Recombinant human elastin with skin damage repairing effect and application thereof

Abstract

The present invention belongs to the field of genetic engineering technology and medical materials, and specifically relates to a recombinant human elastin with skin damage repair effect and its application. The present invention provides a recombinant human elastin, the amino acid sequence of which is completely derived from human elastin, and has a secondary structure and reversible phase transition characteristics similar to natural elastin; the recombinant human elastin is efficiently expressed in Escherichia coli engineering bacteria, and is easy to achieve large-scale production; the recombinant human elastin is cross-linked to form a hydrogel with good mechanical strength, which has good biocompatibility and biological activity; the recombinant human elastin hydrogel has a significant repair effect on skin light damage, and has broad application prospects in the fields of skin repair dressings, implants, artificial skin, medical devices, biomaterials, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant human elastin with skin damage repairing effect and its application. Background Art

[0002] Elastin is an important structural protein in the human body, mainly distributed in elastic tissues such as ligaments, lungs, arteries and skin. Its main function is to give tissues the ability to stretch and deform. Elastin and microfibrils are assembled and combined under enzyme catalysis to form elastic fibers, which are its main form of existence in tissues. Elastin has good biological functions and can regulate cell proliferation and differentiation. Biomaterials prepared with elastin as the main raw material have both high mechanical strength and high biological activity, and have attracted much attention in the field of medical materials.

[0003] Elastin is mainly composed of alternating hydrophobic domains and hydrophilic domains. Its hydrophobic domain is mainly composed of VPGXG as the main repeating unit. Later, various variants such as VAPGVG and VGGVG were discovered, but they are all mainly non-polar hydrophobic amino acids, and are repeated at a lower frequency in the hydrophobic domain. The hydrophilic domain is mainly composed of alanine (A) and lysine (K) randomly alternating and repeating, such as the hydrophilic domain of elastin with sequence number 25, AAAAKSAAKVAAKA; lysine in the hydrophilic domain is an important reaction site in the cross-linking process of elastin. Under the catalysis of lysylase, single-chain tropoelastin cross-links to form a macromolecular mesh structure, providing an ideal scaffold and material exchange site for cell adhesion and tissue growth.

[0004] The special structure of alternating blocks gives elastin the special property of reversible phase transition. When the elastin solution is heated to a certain temperature, the weak binding between the hydrophobic domain and water is destroyed, and then the hydrophobic domains move closer to each other, self-assemble to form hydrogen bonds, and then condense into a precipitate. This thermodynamic phase transition is different from the usual thermal denaturation of proteins. When the elastin undergoing phase transition is cooled to room temperature, it can be redissolved and restored to the state before heating.

[0005] The main methods for preparing elastin are currently animal tissue extraction and recombinant expression. Elastin extracted from animal tissue is prone to excessive degradation, resulting in degradation fragments with low biological activity. The use of genetic engineering technology to recombinantly express elastin has the advantages of low cost and easy large-scale production, so it has become a research hotspot. Invention patent CN202110357328.9 discloses a recombinant elastin-like protein (GVGVAP) expressed by brewer's yeast nInvention patent CN102241747A discloses a recombinant elastin-like protein sequence with (VAPGVG)3S as the basic unit and repeated at a high frequency. The recombinant elastin-like proteins in these patents are simply repeated six amino acids, lacking the important functional sequence of human elastin, and cannot be cross-linked to form a network structure similar to natural elastin.

[0006] In view of the above-mentioned existing problems, the present invention provides a recombinant human elastin (RE) with skin repair efficacy and its application. The amino acid sequence of the recombinant human elastin is completely derived from human elastin, and has a secondary structure and reversible phase transition characteristics similar to those of natural elastin; the recombinant human elastin is efficiently expressed in an engineered bacterium of Escherichia coli, and is easy to achieve large-scale production; the recombinant human elastin is cross-linked to form a hydrogel with good mechanical strength, which has good biocompatibility and biological activity; the recombinant human elastin hydrogel has a significant repair effect on skin photodamage, and has broad application prospects in the fields of skin repair dressings, implants, artificial skin, medical devices, biomaterials, etc. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a recombinant human elastin with skin repair efficacy and its application, and the specific scheme includes:

[0008] In a first aspect, the present invention provides a recombinant human elastin protein, wherein the recombinant human elastin protein comprises one or more elastin domains in the amino acid sequence of the full-length human elastin protein as shown in SEQ ID NO.1.

