Preparation method and application of elastin-like peptide and elastin-like peptide underwater adhesive

Abstract

According to the mussel / barnacle bionic principle, the elastin-like peptide is prepared by alternately introducing lysine residues and tyrosine residues / phenylalanine residues, designing and constructing a target sequence, inserting the target sequence into a plasmid vector and expressing through a host cell. In addition, the invention also discloses a method for preparing an underwater adhesive by adopting the elastin peptide and application of the underwater adhesive. The elastin-like peptide underwater adhesive is obtained by designing and expressing elastin-like protein and combining with methacrylated recombinant collagen to improve the biocompatibility of the elastin-like peptide, and combining with nano filler tannic acid nano cellulose crystals to enhance the adhesion and mechanical properties. And biomedical applications such as hemostasis, tissue regeneration and the like are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a preparation method and application of an elastin-like peptide and an elastin-like peptide underwater adhesive. Background Art

[0002] When adhesives are used in biomedical applications, there are the following problems. First, the curing time is long. Some underwater adhesives require a long curing time to achieve sufficient adhesion strength, which may result in failure to function in time in emergency hemostasis situations. Second, the bonding strength is limited. The bonding performance of adhesives is significantly reduced under wet conditions. The complexity and particularity of the wet environment of body fluids severely limit the widespread development and clinical application of adhesives. This may result in the adhesive being unable to maintain sufficient adhesion. Third, the biocompatibility is poor. Some adhesives may cause irritation or allergic reactions to human tissues, affecting wound healing. Therefore, developing adhesives that can be used underwater and produce strong bonds is a major challenge facing materials engineering.

[0003] To overcome the above difficulties, with the continuous development of the understanding of the natural adhesion mechanism and key elements of aquatic organisms, a variety of medical adhesives have been developed by imitating the adhesion process or utilizing key functional groups, mainly used for clinical needs such as tissue repair, wound dressings and tissue adhesion. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing an elastin-like peptide and an elastin-like peptide underwater adhesive in view of the above-mentioned deficiencies in the prior art. The elastin-like peptide is inspired by the excellent adhesion properties of marine mussel adhesive protein, and the target sequence is designed to be used in pET28 (a + ) is a plasmid vector, the target sequence is inserted into the NcoI and XhoI restriction sites, host cells are selected for expression, and the elastin-like peptide obtained after salting out and isoelectric point purification has excellent underwater adhesion ability.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: an elastin-like peptide, characterized in that the elastin-like peptide utilizes the bionic principle of mussels or barnacles, by alternately introducing lysine residues and tyrosine residues, or lysine residues and phenylalanine residues, designing and constructing a target sequence, inserting the target sequence into a plasmid vector, and expressing it through a host cell.

[0006] The above-mentioned elastin-like peptide is characterized in that the target sequence is composed of a short amino acid sequence of a natural elastin peptide repeated as a repeating unit; the target sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the number of repetitions is 16 times.

[0007] The above-mentioned elastin-like peptide is characterized in that the plasmid vector is pET28 (a + ), inserting the target sequence into the NcoI and XhoI restriction sites, and the host cell is Escherichia coli.

[0008] Furthermore, the present invention provides a method for preparing an elastin-like peptide underwater adhesive using the above-mentioned elastin-like peptide, characterized in that it comprises the following steps:

[0009] Step 1, dissolving the elastin-like peptide and the photoinitiator in deionized water under light-proof conditions to obtain liquid A; dissolving the methacrylylated recombinant collagen in deionized water under water bath conditions to obtain liquid B; dissolving the tannic acid nanocellulose crystals in deionized water to obtain liquid C;

[0010] Step 2: Mix the liquid A, liquid B and liquid C in step 1 to obtain a gel solution.

[0011] The above method is characterized in that it also includes irradiating the gel solution under 365nm light conditions for 0.5min to 1min, and then sterilizing it by Co60 irradiation to obtain an elastin peptide underwater adhesive.

[0012] The above method is characterized in that the preparation method of the methacryloyl recombinant collagen described in step 1 specifically includes: adding glycidyl methacrylate to the recombinant collagen solution, stirring and reacting in a 37°C water bath for 6h to 8h, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen.

