CN113769155A - Biological adhesive based on biological engineering protein and preparation method thereof - Google Patents
Biological adhesive based on biological engineering protein and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0015—Medicaments; Biocides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/08—Polysaccharides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
Abstract
The invention belongs to the technical field of biological adhesives, and discloses a biological adhesive based on bioengineering protein and a preparation method thereof, wherein the biological adhesive based on bioengineering protein comprises, by mass, 30-50 parts of a solvent, 20-35 parts of bioengineering protein, 15-20 parts of chitin whisker, 10-15 parts of parthenocissus tricuspidata sucker polysaccharide, 8-12 parts of tannic acid, 6-8 parts of maleylation hyaluronic acid and 2-4 parts of a catalyst. The bio-adhesive based on the bioengineering protein provided by the invention selects the parthenocissus tricuspidata sucker polysaccharide with good biocompatibility as a matrix, adopts bioengineering protein, tannic acid, maleylation hyaluronic acid and the like as raw materials, has good biocompatibility, small stimulation to biological tissues, can firmly adhere to a bonding part, and has the characteristics of water resistance, antibacterial property, closed hemostasis and no cytotoxicity; the preparation method is simple and suitable for large-scale production.
Description
Technical Field
The invention belongs to the technical field of biological adhesives, and particularly relates to a biological adhesive based on biological engineering protein and a preparation method thereof.
Background
Currently, bioadhesives are biomedical materials that are prepared from materials with excellent biocompatibility, are sterile, non-immunogenic, and are capable of inhibiting bacteria for a certain period of time. The components of the adhesive are free of solvent or the solvent is water, and the adhesive can still generate a certain strength adhesion with body tissues under a wet environment (namely the existence of water, interstitial fluid, blood and the like). The dissolved matters of the biological adhesive and the like do not generate toxicity to organism cells, do not influence the wound healing of organism tissues, are degradable, can be absorbed by the human tissues after meeting the use requirements, and are finally metabolized out of the human body. However, the existing biological adhesive has the defects of low adhesive strength, long preparation time and high price, and has potential virus infection risk. Therefore, a new, simple, efficient, and green-prepared bioadhesive and a preparation method thereof are needed.
Through the above analysis, the problems and defects of the prior art are as follows: the existing biological adhesive has the defects of low adhesive strength, long preparation time and high price, and has potential virus infection risk.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a biological adhesive based on biological engineering protein and a preparation method thereof.
The bio-adhesive based on the bioengineering protein is prepared from 30-50 parts by mass of a solvent, 20-35 parts by mass of the bioengineering protein, 15-20 parts by mass of chitin whiskers, 10-15 parts by mass of boston ivy sucker polysaccharide, 8-12 parts by mass of tannic acid, 6-8 parts by mass of maleylation hyaluronic acid and 2-4 parts by mass of a catalyst.
Further, the solvent is any one of deionized water, water for injection, normal saline, gentamicin normal saline flushing fluid, ringer's solution flushing fluid, mannitol flushing fluid, glucose flushing fluid, chlorhexidine flushing fluid, metronidazole flushing fluid, glycine flushing fluid or physiological balanced fluid.
Further, the bioengineering protein is an elastin-like protein.