[0009] Preferably, the recombinant human elastin consists of one or more elastin domains in the amino acid sequence of the full-length human elastin as shown in SEQ ID NO.1, and one or more of a secretion tag, a histidine tag, a fluorescent protein and a protease cleavage site.

[0010] Preferably, the amino acid sequence of the recombinant human elastin is as shown in SEQ ID NO.2.

[0011] In a second aspect, the present invention provides a nucleotide encoding the recombinant human elastin described in the first aspect.

[0012] Preferably, the nucleotide sequence is as shown in SEQ ID NO.3.

[0013] In a third aspect, the present invention provides a recombinant plasmid, a recombinant vector or a recombinant bacterium containing the nucleotide described in the second aspect.

[0014] Preferably, the vector comprises pCold III.

[0015] Preferably, the recombinant cell is recombinant Escherichia coli BL21.

[0016] In a fourth aspect, the present invention provides a method for preparing the recombinant human elastin according to the first aspect, the method comprising:

[0017] (1) synthesizing a nucleotide sequence encoding the recombinant human elastin described in the first aspect;

[0018] (2) connecting the nucleotide sequence described in step (1) to a vector (plasmid), transforming bacteria, and constructing recombinant bacteria;

[0019] (3) Expressing the recombinant bacteria constructed in step (2), collecting bacterial precipitates, crushing them to obtain supernatant, and purifying them to obtain recombinant elastin.

[0020] Preferably, the method is:

[0021] (1) synthesizing the nucleotide sequence shown in SEQ ID NO.3;

[0022] (2) inserting the nucleotide sequence described in step (1) into the pColdIII plasmid to construct a recombinant vector; and introducing the vector into Escherichia coli BL21 to screen and obtain recombinant bacteria;

[0023] (3) Transfer the recombinant bacteria into LB medium and culture at 37°C for 10-12 hours. 600 When the pH reaches 0.8-1.0, 1 mM IPTG is added and the temperature is lowered to 25°C to induce expression for 12-18 hours. The fermented bacterial precipitate is collected by centrifugation, crushed to obtain the supernatant, and purified to obtain recombinant human elastin.

[0024] In a fifth aspect, the present invention provides a composition comprising the recombinant human elastin described in the first aspect.

[0025] In a sixth aspect, the present invention provides an elastin gel, wherein the elastin gel is prepared by cross-linking the recombinant human elastin described in the first aspect.

[0026] Preferably, the elastin gel raw material comprises 1-50% of the recombinant human elastin described in the first aspect, 0-5% of a cross-linking agent, and the remainder is water, physiological saline or PBS.

[0027] Preferably, the cross-linking agent is selected from one of genipin, glutaraldehyde, N-hydroxysuccinimide (NHS), and tetrakishydroxymethylphosphonium chloride (THPC).

[0028] Preferably, the crosslinking agent is tetrakishydroxymethylphosphonium chloride (THPC).

[0029] Preferably, the elastin gel raw material comprises the following components: 1-50% recombinant human elastin, 0.5-5% tetrakis(hydroxymethyl)phosphonium chloride, and the remaining components are deionized water or physiological saline.

[0030] In a seventh aspect, the present invention provides use of the recombinant human elastin described in the first aspect or the elastin gel described in the sixth aspect in the preparation of a skin damage repair dressing product.

[0031] Preferably, the products include skin care products, skin repair dressings, implants, artificial skin, medical devices, and health foods.

[0032] Preferably, the skin damage is skin ultraviolet light damage.

[0033] The beneficial effects of the present invention are as follows: (1) The present invention provides a recombinant human elastin, the amino acid sequence of which is completely derived from human elastin, and has a secondary structure and reversible phase transition properties similar to those of natural elastin; (2) The recombinant human elastin overcomes the problems of virus transmission and immunogenicity of elastin extracted from animal tissues; (3) The recombinant human elastin has the advantages of good biocompatibility and water solubility, and is convenient for addition and use in cosmetics and skin repair dressings; (4) The recombinant human elastin is efficiently expressed in Escherichia coli engineered bacteria, and is easy to achieve large-scale production; (5) The recombinant human elastin is cross-linked to form a hydrogel with good mechanical strength, which has good biocompatibility and biological activity; (6) The recombinant human elastin hydrogel has a significant repair effect on skin photodamage, and has broad application prospects in the fields of skin repair dressings, implants, artificial skin, medical devices, biomaterials, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 SDS-PAGE results of recombinant human elastin RE before and after purification;

[0035] Figure 2 Circular dichroism spectra of recombinant human elastin RE at different temperatures;