[0013] The above method is characterized in that the recombinant collagen solution is a recombinant collagen solution obtained by dissolving recombinant collagen in deionized water, the mass-volume concentration of the recombinant collagen solution is 1% to 2%; the relative molecular mass of the recombinant collagen is 97KDa.

[0014] The above method is characterized in that the preparation method of tannic acid nanocellulose crystals in step 1 specifically includes: adding tannic acid to a nanocellulose crystal solution with a mass-volume concentration of 1% to 2%, stirring and reacting at room temperature for 6h to 8h, dialyzing, and freeze-drying to obtain tannic acid nanocellulose crystals; the mass ratio of tannic acid to nanocellulose crystals is 1:(2 to 3).

[0015] The above method is characterized in that the mass percentage of elastin-like peptide in the gel solution is 0.2% to 0.6%, the mass percentage of methacrylylated recombinant collagen is 5% to 10%, the mass percentage of photoinitiator is 0.1% to 0.25%, and the mass percentage of tannic acid nanocellulose crystals is 5% to 10%.

[0016] Furthermore, the present invention provides a use of the elastin-like peptide underwater adhesive prepared by the above method in the preparation of medical devices for hemostasis and / or tissue regeneration.

[0017] The present invention is a biomimetic mussel / barnacle designed elastin peptide, lysine and tyrosine residues / phenylalanine residues are introduced to prepare elastin peptide, and then free radical polymerization is initiated by introducing double-bond modified collagen, and then nano fillers or cross-linking agents are added to make the adhesive have high adhesion and mechanical flexibility. In addition, the prepared adhesive has good biocompatibility and is non-toxic and harmless. Rapid adhesion is produced with tissue through hydrogen bonds at the wound, and under the catalytic action of tyrosinase, covalent connection is gradually formed with tissue, and stronger double bond action also enables the adhesive prepared by the present invention to achieve stable wet adhesion. It has good application prospects in biomedical applications such as hemostasis and tissue regeneration.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. The present invention is inspired by the excellent adhesion properties of marine mussel / barnacle adhesion proteins, designs the target sequence, and uses pET28 (a + ) is a plasmid vector, the target sequence is inserted into the NcoI and XhoI restriction sites, host cells are selected for expression, and the elastin-like peptide obtained after salting out and isoelectric point purification has excellent underwater adhesion ability.

[0020] 2. The present invention utilizes elastin-like peptides to cross-link methacrylylated recombinant collagen and tannic acid-coated cellulose nanocrystals (TA@CNC). The photosensitivity of the recombinant collagen modified with glycidyl methacrylate can effectively promote the UV curing process of the adhesive and improve the stability of the adhesive. The tannic acid nanocellulose crystals have high adhesion and mechanical flexibility. The obtained adhesive has the advantages of underwater adhesion, good biocompatibility, and easy use, which is beneficial to biomedical applications such as hemostasis and tissue regeneration.

[0021] 3. The preparation method of the present invention has reliable principle and is conducive to popularization and application.

[0022] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 For pET28 (a + )Original plasmid.

[0024] Figure 2 The elastin-like peptide pET28 (a + )-ELP-K2YV recombinant plasmid.

[0025] Figure 3 This is the protein expression electrophoresis diagram of Example 1 of the present invention.

[0026] Figure 4 This is the electrophoresis diagram of protein purification in Example 1 of the present invention.

[0027] Figure 5 The methacryloyl recombinant collagen prepared in Example 2 of the present invention, wherein a is a schematic diagram of the gelation of the methacryloyl recombinant collagen after ultraviolet light irradiation under the action of a photoinitiator; b is a Fourier transform infrared absorption spectrum analysis diagram of the methacryloyl recombinant collagen; c is a Fourier transform infrared absorption spectrum analysis diagram of the methacryloyl recombinant collagen 1 H-NMR analysis results.

[0028] Figure 6 Schematic diagram of nanocellulose crystals and tannic acid nanocellulose crystals of Example 3 of the present invention, wherein a is a solution image of nanocellulose crystals (CNC); b is a solution image of tannic acid nanocellulose crystals (TA@CNC); c is an atomic force microscope image of nanocellulose crystals (CNC); d is an atomic force microscope image of tannic acid nanocellulose crystals (TA@CNC); e is a scanning electron microscope image of nanocellulose crystals (CNC); f is a scanning electron microscope image of tannic acid nanocellulose crystals (TA@CNC); g is a Fourier transform infrared absorption spectrum analysis image of tannic acid nanocellulose crystals (TA@CNC).

