Method for preparing recombinant elastin-like protein with effect of delaying skin ultraviolet injury

Abstract

The invention belongs to the technical field of biology, and discloses a method for preparing a recombinant elastin with an effect of delaying skin ultraviolet injury, the recombinant elastin contains a human structural domain of a VGVAPG hexapeptide sequence, and has the effect of delaying skin ultraviolet injury. The recombinant elastin disclosed by the invention is separated and purified by utilizing a human-like elastin non-chromatography, so that the recombinant elastin has the advantages of low cost, high expression, easiness in large-scale production and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for preparing recombinant elastin-like protein with the function of delaying skin ultraviolet damage. Background Art

[0002] Photoaging refers to the aging of the skin caused by a variety of exogenous factors, mainly ultraviolet radiation. As the largest organ of the human body exposed to the external environment, the skin plays a protective role for the human body. External and internal aging factors affect the skin and cause skin aging. The gradual reduction and loss of collagen and elastin density in the skin is a sign of intrinsic and extrinsic aging of the skin. Elastin will gradually degrade, cross-link and break down under exposure to harmful factors in the environment. In addition, as the body matures, the production of elastin also decreases. These factors together lead to the loss of skin structural integrity and elasticity.

[0003] Elastin is an extracellular matrix protein that gives skin tissue its unique physiological elasticity. Elastin not only provides mechanical elasticity to the skin, but also acts as a substrate for cell growth, promoting epidermal regeneration and remodeling, and plays an important role in delaying skin aging. Elastin plays an important role in inhibiting the formation of wrinkles and maintaining skin homeostasis, which is essential for the youthfulness, health and normal function of the skin. Elastin has a wide range of applications due to its good mechanical properties and biocompatibility. However, its high insolubility limits its application.

[0004] Elastin-like proteins (ELPs) are artificially synthesized proteins that imitate the amino acid sequence characteristics of natural elastin. They are recombinant proteins prepared by genetic engineering technology, and their amino acid composition simulates the hydrophobic region of elastin. Due to their good biocompatibility and the fact that their environmental reactivity, mechanical properties and cell metabolic pathways can be precisely controlled by recombinant DNA technology design at the genetic level, they have attracted widespread attention because they maintain the excellent properties of elastin and have reversible phase transition characteristics. When the external temperature is lower than the phase transition temperature, ELPs are in a dissolved state. When the external temperature is higher than the phase transition temperature, ELPs automatically aggregate and precipitate from the solution to form a precipitate. This characteristic is called phase transition characteristics and has been widely used in tissue engineering materials, drug targeted delivery and protein purification research. ArgGlyAsp (RGD) has the effect of promoting cell adhesion and division, and ArgGluAspVal (REDV) has the effect of endothelial cell adhesion. These two short peptides have also been used in recombinant protein biomaterials. CN113520899A discloses a recombinant collagen product for repairing skin photodamage, and discloses the amino acid sequence of the recombinant collagen. The recombinant collagen has a good repairing effect on UV-damaged skin, but has a high cost and a low expression level. Summary of the invention

[0005] Human natural elastin usually has more than 700 amino acids. Studies have shown that hexapeptides rich in ValGlyValAlaProGly (VGVAPG) not only have chemotactic activity on monocytes, fibroblasts and tumor cells, but also activate metalloproteinases. Therefore, VGVAPG oligopeptides have been widely used as cosmetic and skin care additives.

[0006] The present invention selects a complete structural unit of human elastin containing VGVAPG, Gln Phe Gly Leu Val Pro GlyVal Gly Val AlaProGly Val Gly Val Ala Pro Gly ValGly Val Pro GlyVal Gly LeuAlaProGly Val Gly Val Ala Pro Gly Val Gly ValAlaPro GlyVal Gly Val AlaPro Gly Ile Gly Pro Gly Gly,

[0007] (QFGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVA PGVGVAPGIGPGG) is recorded as SEQ ID No.1, and RGD and REDV short peptide amino acid sequences are fused at the C-terminus, and a VPGIG sequence of elastin is fused at the N-terminus. The gene encoding the sequence is constructed on the pET26b (+) expression vector after chemical synthesis, and a large amount of soluble expression can be used in Escherichia coli to establish a method for efficiently and quickly producing a recombinant protein with the ability to delay skin ultraviolet damage. It is used for separation and purification, taking into account mechanical properties. Through genetic engineering technology, the gene encoding the recombinant protein sequence is synthesized by a chemical synthesis method and cloned into an Escherichia coli expression vector, and can be mass-produced in Escherichia coli. The recombinant protein can be initially separated and purified by reversible phase change characteristics. It has been verified by nude mice that it has a good effect of delaying skin ultraviolet damage.

