A recombinant fusion elastin, preparation method and application

Abstract

The present invention belongs to the fields of genetic engineering technology and medical materials, and specifically relates to a recombinant fusion elastin, a preparation method and an application. The recombinant fusion elastin comprises an elastin sequence and an Sc12 protein folding domain, and the amino acid sequence of the elastin is completely derived from human elastin; the recombinant fusion elastin is highly expressed in an Escherichia coli engineering bacterium and is easy to achieve large-scale production; the recombinant fusion elastin has a reversible phase change characteristic similar to that of natural elastin; the recombinant fusion elastin crosslinks to form a hydrogel with good mechanical strength, and has high biocompatibility and biological activity; the recombinant fusion elastin hydrogel has a significant repair effect on skin photo-damage and has broad application prospects in the fields of skin repair dressings, implants, artificial skin, biomaterials, medical devices, etc.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering technology and medical materials, and particularly relates to a recombinant fusion elastin, a preparation method and an application thereof. Background Art

[0002] Elastin is a structural protein widely present in the human body, endowing important organs such as blood vessels, ligaments, and skin with elasticity and flexibility. Different from other proteins, elastin has a unique reversible phase transition property, that is, it aggregates to form a precipitate when heated and redissolves after cooling. Lysine in natural elastin undergoes cross-linking under the catalysis of enzymes to form an ordered pore structure. Elastin cross-linked gel can be used as a tissue regeneration scaffold to provide a beneficial microenvironment for cell growth and has attracted much attention in the field of medical materials.

[0003] Animal tissue extraction is one of the main means for preparing elastin at present. The invention patent CN 104710635B discloses a method for extracting animal-derived elastin. By degrading the elastin in bovine neck ligaments, α-elastin fragments are prepared, which destroys the structure and function of natural elastin. At the same time, animal-derived elastin has problems such as immunogenicity and the risk of disease transmission.

[0004] Genetic engineering technology has the advantages of low cost, easy large-scale production, and no risk of virus transmission, so it has been tried to be used to prepare elastin. In the invention patent CN102241747A, an elastin sequence with (VAPGVG) 3 S as the basic unit and highly repeated is constructed, and the expression of this sequence in Escherichia coli ER2566 is realized; however, this elastin sequence is only a simple repetition of VAPGVG, has a huge difference from the amino acid sequence of natural elastin, and does not have the ability to form a gel.

[0005] To solve the above problems, the present invention provides a recombinant fusion elastin (Recombinant Fusion Elastin, RFE), a preparation method and applications thereof. The recombinant fusion elastin comprises an elastin sequence and an Sc12 protein folding domain. The amino acid sequence of the elastin is entirely derived from human elastin. The Sc12 protein folding domain can promote the expression of elastin and help to stabilize the structure of the elastin polymer. The recombinant fusion elastin is highly expressed in Escherichia coli engineering bacteria and is easy to achieve large-scale production. The recombinant fusion elastin has a reversible phase change property similar to that of natural elastin. The recombinant fusion elastin crosslinks to form a hydrogel with good mechanical strength, and has high biocompatibility and biological activity. The recombinant fusion elastin hydrogel has a significant repair effect on skin photo-damage and has broad application prospects in the fields of skin repair dressings, implants, artificial skin, biomaterials, medical devices, etc. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the present invention provides a recombinant fusion elastin, a preparation method and applications thereof. The specific solutions include:

[0007] In a first aspect, the present invention provides a recombinant fusion elastin. The main body of the recombinant fusion elastin is composed of one or more elastin domains in the human full-length elastin amino acid sequence shown in SEQ ID NO.1. At the same time, amino acid sequences for regulating the structure and function of the protein are added to both ends of the sequence to regulate the structure, expression and efficacy of the protein. The amino acid sequences for regulating the structure and function of the protein do not have the characteristics of elastin sequences.

[0008] Preferably, the amino acid sequence for regulating the structure and function of the protein is an Sc12 protein folding domain, and the amino acid sequence of the Sc12 protein folding domain is shown in SEQ ID NO.2.

[0009] Preferably, the recombinant fusion elastin comprises at least one elastin domain in the human full-length elastin shown in SEQ ID NO.1, and an Sc12 protein folding domain is added at the N-terminus. The amino acid sequence of the Sc12 protein folding domain is shown in SEQ ID NO.2.

[0010] Preferably, the elastin domain is composed of the domains numbered 20-24 in the human elastin sequence connected or periodically repeated.

[0011] Preferably, the elastin domain is composed of at least one amino acid sequence shown in SEQ ID NO.3 in series.

[0012] Preferably, the amino acid sequence of the elastin domain is as shown in SEQ ID NO.3.

[0013] Preferably, the recombinant fusion elastin consists of an elastin domain and an Sc12 protein folding domain, and the Sc12 protein folding domain is located at the N-terminus of the elastin domain.

[0014] Preferably, the amino acid sequence of the recombinant fusion elastin is as shown in SEQ ID NO.4.