[0029] Figure 7 This is a test of the adhesion performance of the elastin-like peptide underwater adhesive prepared in Example 4 of the present invention, wherein a is a schematic diagram of the adhesion between the adhesive and pig skin; and b is a schematic diagram of the adhesion performance test.

[0030] Figure 8 This is a schematic diagram of the cytotoxicity test results of the elastin-like peptide underwater adhesive prepared in Example 4 of the present invention. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0032] The elastin-like peptide of the present invention utilizes the principle of mussel / barnacle bionics to design and construct a target sequence by alternately introducing lysine residues and tyrosine residues, or lysine residues and phenylalanine residues, inserting the target sequence into a plasmid vector, and expressing it through a host cell; the target sequence is composed of a short amino acid sequence of a natural elastin peptide as a repeating unit; the target sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the number of repetitions is 16; the plasmid vector is pET28 (a + ), inserting the target sequence into the NcoI and XhoI restriction sites, and the host cell is Escherichia coli.

[0033] The method for preparing the elastin-like peptide underwater adhesive of the present invention comprises the following steps:

[0034] Step 1, dissolving the elastin-like peptide and the photoinitiator in deionized water under light-proof conditions to obtain liquid A; dissolving the methacrylylated recombinant collagen in deionized water under water bath conditions to obtain liquid B; dissolving the tannic acid nanocellulose crystals in deionized water to obtain liquid C;

[0035] The preparation method of the methacryloyl recombinant collagen specifically comprises: adding glycidyl methacrylate to a recombinant collagen solution, stirring in a water bath at 37° C. for a reaction of 6 h to 8 h, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen; the recombinant collagen solution is a recombinant collagen solution obtained by dissolving recombinant collagen in deionized water, and the mass-volume concentration of the recombinant collagen solution is 1% to 2%; the relative molecular mass of the recombinant collagen is 97 KDa;

[0036] The preparation method of the tannic acid nanocellulose crystals specifically comprises: adding tannic acid to a nanocellulose crystal solution with a mass-volume concentration of 1% to 2%, wherein the mass ratio of tannic acid to nanocellulose crystals is 1:(2-3), stirring and reacting at room temperature for 6h to 8h, dialyzing, and freeze-drying to obtain the tannic acid nanocellulose crystals;

[0037] Step 2, mixing the liquid A, liquid B and liquid C in step 1 to obtain a gel solution; the mass percentage of elastin-like peptide in the gel solution is 0.2% to 0.6%, the mass percentage of methacryloyl recombinant collagen is 5%, the mass percentage of photoinitiator is 0.1% to 0.25%, and the content of tannic acid nanocellulose crystals is 5% to 10%;

[0038] Step 3: Irradiate the gel solution in step 2 under 365 nm light conditions for 0.5 min to 1 min, and then sterilize by Co60 irradiation to obtain an elastin peptide underwater adhesive.

[0039] The elastin-like peptide underwater adhesive of the present invention can be used to prepare medical devices for hemostasis and / or tissue regeneration.

[0040] Example 1

[0041] This embodiment provides an elastin-like peptide, which utilizes the principle of mussel biomimetic to design and construct a target sequence by alternately introducing lysine residues and tyrosine residues, inserting the target sequence into a plasmid vector, and expressing it through a host cell; the target sequence is composed of a short amino acid sequence of a natural elastin peptide as a repeating unit; the target sequence is shown in SEQ ID NO: 1, and the number of repetitions is 16; the plasmid vector is pET28 (a + ), inserting the target sequence into the NcoI and XhoI restriction sites, and the host cell is Escherichia coli.