[0008] The purpose of the present invention is to provide a recombinant elastin-like protein having the effect of delaying skin ultraviolet damage, the sequence of which is shown in SEQ ID No. 2:

[0009] MAGLTRGDGGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGV

[0010] PGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGI

[0011] GVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVP

[0012] GIGVPGIGVPGIGVPGIGGSNNNNNNNNNNQFGLVPGVGVAPGV

[0013] GVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGGGVREDVREDVHHHHHH.

[0014] The invention also provides an expression vector pET26b-ELP30 containing the gene encoding the recombinant elastin-like protein.

[0015] The present invention also provides a genetically engineered bacterium with a recombinant elastin-like gene capable of delaying skin ultraviolet damage, comprising the plasmid pET26b-ELP30.

[0016] Furthermore, the genetically engineered bacteria is Escherichia coli.

[0017] The present invention also provides a method for preparing a recombinant elastin-like protein having the effect of delaying skin ultraviolet damage, comprising the following steps: transforming the expression vector into competent cells of Escherichia coli BL21 (DE3), culturing to obtain genetically engineered bacteria, and inducing, expressing and purifying the genetically engineered bacteria to prepare the recombinant elastin-like protein.

[0018] The specific steps include:

[0019] (1) According to the designed recombinant protein sequence and the codon preference of Escherichia coli, the gene encoding the corresponding protein sequence is synthesized and cloned into Figure 1 The expression vector pET26b(+) was shown and named pET26b-ELP30;

[0020] (2) transferring it into a host bacterium, selecting positive monoclones by using kanamycin resistance, collecting the supernatant by centrifugation after induction culture, filtering and purifying to obtain recombinant elastin-like protein, and optimizing protein purification by using the temperature-reversible phase transition characteristics;

[0021] The specific operation of step (2) is as follows:

[0022] Positive monoclonal strains were picked and inoculated into 3 mL LB medium containing Kan (final concentration 50 μg / mL) for shaking culture. The next day, they were inoculated into 400 mL 2 L conical flasks containing automatic induction medium with a final Kan concentration of 50 μg / mL at a ratio of 1:100. After induction expression at 25°C for 24 h, the strains were centrifuged at 8000 rpm for 10 min at 4°C. The bacteria were collected in 50 mL centrifuge tubes and dissolved with 0.2 mol / L salt-free PBS according to the bacterial weight. The strains were stored at -80°C for purification.

[0023] Furthermore, the specific operation of optimizing the purification conditions in step (2) is:

[0024] I. Freeze and thaw the bacterial solution stored at -80℃ at 37℃ and -20℃ twice to completely dissolve the bacteria;

[0025] II. Add lysozyme to a final concentration of 50 μg / mL and place in a 37°C water bath for 30 min to allow the lysozyme to function;

[0026] III. After icing for a while, ultrasonic crushing was carried out in an ice bath for 10 min (conditions: ultrasonic 2s, stop 3s, power 15%);

[0027] IV. Place on ice for 15 minutes, centrifuge at 10,000 g for 10 minutes in a 4°C low-temperature centrifuge, discard the precipitate, and transfer the supernatant to a new tube;

[0028] V. Add DNase to a final concentration of 50 μg / mL and place in an ice bath for 20 minutes;

[0029] VI. Centrifuge at 10,000 g for 10 min in a 4°C low-temperature centrifuge, discard the precipitate, and transfer the supernatant to a new tube;

[0030] VII. Repeat the above steps several times until there is no obvious sediment after centrifugation;

[0031] VIII. Add NaCl solid to the supernatant to a final concentration of 0.5 mol / L, incubate in a 30°C water bath for at least 20 min (until all NaCl is dissolved; the purer the protein, the shorter the water bath time), centrifuge at 30°C and 10,000 g for 10 min, and discard the supernatant;