[0036] Figure 3 Reversible phase transition of recombinant human elastin RE; a is the reversible phase transition of recombinant human elastin RE at different concentrations; b is the phase transition point T t (Transmission Temperature) and the logarithm of protein concentration; c is the reversible phase transition of 100 μM recombinant human elastin RE at different sodium chloride concentrations; d is the relationship curve between the phase transition point and the sodium chloride concentration;

[0037] Figure 4 Characterization of the properties of recombinant human elastin RE cross-linked gel; a is a real photo of recombinant human elastin RE cross-linked gel; b is the microscopic morphology (SEM) of recombinant elastin RE gel; c is the frequency scanning rheology of recombinant human elastin RE gel, and d is the strain scanning rheology of recombinant human elastin RE gel;

[0038] Figure 5 Cell activity of recombinant human elastin RE gel; a is the cytotoxicity of recombinant human elastin RE gel; b is the cell proliferation of recombinant human elastin RE gel; c is the cell adhesion staining imaging of recombinant human elastin RE gel;

[0039] Figure 6 The repair function of recombinant human elastin gel RE on UV-damaged mouse skin; a is the actual photo of the skin of mice in the blank group, control group and experimental group on days 1, 3, 5 and 7; b and c are the HE / Masson staining results of the skin tissue of mice in the blank group, control group and experimental group on days 1, 3, 5 and 7. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to the following.

[0041] The recombinant human elastin described in the following embodiments is a recombinant human elastin obtained in a microbial expression system using transgenic technology and gene recombination technology.

[0042] The recombinant human elastin described in the following examples is recombinant human elastin obtained by Escherichia coli fermentation, but is not limited to the above method and may also be recombinant human elastin prepared by other methods, including recombinant human elastin fermented by Pichia pastoris, cell-free in vitro expressed recombinant protein, etc.

[0043] Specifically, the preparation method of the recombinant human elastin described in the following embodiments includes: constructing an engineered Escherichia coli bacteria for fermentation production, centrifuging and collecting the supernatant after the fermentation is completed, and separating and purifying to obtain the recombinant human elastin.

[0044] The method of preparing the recombinant human elastin gel described in the following embodiments includes: heating a recombinant human elastin solution of a certain concentration to cause the protein to undergo a reversible phase change for prepolymerization, adding a crosslinking agent THPC (tetrakis(hydroxymethyl)phosphonium chloride, the same below) while hot, reacting under certain conditions, removing the reaction residue, and obtaining a recombinant human elastin gel having a certain mechanical strength. However, the method is not limited to the above method, and other crosslinking methods may also be used, including photocrosslinking, thermal crosslinking, and chemical crosslinking represented by genipin crosslinking, BDDE crosslinking, and EDC-NHS crosslinking.

[0045] Example 1 Expression of recombinant human elastin

[0046] Sequence synthesis and expression vector construction: The amino acid sequence of recombinant human elastin as shown in SEQ ID NO.2 was designed, and the gene sequence encoding the corresponding recombinant human elastin was synthesized as shown in SEQ ID NO.3. The gene sequence encoding the recombinant human elastin was inserted into the pColdIII plasmid to obtain a recombinant vector expressing the recombinant human elastin; and introduced into Escherichia coli BL21 to obtain recombinant human elastin Escherichia coli.

[0047] Induced expression: The recombinant E. coli was pre-expressed and screened, and the selected single colony was inoculated into LB medium (200 mL, added with 50 mg / L), cultured at 37°C overnight, and then the strain was transferred into 4×1L LB medium and expanded at 37°C for 10-12 hours. The absorbance (OD 600 ) monitor the growth density of bacteria, when OD 600 When the concentration reaches 0.8-1.0, add 1 mM IPTG and cool to 25°C to induce expression for 12-18 hours; collect the fermented bacteria by centrifugation.

[0048] Nickel column purification of recombinant human elastin: resuspend the cells with phosphate buffer (phosphate buffer, 0.02M, pH = 7.4), break the cells with a high-pressure homogenizer under low-temperature water cooling circulation conditions, then centrifuge at 10000rpm for 20min at 4°C to remove cell debris, and then filter the supernatant with a microporous filter to remove impurities; inject the sample into a His-trap nickel ion affinity chromatography column (25mL), and then wash it with an eluent (0.02M PB, 0.5M NaCl, imidazole) for 5-8 column volumes. The imidazole content in the elution buffer increases in a step-like manner (20mM, 50mM and 500mM) to elute the protein, and collect the peak protein at a concentration of 500mM imidazole. The resulting protein solution is dialyzed with 0.02M PB buffer or ultrapure water at 4°C, and the target protein with a molecular weight cutoff greater than 8000 is retained. The imidazole, NaCl and other components in the protein solution are removed, and lyophilized.