[0042] The specific preparation method includes:

[0043] Step 1: Use pET28 (a + ) is a plasmid vector, the target sequence SEQ ID NO: 1 is inserted into the NcoI and XhoI restriction sites, and Escherichia coli is selected as the host cell for expression;

[0044] Step 2, collecting the protein solution and preliminarily purifying it by isoelectric point method; the preliminarily purifying by isoelectric point method is to adjust the pH of the protein solution to 2-11 by gradient, determine the appropriate pH according to the result of protein electrophoresis, and then collect the supernatant solution by centrifugation at 12000 rpm for 10 min;

[0045] Step 3, collecting the supernatant solution of step 2, and further purifying it by salting out method; the further purification by salting out method is to add different concentrations of ammonium sulfate to the supernatant solution, the ammonium sulfate concentration is 10% to 100%, and the appropriate ammonium sulfate concentration is determined according to the result of protein electrophoresis, and then centrifuging at 12000rpm for 10min to collect the precipitated product;

[0046] Step 4: The collected precipitate product is dialyzed in deionized water for 3 consecutive days to remove salt, and freeze-dried to obtain elastin-like peptide; the dialysis bag has a molecular weight cutoff of 3500Da.

[0047] Example 2

[0048] This embodiment provides a method for preparing methacrylylated recombinant collagen, which specifically includes:

[0049] Step 1, dissolving 1g to 2g (preferably 1g, 1.5g or 2g) of recombinant collagen in 100mL of deionized water to obtain a recombinant collagen solution with a mass-volume concentration of 1% to 2%; the recombinant collagen has a relative molecular mass of 97KDa; the recombinant collagen CF-1552 is obtained by reverse transcription of human collagen mRNA to generate cDNA, and then expressed in Escherichia coli BL21. Its structure and acquisition method can be referred to, for example, the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method";

[0050] Step 2: add 30 mL of glycidyl methacrylate dropwise to the recombinant collagen solution, stir in a 37° C. water bath for 6 to 8 hours (preferably 6 hours, 7 hours or 8 hours), and collect the product;

[0051] Step 3: dialyze the product in deionized water for 7 consecutive days and freeze-dry to obtain methacrylylated recombinant collagen; the dialysis bag has a molecular weight cutoff of 3500 Da.

[0052] Example 3

[0053] The present embodiment provides a method for preparing tannic acid nanocellulose crystals, which specifically comprises: adding tannic acid to a nanocellulose crystal solution having a mass-volume concentration of 1% to 2% (preferably 1%, 1.5% or 2%), wherein the mass ratio of tannic acid to nanocellulose crystals is 1:(2 to 3) (preferably 1:2, 1:2.5 or 1:3), stirring the reaction at room temperature for 6h to 8h (preferably 6h, 7h or 8h), dialyzing, and freeze-drying to obtain tannic acid nanocellulose crystals.

[0054] Example 4

[0055] In this embodiment, the elastin-like peptide of embodiment 1, the methacrylylated recombinant collagen of embodiment 2 and the tannic acid nanocellulose crystals of embodiment 3 are used to prepare the elastin-like peptide underwater adhesive. The preparation method specifically includes:

[0056] Step 1: Place 10 mg of elastin peptide and 12.5 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2 mL of deionized water, cover with tin foil to avoid light, and stir at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 2 hours;

[0057] Place 250 mg of methacryloyl recombinant collagen in 1.5 mL of deionized water and stir in a 37°C water bath until completely dissolved to obtain solution B; the stirring time in the water bath can be 2 hours;

[0058] Dissolve 250 mg of tannic acid nanocellulose crystals in 1 mL of deionized water, and sonicate until completely dissolved to obtain liquid C; the sonication time may be 1 hour;

[0059] Step 2: Mix the liquid A, liquid B and liquid C in step 1, and stir for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is light yellow;

[0060] Step 3: Place the gel solution in step 2 in a UV curing box, irradiate at 365 nm for 0.5 min, and then sterilize by Co60 irradiation to obtain an elastin peptide underwater adhesive.