[0032] IX. Resuspend the pellet in 20 mL of 0.02 mol / L salt-free PBS, place on ice for 15 min, centrifuge at 10,000 g for 10 min at 4 °C, discard the pellet and transfer the supernatant to a new tube;

[0033] X. Add NaCl solid to the supernatant to a final concentration of 0.5 mol / L, incubate in a 30°C water bath for 15 min, centrifuge at 30°C and 10,000 g for 10 min, and discard the supernatant;

[0034] XI. Resuspend the pellet in 20 mL of 0.02 mol / L salt-free PBS, place on ice for 15 min, centrifuge at 10,000 g for 10 min in a 4°C cold centrifuge, discard the pellet and transfer the supernatant to a new tube;

[0035] XII. Add NaCl solid to the supernatant to a final concentration of 0.5 mol / L, incubate in a 30°C water bath for 10 min, centrifuge at 10000 g for 10 min at 30°C, and discard the supernatant;

[0036] XIII. Resuspend the precipitate in 2-5 mL 0.02 mol / L salt-free PBS, place on ice for 15 min, centrifuge at 10,000 g for 8 min in a 4°C low-temperature centrifuge, and transfer the supernatant into new centrifuge tubes for storage.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention designs an elastin-like protein, comprising a human elastin protein rich in VGVAPG sequence, RGD and REDV sequence, and fused with a VPGIG sequence at the N-terminus of the sequence, and constructing the gene encoding the sequence on a pET26b (+) expression vector through chemical synthesis, and can be expressed in large quantities by Escherichia coli, thereby establishing a method for efficiently and quickly producing a recombinant elastin-like protein with the function of delaying ultraviolet damage to the skin. The recombinant elastin-like protein can be initially separated and purified through the reversible phase change characteristic.

[0039] The recombinant elastin prepared by the present invention utilizes the non-chromatographic separation and purification technology of human-like elastin, so that it has the advantages of low cost, high expression, and easy large-scale production. During the preparation process, the yield is increased by reducing the salt concentration, because high concentration of salt has a negative impact on enzyme activity. It can be applied to the research and development of related recombinant protein biological products, production and quality evaluation system establishment, and the expression product has the function of delaying aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a map of the recombinant elastin-like protein expression vector;

[0041] Figure 2 This is a comparison chart of changes in skin tissue structure detected by HE;

[0042] Figure 3 This is the distribution and morphology of collagen fibers;

[0043] Figure 4 This is a diagram showing the distribution and morphological observation results of elastic fibers. DETAILED DESCRIPTION

[0044] The present invention is further illustrated by the following examples, but is not intended to be limiting of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. The sources or compositions of the materials involved in each example are as follows:

[0045] The formula of LB liquid culture medium is as follows: tryptone: 4 g; yeast extract: 2 g; sodium chloride 4 g; RO water is added to make up to 400 mL.

[0046] The formula of LB solid culture medium is as follows: agar powder: 4-5 g; tryptone: 3 g; yeast extract: 1.5 g; sodium chloride (NaCl): 3 g; RO water is added to make up to 300 mL.

[0047] The auto-induction medium formula is as follows:

[0048] 20×NPS: disodium hydrogen phosphate (Na2HPO4) 22.736 g, dipotassium hydrogen phosphate (K2HPO4) 6.8045 g; adjust pH to about 7.0-7.2; ammonium sulfate ((NH4)2SO4): volume × 0.5 × 132.14.

[0049] 1MMgSO4: 4.81g magnesium sulfate (MgSO4); RO water to 40mL;

[0050] 50×5052: 12.5g glycerol; 1.25g glucose: 5g; α-lactose; dilute to 50mL with RO water.

[0051] ZY medium: Tryptone: 4 g; Yeast powder: 2 g; RO water to 400 mL.

[0052] Example 1

[0053] The method for constructing the recombinant elastin-like protein expression vector comprises the following steps:

[0054] Firstly, the gene sequence encoding the recombinant elastin-like protein was designed and artificially synthesized: MAGLTRGDGGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGGSNNNNNNNNNNQFGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGVGVAPGIGPGGGVREDVREDVHHHHHH. Amino acids: M: methionine; A: alanine; G: glycine; L: leucine; T: threonine; R: arginine; D: aspartic acid; V: valine; P: proline; I: isoleucine; S: serine; N: asparagine; Q: glutamine; F: phenylalanine; E: glutamic acid; H: histidine. Plasmid pET26b-ELP30 see Figure 1 , which carries sequences encoding VGVAPG, RGD and REDV, and carries a gene sequence encoding a tag that can be used for reversible purification of elastin.

