significantly higher than that of the blank group. As shown in fig. 3C, the live/dead staining image further showed that the Elastin C group cells remained continuously growing after 5 days of culture. Taken together, the results show that the recombinant humanized elastin of the present invention has excellent cell proliferation promoting activity.

(3) Cell adhesion experiments

The recombinant humanized Elastin C prepared in example 1 was formulated into a 1.0 mg/mL solution with PBS, heat denatured 1% BSA as a negative control, the formulated Elastin C solution was added to a 24-well plate to adsorb 24h at 4℃and then the solution was aspirated. Human skin fibroblasts HFF-1 cells were diluted to a density of 1X10 ⁵ cells/mL with DMEM high-sugar medium, 500mL cells were added to each well, and cell adhesion was observed under a microscope after 6 h.

As shown in FIG. 3D, HFF-1 cells in the BSA group are round, which indicates that the adhesion capacity is weak, while Elastin C group cells are in a typical spindle shape, which indicates that the recombinant humanized Elastin of the invention has excellent cell adhesion performance.

(4) Cell migration experiments

HFF-1 cells were seeded at a density of 5X 10 ⁵ cells/well in 6-well plates and routinely cultured for 24 hours to confluence of cell monolayers. The linear scratches were streaked vertically along the pre-labeled lines at the bottom of the well plate using a 10 μl pipette tip, followed by gentle washing 3 times with PBS to remove the shed cells. The experimental group was added with DMEM medium containing 1.0 mg/mL of recombinant humanized Elastin C prepared in example 1, and the blank group was added with DMEM medium only. The well plate was placed in a 37 ℃ 5% CO ₂ wet incubator for incubation, and images of the scratch areas were taken using an inverted microscope at 0 hours and 18 hours, respectively, and analyzed for cell migration.

As shown in figure 3E, the scratches of the 0-hour space-time group and the Elastin C group are clear and uniform, and after the culture for 18 hours, the cell migration rate of the Elastin C group is obviously higher than that of the blank group, which indicates that the recombinant humanized Elastin can effectively promote the HFF-1 cell migration.

Example 4 characterization of skin aging repair Properties of recombinant humanized elastin

In a controlled experimental environment (relative humidity 60% RH, temperature 25.+ -. 2 ℃), mice were kept under a circadian rhythm of 12 h light/12 h darkness with free ingestion of drinking water. After 7 days of adaptation, the back hair of the mice was shaved using a shaver and the hair was further removed using a depilatory cream, exposing a skin area of about 3 cm x 3 cm. To ensure that the depilatory cream does not affect the integrity of the epidermis, the skin is stabilized by resting 24h after depilation. Thereafter, the mice were randomly divided into five groups of 6 mice each. Blank (Blank) was healthy mice subcutaneously injected daily with 0.3 mL saline, and the remaining four groups subcutaneously injected daily with D-galactose (D-gal, 1000 mg/kg in saline) for 8 consecutive weeks. The four groups were model, positive control (PC, 5% vitamin E emulsion), elastin C (recombinant Elastin C prepared in 1000 mg/L PBS-dissolved example 1) and Elastin M (commercial group, 1000 mg/L PBS-dissolved commercial Elastin). The blank group was topically applied for 8 weeks at 0.1 mL PBS a day throughout the experiment. All topical treatments (including experimental treatment solutions and vitamin E emulsions) were applied uniformly to the exposed back skin area and rubbed thoroughly until no visible residue was present. The topical treatment was continued for 6 weeks from week 3 of the experiment. At the set experimental time points (week 2, week 4 and week 8), mice were subjected to back skin test under anesthesia, back hair was shaved before the test, and skin status was evaluated using the dermlab Combo system. Mice were sacrificed by cervical dislocation at the end of the assessment and back skin tissue was immediately taken for subsequent analysis. The skin tissue sections were stained HE, masson, VEG for each, and skin aging and recovery were observed under a microscope.