[0061] Example 5

[0062] In this embodiment, the elastin-like peptide of embodiment 1, the methacrylylated recombinant collagen of embodiment 2 and the tannic acid nanocellulose crystals of embodiment 3 are used to prepare the elastin-like peptide underwater adhesive. The preparation method specifically includes:

[0063] Step 1, 20 mg of elastin peptide and 5 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) are placed in 2 mL of deionized water, sealed with tin foil to avoid light, and stirred at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature can be 2 hours;

[0064] Place 400 mg of methacryloyl recombinant collagen in 1.3 mL of deionized water and stir in a 37°C water bath until completely dissolved to obtain solution B; the stirring time in the water bath can be 2 hours;

[0065] Dissolve 300 mg of tannic acid nanocellulose crystals in 1 mL of deionized water, and perform ultrasonication until completely dissolved to obtain liquid C; the ultrasonication time may be 1 h;

[0066] Step 2: Mix the liquid A, liquid B and liquid C in step 1, and stir for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is light yellow;

[0067] Step 3: Place the gel solution in step 2 in a UV curing box, irradiate at 365 nm for 0.8 min, and then sterilize by Co60 irradiation to obtain an elastin peptide underwater adhesive.

[0068] Example 6

[0069] In this embodiment, the elastin-like peptide of embodiment 1, the methacrylylated recombinant collagen of embodiment 2 and the tannic acid nanocellulose crystals of embodiment 3 are used to prepare the elastin-like peptide underwater adhesive. The preparation method specifically includes:

[0070] Step 1: Place 30 mg of elastin peptide and 10 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2 mL of deionized water, cover with tin foil to avoid light, and stir at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 2 hours;

[0071] Place 500 mg of methacryloyl recombinant collagen in 1 mL of deionized water and stir in a 37°C water bath until completely dissolved to obtain solution B; the stirring time in the water bath can be 2 hours;

[0072] Dissolve 500 mg of tannic acid nanocellulose crystals in 1 mL of deionized water and perform ultrasonication until completely dissolved to obtain liquid C; the ultrasonication time may be 1 h;

[0073] Step 2: Mix the liquid A, liquid B and liquid C in step 1, and stir for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is light yellow;

[0074] Step 3: Place the gel solution in step 2 in a UV curing box, irradiate at 365 nm for 1 min, and then sterilize by Co60 irradiation to obtain an elastin peptide underwater adhesive.

[0075] Performance Evaluation

[0076] Figure 1 For Example 1 pET28 (a + )Original plasmid vector map. Figure 2 For the elastin peptide of Example 1, pET28 (a+) was used as a plasmid vector, and the target sequence was inserted into the NcoI and XhoI restriction sites.

[0077] Figure 3 The following is a protein gel electrophoresis diagram of the protein induced by different IPTG concentrations. Figure 3 It can be seen that after IPTG induction, there is obvious protein expression at 37Kda. Figure 4 The gel electrophoresis diagram of the protein after salting out and isoelectric point purification is shown in FIG. The purification method includes: ultrasonically crushing the E. coli expressing the protein, centrifuging to obtain the whole protein supernatant, first adjusting the pH to 5 so that the target protein is dissolved in the solution and the impurity protein is precipitated, centrifuging to obtain the preliminary purified protein supernatant, adjusting the ammonium sulfate concentration to 50% so that the target protein is precipitated, centrifuging to purify the protein precipitate, and re-dissolving and freeze-drying to obtain the elastin-like peptide. Figure 4 It can be seen that after purification by salting out and isoelectric point, there is an obvious protein band at 37Kda but no obvious mixed bands.

[0078] Figure 5 a is a schematic diagram of the formation of gel after irradiation of ultraviolet light by methacrylylated recombinant collagen in Example 2 under the action of a photoinitiator. Figure 5a It can be seen that the methacrylylated recombinant collagen of the present invention can form gel under light conditions. Figure 5 b is the Fourier transform infrared absorption spectrum analysis diagram of methacrylylated recombinant collagen in Example 2, according to Figure 5 b is visible at 1653cm -1 The peak at the center is the stretching vibration peak of the carbon-carbon double bond, indicating that the recombinant collagen was successfully methacrylated. Figure 5 c is the methacrylylated recombinant collagen of Example 2 1 H-NMR analysis results. Figure 5 c As can be seen, there are olefin double peaks at 6.08 ppm and 5.70 ppm, confirming that the recombinant collagen was successfully grafted with double bond groups.