[0015] In a second aspect, the present invention provides a nucleotide encoding the recombinant fusion elastin described in the first aspect above.

[0016] Preferably, the nucleotide sequence is as shown in SEQ ID NO.5.

[0017] In a third aspect, the present invention provides a recombinant plasmid, recombinant vector or recombinant cell containing the nucleotide described in the second aspect above.

[0018] Preferably, the vector includes pCold III.

[0019] Preferably, the recombinant cell is recombinant Escherichia coli BL21.

[0020] In a fourth aspect, the present invention provides a method for preparing the recombinant fusion elastin described in the first aspect above, and the method includes the following steps:

[0021] (1) Synthesize the nucleotide sequence encoding the recombinant fusion elastin described in the first aspect above;

[0022] (2) Connect the gene described in step (1) with a vector / plasmid, transform bacteria, and construct a recombinant genetically engineered bacterium;

[0023] (3) Express the recombinant genetically engineered bacterium constructed in step (2), collect the cell precipitate, break it to obtain the supernatant, and purify to obtain the recombinant fusion elastin.

[0024] Preferably, the method includes the following steps:

[0025] (1) Synthesize the nucleotide sequence shown in SEQ ID NO.5;

[0026] (2) Insert the nucleotide sequence described in step (1) into the pColdIII plasmid to construct a recombinant vector; and introduce it into Escherichia coli BL21, and screen to obtain recombinant bacteria;

[0027] (3) Transfer the recombinant bacteria into an LB medium, and culture them at 37°C for 10 - 12 h. When the OD 600When it reaches 0.8 - 1.0, add 1 mM IPTG, and at the same time cool down to 25 °C, and induce expression for 12 - 18 h; centrifuge to collect the fermentation bacterial cell precipitate, break it to obtain the supernatant, and purify to obtain the recombinant fusion elastin.

[0028] In the fifth aspect, the present invention provides the application of the recombinant fusion elastin described in the first aspect above in the preparation of skin care products, skin repair dressings, implants, artificial skin, medical devices, and health foods.

[0029] In the sixth aspect, the present invention provides a recombinant fusion elastin material, and the recombinant fusion elastin material comprises the following components in mass fractions: 1 - 50% of the recombinant fusion elastin described in the first aspect above; 0 - 5% of a crosslinking agent, and the remaining components are water or excipients.

[0030] Preferably, the crosslinking agent includes glutaraldehyde, N - hydroxysulfosuccinimide (NHS), tetrakis(hydroxymethyl)phosphonium chloride, and genipin.

[0031] Preferably, the crosslinking agent is tetrakis(hydroxymethyl)phosphonium chloride.

[0032] Preferably, the excipients include hyaluronic acid, agarose, and chitosan.

[0033] In the seventh aspect, the present invention provides the application of the recombinant fusion elastin material described in the sixth aspect above in the preparation of skin injury repair products.

[0034] In the eighth aspect, the present invention provides a recombinant fusion elastin gel, and the recombinant fusion elastin gel comprises the following components: 1 - 50% of the recombinant fusion elastin described in the first aspect above, 0.5 - 5% of tetrakis(hydroxymethyl)phosphonium chloride, and the remaining components are deionized water or normal saline.

[0035] In the ninth aspect, the present invention provides the application of the recombinant fusion elastin gel described in the eighth aspect above in the preparation of skin injury repair products.

[0036] The beneficial effects of the present invention are as follows:

[0037] (1) The present invention constructs and prepares a recombinant fusion elastin, and its sequence includes a human elastin sequence and an Sc12 folding domain; the Sc12 folding domain can promote the expression of elastin, increase the order degree of the secondary structure of elastin, and help to stabilize the structure of the elastin polymer.

[0038] (2) The recombinant fusion elastin has a reversible phase transition property similar to that of natural elastin, and the phase transition temperature is close to that of human elastin.

[0039] (3) The recombinant fusion elastin overcomes the problems such as the risk of virus transmission and immunogenicity in the extraction of elastin from animal tissues.

[0040] (4) The recombinant elastin fusion protein has good biocompatibility and water solubility, which is convenient for addition and use in cosmetics and skin repair dressings.

[0041] (5) The recombinant elastin fusion protein is highly expressed in Escherichia coli engineering bacteria and is easy to achieve large-scale production.

[0042] (6) The present invention develops a recombinant fusion elastin gel. The preparation method of the gel is simple and the conditions are mild, which is suitable for large-scale batch production.