(1) DermaLab Combo System evaluation

As shown in fig. 4A, at week 2, the skin of the back of each group of mice except the blank group was aged with no significant difference. At week 4, the model group had severe skin aging and increased wrinkles, and the positive control group, elastin C group and the commercial group had reduced wrinkles and the skin became smoother. At week 8, the skin of the back of the mice in the model group showed more pronounced wrinkles, the skin of the positive control group, the skin of the Elastin C group and the skin of the commercially available group were substantially restored to normal state, and the skin of the Elastin C group was smoother than the skin of the positive control group and the skin of the commercially available group. The recombinant humanized elastin disclosed by the invention is favorable for repairing aged skin, and the effect of the recombinant humanized elastin is better than that of a commercially available product.

The ability of Elastin C to promote aged skin repair was visually assessed by observing the density and thickness of mouse skin in the in vivo state using ultrasound imaging of Combo, and the results are shown in fig. 4B. The results showed that at week 2, epidermis was thinned except for the blank group, and dermis density was reduced. At week 4, the dermis layer densities of the positive control, elastin C, and commercial groups began to increase, the epidermis began to thicken, and the dermis layer densities of the model group were not significantly different from week 2. At week 8, the epidermis thickness and dermis density of the positive control, elastin C and commercial groups recovered to normal skin levels, and the Elastin C group was superior to the positive control and commercial groups, with no significant difference in the model epidermis and dermis densities compared to weeks 2 and 4. The recombinant humanized elastin disclosed by the invention is favorable for recovering the epidermis thickness and dermis density of aged skin to normal levels, and the effect of the recombinant humanized elastin is better than that of a commercially available product.

The density of the back skin of the mice was examined in a living state using a Combo ultrasonic probe, and the ability of recombinant humanized Elastin C to promote recovery of skin density was evaluated, and the results are shown in fig. 4C. The results showed that at week 2, the skin densities of the model, positive control, elastin C, and commercial groups were all reduced, with no significant difference between the four, and the blank group had significantly higher skin densities than the latter four. Beginning at week 3, galactose was injected except for the blank group, and the positive control group, elastin C group, and the commercial group were administered simultaneously. At week 4, the increase in skin density of the positive control, elastin C and commercial groups began to increase, and the skin density of the Elastin C group was higher than that of the commercial group, and the skin density of the positive control, elastin C and commercial groups was significantly higher than that of the model group and lower than that of the blank group. At week 8, the skin densities of the positive control and Elastin C groups recovered to normal skin levels, and the commercial skin densities were still significantly below normal.

The moisture content of the skin of the back of the mice was examined in a living state using a moisture probe of Combo, and the ability of recombinant humanized Elastin C to promote skin moisture recovery was evaluated, and the results are shown in fig. 4D. The results showed that at week 2, the skin moisture content was reduced in the model, positive control, elastin C, and commercial groups, all without significant differences and significantly lower than the blank group. At week 4, the skin moisture content of the Elastin C and positive control groups increased, and were significantly higher than in the commercial group, all three were significantly higher than in the model group. At week 8, the skin moisture content of the Elastin C and positive control groups returned to normal skin levels, while the skin moisture content of the commercial group was still significantly below normal.

The ability of Elastin-recombinant humanized Elastin-C to repair skin barriers was assessed using a Combo TEWL probe to detect transepidermal water loss from the back skin of mice in a living state and the results are shown in figure 4E. The results showed that at week 2, the skin barrier function was disrupted and TEWL values were elevated for the model, positive control, elastin C, and commercial groups, which were not significantly different and significantly lower than the blank group. At week 4, TEWL was decreased for the positive control, elastin C, and the commercial groups, and TEWL was lower for both the Elastin C and positive control than the commercial group, with all three significantly lower than the model group and higher than the blank group. At week 8, TEWL was restored to normal skin levels in the positive control and Elastin C groups, with commercial TEWL still being higher than normal.

The elasticity of the back skin of the mice was examined in a living state, and the ability of recombinant humanized Elastin C to promote elastic recovery of the skin was evaluated. As shown in fig. 4F, at week 2, the skin contraction time was increased for the model, positive control, elastin C, and commercial groups, which were not significantly different and significantly higher than the blank group. At week 4, the skin contraction time was reduced for the positive control, elastin C, and the commercial group, all of which were significantly lower than for the model group. At week 8, the skin contraction times of the positive control group and the Elastin C group were restored to normal skin levels, and the skin contraction time of the commercial group was still significantly higher than the normal level.