[0079] Figure 6 a is the aqueous solution of nanocellulose crystals in Example 3. Figure 6 b is the aqueous solution of tannic acid nanocellulose crystals prepared in Example 3. Figure 6 c is the atomic force microscopy image of the nanocellulose crystals in Example 3. Figure 6 d is an atomic force microscope image of tannic acid nanocellulose crystals in Example 3. Figure 6 As can be seen in c and 6d, the AFM images show that after TA is coated on CNC, the tube wall is obviously thickened. Figure 6 e is a scanning electron microscope image of the nanocellulose crystals in Example 3. Figure 6 f is a scanning electron microscope image of tannic acid nanocellulose crystals in Example 3. Figure 6 e, SEM images in 6f show that the size of TA@CNC is significantly increased compared with that of natural CNC, further verifying the successful coating of TA on CNC. Figure 6 g is the Fourier transform infrared absorption spectrum analysis diagram of tannic acid nanocellulose crystals in Example 3, according to Figure 6 g is visible at 755cm -1 The out-of-plane bending vibration of the CH of the phenyl group is 2897 cm -1 The CH stretching vibration on the benzene ring is 1158cm -1 The stretching vibration of polyol CO is shown in Figure 2, which indicates that TA@CNC was successfully synthesized.

[0080] Figure 7 a is a schematic diagram of the adhesion of the adhesive to pig skin in Example 4. Figure 7 b is a schematic diagram of the adhesive bonding different weights such as magnets, weights, rubber stoppers, small beakers and underwater magnets in Example 4. Figure 7 It can be seen from Fig. 7a and 7b that the adhesive exhibits excellent adhesion performance.

[0081] Figure 8The figure is a schematic diagram of the cytotoxicity test results of the adhesive in Example 4, and the test method includes:

[0082] The 1640 complete culture medium is prepared, specifically comprising: adding fetal bovine serum and double antibodies to the 1640 basic culture medium to obtain the 1640 complete culture medium; in the 1640 complete culture medium, the mass percentage of the fetal bovine serum is 10%, the mass percentage of the double antibodies is 1%, and the double antibodies are streptomycin and penicillin;

[0083] The adhesive extract is prepared, specifically comprising: placing the adhesive of Example 4 in a 50 mL sterile centrifuge tube, adding 1 mL of 1640 complete culture medium per 0.1 g of the adhesive, placing the centrifuge tube in an incubator at 37° C. and 5% CO 2 for 72 hours to obtain an adhesive extract;

[0084] L929 is 1×10 4 The cells / well were inoculated in a 96-well plate at a density of 1.347 μg / well and cultured in an incubator at 37°C and 5% CO2 for 24 h. The old culture medium was removed and the adhesive extract was added to continue the culture. The 1640 complete culture medium was used as the control group. After culturing for 24 h and 48 h, CCK-8 solution was added to the 96-well plate at 50 μL / well. After continuing to culture for 2-4 h, the supernatant was removed and the OD value of each well was measured at 450 nm using an enzyme marker. The cell survival rate was calculated based on the OD value. The cell viability was calculated as follows:

[0085]

[0086] The cell viability calculation results are as follows Figure 8 As shown, according to Figure 8 It can be seen that the adhesive prepared by the present invention has no obvious cytotoxicity and can be used in organisms.

[0087] Example 7

[0088] This embodiment provides an elastin-like peptide, which utilizes the barnacle biomimetic principle to design and construct a target sequence by alternately introducing lysine residues and phenylalanine residues, inserting the target sequence into a plasmid vector, and expressing it through a host cell; the target sequence is composed of a short amino acid sequence of a natural elastin peptide as a repeating unit; the target sequence is shown in SEQ ID NO: 2, and the number of repetitions is 16; the plasmid vector is pET28 (a + ), inserting the target sequence into the NcoI and XhoI restriction sites, and the host cell is Escherichia coli.