[0043] (7) The recombinant fusion elastin gel has a significant repair effect on the ultraviolet damage of the skin and can significantly reduce the inflammatory symptoms of photo-damaged skin; the gel is prepared into drugs, medical devices or cosmetics such as dressings and patches that are easy to apply to the skin surface, which is convenient for storage and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 SDS-PAGE of recombinant fusion elastin;

[0045] Figure 2 Effect of Sc12 protein folding domain on the expression and aggregation behavior of recombinant fusion elastin; where a is the SDS-PAGE of recombinant fusion elastin RFE and recombinant elastin RE without Sc12 protein folding domain; b is the native electrophoresis comparison of recombinant fusion elastin RFE and recombinant elastin RE at different temperatures;

[0046] Figure 3 Circular dichroism spectra of recombinant fusion elastin RFE at different temperatures;

[0047] Figure 4 Reversible phase transition of recombinant fusion elastin RFE; where a is the reversible phase transition curve of recombinant fusion elastin RFE at different concentrations; b is the relationship curve between the phase transition point T t and the logarithm of protein concentration;

[0048] Figure 5 Characterization of the properties of recombinant fusion elastin RFE gel; where a is the physical photo of the cross-linked gel of recombinant fusion elastin RFE; b is the scanning electron microscope image of the recombinant fusion elastin gel; c is the frequency sweep rheology of the recombinant fusion protein gel, d is the strain sweep rheology of the recombinant fusion elastin gel;

[0049] Figure 6Cell viability of recombinant fusion elastin RFE gel; where a is the cytotoxicity of recombinant fusion elastin RFE gel; b is the cell proliferation of recombinant fusion elastin RFE gel; c is the cell adhesion staining imaging of recombinant fusion elastin RFE gel.

[0050] Figure 7 It is the repair function of recombinant fusion elastin RFE gel on ultraviolet-damaged skin of mice; where a is the control group, and the skin of blank group, experimental group mice is macroscopically photographed on the 1st, 3rd, 5th, and 7th days; b is the HE staining results of skin tissues of blank group, control group, and experimental group mice on the 1st, 3rd, 5th, and 7th days; c is the Masson staining results of skin tissues of blank group, control group, and experimental group mice on the 1st, 3rd, 5th, and 7th days. Specific implementation mode

[0051] The following further describes the technical solution of the present invention in combination with specific embodiments, but the protection scope of the present invention is not limited to the following.

[0052] The recombinant fusion elastin described in the following embodiments is a fusion protein composed of elastin and proteins with other functional types obtained in a microbial expression system by using transgenic technology and gene recombination technology. This protein retains the characteristic properties of elastin.

[0053] The recombinant fusion elastin described in the following embodiments is the recombinant fusion elastin obtained by Escherichia coli fermentation, but is not limited to the above method, and can also be a fusion protein mainly composed of elastin sequences prepared by other methods, including recombinant fusion elastin fermented by Pichia pastoris, recombinant fusion elastin expressed in vitro by cell-free (Cell-Free), etc.

[0054] Specifically, the preparation method of the recombinant fusion elastin described in the following embodiments includes: constructing an Escherichia coli engineering bacterium for fermentation production, centrifuging to obtain the supernatant after fermentation, and separating and purifying to obtain the recombinant fusion elastin.

[0055] The method of the recombinant fusion elastin gel described in the following embodiments includes: heating a recombinant fusion elastin solution with a certain concentration to cause the protein to undergo a reversible phase change characteristic of elastin for prepolymerization, adding a crosslinking agent THPC (tetrakis(hydroxymethyl)phosphonium chloride, the same below) while it is hot, reacting under certain conditions, and removing the reaction residues to obtain an elastin gel with a certain mechanical strength; but it is not limited to the above method, and other crosslinking methods can also be used, including photocrosslinking, thermal crosslinking, and chemical crosslinking represented by genipin crosslinking, BDDE crosslinking, and EDC-NHS crosslinking.

[0056] Example 1 Preparation of recombinant fusion elastin

[0057] 1. Construction of expression vector for recombinant fusion elastin sequence

[0058] Synthesize the nucleotide sequence of recombinant fusion elastin encoding as shown in SEQ ID NO.5, construct the pCold III recombinant plasmid into which the above nucleic acid is introduced, and confirm the successful synthesis of the plasmid by DNA sequencing; transform the recombinant plasmid into Escherichia coli BL21-DE3 strain to obtain the recombinant fusion elastin expression strain. After adding glycerol to the successfully transformed strain, store it in a -80 °C refrigerator.

[0059] 2. Induced expression of recombinant fusion elastin in Escherichia coli

[0060] Inoculate Escherichia coli containing the recombinant fusion elastin expression gene onto a solid LB medium containing 50 mg / L kanamycin sulfate for pre-expression and screening of recombinant Escherichia coli. The selected single colonies are inoculated into a liquid LB medium (200 mL supplemented with 50 mg / L kanamycin sulfate), cultured in a constant temperature shaker at 37 °C and 250 rpm for 12 h, and then the bacterial strain is transferred into 4 × 1 L of LB medium and cultured at 37 °C and 250 rpm for 10 - 12 h. By sampling and measuring the absorbance (OD 600 ) of the medium at a wavelength of 600 nm, the growth density of the bacterial strain is monitored. When OD 600 reaches 0.8 - 1.0, add IPTG with a final concentration of 1 mM, and at the same time cool down to 25 °C and induce expression at 250 rpm for 12 - 18 h. Centrifuge to collect the fermented bacterial cells.