In conclusion, the recombinant humanized elastin of the present invention helps to restore the epidermis thickness and dermis density of the aged skin to normal levels, restore the barrier of the aged skin, restore the moisture content and elasticity of the skin to normal levels, and has significantly better effects than commercially available products.

(2) Tissue section staining

The ability of recombinant humanized Elastin C to promote the repair of aged skin was studied from the tissue level by HE staining. As shown in fig. 5A and 5B, the back skin of the mice in the blank group was in a healthy state during the experiment, the epidermis layer was thicker and continuous and intact, the cells of each layer of epidermis were orderly arranged, and the fiber arrangement in the dermis layer was continuous and dense. At week 2, the epidermis of the back skin of each group of mice except the blank group was thinned. At week 4, the epidermis of the model group tended to thin, and the epidermis of the positive control group, elastin C group and the commercially available group gradually thickened. At week 8, the epidermis thicknesses of the positive control, elastin C and commercial groups recovered to normal state, and the Elastin C group was superior to the commercial group, with no significant improvement in the model group epidermis compared to week 4.

The ability of recombinant humanized Elastin C to promote repair and regeneration of collagen fibers in aged skin was studied by Masson staining. As shown in fig. 5C and 5D, the back skin of the mice in the blank group was in a healthy state during the experiment, the arrangement of fibers in the dermis layer was continuous and dense, the collagen fibers in the papillary layer were fine long, the collagen fibers in the reticular layer were coarse, and the collagen fibers were arranged approximately in parallel along the epidermis layer. At week 2, the collagen fibers in the dermis layer of the back skin of each group of mice except the blank group were broken and broken, and the overall fiber arrangement was disordered. At week 4, the dermis layer collagen fiber breaks of the positive control, elastin C and commercial groups began to grow new collagen fibers, and the fiber arrangement began to become ordered, but a small portion of the collagen fibers remained disordered. At week 8, the dermis collagen fibers of the positive control, elastin C and commercial groups were substantially restored to normal condition with ordered fiber arrangement, with Elastin C being better than commercial groups and with chaotic fiber arrangement in the model group.

The ability of recombinant humanized Elastin C to promote regeneration of aged skin elastic fibers was studied by VEG staining as shown in figures 5E and 5F, with the back skin of the mice in the blank group in a healthy state and with elastic fibers densely arranged during the experiment. At week 2, the mice in each group except the blank group had reduced elastic fiber in the back skin dermis layer. At week 4, the model mice continued to decrease in elastic fiber, and the positive control, elastin C, and commercial groups began to regenerate elastic fiber. At week 8, the positive control, elastin C, and commercial groups of dermal layer elastane fibers were substantially restored to normal condition, and Elastin C was superior to the commercial group.

EXAMPLE 5 characterization of antioxidant stress Properties of recombinant humanized elastin

The level of superoxide dismutase (SOD), catalase (CAT) activity, total Glutathione (GSH) content and Malondialdehyde (MDA) of the antioxidant enzyme is detected by using a colorimetric kit. The skin tissue was weighed, added with the extract at a ratio of 0.1 g/mL, homogenized under ice bath conditions, and centrifuged at 8X 10 ³ Xg at 10min (4 ℃) to obtain the supernatant for detection. And finally, respectively measuring SOD, MDA, CAT and GSH content by an enzyme-labeled instrument.

MDA, SOD, GSH and CAT are used as critical antioxidant substances in cells and play an important role in the aspect of antioxidant stress. MDA is an important indicator of the degree of lipid peroxidation, and MDA content in skin increases significantly with aging. Skin aging can lead to an increase in active oxygen, including superoxide hydrogen ions, hydrogen peroxide, hydroxyl radicals, etc., thereby compromising the skin's oxidative/antioxidant balance.