[0049] Weigh a small amount of the freeze-dried protein sample obtained in the previous step, re-dissolve it in water, and determine the protein molecular weight and purity by SDS-PAGE. Figure 1 As shown, a single band was detected in the protein sample lane, and the Marker indicated that the molecular weight of the target protein was slightly less than 25 kDa, which was consistent with the theoretical molecular weight (23.1 kDa). The results showed that the recombinant human elastin (RE) was successfully expressed and purified.

[0050] Example 2 Characterization of the properties of recombinant human elastin

[0051] 1. Recombinant elastin secondary structure

[0052] The recombinant human elastin solid obtained in Example 1 was prepared into a 1.0 mg / mL aqueous solution, and then allowed to stand at 4°C for more than 24 h. A circular dichroism spectrum was scanned at 4, 25, 30, 37, and 50°C using a 1 mm cuvette, with the wavelength ranging from 190 nm to 260 nm, with a wavelength interval of 1 nm, and the solution was kept at each wavelength for 5 s.

[0053] The results are as follows Figure 2 As shown, the recombinant human elastin has a strong negative absorption peak mainly near 200nm, and the proportion of various conformations in the spectrum peak is analyzed by software (see Table 1). It can be seen from the conformational analysis results that when the temperature is in the range of 4-50°C, as the temperature increases, the disordered structure in the recombinant human elastin conformation is basically stable at about 70%, and the proportion of α helix decreases from 7% to 4%. When the temperature rises from 4°C to 25°C, the proportion of β helix conformation decreases from 14% to 11%, and at the same time, the β turn conformation increases from 12% to 16%. In the process of further increasing the temperature from 25°C to 50°C, the proportion of β helix conformation increases from 11% to 21%, and the proportion of β turn conformation decreases from 16% to 5%. The above structural changes are basically consistent with the conformational changes of natural elastin during heating in the literature, indicating that the recombinant human elastin designed by the present invention has a structure similar to that of natural elastin.

[0054] Table 1 Relationship between the proportion of various conformations of recombinant human elastin and temperature

[0055] Temperature(℃) α-helix(%) β-spiral(%) β-turn(%) Disordered(%) 4 7.2±2.5 13.5±1.5 12.0±3.7 68.2±7.7 25 6.4±2.1 10.6±3.1 15.8±4.4 70.4±4.9 30 5.4±1.7 16.7±3.6 11.3±1.8 72.1±5.6 37 5.2±2.0 21.5±4.7 7.8±3.6 69.1±4.1 50 3.7±2.9 20.7±2.9 5.4±0.9 73.1±5.7

[0056] 2. Reversible phase transition of recombinant human elastin

[0057] (1) Reversible phase transition at different protein concentrations:

[0058] The recombinant human elastin solid prepared in Example 1 was configured into 100, 50, 25, 10, and 5 μM solutions. The other components of the solution included: 0.5M NaCl, 0.1M CaCl2, 50mM tris, 0.02M PB, pH = 7.4. The above solution was placed in a constant temperature water bath and heated at room temperature (25°C) as the starting temperature. After the solution reached a constant temperature, the protein solution at room temperature was used as a reference to measure the absorbance of the protein solution at a wavelength of 350nm after reaching the constant temperature. The temperature was raised at an interval of 2.5°C until the absorbance basically stopped increasing. The heated solution was cooled, the protein was redissolved, and restored to the state before heating, completing a reversible phase change cycle.

[0059] The absorbance-temperature relationship curves of recombinant human elastin solutions with different concentrations are shown in Figure 3 As shown in a, the temperature corresponding to the maximum absorbance of the solution during the phase transition is the reversible phase transition temperature (T t ), the reversible phase transition temperature of 100μM recombinant human elastin is about 39℃, the reversible phase transition temperature of 50μM recombinant human elastin is about 42℃, the reversible phase transition temperature of 25μM recombinant human elastin is about 44℃, the reversible phase transition temperature of 10μM recombinant human elastin is 48℃, and the reversible phase transition temperature of 5μM recombinant human elastin is 53℃. Plot the logarithm of phase transition temperature and protein concentration ( Figure 3 The results show that the recombinant human elastin has similar thermal properties to natural elastin.