[0061] 3. Nickel column purification of recombinant fusion elastin

[0062] Suspend the Escherichia coli cells obtained in step 2 above in phosphate buffer (phosphate Buffer, 0.02 M, pH = 7.4) at a volume ratio of 1:15. Under the condition of low-temperature water cooling cycle, use a high-pressure homogenizer to break the cells, so that the recombinant protein inside Escherichia coli is released into the buffer solution. Centrifuge at 4 °C and 10,000 rpm for 20 min to separate the cell debris precipitate, retain the supernatant, and filter through a microporous membrane to remove residual insoluble impurities; add an equal volume of the sample to a His-trap nickel ion affinity chromatography column (25 mL), shake the column body to make the target protein with a 6×His tag fully bind to the column packing, and then rinse with an elution buffer (0.02 M PB, 0.5 M NaCl, imidazole) for 5 - 8 column volumes. The content of imidazole in the elution buffer increases stepwise (20 mM, 50 mM, and 500 mM) to elute the protein. Collect the protein eluted at a 500 mM imidazole concentration. The obtained protein solution is dialyzed at 4 °C with 0.02 M PB buffer or ultrapure water, and the target protein with a molecular weight cut-off greater than 8000 is retained to remove components such as imidazole and NaCl in the protein solution, and then freeze-dry.

[0063] The SDS-PAGE detection results of the purified protein sample are as Figure 1 shown, presenting a single band, indicating that a protein sample with high purity was prepared. According to the Marker indication, the molecular weight of the target protein is between 25-35 kDa, which is consistent with the theoretical molecular weight. These results indicate that a recombinant fusion elastin (RFE) with high purity was successfully prepared.

[0064] Example 2 Effect of the Sc12 protein folding domain on the properties of recombinant fusion elastin

[0065] 1. Effect of the Sc12 protein folding domain on protein expression level

[0066] According to the Escherichia coli culture method in Example 1, under the same conditions, Escherichia coli transfected with recombinant plasmid RFE and Escherichia coli transfected with RE (Recombinant Elastin, only containing the elastin domain) were cultured, induced for expression, and purified. SDS-PAGE analysis was performed on the total proteins of the two types of Escherichia coli after induction and the highly purified recombinant elastin obtained by the same purification method to compare the differences in the expression levels of the two recombinant proteins.

[0067] The results are as Figure 2 shown in a of the figure. The expression level of recombinant fusion elastin (RFE) is significantly higher than that of recombinant elastin (RE) without the Sc12 protein folding domain, indicating that the Sc12 protein folding domain has a significant promoting effect on the expression of elastin.

[0068] 2. Native electrophoresis of recombinant fusion elastin

[0069] Native-PAGE was used to analyze recombinant fusion elastin RFE incubated at different temperatures and then restored to room temperature. At the same time, recombinant elastin RE under the same conditions was used as a control to confirm the effect of the Sc12 protein folding domain on regulating the aggregation behavior of recombinant fusion elastin by comparing the changes in molecular weight at different temperatures of the two.

[0070] The Native-PAGE results are shown in Figure 2As shown in Figure b, after the recombinant fusion elastin RFE was incubated at low temperature and then restored to room temperature, it showed a single band, which was consistent with the molecular weight of the single chain. As the incubation temperature increased, a new band with a larger molecular weight appeared in the RFE, which was consistent with the molecular weight of the trimer, and at the same time, bands of polymers with higher molecular weights appeared. The intensities of the corresponding bands of the trimer and polymers increased continuously with the increase of the incubation temperature. At the same time, the intensity of the single molecular weight band of RFE decreased continuously. On the contrary, the elastin RE without the Sc12 protein folding domain maintained a single molecular band after incubation at different temperatures and restoration to room temperature. These results indicate that the recombinant fusion elastin formed trimers and other polymers during heating, and these aggregates could stably exist after restoration to room temperature, indicating that the Scl2 protein folding domain has the function of stabilizing the aggregates of the recombinant fusion elastin.

[0071] Example 3 Study on the Properties of Recombinant Fusion Elastin

[0072] 1. Circular Dichroism of Recombinant Fusion Elastin

[0073] The solid recombinant fusion elastin obtained in Example 1 was formulated into an aqueous solution with a concentration of 1.0 mg / mL, and then allowed to stand at 4 °C for more than 24 h. A 1-mm cuvette was used for full-wavelength scanning of the circular dichroism at 4, 25, 30, 37, and 50 °C. The wavelength range was from 190 nm to 260 nm, the wavelength interval was 1 nm, and the residence time at each wavelength was 5 s.

[0074] The results are as Figure 3 shown. At lower temperatures, the designed recombinant fusion elastin had a strong negative peak near 200 nm and a medium-intensity positive peak near 220 nm. As the temperature increased, the intensities of both characteristic absorption peaks decreased continuously. The proportion of various secondary structures in the spectral peaks was analyzed by software (see Table 1).