[0055] Using the XhoI and NdeI restriction sites, the target gene-ELP tag gene fragment was connected to the expression vector to form a recombinant expression vector pET26b-ELP30.

[0056] These two restriction sites are designed according to the expression requirements of the vector pET-26b, but are not limited to these two restriction sites. Other restriction site sequences can be designed according to the restriction sites required for connection to different vectors to construct different expression vectors.

[0057] Example 2: Expression and purification methods

[0058] 2.1: Transformation of E. coli BL21 (DE3) competent cells: Slowly thaw the competent bacteria on ice (freshly prepared competent cells can be used directly), add 2-2.5 μL of plasmid, gently flick the centrifuge tube with your fingers to mix the bacteria and plasmid ligation product, place on ice for 20 minutes, heat shock in a 42°C water bath for 80 seconds, add 200 μL SOC, culture in a constant temperature shaker at 37°C and 220 rpm for half an hour, and then shake in a constant temperature shaker at 37°C and 220 rpm for one hour. Resuspend the supernatant and mix well, then spread it on an LB plate containing kanamycin resistance, and culture it upside down at 37°C overnight.

[0059] 2.2: The culture and expression conditions described in the present invention are as follows: Pick the positive monoclonal strain and inoculate it into 3mL LB medium containing Kan (final concentration 50μg / mL) for shaking culture. The next day, inoculate it into 400mL of 2L conical flask containing automatic induction medium with a final Kan concentration of 50μg / mL at a volume ratio of 1:100, and culture it. After inducing expression at 25℃ for 24h, centrifuge at 8000rpm4℃ for 10min, collect the bacteria in a 50mL centrifuge tube, dissolve it with 0.2mol / L salt-free PBS according to the weight of the bacteria, and store it at -80℃ for purification.

[0060] 2.3: Purify the bacterial solution:

[0061] I. Freeze and thaw the bacterial solution stored at -80℃ at 37℃ and -20℃ twice to completely dissolve the bacteria;

[0062] II. Add lysozyme to a final concentration of 50 μg / mL and place in a 37°C water bath for 30 min to allow the lysozyme to function;

[0063] III. After icing for a while, ultrasonic crushing was carried out in an ice bath for 10 min (conditions: ultrasonic 2s, stop 3s, power 15%);

[0064] IV. Place on ice for 15 minutes, centrifuge at 10,000 g for 10 minutes in a 4°C low-temperature centrifuge, discard the precipitate, and transfer the supernatant to a new tube;

[0065] V. Add DNase to a final concentration of 50 μg / mL and place in an ice bath for 20 minutes;

[0066] VI. Centrifuge at 10,000 g for 10 min in a 4°C low-temperature centrifuge, discard the precipitate, and transfer the supernatant to a new tube;

[0067] VII. Repeat the above steps several times until there is no obvious sediment after centrifugation;

[0068] VIII. Add NaCl solid to the supernatant to a final concentration of 0.5 mol / L, incubate in a 30°C water bath for at least 20 min (until all NaCl is dissolved; the purer the protein, the shorter the water bath time), centrifuge at 30°C and 10,000 g for 10 min, and discard the supernatant;

[0069] IX. Resuspend the pellet in 20 mL of 0.02 mol / L salt-free PBS, place on ice for 15 min, centrifuge at 10,000 g for 10 min at 4 °C, discard the pellet and transfer the supernatant to a new tube;

[0070] X. Add NaCl solid to the supernatant to a final concentration of 0.5 mol / L, incubate in a 30°C water bath for 15 min, centrifuge at 30°C and 10,000 g for 10 min, and discard the supernatant;

[0071] XI. Resuspend the pellet in 20 mL of 0.02 mol / L salt-free PBS, place on ice for 15 min, centrifuge at 10,000 g for 10 min in a 4°C cold centrifuge, discard the pellet and transfer the supernatant to a new tube;

[0072] XII. Add NaCl solid to the supernatant to a final concentration of 0.5 mol / L, incubate in a 30°C water bath for 10 min, centrifuge at 10000 g for 10 min at 30°C, and discard the supernatant;

[0073] XIII. Resuspend the precipitate in 2-5 mL 0.02 mol / L salt-free PBS, place on ice for 15 min, centrifuge at 10,000 g for 8 min in a 4°C low-temperature centrifuge, and transfer the supernatant into new centrifuge tubes for storage.