Another object of the present invention is to provide a method for preparing a bio-engineered protein-based bio-adhesive using the bio-engineered protein-based bio-adhesive, the method comprising the steps of:
step one, preparing bioengineering protein: converting the ELP-like carrier plasmid into a pichia pastoris or escherichia coli expression strain, selecting a monoclonal colony, and carrying out overnight culture by using LB culture solution; adding the overnight activated expression seed liquid into a TB culture medium, adding an inducer isopropyl-beta-D-thiogalactopyranoside when the bacterial liquid reaches an OD600 value of 0.8-1.0, and cooling to 30 ℃ for overexpression; inducing for 10-12 h, collecting thalli, resuspending with lysis buffer, adding mixed microbial inoculum, crushing thalli, centrifuging at 10000-12000 rpm, collecting supernatant, and purifying by HPLC to obtain elastin-like protein (ELP) for later use;
step two, preparing the chitin whisker: weighing shrimp and crab shells, crushing the shrimp and crab shells into powder, dispersing the powder in 3M concentrated sulfuric acid, stirring the powder in a water bath at 85-95 ℃ for 10-12 hours, diluting the powder with deionized water, centrifuging the diluted solution to remove supernatant, and repeating the step for 3-5 times to obtain precipitate; resuspending the obtained precipitate with deionized water, transferring the precipitate into a dialysis bag with the molecular weight cutoff of 8000-14000 Da, and fully dialyzing until the pH value of the suspension in the dialysis bag is 6.0; performing ultrasonic treatment at the power of 600-800W for 10-30 min to completely suspend the precipitate, centrifuging the obtained suspension at 7000-7500 rpm, and freeze-drying for 5-20 min to obtain chitin whiskers for later use;
step three, preparing the parthenocissus tricuspidata sucker polysaccharide: collecting the parthenocissus tricuspidata sucker, washing with deionized water, airing in the sun for 6-8 h, then putting into an airflow crusher for crushing, and sieving with a 80-120-mesh sieve to obtain parthenocissus tricuspidata sucker powder; adding the parthenocissus tricuspidata sucker powder into an ethanol solution, heating in a water bath, performing reflux extraction for 2-4 h, and distilling at 60-70 ℃ to recover the ethanol solution to obtain degreased sucker powder; adding hot water with the temperature of 90-100 ℃ which is 10-20 times that of the degreased sucker powder, leaching for 3-5 hours, and performing centrifugal concentration to obtain a polysaccharide concentrated solution of the parthenocissus tricuspidata sucker for later use;
step four, preparation of maleylation hyaluronic acid is carried out: placing hyaluronic acid and dimethylformamide into deionized water, and stirring at room temperature for 8-24 hours to obtain a mixed solution A; adding maleic anhydride into the mixed solution A, uniformly stirring, and reacting for 12-48 h at 35-80 ℃ to obtain a mixed solution B; adding 1mol/L NaHCO into the mixed solution B3Adjusting the pH value of the mixed solution to 7-8, dialyzing, and freeze-drying to obtain maleylation hyaluronic acid for later use;
step five, weighing a solvent, a biological engineering protein, chitin whiskers, parthenocissus tricuspidata sucker polysaccharide, tannic acid, maleylation hyaluronic acid and a catalyst in sequence according to parts by weight for later use;
step six, adding 50-70 times of deionized water into the bioengineering protein, dissolving the protein under the condition of heating and stirring at 60 ℃, adding 12mol/L sodium hydroxide solution to adjust the pH value to 8.5, adding tannic acid, fully stirring and dissolving, and continuously introducing air;
and seventhly, sequentially adding the chitin whisker, the parthenocissus tricuspidata sucker polysaccharide, the maleylation hyaluronic acid and the catalyst, fully stirring and dissolving, adjusting the pH to 7.4 by using 0.5mol/L diluted hydrochloric acid after the reaction is finished, cooling the reactant to room temperature, dialyzing, and freeze-drying to obtain the biological adhesive based on the biological engineering protein.
Further, in the first step, the molar ratio of the elastin-like protein to the alkyl anionic surfactant is 1: (1.5 to 3).
Further, in the step one, the mixed microbial inoculum comprises a protease inhibitor, a DNase and lysozyme.
Further, in the second step, the mixing ratio of the shrimp and crab shells to the concentrated sulfuric acid solution is 3-5% w/v.
Further, in the third step, the mass concentration of the ethanol solution is 95%.
Further, in the fourth step, the freeze-drying conditions are as follows: and (3) placing the mixed solution at-50 ℃ and under the pressure of 5-20 Pa, and freeze-drying for 48-72 h.
Further, in step seven, the dialysis is:
and carrying out deionized water dialysis on the reactant for 3-5 times by using a dialysis bag with a molecular weight cut-off value of 3500 Da.
By combining all the technical schemes, the invention has the advantages and positive effects that: the biological adhesive based on the bioengineering protein selects the saccharide macromolecules with good biocompatibility, namely the parthenocissus tricuspidata sucker polysaccharide as a matrix, adopts the bioengineering protein, the tannic acid, the maleylation hyaluronic acid and the like as raw materials, has good biocompatibility, small stimulation to organism tissues, can firmly adhere to a combined part, and has the characteristics of water resistance, antibacterial property, closed hemostasis and no cytotoxicity; the preparation method is simple, the production process is pollution-free, the raw materials are easy to obtain, the cost is low, and the preparation method is suitable for large-scale production.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a method for preparing a bio-adhesive based on bioengineered proteins according to embodiments of the invention.
FIG. 2 is a flow chart of a method for preparing a bioengineered protein according to an embodiment of the invention.
Fig. 3 is a flow chart of a method for preparing chitin whiskers provided by an embodiment of the invention.
FIG. 4 is a flow chart of a method for preparing the parthenocissus tricuspidata sucker polysaccharide according to an embodiment of the present invention.