[0075] Table 1 Proportion of Various Secondary Structures in Recombinant Fusion Elastin at Different Temperatures

[0076] Temperature(℃) α-helix(%) β-spiral(%) β-turn(%) Disordered(%) 4 9.1±3.5 14.2±0.3 32.0±3.7 43.2±6.7 25 8.4±2.1 22.6±3.1 25.8±6.4 45.4±5.8 30 7.4±2.9 28.7±1.5 18.3±1.8 46.1±4.0 37 6.9±2.0 31.8±4.6 13.8±5.8 48.1±5.2 50 6.7±3.5 33.7±2.9 11.4±1.9 51.1±4.3

[0077] From the conformational analysis results, it can be seen that when the temperature is in the range of 4 - 50 °C, with the increase of temperature, the proportion of disordered structures in the conformation of the recombinant fusion elastin remains basically unchanged, the proportion of β-turns decreases from 32% to 11%, and the proportion of β-helix conformations increases from 14% to 34%. Compared with the results reported in the literature, the proportion of ordered structures (β-sheet, β-turn) is significantly higher than that of natural elastin. This indicates that the Sc12 protein folding domain increases the order of the secondary structure of the recombinant fusion elastin.

[0078] 2. Reversible Phase Transition of Recombinant Fusion Elastin

[0079] The recombinant fusion elastin RFE prepared in Example 1 was dissolved in a phase transition buffer solution (0.5 M NaCl, 0.1 M CaCl 2 , 50m M tris, 0.02M PB, pH = 7.4), so that the final protein concentration is 5, 25, 50, 75, 100mM, respectively, the recombinant fusion elastin phase change solution is placed in a constant temperature water bath, heated at room temperature (25°C) as the starting temperature, after the solution reaches a stable temperature, the protein solution at room temperature is used as a reference, and the absorbance of the protein solution at a wavelength of 350nm after reaching a constant temperature is measured, and then the temperature is increased, the temperature is increased by 2.5°C, and the balance is 5min, until the absorbance basically no longer increases. The heated solution is cooled, the protein is redissolved, and restored to the state before heating, completing a reversible phase change cycle.

[0080] The absorbance-temperature relationship curves of recombinant fusion elastin solutions with different concentrations are shown in Figure 2. Figure 4 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 fusion elastin is about 34℃, the reversible phase transition temperature of 50μM recombinant fusion elastin is about 38℃, the reversible phase transition temperature of 25μM recombinant fusion elastin is about 41℃, the reversible phase transition temperature of 10μM recombinant fusion elastin is 45℃, and the reversible phase transition temperature of 5μM recombinant fusion elastin is 46℃. Plot the logarithm of phase transition temperature and protein concentration ( Figure 4 The results show that the recombinant fusion elastin has similar thermal properties to natural elastin.

[0081] Example 4 Preparation and Application of Recombinant Fusion Elastin Gel

[0082] 1. Gel Preparation

[0083] Weigh a certain amount of the recombinant fusion elastin obtained in Example 1, dissolve it in 0.02M PBS buffer, and prepare an elastin aqueous solution with a final concentration of 100μM. Incubate it at 37°C to allow the elastin to aggregate due to heat, and the overall absorbance of the solution to increase. Oscillate the turbid solution to disperse the protein evenly, then inject it into the mold, add THPC with a final concentration of 0.75% while it is hot, and let it stand at room temperature to form a gel. Soak the gel in ultrapure water, let it stand for 12 hours, and change the solution 3-5 times to remove the residual unreacted cross-linking agent and protein. The macroscopic physical picture of the prepared gel is as follows: Figure 5 As shown in a.

[0084] 2. SEM of Gel

[0085] The gel prepared in Step 1 above was freeze-dried, sliced, adhered to the sample plate, and observed under a Hitachi S-4800 scanning electron microscope. The results are as Figure 5 shown in b of Figure 5 . The gel has an orderly arranged fiber mesh pore structure with a pore size of about 50 - 100 μm, providing a beneficial growth environment for cells.

[0086] 3. Rheomechanics of the gel

[0087] The reacted gel was carefully taken out of the mold and placed on the sample stage of an Anton Paar rheometer. Frequency sweep: Set the strain parameter of the rheometer to 1%, and the stress to 1 N. Measure the storage modulus G’ (Pa) and loss modulus G” (Pa) of the gel as the scanning frequency changes. Strain sweep: Fix the scanning frequency at 1 rad / s and measure the storage modulus G’ (Pa) as the shear strain changes.