[0074] 2.4: SDS-PAGE electrophoresis identification

[0075] Example 3: Effects of different salt concentrations on elastin-like proteins

[0076] I. Freeze and thaw the bacterial solution stored at -80℃ at 37℃ and -20℃ twice to completely dissolve the bacteria;

[0077] II. Add lysozyme to a final concentration of 50 μg / mL and place in a 37°C water bath for 30 min to allow the lysozyme to function;

[0078] III. After icing for a while, ultrasonic crushing was carried out in an ice bath for 10 min (conditions: ultrasonic 2s, stop 3s, power 15%);

[0079] IV. Place on ice for 15 minutes, centrifuge at 10,000 g for 10 minutes in a 4°C low-temperature centrifuge, discard the precipitate, and transfer the supernatant to a new tube;

[0080] V. Add DNase to a final concentration of 50 μg / mL and place in an ice bath for 20 minutes;

[0081] VI. Centrifuge at 10,000 g for 10 min in a 4°C low-temperature centrifuge, discard the precipitate, and transfer the supernatant to a new tube;

[0082] VII. Repeat the above steps several times until there is no obvious sediment after centrifugation;

[0083] VIII. Add NaCl solid to the supernatant to a final concentration of 2 mol / L, 1.5 mol / L, 1 mol / L, 0.5 mol / L, and 0.3 mol / L, and incubate in a 30°C water bath for at least 20 min (until all NaCl is dissolved; the purer the protein, the shorter the water bath time), centrifuge at 30°C and 10,000 g for 10 min, and discard the supernatant;

[0084] IX. Resuspend the pellet in 20 mL of 0.02 mol / L salt-free PBS, place on ice for 15 min, centrifuge at 10,000 g for 10 min at 4 °C, discard the pellet and transfer the supernatant to a new tube;

[0085] X. Add NaCl solid to the supernatant to a final concentration of 2 mol / L, 1.5 mol / L, 1 mol / L, 0.5 mol / L, and 0.3 mol / L, incubate in a 30°C water bath for 15 min, centrifuge at 30°C and 10,000 g for 10 min, and discard the supernatant;

[0086] XI. Resuspend the pellet in 20 mL of 0.02 mol / L salt-free PBS, place on ice for 15 min, centrifuge at 10,000 g for 10 min in a 4°C cold centrifuge, discard the pellet and transfer the supernatant to a new tube;

[0087] XII. Add NaCl solid to the supernatant to a final concentration of 2 mol / L, 1.5 mol / L, 1 mol / L, 0.5 mol / L, and 0.3 mol / L, incubate in a 30°C water bath for 10 min, centrifuge at 10000 g for 10 min at 30°C, and discard the supernatant;

[0088] XIII. Resuspend the precipitate in 2-5 mL 0.02 mol / L salt-free PBS, place on ice for 15 min, centrifuge at 10,000 g for 8 min in a 4°C low-temperature centrifuge, and transfer the supernatant into new centrifuge tubes for storage.

[0089] Example 4 Construction of skin photoaging damage model

[0090] Six-week-old female nude mice were selected as experimental animals and divided into five groups, with six mice in each group. Photoaging modeling was performed, and 20W UVB narrow-wave ultraviolet lamps were used for daily irradiation. The limbs of the nude mice in the model group were fixed, and important body parts were physically protected, with only the back skin exposed. The light source was about 30cm away from the irradiated skin. Irradiation was performed for 10 minutes every day, and samples were collected after 6W of irradiation. In the fourth week of irradiation, it was seen that the back skin showed obvious roughness, irregular thickening, partial shallow wrinkles, pigmentation, etc. In the sixth week of irradiation, the skin elasticity was significantly reduced after ultraviolet irradiation, and the photoaging modeling of nude mice was successful.