As shown in FIG. 6, the expression of SOD, CAT and GSH in the model group is obviously reduced, the expression of MDA is obviously increased, the MDA content in the Elastin C group is obviously reduced, the activity of SOD, GSH and CAT is increased, and the recombinant humanized Elastin has no obvious difference from a blank group, so that the recombinant humanized Elastin can relieve skin oxidative stress and delay aging by reducing the conversion of superoxide anions into hydrogen peroxide, inhibiting the generation of hydrogen peroxide and lipid peroxide and other ways.

EXAMPLE 6 Zebra fish experiments with recombinant humanized elastin

(1) Anti-wrinkle experiment

Healthy wild type AB strain zebra fish of 3 days of age were randomly allocated to 6 well plates, 15 tails per well, and divided into 4 groups, a blank group, a positive control group (PC, 0.5 mg/mL tea polyphenol treatment), a model group (water treatment), and a recombinant humanized Elastin C group (1 mg/mL). At the beginning of the experiment, standard dilution water in the well plate was removed quickly to avoid injury to the young fish, and then 5mL of the test solution was added to each well and incubated 2 h. The experimental group received three light stimuli during the incubation, 15 min each time, 30 min intervals, with a total dose of 2.0-2.5J/cm ². Following irradiation, zebra fish were incubated at 28.5±1 ℃ for a further 22 h. The blank group did not undergo any treatment.

The zebra fish tail fin image was taken using a visible light microscope and the tail fin area (in pixels) was automatically measured and calculated by ImageJ software. The skein wrinkle inhibition rate of the test substance was calculated as follows:

Wherein, the

Compared with a model control group, the inhibition rate of tail fin shrinkage of the zebra fish test group is shown as follows;

a1, average tail fin area (pixels) of the treatment group;

A2, average tail fin area (pixels) of the model group;

a3 average skeg area (pixels) of normal control group.

The anti-wrinkle efficacy of recombinant humanized Elastin C was evaluated using the uv-induced interference model of zebra fish tail fin structure as shown in fig. 7A and 7B, with significant reduction in model group tail fin area after 22 h uv irradiation, being 224,945.42 ± 13,876.02 pixels, with highly significant differences (P < 0.0001) compared to 422,397.51 ± 23,997.33 pixels of the blank group, verifying successful model establishment, as evidenced by significant shrinkage and deformation of tail fins. In the PC group and the Elastin C group, the tail fin areas were about 401,919.24 + -14,550.44 pixels and 419,208.91 + -20,115.49 pixels, respectively, and the tail fin shrinkage inhibition rates were 40.88% and 51.62%, respectively. The result shows that the recombinant humanized elastin has obvious anti-wrinkle effect, and can effectively reduce shrinkage and deformation of zebra fish tail fins.

(2) Reactive oxygen scavenging experiments

Reactive Oxygen Species (ROS) levels in young zebra fish were detected by the cell permeable fluorescent probe H ₂ DCFDA. Young zebra fish of 3 days of age are selected and placed in 6-well plates with 10 tails per well. Zebra fish are divided into 5 groups, namely a blank group, a model group, a positive control group (PC, 0.5 mg/mL dipotassium glycyrrhizinate) and a recombinant humanized Elastin C group (1 mg/mL). The blank group was not subjected to any treatment. The treatment mode of the recombinant humanized Elastin C group juvenile fish is adding 1 mg/mL recombinant humanized Elastin C solution 4 mL, and the effect is 1 h. The model group received only CuSO ₄ treatment and the positive control group received 0.5 mg/mL dipotassium glycyrrhizinate treatment. After treatment, all groups were induced for oxidative stress by exposure to 30 μm CuSO ₄ in 20: 20 min.

As shown in fig. 7C and 7D, the fluorescence intensity of the model group was significantly increased to 106.24 ±15.68 compared to the blank group (18.91±1.67), indicating that the experimental model construction was successful. In contrast, the positive control group (PC group) significantly reduced ROS production to 27.92 ±3.94. Likewise, the Elastin C group also showed a significant reduction in ROS levels with a fluorescence intensity of 25.77 ±2.40, i.e. the use of recombinant humanized Elastin C significantly inhibited ROS production. The result shows that the recombinant humanized elastin can effectively relieve the reactive oxygen species generation induced by CuSO ₄.

