A medical tissue adhesive based on mussel mucin and hyaluronic acid and a preparation method thereof

Abstract

The invention discloses a medical tissue adhesive based on mussel mucin and hyaluronic acid and a preparation method thereof, and belongs to the technical field of medical tissue adhesive. The method uses recombinant mussel mucin fp151 containing a DOPA group and dopamine-grafted hyaluronic acid as raw materials, and specifically comprises the following steps: obtaining recombinant mussel mucin fp151, realizing DOPAization of recombinant mussel mucin fp151 by enzyme catalysis; oxidizing hyaluronic acid to obtain aldehyded hyaluronic acid, and reacting aldehyded hyaluronic acid with dopamine hydrochloride by Schiff base to obtain dopamine-grafted hyaluronic acid; preparing a coacervate by electrostatic interaction between recombinant mussel mucin fp151 containing a DOPA group and dopamine-grafted hyaluronic acid, and encapsulating THY3. The adhesive of the invention has good biocompatibility, and the wet adhesion strength of the tissue adhesive can still reach 0.1MPa when sodium periodate is not used to oxidize DOPA.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical tissue adhesives, and in particular, relates to a medical tissue adhesive based on mussel mucin and hyaluronic acid with high biocompatibility and a preparation method thereof. Background Art

[0002] At present, people widely use surgical sutures to reconnect large wounds after surgery. However, this common method has many problems. For example, it will cause secondary damage to the wound tissue, is inconvenient to operate, is prone to infection, and easily leaves scars after healing, and will significantly increase the patient's pain. Tissue adhesives with adhesive properties have gradually entered the clinic and been used as a substitute or auxiliary material for surgical sutures. This adhesive is easy to operate, can seal the wound and prevent tissue fluid from leaking, and has little damage to surrounding tissues.

[0003] An ideal tissue adhesive needs to meet certain safety and practical requirements: good biocompatibility, no inflammatory response; high adhesion strength, maintaining high adhesion strength in the high humidity environment of human body fluids; effective promotion of wound healing, meeting patients' pursuit of beauty; biodegradable, and the degradation products are non-toxic and harmless to the human body; with a certain fluidity, easy to operate, and can be accurately applied to the wound, etc. At present, tissue adhesives that meet the above requirements are still in urgent need of exploration. Summary of the invention

[0004] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a medical tissue adhesive based on mussel mucin and hyaluronic acid and a preparation method thereof. The medical tissue adhesive provided by the present invention can effectively promote wound healing and can still achieve high wet adhesion while avoiding the use of strong oxidants such as sodium periodate.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, a method for preparing a medical tissue adhesive based on mussel mucin and hyaluronic acid is provided, wherein the method uses recombinant mussel mucin fp151 containing a DOPA group and dopamine-grafted hyaluronic acid as raw materials.

[0007] The amino acid sequence of the recombinant mussel mucin fp151 is shown in SEQ ID NO:1.

[0008] The preparation method of the medical tissue adhesive based on mussel mucin and hyaluronic acid specifically comprises the following steps:

[0009] Step 1: obtaining recombinant mussel mucin fp151, and realizing DOPAization of the recombinant mussel mucin fp151 by enzyme catalysis to obtain recombinant mussel mucin fp151 containing DOPA groups;

[0010] Step 2: Weigh hyaluronic acid and dopamine hydrochloride, oxidize the hyaluronic acid to obtain aldehyde-modified hyaluronic acid, and react the aldehyde-modified hyaluronic acid with dopamine hydrochloride through Schiff base to obtain dopamine-grafted hyaluronic acid;

[0011] Step 3: The recombinant mussel mucin fp151 containing DOPA groups obtained in step 1 and the dopamine-grafted hyaluronic acid obtained in step 2 are used to prepare a coacervate through electrostatic interaction. In this process, a variety of bioactive ingredients including cockroach thymosin can be encapsulated to form a medical tissue adhesive with strong adhesion and bioactivity in a humid or underwater environment.

[0012] In a second aspect, a medical tissue adhesive based on mussel mucin and hyaluronic acid is obtained by the above preparation method.

[0013] The medical tissue adhesive provided by the present invention can effectively promote wound healing and can still achieve high wet adhesion without using strong oxidants such as sodium periodate. In a third aspect, the medical tissue adhesive based on mussel mucin and hyaluronic acid is used in the medical field.