[0091] Example 5 Detection of the effect of resisting photoaging damage

[0092] 1. Sampling: Nude mice were anesthetized with 5% sodium pentobarbital and 1 cm thick tissue was removed from the back using ophthalmic scissors. 2 x1cm 2 Skin tissue

[0093] 2. Fixation: With the help of scissors and tweezers, spread the skin on a small piece of filter paper and quickly place it in a six-well plate pre-added with a lot of formaldehyde, and fix it overnight in the dark.

[0094] 3. Flushing: Take out the fixed tissue block and put it into a plastic embedding box, string it with a rope and put it into a container. Move the container under the faucet and flush it with low-speed water for one day.

[0095] 4. Dehydration: Use 75%, 85%, 95%, and 100% ethanol concentrations for dehydration for 2 minutes respectively.

[0096] 5. Transparency: Prepare a 1:1 solution of ethanol and xylene for 45 minutes, xylene 1 for 30 minutes, and xylene 2 for 30 minutes.

[0097] 6. Wax immersion: Paraffin: xylene immersion for 30 minutes, paraffin 1 for 30 minutes, paraffin 2 for 2 hours, paraffin 3 for 2 hours.

[0098] 7. Embedding: First add a little melted wax to the embedding lead box, then quickly put the trimmed tissue block in, adjust the tissue position, and then fill the lead box with wax. After embedding, move it aside to cool, wait for the wax block to completely solidify, and store it in a -20℃ refrigerator.

[0099] 8. Slicing: Take the lead box out of the refrigerator before slicing. The frozen wax block can be easily separated from the lead box. Slightly trim the wax block and then slice it using a microtome. Control the thickness of the slice to 4-5μm.

[0100] 9. Spreading: Spread the paraffin thin slices in a 42℃ water bath of the spreading machine.

[0101] 10. Baking: Pick up the slices with a glass slide, absorb excess moisture with filter paper, and place them on a baking sheet to dry at 62℃ for 2 hours.

[0102] 11. HE staining:

[0103] (1) Dewaxing of paraffin sections to water: Place the sections into an environmentally friendly dewaxing solution in turn.

[0104] Ⅰ20min—Environmentally friendly dewaxing liquid Ⅱ20min—Anhydrous ethanol Ⅰ5min—Anhydrous ethanol

[0105] Ⅱ5min—75% alcohol for 5min, wash with tap water.

[0106] (2) Pretreatment: The sections were treated in high-definition constant stain pretreatment solution for 1 min.

[0107] (3) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0108] (4) Eosin staining: Dehydrate the sections in 95% alcohol for 1 min and then stain them in eosin solution for 15 s.

[0109] (5) Dehydration and sealing: The sections were placed in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, and xylene II for 2 min, and then sealed with transparent neutral gum.

[0110] 12.Masson staining:

[0111] (1) The operation steps are the same as the first step of HE staining.

[0112] (2) Soak the slices in Masson A solution overnight and rinse with running water.

[0113] (3) Slice into a dye solution consisting of a mixture of Masson B and Masson C in equal proportions for 1 min, rinse with tap water, differentiate with differentiation solution for a few seconds, and rinse with tap water.

[0114] (4) Immerse the sections in Masson D solution for 6 min and rinse with tap water.

[0115] (5) Dye with Masson E solution for 1 min.

[0116] (6) Without washing, drain slightly and dye directly in Masson F solution for 2-30 seconds.

[0117] (7) The sections were rinsed and differentiated with 1% acetic acid and dehydrated with two cylinders of anhydrous ethanol.

[0118] (8) Transparent sealing: Put the slices into the third cylinder of anhydrous ethanol for 5 minutes, xylene for 5 minutes to make them transparent, and then seal them with neutral gum.

[0119] 13.Weigert staining

[0120] (1) The operation steps are the same as the first step of HE staining.

[0121] (2) Resorcinol alkaline fuchsin staining: Immerse the sections in resorcinol fuchsin stain solution A and cover. Incubate at room temperature for 4 hours. Remove the sections and rinse with running water until the water on the slide turns colorless.

[0122] (3) Background differentiation: The slices were placed in 1% hydrochloric acid alcohol differentiation solution for rapid differentiation for 2 seconds, washed with tap water for 5 seconds, and examined under a microscope. Rapid differentiation, water washing, and microscopic examination can be repeated until clear purple elastic fibers are observed and the background is light purple to almost colorless.