[0060] (2) Reversible phase transition of recombinant human elastin at different salt concentrations:

[0061] A certain mass of recombinant human elastin RE lyophilized powder was weighed and dissolved in a phase change buffer solution not containing NaCl. A certain amount of saturated NaCl solution was added and the volume was fixed with a phase change buffer solution not containing NaCl to obtain a protein solution with a protein concentration of 100 μM and NaCl concentrations of 1.5 M, 1.0 M, 0.75 M, 0.5 M and 0.25 M, respectively. Referring to the method in Example 2.2 (1), the reversible phase change behavior of 100 μM recombinant elastin under different sodium chloride concentrations was determined.

[0062] The reversible phase transition of 100 μM recombinant human elastin RE under different sodium chloride concentrations is shown in Figure 3As shown in c, the reversible phase transition, a thermosensitive aggregation behavior, can occur in solutions with a sodium chloride concentration of 0.25-1.5M. When the NaCl concentration is 1.5M, the phase transition temperature is about 31°C, when the NaCl concentration is 1.0M, the phase transition temperature is about 33°C, when the NaCl concentration is 0.75M, the phase transition temperature is about 36°C, when the NaCl concentration is 0.5M, the phase transition temperature is about 43°C, and when the NaCl concentration is 0.25M, the phase transition temperature is about 47°C. As the salt ion concentration continues to increase (0.25M-1.5M NaCl), the phase transition temperature of the recombinant human elastin gradually decreases. Plot the phase transition point and sodium chloride concentration ( Figure 3 (d) The two show a linear negative correlation, which is consistent with the theoretical model of elastin phase transition reported in relevant literature.

[0063] Example 3 Preparation of recombinant human elastin gel

[0064] 1. Preparation of recombinant human elastin gel

[0065] The recombinant human elastin obtained in Example 1 was prepared into a 100 μM protein aqueous solution and incubated at 40°C to make the elastin undergo a phase change; after oscillation to evenly disperse the protein, it was injected into a mold, and THPC was added at a final concentration of 0.2%. The solution was allowed to stand at room temperature, and the recombinant human elastin and THPC formed a gel. The gel was soaked in ultrapure water, left to stand overnight, and the immersion solution was changed 3-5 times to remove the residual unreacted crosslinker and protein. The macroscopic physical picture of the prepared gel is shown in the figure below: Figure 4 As shown in a.

[0066] 2. SEM of recombinant human elastin gel

[0067] The prepared gel was freeze-dried and sliced, adhered to a sample plate, and observed under a Hitachi S-4800 scanning electron microscope. The results are as follows Figure 4 As shown in b, the interior of the gel is arranged in a dense honeycomb mesh structure with a mesh unit diameter of 30-80 μm; inside the mesh, the proteins are arranged in an orderly fibrous shape.

[0068] 3. Rheological properties of recombinant human elastin gel

[0069] Carefully remove the reacted gel from the mold and place it on the sample stage of the Anton Paar rheometer. Frequency sweep: Set the strain parameter of the rheometer to 1% and the stress to 1N. Test the storage modulus G'(Pa) and loss modulus G" (Pa) of the gel as the scanning frequency changes. Strain sweep: Fix the scanning frequency to 1rad / s and test the storage modulus G'(Pa) as it changes with shear strain.

[0070] Frequency sweep( Figure 4The storage modulus (G') and compression modulus (G") of the recombinant human elastin RE gel in (c) both increase gradually with the increase of shear frequency, and the slope of the modulus curve increases continuously, showing the typical strain characteristics of hydrogel materials. At a shear frequency of 80 rad / s or higher, the storage modulus can reach 1.0×10 3 pa, proving that the gel has good mechanical strength. In the strain sweep experiment ( Figure 4 As shown in (d), with the continuous increase of shear strain, the storage modulus of the recombinant elastin RE gel gradually decreases. When the shear strain exceeds 40%, the storage modulus tends to be stable, which is consistent with the typical characteristics of the shear thinning behavior of the gel.

[0071] 4. Cellular Activity of Recombinant Human Elastin Gel

[0072] (1) Cytotoxicity of gel: In a 96-well plate without TC treatment, 100 μL of a sterile aqueous solution of recombinant human elastin (100 μM) was added to each well, and then THPC was added at a final concentration of 0.1-2%. After thorough mixing, the plate was allowed to stand until a 1 mm thick uniform recombinant human elastin gel layer was coated on the bottom. Deionized water, 0.02 M PBS, and TEMED culture medium were added in sequence to infiltrate the gel to remove residual unreacted proteins and cross-linking agents. A certain number of HFF cells were inoculated in each well and incubated at 37°C for 24 h. Cell Counting Kit-8 (CCK-8, the same below) was added. After binding for 30 min, the absorbance was measured using an ELISA reader and compared with the control group without gel. The cell survival rate of the recombinant human elastin gel formed under different cross-linking agent concentrations was determined.