[0014] The beneficial effects of the present invention are:

[0015] 1. The present invention provides a method for preparing a tissue adhesive composed of mussel mucin and aldehyde-modified hyaluronic acid. The adhesive is composed of components with good biocompatibility. No strong oxidants such as sodium periodate that can improve adhesion are used in the preparation process. The obtained product still has a very high wet adhesion strength. In the case where sodium periodate is not used to oxidize DOPA, the wet adhesion strength of the tissue adhesive can still reach 0.1 MPa.

[0016] 2. The present invention oxidizes hyaluronic acid to obtain aldehyde-modified hyaluronic acid. The aldehyde group can react with the amino group to form a dynamic cross-linking bond. Dopamine can be grafted onto hyaluronic acid by using the Schiff base reaction, and DOPA can be further introduced into the tissue adhesive. At the same time, the aldehyde group can also react with the amino group in fp151 to enhance the cohesive force of the tissue adhesive. The aldehyde group can also react with the amino group on the skin surface to enhance the adhesion of the tissue adhesive. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 This is a wet adhesion graph of the tissue adhesive prepared in Example 4 of the present invention adhered to pig skin and soaked in PBS for 24 hours;

[0019] Figure 2 This is a diagram showing the wet adhesion of the tissue adhesive prepared in Example 4 of the present invention and cellulose protein as a tissue adhesive to adhere pig skin after being immersed in PBS for different time periods;

[0020] Figure 3 Figure 1 shows the healing status of rat back skin at 0 day and 7 days after surgery with no wound treatment (a), surgical sutures (b), tissue adhesive without THY (c), and tissue adhesive with THY (d). DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0022] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0023] In view of the fact that there is no safe and practical tissue adhesive among the existing medical adhesives to meet daily needs, the present invention proposes a medical tissue adhesive based on mussel mucin and hyaluronic acid and a preparation method thereof.

[0024] A typical embodiment of the present invention provides a method for preparing a medical tissue adhesive based on mussel mucin and hyaluronic acid, wherein the method uses recombinant mussel mucin fp151 containing a DOPA group and dopamine-grafted hyaluronic acid as raw materials.

[0025] The preparation method of the medical tissue adhesive based on mussel mucin and hyaluronic acid specifically comprises the following steps:

[0026] Step 1: obtaining recombinant mussel mucin fp151, and realizing DOPAization of the recombinant mussel mucin fp151 by enzyme catalysis to obtain recombinant mussel mucin fp151 containing DOPA groups;

[0027] Step 2: Weigh hyaluronic acid and dopamine hydrochloride, oxidize the hyaluronic acid to obtain aldehyde-modified hyaluronic acid, and react the aldehyde-modified hyaluronic acid with dopamine hydrochloride through Schiff base to obtain dopamine-grafted hyaluronic acid;

[0028] Step 3: The recombinant mussel mucin fp151 containing DOPA groups obtained in step 1 and the dopamine-grafted hyaluronic acid obtained in step 2 are used to prepare a coacervate through electrostatic interaction, and in this process, the cockroach thymosin THY3 is encapsulated to form a medical tissue adhesive with strong adhesion and biological activity in a humid or underwater environment.

[0029] In some examples of this embodiment, the amino acid sequence of the recombinant mussel mucin fp151 is as shown in SEQ ID NO:1.

[0030] Preferably, the method for obtaining the recombinant mussel mucin fp151 in step 1 is:

[0031] The plasmid pet28a-fp151 carrying the recombinant mussel mucin fp151 DNA sequence was transformed into E. coli BL21 competent cells, single clones were selected with kanamycin, BL21 / pet28a-fp151 strain seed solution was prepared, inoculated, induced, fermented to obtain fermentation broth, and the cells were collected by centrifugation; the inclusion bodies were lysed, washed and dissolved with 40 times the volume of 25% acetic acid solution, the supernatant was dialyzed against acetic acid solution and deionized water by stepwise dialysis to obtain the recombinant mussel mucin fp151, and the lyophilized solution was used for later use;

[0032] Or, in step one, the enzyme catalysis is: preparing a tyrosinase reaction buffer; dissolving the recombinant mussel mucin fp151 in the buffer; after the recombinant mussel mucin fp151 is completely dissolved, adding tyrosinase to the buffer and stirring at room temperature for 12 hours, dialyzing and freeze-drying to obtain the recombinant mussel mucin fp151 containing the DOPA group.