[0088] The frequency sweep test results of the gel rheomechanics are as Figure 5 shown in c of Figure 5 . As the shear frequency increases continuously, both the storage modulus (G’) and compression modulus (G”) of the recombinant fusion elastin RFE gel gradually increase, and the modulus growth rate also increases continuously, showing the strain characteristics of typical hydrogel materials. At a shear frequency of 90 rad / s or higher, the storage modulus can reach 2×10 3 Pa, proving that the gel has good mechanical strength. The strain sweep test results are as Figure 5 shown in d of Figure 5 . The storage modulus of the recombinant fusion elastin RFE gel gradually decreases, and when the shear strain exceeds 60%, the storage modulus tends to be stable. The results show that as the strain increases, the viscosity of the material continuously decreases, which conforms to the typical characteristics of the shear thinning behavior of the gel.

[0089] 4. Cell viability of the gel

[0090] (1) Cytotoxicity

[0091] Weigh a certain amount of the recombinant fusion elastin (RFE) freeze-dried powder, dissolve it in a small amount of sterile water to prepare a protein concentrated solution, then filter the protein concentrated solution through a 0.22-μm aqueous filter membrane. After that, add a certain amount of DMEM cell culture medium for dilution to make the final protein concentration 100 μM. Add this recombinant fusion elastin solution to a TC-treated 96-well plate to evenly cover the bottom of the plate, and then add a sterile aqueous solution of THPC for crosslinking to make the final concentration of THPC 0.1 - 2.0%. Place the plate in a laminar flow hood at room temperature for crosslinking. After the protein at the bottom layer of the plate is completely solidified into a gel, wash the gel 3 times with 100 μL of 0.067 M PBS and set it aside. After infiltrating with DMEM cell culture medium for 3 h, add 100 μL of HFF-1 cells that have been digested with trypsin and dispersed evenly at a density of 5000 cells / mL. Place the plate in a cell incubator and culture at 37 °C for 24 h. After incubating with CCK-8 for 30 min, quantitatively determine the number of cells by measuring the absorbance of each well with an enzyme-linked immunosorbent assay (ELISA) reader. The blank group is the gel without adding cells, and the control group is the same amount of cell culture medium (containing cells) without gel at the bottom.

[0092] The results of cytotoxicity are shown in Figure 6 a. Compared with the control group, on the surfaces of gels crosslinked with different concentrations of THPC, the cell survival rate of each group is greater than 95%, indicating that the recombinant fusion elastin gel has no cytotoxicity and has good biocompatibility.

[0093] (2) Cell proliferation

[0094] Weigh a certain amount of the recombinant fusion elastin freeze-dried powder, dissolve it in a small amount of sterile water to prepare a protein concentrated solution, then filter the protein concentrated solution through a 0.22-μm aqueous filter membrane. After that, add a certain amount of DMEM cell culture medium for dilution to make the final protein concentration 100 μM. Add 100 μL of this recombinant elastin solution to a TC-treated 96-well plate to evenly cover the bottom of the plate, and then add a sterile aqueous solution of THPC for crosslinking to make the final concentration of THPC 0.75%. Add deionized water, 0.02 M PBS, and TEMED culture medium to infiltrate the gel in portions to remove the residual unreacted protein and crosslinking agent; inoculate 500 HFF cells in each well, place them in an incubator and culture at 37 °C, and change the cell culture medium every 48 h to prevent the accumulation of cell metabolites. Add CCK-8 every 24 h to quantitatively determine the number of living cells on the surface of each group of gels.

[0095] The results of cell proliferation are as shown in Figure 6As shown in b, compared with the control group without gel, the proliferation rate of the experimental group with recombinant fusion elastin gel was slightly greater than 100% on the 1st - 2nd day. On the 3rd - 4th day, the cell proliferation rate increased to more than 150%, and on the 5th - 7th day, the cell proliferation rate exceeded 200%. This indicates that the recombinant fusion elastin gel has a significant promoting effect on cell proliferation.

[0096] (3) Cell adhesion staining

[0097] Pour the DMEM medium solution containing 100 μM recombinant fusion elastin into several untreated glass - bottom culture dishes, make the protein solution completely cover the bottom of the culture dish, add the THPC solution with a final concentration of 0.75%, and let it stand in the laminar flow hood for 3 h; after cross - linking, add PBS and rinse three times, then add DMEM medium and soak for use. Add 100 μL of HFF - 1 cells digested with trypsin and dispersed evenly at a density of 5000 cells / mL. Culture in a cell incubator at 37 °C, and change the cell culture medium every 48 h to prevent the accumulation of cell metabolites. Take samples on the 1st, 4th, and 7th days respectively, add 4% formaldehyde to fix for 10 min and then aspirate, permeabilize with 0.1% Triton - X for 5 min, block with 1% BSA for 30 min, add phalloidin to stain the cytoplasm, and then add Hoechest33258 to stain the cell nucleus; coat the surface of the gel with a mounting medium and observe the number and morphology of HFF - 1 cells adhered to the gel surface under a fluorescence microscope.