[0073] Cytotoxicity results such as Figure 5 As shown in a, compared with the control group, the cell survival rates of the gels in each group cross-linked at different THPC concentrations were all greater than 98%, indicating that the recombinant human elastin gel designed in the present invention had no cytotoxicity.

[0074] (2) Cell proliferation in recombinant human elastin gel: The preparation and pretreatment methods of sterile gel that can be used for cell experiments refer to the cytotoxicity test. After the gel is infiltrated with DMEM in the well plate, 100 μL of 5000 / mL HFF-1 cells that have been digested with trypsin and evenly dispersed are added to the upper layer. The cells are cultured in a cell culture incubator at 37°C, and the cell culture medium is replaced every 48 hours to prevent the accumulation of cell metabolites. One group is taken out every 24 hours, CCK-8 is added, and the number of live cells on the surface of each group of gel is quantified. The blank group is the gel without cells added, and the control group is the same amount of cell culture medium (containing cells) without gel at the bottom.

[0075] Cell proliferation results Figure 5As shown in b. Compared with the control group, the recombinant human elastin gel has a significant ability to promote the proliferation of fibroblasts. The cell proliferation rate on days 1-2 is close to 100%. On day 3, the cell proliferation rate increased significantly (140%), and on day 4, the cell proliferation rate increased to more than 170%. On day 5, the cell proliferation rate reached 210%, and on days 6-7, the cell proliferation rate was 270%. The above results show that the recombinant human elastin gel has a significant promoting effect on cell proliferation.

[0076] (3) Staining of cell adhesion on the surface of recombinant human elastin gel: Pour the DMEM culture medium solution containing 100 μM recombinant human elastin into several glass-bottomed culture dishes that have not been treated with TC, so that the protein solution completely covers the bottom of the culture dish, add THPC solution with a final concentration of 1%, and let it stand in the clean bench for 3 hours; after cross-linking is completed, add PBS to rinse three times, and then add DMEM culture medium to soak for use. Add 100 μL 5000 / mL HFF-1 cells that have been digested with trypsin and evenly dispersed. Culture in a cell culture incubator at 37°C, and replace the cell culture medium every 48 hours to prevent the accumulation of cell metabolites. The cells were taken out on the 1st, 4th and 7th days respectively, fixed with 4% formaldehyde for 10 min and then aspirated out, permeabilized with 0.1% Triton-X for 5 min, blocked with 1% BSA for 30 min, and then phalloidin was added to stain the cytoplasm, and then Hoechest 33258 was added to stain the cell nucleus; the surface of the gel was coated with mounting medium, and the number and morphology of HFF-1 cells on the surface of the gel were observed under a fluorescence microscope.

[0077] The results are as follows Figure 5 As shown in c. It was determined that the fibroblasts in each experimental group successfully attached to the wall and stretched on the surface of the gel layer within 24 hours; as time went by, the number of cells continued to increase, and the cell morphology was intact. The above results show that fibroblasts can survive and grow normally on the surface of recombinant human elastin gel.

[0078] Example 4 Animal injury repair experiment of elastin gel

[0079] 45 male Kunming mice of SPF grade weighing 20±2g were selected. The backs of the mice were shaved with a Philips hair clipper, and then depilatory cream was applied to remove hair, so that the back epidermis was completely exposed. The depilatory area was about 2×4cm. After the depilatory was completed, it was stabilized for 24 hours to eliminate the effect of the depilatory cream on epidermal damage. The mice were randomly divided into three groups. The blank group was not treated. The mice in the experimental group and the control group were irradiated with continuous wavelength ultraviolet lamps A (320-440nm) and B (280-320nm) on the back skin. The ultraviolet irradiator was used to quantify the radiation, which was 200mJ / cm 2After irradiation, obvious local redness and swelling occurred on the back of the mice, indicating that acute skin inflammation in mice was successfully induced. The recombinant human elastin gel prepared in Example 2 was applied to the UV-irradiated area of ​​the back skin of the mice in the experimental group, covered with gauze, and then fixed with a bandage to prevent the gel from falling off during the activities of the mice. The gel patch was replaced and re-fixed every 48 hours; the control group applied physiological saline to the back of the mice, covered with gauze, and fixed with a bandage; the blank group was not irradiated with UV light or covered with gauze. Starting from the first day after modeling, the mice were dislocated and killed every 48 hours, and the back skin was taken, and the skin sections were stained with HE and Masson respectively. Skin damage and recovery were observed under a microscope.