[0123] (4) Re-staining VG: 9 mL of resorcinol basic fuchsin dye solution B was added with 1 mL of resorcinol basic fuchsin dye solution C to make VG dye solution (prepare the required amount according to the ratio), dye for 1 min, quickly wash with water, and quickly dehydrate in three cylinders of anhydrous ethanol for 3 s, 5 s, and 30 s, respectively.

[0124] (5) Transparent sealing: Use two cylinders of clean xylene for 20 seconds and 5 minutes respectively (xylene is dedicated to xylene and should not be used with other xylenes), and then seal the slides with neutral gum.

[0125] 14. Observation of results

[0126] (1) Observation of HE epidermal tissue structure

[0127] The common histopathological changes of HE in photoaged skin are an increase in the number of epidermal cell layers with unclear stratification and incomplete structure; disappearance of dermal papillae; disordered arrangement of dermal tissue, dilated and congested blood vessels, and thickened vessel walls; accompanied by dilated hair follicles, atrophy of sebaceous glands, increased glands, and disordered arrangement; infiltration of inflammatory cells, etc.

[0128] (2) Masson test: Observation of the distribution and morphology of collagen fibers

[0129] The blue collagen fibers under photoaged skin often show abnormal changes such as sparse distribution, significantly reduced number, disordered arrangement, and breakage.

[0130] (3) Weigert test: observation of the distribution and morphology of elastic fibers

[0131] The slice results showed that the elastic fibers were blue-black, the cell nuclei were blue, and the collagen fibers were red-purple. In the photoaging model, the blue-black elastic fibers increased, became thicker, and twisted.

[0132] The embodiments of the present invention are not limited to this. According to the above contents of the present invention, according to the common technical knowledge and common methods in the art, without departing from the above basic technical ideas of the present invention, the present invention may also have other embodiments. For example, a variety of recombinant elastin proteins may be used for expression and application. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A recombinant elastin-like protein having the effect of delaying skin ultraviolet damage, characterized in that: The amino acid sequence is shown in SEQ ID No. 2: MAGLTRGDGGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGV PGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGGSNNNNNNNNNNQFGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGGGVREDVREDVHHHHHH.

2. The recombinant elastin-like protein having the effect of delaying skin ultraviolet damage according to claim 1, characterized in that: The plasmid pET26b-ELP30 contains the gene encoding the recombinant elastin.

3. The recombinant elastin-like protein having the effect of delaying skin ultraviolet damage according to claim 2, characterized in that: A genetically engineered bacterium having a recombinant protein gene capable of delaying skin ultraviolet damage, and a plasmid containing the gene.

4. The recombinant elastin-like protein having the effect of delaying skin ultraviolet damage according to claim 3, characterized in that: The genetically engineered bacteria is Escherichia coli.

5. A method for preparing a recombinant elastin-like protein having the effect of delaying skin ultraviolet damage, characterized in that: The following steps are involved: The expression vector is transformed into competent cells of Escherichia coli BL21 (DE3), and genetically engineered bacteria are obtained after culture. The genetically engineered bacteria are induced, expressed, and purified to prepare recombinant elastin.

6. The method for preparing a recombinant elastin containing the effect of delaying skin ultraviolet damage according to claim 5, characterized in that: The specific steps include: (1) constructing the expression vector pET26b-ELP30 as shown in FIG. 1 ; (2) The protein was transferred into the host bacteria, and positive monoclones were selected by using kanamycin resistance. After induction culture, the supernatant was collected by centrifugation, and the recombinant elastin was obtained by filtration and purification. The temperature-reversible phase transition characteristics were used to optimize the protein purification.

7. The method for preparing a recombinant elastin-like protein having the effect of delaying skin ultraviolet damage according to claim 6, characterized in that: The specific operation of step (2) is: Positive monoclonal strains were picked and inoculated into 3 mL LB medium containing Kan (final concentration 50 μg / mL) for shaking culture. The next day, they were inoculated into 400 mL 2 L conical flasks containing automatic induction medium with a final Kan concentration of 50 μg / mL at a ratio of 1:

100. After induction expression at 25°C for 24 h, the strains were centrifuged at 8000 rpm for 10 min at 4°C. The bacteria were collected in 50 mL centrifuge tubes and dissolved with 0.2 mol / L salt-free PBS according to the bacterial weight. The strains were stored at -80°C for purification.

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