[0098] The results of cell adhesion staining are as Figure 6 shown in c. On the 1st day after adding cells to the upper layer of the gel, fibroblasts adhered and spread normally on the gel surface. Cultured until the 4th day, the number of cells increased significantly compared with the 1st day, the cell distribution was relatively uniform, and the morphology was intact. On the 7th day, the cell density on the gel surface further increased, and almost all of it was covered by cells. These results indicate that the recombinant fusion elastin gel has good biological activity.

[0099] 5. Animal injury repair experiment of the gel

[0100] Take 45 male SPF - grade Kunming mice weighing 20 ± 2 g. Use a Philips hair clipper to shave the hair on the backs of the mice, and then apply depilatory cream to remove the hair, making the back epidermis completely bare. The depilated area is about 2 × 4 cm. After depilation, stabilize for 24 h to exclude the influence of depilatory cream on epidermal damage. Randomly divide the mice into three groups. The blank group is not treated. The experimental group and the control group are irradiated on the backs of the mice with ultraviolet lamps A (320 - 440 nm) and B (280 - 320 nm) with continuous wavelengths. Use an ultraviolet irradiance meter to quantify the radiation, which is 200 mJ / cm 2, after ultraviolet irradiation, large areas of redness and swelling appeared in the depilated areas on the backs of the mice, indicating that acute skin inflammation had been successfully induced in the mice. The gel was applied to the ultraviolet lamp-irradiated areas of the back skin of the experimental group mice, covered with gauze, and then fixed with a bandage to prevent the gel from falling off during the movement of the mice. The gel patch was replaced and refixed every 48 h. After applying normal saline to the backs of the control group mice, they were covered with gauze and fixed with a bandage. The blank group was not irradiated with ultraviolet light or covered with gauze. Starting from the first day after modeling, the mice were sacrificed by dislocation every 48 h, and the skin at the depilated areas on the back was taken and stored in formalin solution. HE staining and Masson staining were performed on the skin sections respectively. The skin damage and recovery were observed under a microscope.

[0101] The macroscopic physical pictures of the skin of the blank group, the control group, and the experimental group are shown in Figure 7 a as shown. The skin of the blank group mice remained healthy throughout the 1st - 7th days, with a smooth and flat surface and no red and swollen areas. Local uneven redness and swelling occurred on the back skin of the control group mice on the 1st day; a large number of red papules appeared on the 3rd - 5th days; there was still a small amount of local redness and swelling on the 7th day, accompanied by local dry cracking and peeling. Partial redness and swelling appeared on the back skin of the experimental group mice on the 1st day, the area of the red and swollen area decreased on the 3rd day, wrinkles appeared on the surface, and at the same time, a small amount of erythematous papules were accompanied. The redness and swelling basically subsided on the 5th day, the skin wrinkle condition improved significantly, and at the same time, local epidermal exfoliation occurred. The skin recovery was in good condition on the 7th day, with a smooth and flat surface, and there was no obvious difference from the skin of the normal group mice. These results indicate that recombinant fusion elastin has an obvious repair effect on the skin photo-damage of mice and can significantly shorten the repair cycle of photo-damaged skin.

[0102] The HE staining results of the blank group, the control group, and the experimental group are shown in Figure 7As shown in Figure b, in the blank group of mice, the epidermis and the entire dermis layer were visible from day 1 to day 7, with a complete structure, normal numbers and morphologies of each appendage, and uniform distribution of tissue fibers. On day 1 in the control group, partial inflammatory reactions occurred in the reticular layer, and the epidermis layer was significantly damaged and thinned; on day 3, the fibers in the epidermis layer were significantly thickened and the number decreased, and at the same time, some fibers showed irregular fractures; local edema occurred in the reticular layer, accompanied by a large number of inflammatory cell infiltrations; on day 5, it began to recover slowly, but the damage to the skin tissue was still relatively obvious; on day 7, the overall damage to the skin tissue was still relatively obvious, accompanied by abnormal hyperplasia of the local epidermis layer, local fiber fractures and abnormal depositions. On day 1 of the dorsal skin of the experimental group of mice, the inflammatory reaction in the reticular layer and the degree of damage to the epidermis layer were both reduced compared with the control group; on day 3, the degree of damage to the epidermis layer of the mice was further deepened compared with day 1, the structure was incomplete, the local basal layer was damaged, the dermis was locally loose and edematous, with a small amount of inflammatory cell infiltration, and the hair follicles were damaged; on day 5, the damage to the epidermis layer was reduced, the integrity of the structure was improved compared with day 3, the degree of dermal looseness was reduced, and the number of inflammatory cells decreased; on day 7, the skin tissue of the mice basically returned to normal, the epidermis and the entire dermis layer were visible, the structure was complete, the numbers and morphologies of each appendage were normal, and the distribution of tissue fibers was uniform, showing no significant difference from the normal skin tissue structure.