[0080] The actual photo of mouse skin is as follows Figure 6 As shown in a, the skin of the blank group mice was in good shape within 1-7 days, with a smooth and flat surface and no red and swollen areas. After quantitative ultraviolet irradiation, the skin of the control group mice showed a large amount of local redness and swelling on the first day; the red and swollen area further expanded on the third day, and irregular cracks appeared on the outer layer of the epidermis; the local redness and swelling improved on the fifth day, but a large number of erythematous papules appeared on the skin surface, accompanied by large-scale epidermal shedding; the local redness and swelling basically subsided on the seventh day, and the epidermal erythema began to scab, but there was still a small amount of epidermal shedding. After ultraviolet irradiation and application of recombinant human elastin gel, the mice in the experimental group showed partial redness and swelling on the first day; the red and swollen area was reduced on the third day, wrinkles appeared on the skin surface, and there was a small amount of erythematous papules; the redness and swelling basically subsided on the fifth day, the skin wrinkles were significantly improved, and there was a local epidermal shedding phenomenon; the skin of the mice recovered well on the seventh day, with a smooth and flat surface, and there was no significant difference from the skin of the normal group mice. These results indicate that the recombinant human elastin gel designed in this patent has a significant repairing effect on photodamaged skin of mice, can effectively reduce the degree of skin damage, and shorten the repair cycle of photodamaged skin.

[0081] Mouse skin HE staining results are as follows Figure 6As shown in middle b, the mice in the blank group were generally healthy and normal within 1-7 days, specifically: the upper layer of the epidermis was uniform and of moderate thickness, the lower layer was wavy, the fibers in the dermis were densely arranged without obvious breaks, the cell nuclei were in good morphology, there was no inflammatory cell infiltration, the appendages were intact, and the subcutaneous fat and the entire layer of reticular fibers were visible. On the first day, the epidermis of the mice in the control group was defective, the thickness increased, edema occurred in part of the basal layer, the local structure was damaged, the dermis was loose, the blood vessels were dilated and congested, inflammatory cells infiltrated, and the hair follicle fiber sheath atrophy; on the third day, the epidermal structure defect was more serious, and the damaged area expanded to the basal layer. At the same time, the basal layer was loose, the dermis was loose and edema began to appear, the fibers thickened, the number of appendages decreased sharply, the hair follicle fiber sheath disappeared in large quantities, and the degree of inflammatory cell infiltration was further deepened; on the fifth day, the epidermal structure defect was slightly alleviated but still discontinuous, the area of ​​the damaged area was reduced, and new hair follicle fiber sheaths began to form, the edema of the dermis was alleviated, the fibers further broke and thickened, the number of inflammatory cells decreased, the blood vessels were dilated and congested, and the number of appendages increased slightly compared with the third day; on the seventh day, the epidermal structure of the mice tended to be complete but still discontinuous, and there were still local breaks, the edema of the dermis basically disappeared, the number of inflammatory cells further decreased, the morphology of the new appendages was complete, and the hair follicle fiber sheath grew further, but there was still a big difference from the normal morphology of the blank group. On the first day, the skin of the mice in the experimental group showed full-thickness epidermis defect, partial basal edema, local structural damage, loose dermis with a small amount of inflammatory cell infiltration, and atrophy of the hair follicle fiber sheath; on the third day, the epidermal structural defect did not expand further, but vascular dilation and congestion and partial thickening of the epidermis appeared, the dermis structure was loose and partially edematous, some fibers were thickened and broken, and the number of appendages decreased to a certain extent; on the fifth day, the epidermal structure was close to complete but still partially discontinuous, with local breaks, the dermis edema basically subsided, and the degree of fiber thickening and breakage was slightly reduced compared with the third day; on the seventh day, the mouse epidermis structure was intact and the whole layer was visible, with obvious stratification, the dermis fibers were arranged in order, the number and morphology of the appendages were not significantly different from those of normal skin, and there was no edema or vascular congestion and dilation.