[0033] Further preferably, the tyrosinase is mushroom tyrosinase.

[0034] The present invention uses recombinant protein technology to express a mussel mucin, recombinant mussel mucin fp151, using E. coli. E. coli does not have the ability of post-translational modification. The present invention uses tyrosinase in vitro modification to convert tyrosine in the protein into an amino acid L-3,4-dihydroxyphenylalanine, or DOPA, which plays a key role in wet adhesion. The structures of DOPA and tyrosine are as follows:

[0035]

[0036] In some examples of this embodiment, in step 2, the specific steps of oxidation are:

[0037] 1) Completely dissolve hyaluronic acid in PBS buffer at pH 5.0;

[0038] 2) adding sodium periodate to the solution obtained in step 1) and reacting for 6 to 10 hours, preferably 8 hours, in the dark;

[0039] 3) adding ethylene glycol to the solution of step 2) to remove excess sodium periodate;

[0040] 4) The solution obtained in step 3) is placed in a dialysis bag with a molecular weight cutoff of 8KD-14KD and dialyzed with deionized water for multiple times, preferably 2 times; the solution is frozen at -80°C and freeze-dried to obtain aldehyded hyaluronic acid;

[0041] The mass ratio of hyaluronic acid to sodium periodate is 1 to 3:1, preferably 2:1;

[0042] Or, the specific steps of the Schiff base reaction are:

[0043] Take the aldehyde-modified hyaluronic acid and dissolve it in PBS buffer added with ascorbic acid, and adjust the pH to 5.0; add a certain amount of dopamine hydrochloride for reaction; after the reaction, put the reaction solution into a dialysis bag with a molecular weight cutoff of 8KD-14KD and dialyze it twice with deionized water; freeze the solution at -80℃ and freeze-dry it to obtain dopamine-grafted hyaluronic acid.

[0044] The reaction process of step 2 is as follows:

[0045]

[0046] The present invention oxidizes hyaluronic acid to obtain aldehyde-modified hyaluronic acid. The aldehyde group can react with the amino group to form a dynamic cross-linking bond. Dopamine can be grafted onto hyaluronic acid by using the Schiff base reaction, and DOPA can be further introduced into the tissue adhesive. At the same time, the aldehyde group can also react with the amino group in fp151 to enhance the cohesive force of the tissue adhesive. The aldehyde group can also react with the amino group on the skin surface to enhance the adhesion of the tissue adhesive.

[0047] In some examples of this embodiment, in step 3, the electrostatic interaction is specifically:

[0048] 1) dissolving the recombinant mussel mucin fp151 containing DOPA group in 100 mM sodium acetate buffer at pH 3.8 at 5-15 mg / mL, preferably 10 mg / mL;

[0049] 2) dissolving dopamine-grafted hyaluronic acid in 100 mM sodium acetate buffer at pH 3.8 at 5-15 mg / mL, preferably 10 mg / mL;

[0050] 3) mixing the solution of step 1) and the solution of step 2) in a centrifuge tube at a volume ratio of 3 to 5:1, preferably 4:1;

[0051] 4) The solution of step 3) is placed at room temperature for 5 to 15 minutes, preferably 10 minutes, and the coagulated phase is collected by centrifugation.

[0052] In some examples of this embodiment, the coacervate in step 3 also encapsulates proteins and other small molecule drugs, thereby effectively promoting wound healing;

[0053] Preferably, the protein drug is American cockroach thymosin THY;

[0054] Preferably, the package specifically includes:

[0055] 1) dissolving the recombinant mussel mucin fp151 containing DOPA group in 100 mM sodium acetate buffer at pH 3.8 at 5-15 mg / mL, preferably 10 mg / mL;

[0056] 2) adding the American cockroach thymosin to the solution in step 1);

[0057] 3) dissolving dopamine-grafted hyaluronic acid in 100 mM sodium acetate buffer at pH 3.8 at 5-15 mg / mL, preferably 10 mg / mL;

[0058] 3) mixing the solution of step 2) and the solution of step 3) in a centrifuge tube at a volume ratio of 3 to 5:1, preferably 4:1;

[0059] 4) The solution of step 3) is placed at room temperature for 5 to 15 minutes, preferably 10 minutes, and the coagulated phase is collected by centrifugation.