[0089] The specific preparation method includes:

[0090] Step 1: Use pET28 (a +) is a plasmid vector, the target sequence SEQ ID NO: 2 is inserted into the NcoI and XhoI restriction sites, and Escherichia coli is selected as the host cell for expression;

[0091] Step 2, collecting the protein solution and preliminarily purifying it by isoelectric point method; the preliminarily purifying by isoelectric point method is to adjust the pH of the protein solution to 2-11 by gradient, determine the appropriate pH according to the result of protein electrophoresis, and then collect the supernatant solution by centrifugation at 12000 rpm for 10 min;

[0092] Step 3, collecting the supernatant solution of step 2, and further purifying it by salting out method; the further purification by salting out method is to add different concentrations of ammonium sulfate to the supernatant solution, the ammonium sulfate concentration is 10% to 100%, and the appropriate ammonium sulfate concentration is determined according to the result of protein electrophoresis, and then centrifuging at 12000rpm for 10min to collect the precipitated product;

[0093] Step 4: The collected precipitate product is dialyzed in deionized water for 3 consecutive days to remove salt, and freeze-dried to obtain elastin-like peptide; the dialysis bag has a molecular weight cutoff of 3500Da.

[0094] In this embodiment, tyrosine residues are replaced with phenylalanine residues, that is, lysine residues and phenylalanine residues are alternately introduced to design and synthesize elastin peptides that mimic barnacles. Barnacle cement protein containing a large amount of cationic lysine (Lys) and amino acids is the main component of the barnacle cement-substrate boundary, in which the protein folds its amino acid chain into a β-sheet to form a large number of hydrogen bond cross-linking sites. It acts with tannic acid nanocellulose crystals, providing adhesion while having mechanical stability. It can still exert adhesion capabilities similar to those of bionic mussels.

[0095] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. An elastin-like peptide, characterized in that: The elastin-like peptide utilizes the bionic principle of mussels or barnacles, and designs and constructs a target sequence by alternately introducing lysine residues and tyrosine residues, or lysine residues and phenylalanine residues, inserting the target sequence into a plasmid vector, and expressing it through a host cell.

2. The elastin-like peptide according to claim 1, characterized in that: The target sequence is composed of a short amino acid sequence of a natural elastin peptide repeated as a repeating unit; the target sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the number of repetitions is 16 times.

3. The elastin-like peptide according to claim 1, characterized in that: The plasmid vector is pET28 (a + ), inserting the target sequence into the NcoI and XhoI restriction sites, and the host cell is Escherichia coli.

4. A method for preparing an elastin-like peptide underwater adhesive using the elastin-like peptide as claimed in claim 1, characterized in that: The following steps are involved: Step 1, dissolving the elastin-like peptide and the photoinitiator in deionized water under light-proof conditions to obtain liquid A; dissolving the methacrylylated recombinant collagen in deionized water under water bath conditions to obtain liquid B; dissolving the tannic acid nanocellulose crystals in deionized water to obtain liquid C; Step 2: Mix the liquid A, liquid B and liquid C in step 1 to obtain a gel solution.

5. The method according to claim 4, characterized in that The method also includes irradiating the gel solution under 365nm light conditions for 0.5min to 1min, and then sterilizing it by Co60 irradiation to obtain an elastin peptide underwater adhesive.

6. The method according to claim 4, characterized in that The method for preparing the methacryloyl recombinant collagen described in step 1 specifically comprises: adding glycidyl methacrylate to the recombinant collagen solution, reacting in a 37° C. water bath with stirring for 6 to 8 hours, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen.

7. The method according to claim 6, characterized in that The recombinant collagen solution is a recombinant collagen solution obtained by dissolving recombinant collagen in deionized water. The mass-volume concentration of the recombinant collagen solution is 1% to 2%; the relative molecular mass of the recombinant collagen is 97KDa.

8. The method according to claim 4, characterized in that The preparation method of tannic acid nanocellulose crystals described in step 1 specifically includes: adding tannic acid to a nanocellulose crystal solution with a mass-volume concentration of 1% to 2%, stirring and reacting at room temperature for 6h to 8h, dialyzing, and freeze-drying to obtain tannic acid nanocellulose crystals; the mass ratio of tannic acid to nanocellulose crystals is 1:(2 to 3).

9. The method according to claim 4, characterized in that The mass percentage of elastin-like peptide in the gel solution is 0.2% to 0.6%, the mass percentage of methacrylylated recombinant collagen is 5% to 10%, the mass percentage of photoinitiator is 0.1% to 0.25%, and the mass percentage of tannic acid nanocellulose crystals is 5% to 10%.

10. Use of the elastin-like peptide underwater adhesive prepared by the method of claim 4 in preparing medical devices for hemostasis and / or tissue regeneration.

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