[0103] The Masson staining results of the blank group, the control group, and the experimental group are as Figure 7As shown in Figure C, on the 1st - 7th day, the collagen fibers in the dermal papillary layer of the skin of the blank - group mice were slender, intertwined with each other to form a network, and each secretory gland and blood vessel was surrounded by a fibrous - reticular collagen. The collagen fiber bundles in the reticular layer were thick and showed wavy undulations, extending in all directions on the same horizontal plane, and fibroblasts were evenly distributed in the gaps between the collagen fibers. On the 1st day of the control - group mice, the arrangement of the collagen fibers in the dermis was disordered, the number decreased sharply, the fibers became thicker, curled and knotted, and some aggregated into clusters. At the same time, a large number of breaks and fragments occurred, and inflammatory cells infiltrated into the fiber gaps. On the 3rd day, the damage to the dermis was aggravated, the fibers became further thicker and aggregated into blocks, the breaks were more serious, and the number of infiltrating inflammatory cells increased further. On the 5th day, the damage to the collagen fibers in the dermis was alleviated to some extent, but there were still some phenomena of aggregation into blocks and breaks. The fiber arrangement was loose, the broken parts began to repair, but the whole was still severely defective. On the 7th day, the number of collagen fibers in the dermis began to increase, and the newly - formed fibers began to extend in all directions on the same horizontal plane, but there were still some defects in the overall skin tissue. On the 1st day of the experimental - group mice, the arrangement of some collagen fibers in the dermis was disordered, the number decreased, some fibers became thicker, curled and knotted, some aggregated into clusters. At the same time, a large number of breaks and fragments occurred, and inflammatory cells infiltrated into the fiber gaps. On the 3rd day, the damage to the dermis was slightly reduced, the arrangement of some collagen fibers was disordered, and there were a small number of phenomena of thickening and aggregation of collagen fibers into blocks. The degree of infiltration of inflammatory cells was basically the same as that on the 1st day. On the 5th day, the damage to the dermis was further reduced, slender newly - formed 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 degree of loose fiber arrangement was reduced. On the 7th day, the collagen fibers in the dermal papillary layer were slender, intertwined with each other to form a network, and each secretory gland and blood vessel was surrounded by a fibrous - reticular collagen. The collagen fiber bundles in the reticular layer were thick and showed wavy undulations, extending in all directions on the same horizontal plane, and fibroblasts were evenly distributed in the gaps between the collagen fibers.

[0104] In summary, the recombinant fusion elastin gel described in the present invention can inhibit the inflammatory reaction of the skin, promote the regeneration of collagen in damaged skin, and has a good repair effect on ultraviolet - damaged skin. The light damage in this experiment was all caused by ultraviolet radiation of 200mJ / cm 2 We can infer that injuries caused by other types, at least of the same intensity, can be treated with this gel to reduce the degree of injury and accelerate the recovery of skin tissue, such as electromagnetic radiation from different sources like lasers, red light, and blue light.

[0105] The above - mentioned are only the details of individual exemplary embodiments of the present invention. For those skilled in the relevant art, the present invention can have various changes and modifications according to specific preparation conditions during the actual application process, and is not used to limit the present invention. All those within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant fusion elastin, characterized in that, the recombinant fusion elastin is composed of a domain of elastin and a Sc12 protein folding domain, and the Sc12 protein folding domain is located at the N-terminal of the elastin domain; the amino acid sequence of the recombinant fusion elastin is shown in SEQ ID NO.

4.

2. A nucleotide encoding the recombinant fusion elastin according to claim 1.

3. The nucleotide according to claim 2, characterized in that, the nucleotide sequence is shown in SEQ ID NO.

5.

4. A recombinant plasmid, recombinant vector or recombinant cell containing the nucleotide according to claim 2 or 3.

5. The method for preparing the recombinant fusion elastin according to claim 1, characterized in that, the method comprises the following steps: (1) Synthesize the nucleotide sequence encoding the recombinant fusion elastin according to claim 1; (2) Connect the nucleotide sequence in step (1) with a vector / plasmid, transform bacteria, and construct a recombinant genetically engineered bacterium; (3) Culture the recombinant genetically engineered bacterium constructed in step (2), induce expression, collect the cell precipitate, break it to obtain the supernatant, and purify to obtain the recombinant fusion elastin.

6. A recombinant fusion elastin material, characterized in that, the recombinant fusion elastin material comprises the following components by mass fraction: 1-50% of the recombinant fusion elastin according to claim 1; 0-5% of a cross-linking agent, and the remaining components are water or excipients.

7. A recombinant fusion elastin gel, characterized in that, the recombinant fusion elastin gel comprises the following components: 1-50% of the recombinant fusion elastin according to claim 1, 0.5-5% of tetrakis(hydroxymethyl)phosphonium chloride, and the remaining components are deionized water or physiological saline.

8. The use of the recombinant fusion elastin according to claim 1, or the recombinant fusion elastin material according to claim 6, or the recombinant fusion elastin gel according to claim 7 in the preparation of skin injury repair products; the products include skin care products, skin repair dressings, implants, artificial skin, medical devices, biological materials.

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