[0060] The present invention encapsulates an American cockroach thymosin (THY3) in a tissue adhesive. Thymosin is a lymphocyte growth factor extracted from bovine thymus and is divided into three categories: α, β, and γ. The β-thymosin has been studied the most, and Tβ4 is the main member of the β-thymosin, which has multiple functions, such as promoting angiogenesis and wound healing, and promoting cell migration. THY3 has similar biological functions to the β-thymosin and has good biological activity in promoting tissue damage repair. THY3 was obtained by the applicant through recombinant expression and purification. The American cockroach thymosin is encapsulated in a tissue adhesive, and the prepared tissue adhesive can effectively promote wound healing and significantly reduce the formation of scars. Similarly, in the preparation process of the coagulant, the adhesive can also encapsulate a variety of biologically active ingredients including proteins and other small molecules.

[0061] Another typical embodiment of the present invention is a medical tissue adhesive based on mussel mucin and hyaluronic acid, which is obtained by the above-mentioned preparation method.

[0062] A third typical embodiment of the present invention provides a medical tissue adhesive based on mussel mucin and hyaluronic acid for use in the medical field.

[0063] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0064] Example 1: Preparation method of recombinant mussel mucin fp151

[0065] (I) The amino acid sequence of recombinant mussel adhesive protein fp151 is as shown in SEQ ID NO: 1. The plasmid pet28a-fp151 carrying the recombinant mussel adhesive protein DNA sequence is transformed into E. coli BL21 competent cells, and single clones are selected with kanamycin to prepare the BL21 / pet28a-fp151 strain seed liquid, which is inoculated in a fermenter, induced, and fermented to obtain a fermentation liquid, and the bacteria are collected by centrifugation. The inclusion bodies are lysed by cell enzymatic hydrolysis and mechanical lysis, and then washed and dissolved with 40 times the volume of 25% acetic acid solution. The supernatant is dialyzed and renatured in 10%, 5%, and 0.5% acetic acid solutions and deionized water by stepwise dialysis, and then freeze-dried for use.

[0066] (ii) Tyrosinase modification of recombinant mussel mucin fp151.

[0067] Tyrosine residues in the recombinant mussel mucin FP151 are converted to DOPA by mushroom tyrosinase.

[0068] 1) Prepare tyrosinase reaction buffer, 50 mM sodium acetate, 10 mM boric acid, 25 mM ascorbic acid, and adjust the pH to 5.5.

[0069] 2) The lyophilized recombinant mussel mucin fp151 was completely dissolved in the reaction buffer of step 1) to a concentration of 2 mg / mL.

[0070] 3) Add mushroom tyrosinase to the solution in step 2) at a concentration of 0.01 mg / mL.

[0071] 4) The solution of step 3) was stirred at room temperature for 12 hours.

[0072] 5) The solution obtained in step 4) was dialyzed against 25 mM ascorbic acid, 50 mM sodium acetate solution, and then dialyzed against deionized water overnight.

[0073] 6) The solution obtained in step 5) is freeze-dried to obtain the recombinant mussel mucin fp151 containing the DOPA group.

[0074] Example 2: Synthesis of DOPA-grafted hyaluronic acid

[0075] (I) Synthesis of aldehyde-modified hyaluronic acid by oxidation reaction:

[0076] 1) Dissolve 1.0 g of hyaluronic acid completely in 100 ml of PBS buffer (pH 5.0).

[0077] 2) Add 0.5 g of sodium periodate to the solution obtained in step 1) and react for 8 hours in the dark.

[0078] 3) 1 ml of ethylene glycol was added to the solution in step 2) to remove excess sodium periodate.

[0079] 4) The solution obtained in step 3) is placed in a dialysis bag with a molecular weight cut-off of 8KD-14KD and dialyzed twice with deionized water. The solution is frozen at -80°C and freeze-dried to obtain the product.

[0080] (II) Synthesis method of DOPA-grafted hyaluronic acid:

[0081] 1) Take 1.0 g of aldehyded hyaluronic acid and dissolve it in 100 ml of PBS buffer with 25 mM ascorbic acid added, and adjust the pH to 5.0.

[0082] 2) Add a certain amount of dopamine hydrochloride and react for a specific time.

[0083] 3) After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8KD-14KD and dialyzed twice with deionized water. The solution was frozen at -80°C and freeze-dried to obtain the product.

[0084] Example 3: Synthesis of dopamine-grafted hyaluronic acid with different dopamine grafting amounts

[0085] The invention can effectively control the grafting amount of dopamine in the product by changing the feed ratio of dopamine to aldehyded hyaluronic acid.