[0082] The results of Masson staining of mouse skin are shown in Figure 6As shown in c. In the blank group, the collagen fibers in the papillary layer of the dermis of mice skin were long and interwoven into a network within 1-7 days. The secretory glands and blood vessels were wrapped and surrounded by fibrous reticular collagen. The reticular collagen fiber bundles were thick and wavy, extending in all directions along the same horizontal plane, and fibroblasts were evenly distributed in the gaps between collagen fibers. On the first day, the collagen fibers in the dermis of the control group mice skin were disordered, the number decreased sharply, the fibers became thicker, curled and tangled, and some of them gathered into clusters. At the same time, a large number of fractures and fragments occurred, and inflammatory cells infiltrated in the gaps between the fibers; on the third day, the damage to the dermis was aggravated, the fibers further thickened and gathered into blocks, the fractures became more serious, and the number of inflammatory cell infiltrations increased further. On the fifth day, the damage to the collagen fibers in the dermis was alleviated, but there were still some aggregations and fractures. The fibers were loosely arranged, and the broken parts began to repair, but the overall defect was still serious; on the seventh day, the number of collagen fibers in the dermis began to increase, and the new fibers began to extend along all directions on the same horizontal plane, but the skin tissue as a whole still had some defects. On the first day, a large number of collagen fibers in the dermis of the skin tissue of the mice in the experimental group were arranged in a disordered manner, with a reduced number of fibers. The fibers became thicker, curled and kinked, and some of them aggregated into clumps. At the same time, there were fractures and breakages, and inflammatory cells infiltrated in the interfiber spaces. On the third day, the damage to the dermis was alleviated, some collagen fibers were arranged in a disordered manner, and a small number of collagen fibers thickened and aggregated into clumps. The degree of inflammatory cell infiltration was basically the same as that on the first day. On the fifth day, the damage to the dermis was further alleviated, and thin new collagen fibers appeared at the broken parts of the collagen fibers and extended in all directions on the same horizontal plane. The number of inflammatory cells decreased, and the looseness of the fiber arrangement was reduced. On the seventh day, the collagen fibers in the papillary layer of the dermis were slender and interwoven into a network. The secretory glands and blood vessels were wrapped and surrounded by fibrous reticular collagen. The collagen fiber bundles in the reticular layer were thick and wavy, extending in all directions along the same horizontal plane, and fibroblasts were evenly distributed in the gaps between the collagen fibers.

[0083] In summary, the recombinant human elastin gel described in the present invention can inhibit the formation of wrinkles and cracks on the surface of photodamaged skin, reduce the inflammatory response of the skin, promote the regeneration of collagen in damaged skin, and has a good repair effect on ultraviolet light damaged skin. The light damage in this experiment was 200mJ / cm 2 From this, we can infer that other types of damage, at least of equal intensity, can be treated with this gel to reduce the degree of damage and accelerate the recovery of skin tissue, such as electromagnetic radiation from different sources such as laser, red light, and blue light.

[0084] The above description is only the details of individual exemplary implementation cases of the present invention. For those skilled in the art, the present invention may have various modifications and changes according to specific preparation conditions during actual application, and is not intended to limit the present invention. Anything within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

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

2.

2. A nucleic acid encoding the recombinant human elastin according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleic acid sequence is shown as SEQ ID NO.

3.

4. A recombinant plasmid, recombinant vector or recombinant bacterium containing the nucleic acid according to claim 2 or 3.

5. The method for preparing recombinant human elastin according to claim 1, characterized in that: The method comprises: (1) synthesizing a nucleic acid sequence encoding the recombinant human elastin protein according to claim 1; (2) connecting the nucleic acid sequence described in step (1) to a vector / plasmid, transforming bacteria, and constructing a recombinant engineered bacterium; (3) Expressing the recombinant Escherichia coli constructed in step (2), collecting bacterial precipitates, crushing the supernatant, and purifying the recombinant human elastin.

6. A recombinant human elastin gel prepared by cross-linking the recombinant human elastin according to claim 1.

7. The recombinant human elastin gel according to claim 6, wherein the recombinant human elastin gel raw material comprises 1-50% of the recombinant human elastin according to claim 1, 0-5% of a cross-linking agent, and the remainder is water or an excipient; the cross-linking agent is selected from one of genipin, glutaraldehyde, N-hydroxysuccinimide (NHS), and tetrakishydroxymethylphosphonium chloride (THPC).

8. Use of the recombinant human elastin according to claim 1 or the recombinant human elastin gel according to any one of claims 6 to 7 in the preparation of skin care products, medical devices and biomaterials.

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