In conclusion, the high bioactivity recombinant humanized elastin provided by the invention has an amino acid sequence 100% derived from human elastin, has a typical beta-helical hydrophobic domain and a lysine-rich hydrophilic domain, shows excellent biosafety and bionic function characteristics, has remarkable proliferation, adhesion promotion and migration promotion activities on human skin fibroblasts (HFF-1), remarkably improves skin thickness and dermis density, increases stratum corneum water content, reduces transdermal water loss rate (TEWL) and recovers skin elasticity in a D-galactose-induced skin aging mouse model, can effectively lighten tail fin shrinkage induced by ultraviolet rays and inhibit ROS production in zebra fish and active oxygen inhibition experiments, can remarkably reduce MDA content and promote superoxide dismutase (SOD), catalase (CAT) and Glutathione (GSH) activities, and has favorable skin oxidative stress relieving and aging resistance by reducing conversion of superoxide anions into hydrogen peroxide, inhibiting lipid peroxide production and the like, has good skin dissolution stability and good skin aging stability, and is suitable for being implanted in the fields of skin-aging-promoting systems, and has wide application in the fields of skin-aging-promoting cosmetics.

The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.

Claims

1. A recombinant humanized elastin protein with high biological activity, characterized in that the amino acid sequence of the recombinant humanized elastin protein comprises the amino acid sequence as shown in SEQ ID NO. 1, or an amino acid sequence as shown in SEQ ID NO. 1 in which one or more amino acids are substituted, deleted, and/or added, and which has equivalent function to a protein composed of the amino acid sequence as shown in SEQ ID NO. 1.

2 . The recombinant humanized elastin with high biological activity according to claim 1 , wherein the recombinant humanized elastin is obtained by treating precursor elastin with a protease.

3. The highly bioactive recombinant humanized elastin protein according to claim 2, characterized in that the amino acid sequence of the precursor elastin protein comprises the amino acid sequence shown in SEQ ID NO. 2, or an amino acid sequence shown in SEQ ID NO. 2 in which one or more amino acids are substituted, deleted, and/or added, and which has equivalent functions to a protein composed of the amino acid sequence shown in SEQ ID NO. 2.

4 . The recombinant humanized elastin with high biological activity according to claim 2 , wherein the protease comprises thrombin, pepsin, trypsin or bromelain.

5. The method for preparing the recombinant humanized elastin according to any one of claims 1 to 4, comprising the following steps:

(1) synthesizing a gene sequence encoding the precursor elastin of the recombinant humanized elastin;

(2) Connecting the gene sequence to a vector, transforming genetically engineered bacteria, and constructing recombinant genetically engineered bacteria;

(3) Expressing the constructed recombinant genetically engineered bacteria, collecting the bacteria, crushing them to obtain the supernatant, and purifying the precursor elastin;

(4) Add the precursor elastin obtained by protease treatment and purify it to obtain recombinant humanized elastin.

6. A gene encoding a precursor elastin of the recombinant humanized elastin according to any one of claims 1 to 4, characterized in that the gene sequence encoding the precursor elastin comprises the gene sequence shown in SEQ ID NO. 3, or a gene sequence in which one or more nucleotides are substituted, deleted, and/or added to the gene sequence shown in SEQ ID NO. 3 and which expresses the same protein as the gene sequence shown in SEQ ID NO. 3.

7. A recombinant vector or recombinant genetically engineered bacterium carrying the gene according to claim 6.

8. The recombinant vector or recombinant genetically engineered bacterium according to claim 7, wherein the recombinant vector comprises pCold or pET.

9. The recombinant vector or recombinant genetically engineered bacterium according to claim 7, wherein the recombinant genetically engineered bacterium comprises Escherichia coli.

10. Use of the recombinant humanized elastin according to any one of claims 1 to 4 in the preparation of skin care products, dressings, implants, artificial skin, artificial blood vessels, medical devices, and biomaterials.