[0086] In step 2) of part (ii) of Example 2, 0.25 g of dopamine hydrochloride was added and reacted for two hours to prepare dopamine-grafted hyaluronic acid with a low grafting amount.

[0087] In step 2) of part (ii) of Example 2, 10 g of dopamine hydrochloride was added and reacted for twelve hours to prepare dopamine-grafted hyaluronic acid with a high grafting amount.

[0088] The present invention can effectively control the grafting amount of dopamine in the product by feed ratio and reaction time, and achieve high grafting. The present invention utilizes the Schiff base reaction between the aldehyde group on hyaluronic acid and the amino group of dopamine to achieve dopamine grafting. Compared with traditional carbodiimide / imide chemistry, this method can shorten the reaction time and effectively avoid side reactions.

[0089] Example 4: Preparation of tissue adhesive composed of recombinant mussel mucin fp151 containing DOPA groups and low-grafted DOPA-grafted hyaluronic acid

[0090] 1) The recombinant mussel mucin fp151 containing DOPA groups prepared in Example 1 was dissolved in a 100 mM sodium acetate buffer at pH 3.8 at a concentration of 10 mg / mL.

[0091] 2) The low-grafted DOPA-grafted hyaluronic acid prepared in Example 3 was dissolved in 100 mM sodium acetate buffer at pH 3.8 at a concentration of 10 mg / mL.

[0092] 3) The solution of step 1) and the solution of step 2) were mixed in a centrifuge tube at a volume ratio of 4:1.

[0093] 4) The solution of step 3) was placed at room temperature for 10 min, and the coacervate phase was collected by centrifugation (8000×g, 10 min, 4° C.) and labeled as fp+DAHA-L.

[0094] Example 5: Preparation of tissue adhesive composed of recombinant mussel mucin fp151 containing DOPA groups and high grafting amount of DOPA-grafted hyaluronic acid

[0095] 1) The recombinant mussel mucin fp151 containing DOPA groups prepared in Example 1 was dissolved in a 100 mM sodium acetate buffer at pH 3.8 at a concentration of 10 mg / mL.

[0096] 2) The DOPA-grafted hyaluronic acid with a high grafting amount prepared in Example 3 was dissolved in 100 mM sodium acetate buffer at pH 3.8 at a concentration of 10 mg / mL.

[0097] 3) The solution of step 1) and the solution of step 2) were mixed in a centrifuge tube at a volume ratio of 4:1.

[0098] 4) The solution of step 3) was placed at room temperature for 10 min, and the coacervate phase was collected by centrifugation (8000×g, 10 min, 4° C.) and labeled as fp+DAHA-H.

[0099] Comparative Example 1

[0100] Different from Example 5, the tissue adhesive was prepared using recombinant mussel mucin fp151 containing DOPA groups and hyaluronic acid, and the obtained adhesive was labeled as fp+HA.

[0101] Comparative Example 2

[0102] Different from Example 5, the tissue adhesive was prepared by using recombinant mussel mucin fp151 containing DOPA group and fruit acid AHA, and the obtained adhesive was labeled as fp+AHA

[0103] The coacervate phase of the present invention is prepared in a sodium acetate buffer at pH 3.8. The acidic environment is conducive to avoiding premature oxidation of DOPA and retaining its high adhesion. At the same time, combined with the high grafting amount obtained by the Schiff base reaction method, the obtained tissue adhesive can still exhibit high wet adhesion without using a strong oxidant. Its wet adhesion strength is as follows Figure 1 , 2 shown.

[0104] Example 6: Expression and purification of American cockroach thymosin THY

[0105] The amino acid sequence of thymosin THY from American cockroach is shown in SEQ ID NO:2.

[0106] Use E.coli BL21 (DE3) for recombinant expression, collect the cells, sonicate the lysate, centrifuge to obtain the protein supernatant, and filter with a 0.2μm filter membrane. After equilibrating the nickel ion column with 10 times the volume of 10mM imidazole buffer, load the target protein to bind to the nickel ion column. Wash with 20 column volumes of buffer containing 20mM imidazole. Elute the target protein with 400mM imidazole buffer and collect the eluate. Place the eluate in a dialysis bag with a molecular weight cutoff of 8KD-14KD and dialyze in deionized water until the buffer is completely replaced.

[0107] Example 7: Preparation of tissue adhesive containing American cockroach thymosin THY

[0108] 1) The recombinant mussel mucin fp151 containing DOPA groups prepared in Example 1 was dissolved in a 100 mM sodium acetate buffer at pH 3.8 at a concentration of 10 mg / mL.

[0109] 2) Add 1 mg of American cockroach thymosin to the solution in step 1).

[0110] 3) The DOPA-grafted hyaluronic acid with a high grafting amount prepared in Example 3 was dissolved in 100 mM sodium acetate buffer at pH 3.8 at a concentration of 10 mg / mL.

[0111] 4) The solution from step 2) and the solution from step 3) were mixed in a centrifuge tube at a volume ratio of 4:1.

[0112] 5) The solution of step 4) was placed at room temperature for 10 min, and the coacervate phase was collected by centrifugation (8000×g, 10 min, 4° C.).

[0113] The coagulant of the present invention is composed of hyaluronic acid and mussel mucin. This material with excellent biocompatibility can effectively promote wound healing. Combined with American cockroach thymosin, it can further promote wound healing and regeneration of new tissue. In the rat back wound healing experiment, the coagulant has a promoting effect on wound healing. Figure 3 shown. Figure 3 (c) uses the tissue adhesive prepared in Example 5, which does not contain THY; (d) uses the tissue adhesive prepared in Example 7, which contains THY. Figure 3 It can be seen that the tissue adhesive of the present invention can effectively promote wound healing, and combined with American cockroach thymosin, it can further promote wound healing and regeneration of new tissue.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a medical tissue adhesive based on mussel mucin and hyaluronic acid, characterized in that: The method uses recombinant mussel mucin fp151 containing DOPA groups and dopamine-grafted hyaluronic acid as raw materials; The preparation method specifically comprises the following steps: Step 1: obtaining recombinant mussel mucin fp151, and realizing DOPAization of the recombinant mussel mucin fp151 by enzyme catalysis to obtain recombinant mussel mucin fp151 containing DOPA groups; Step 2: Weigh hyaluronic acid and dopamine hydrochloride, oxidize the hyaluronic acid to obtain aldehyde-modified hyaluronic acid, and react the aldehyde-modified hyaluronic acid with dopamine hydrochloride through Schiff base to obtain dopamine-grafted hyaluronic acid; Step 3: preparing a coacervate through electrostatic interaction of the recombinant mussel mucin fp151 containing DOPA groups obtained in step 1 and the dopamine-grafted hyaluronic acid obtained in step 2, and encapsulating the cockroach thymosin THY3 in the process to form a medical tissue adhesive; The method for obtaining the recombinant mussel mucin fp151 in step 1 is: The plasmid pet28a-fp151 carrying the recombinant mussel mucin fp151 DNA sequence was transformed into E. coli BL21 competent cells, single clones were selected with kanamycin, BL21 / pet28a-fp151 strain seed solution was prepared, inoculated, induced, fermented to obtain fermentation broth, and the cells were collected by centrifugation; the inclusion bodies were lysed, washed and dissolved with 40 times the volume of 25% acetic acid solution, the supernatant was dialyzed and renatured in 10%, 5%, 0.5% acetic acid solution and deionized water by stepwise dialysis method to obtain the recombinant mussel mucin fp151, and lyophilized for later use; In step 2, the mass ratio of the aldehyde-modified hyaluronic acid to dopamine hydrochloride is 1:0.25-10; In step 3, the electrostatic interaction is specifically: 1) Dissolve the recombinant mussel mucin fp151 containing DOPA groups at 5-15 mg / mL in 100 mM sodium acetate buffer at pH 3.8; 2) Dissolve dopamine-grafted hyaluronic acid at 5-15 mg / mL in 100 mM sodium acetate buffer at pH 3.8; 3) Mix the solution of step 1) and the solution of step 2) in a volume ratio of 3-5:1 in a centrifuge tube; 4) Step 3) The solution was placed at room temperature for 5-15 min and the condensed phase was collected by centrifugation.

2. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid as claimed in claim 1, characterized in that: The amino acid sequence of the recombinant mussel mucin fp151 is shown in SEQ ID NO:

1.

3. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid as claimed in claim 1, characterized in that: In step one, the enzyme catalysis is as follows: preparing a tyrosinase reaction buffer; dissolving the recombinant mussel mucin fp151 in the buffer; after the recombinant mussel mucin fp151 is completely dissolved, adding tyrosinase to the buffer and stirring at room temperature for 12 hours, dialyzing and freeze-drying to obtain the recombinant mussel mucin fp151 containing the DOPA group.

4. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid as claimed in claim 3, characterized in that: The tyrosinase is mushroom tyrosinase.

5. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid as claimed in claim 1, characterized in that: In step 2, the specific steps of oxidation are: 1) Completely dissolve hyaluronic acid in PBS buffer at pH 5.0; 2) Add sodium periodate to the solution obtained in step 1) and react for 6 to 10 hours in the dark; 3) Adding ethylene glycol to the solution of step 2) to remove excess sodium periodate; 4) The obtained solution in step 3) is placed in a dialysis bag with a molecular weight cutoff of 8 KD-14 KD and dialyzed twice with deionized water; the solution is frozen at -80°C and freeze-dried to obtain aldehyded hyaluronic acid; The mass ratio of hyaluronic acid to sodium periodate is 1-3:1; Or, the specific steps of the Schiff base reaction are: Take the aldehyde-modified hyaluronic acid and dissolve it in PBS buffer added with ascorbic acid, adjust the pH to 5.0; add a certain amount of dopamine hydrochloride to react; after the reaction, put the reaction solution into a dialysis bag with a molecular weight cutoff of 8 KD-14 KD and dialyze it twice with deionized water; freeze the solution at -80 ℃ and freeze-dry it to obtain dopamine-grafted hyaluronic acid; The mass ratio of the aldehyde-modified hyaluronic acid to dopamine hydrochloride is 1:0.25-10.

6. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid as claimed in claim 5, characterized in that: In step 2, sodium periodate is added to the obtained solution and reacted for 8 hours in the dark; The obtained solution was placed in a dialysis bag with a molecular weight cutoff of 8 KD-14 KD and dialyzed twice with deionized water; the mass ratio of the hyaluronic acid to sodium periodate was 2:

1.

7. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid as claimed in claim 1, characterized in that: In step 3, the electrostatic interaction is specifically: 1) Dissolve the recombinant mussel mucin fp151 containing DOPA groups at 10 mg / mL in 100 mM sodium acetate buffer at pH 3.8; 2) Dissolve dopamine-grafted hyaluronic acid at 10 mg / mL in 100 mM sodium acetate buffer at pH 3.8; 3) Mix the solution in step 1) and the solution in step 2) in a volume ratio of 4:1 into a centrifuge tube; 4) Step 3) The solution was placed at room temperature for 10 min and the condensed phase was collected by centrifugation.

8. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid according to claim 1 or any one of claims 3 to 7, characterized in that: The coacervate in step three also encapsulates the protein drug.

9. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid as claimed in claim 8, characterized in that: The protein drug is American cockroach thymosin THY; The amino acid sequence of the American cockroach thymosin THY is shown in SEQ ID NO:

2.

10. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid as claimed in claim 8, characterized in that: The package is specifically: 1) Dissolve the recombinant mussel mucin fp151 containing DOPA groups at 5-15 mg / mL in 100 mM sodium acetate buffer at pH 3.8; 2) Add American cockroach thymosin to the solution in step 1); 3) Dissolve dopamine-grafted hyaluronic acid at 5-15 mg / mL in 100 mM sodium acetate buffer at pH 3.8; 4) Mix the solution in step 2) and the solution in step 3) in a volume ratio of 3-5:1 in a centrifuge tube; 5) Step 4) The solution was placed at room temperature for 5-15 min and the coacervate phase was collected by centrifugation.

11. The method for preparing the medical tissue adhesive based on mussel mucin and hyaluronic acid as claimed in claim 10, characterized in that: The package is specifically: 1) Dissolve the recombinant mussel mucin fp151 containing DOPA groups at 10 mg / mL in 100 mM sodium acetate buffer at pH 3.8; 2) Add American cockroach thymosin to the solution in step 1); 3) Dissolve dopamine-grafted hyaluronic acid at 10 mg / mL in 100 mM sodium acetate buffer at pH 3.8; 4) Mix the solution in step 2) and the solution in step 3) in a volume ratio of 4:1 into a centrifuge tube; 5) Step 4) The solution was placed at room temperature for 10 min and the condensed phase was collected by centrifugation.

12. A medical tissue adhesive based on mussel mucin and hyaluronic acid, characterized in that: Obtained by the preparation method according to any one of claims 1 to 11.

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