FIG. 5 is a flow chart of a method for preparing maleylated hyaluronic acid according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In view of the problems of the prior art, the present invention provides a bio-adhesive based on bio-engineered proteins and a method for preparing the same, and the present invention is described in detail below with reference to the accompanying drawings.
The bio-adhesive based on bioengineering protein provided by the embodiment of the invention comprises, by mass, 30-50 parts of a solvent, 20-35 parts of bioengineering protein, 15-20 parts of chitin whisker, 10-15 parts of parthenocissus tricuspidata sucker polysaccharide, 8-12 parts of tannic acid, 6-8 parts of maleylation hyaluronic acid and 2-4 parts of a catalyst.
The solvent provided by the embodiment of the invention is any one of deionized water, water for injection, normal saline, gentamicin normal saline flushing fluid, ringer's solution flushing fluid, mannitol flushing fluid, glucose flushing fluid, chlorhexidine flushing fluid, metronidazole flushing fluid, glycine flushing fluid or physiological balance fluid.
The bioengineering protein provided by the embodiment of the invention is elastin-like protein.
As shown in fig. 1, the preparation method of the bio-adhesive based on the bio-engineered protein provided by the embodiment of the invention comprises the following steps:
s101, respectively preparing bioengineering protein, chitin whisker, parthenocissus tricuspidata sucking disc polysaccharide and maleylation hyaluronic acid;
s102, weighing a solvent, a biological engineering protein, chitin whiskers, parthenocissus tricuspidata sucker polysaccharide, tannic acid, maleylation hyaluronic acid and a catalyst in sequence by mass for later use;
s103, adding 50-70 times of deionized water into the bioengineering protein, dissolving at 60 ℃ under heating and stirring conditions, adding 12mol/L sodium hydroxide solution to adjust the pH value to 8.5, adding tannic acid, fully stirring and dissolving, and continuously introducing air;
s104, sequentially adding chitin whisker, parthenocissus tricuspidata sucker polysaccharide, maleylation hyaluronic acid and a catalyst, fully stirring and dissolving, and adjusting the pH to 7.4 by using 0.5mol/L diluted hydrochloric acid after the reaction is finished;
and S105, cooling the reactant to room temperature, dialyzing, and freeze-drying to obtain the biological adhesive based on the biological engineering protein.
As shown in fig. 2, in step S101 provided in the embodiment of the present invention, the preparing of the bioengineered protein includes:
s201, converting an elastin-like protein ELP vector plasmid into a pichia pastoris or escherichia coli expression strain, selecting a monoclonal colony, and carrying out overnight culture by using LB culture solution;
s202, adding the overnight activated expression seed liquid into a TB culture medium, adding an inducer isopropyl-beta-D-thiogalactopyranoside when the bacterial liquid reaches an OD600 value of 0.8-1.0, and cooling to 30 ℃ for overexpression;
s203, inducing for 10-12 h, collecting thalli, carrying out heavy suspension by using a lysis buffer solution, adding a mixed microbial inoculum, crushing the thalli, centrifuging at 10000-12000 rpm, collecting supernate, and carrying out HPLC (high performance liquid chromatography) purification to obtain the elastin-like protein ELP.
The molar ratio of the elastin-like protein to the alkyl anionic surfactant provided by the embodiment of the invention is 1: (1.5 to 3).
The mixed microbial inoculum provided by the embodiment of the invention comprises a protease inhibitor, DNA enzyme and lysozyme.
As shown in fig. 3, in step S101 provided in the embodiment of the present invention, the preparing of the chitin whisker includes:
s301, weighing shrimp and crab shells, crushing the shrimp and crab shells into powder, dispersing the powder in 3M concentrated sulfuric acid, stirring the powder in a water bath at the temperature of 85-95 ℃ for 10-12 hours, diluting the powder with deionized water, centrifuging to remove supernatant, and repeating the steps for 3-5 times to obtain precipitate;
s302, resuspending the obtained precipitate with deionized water, transferring the precipitate into a dialysis bag with the molecular weight cutoff of 8000-14000 Da, and fully dialyzing until the pH value of the suspension in the dialysis bag is 6.0;
s303, carrying out ultrasonic treatment for 10-30 min under the power of 600-800W to completely suspend the precipitate, centrifuging the obtained suspension at 7000-7500 rpm, and freeze-drying for 5-20 min to obtain the chitin whisker.
The mixing ratio of the shrimp and crab shells to the concentrated sulfuric acid solution provided by the embodiment of the invention is 3-5% w/v.
As shown in fig. 4, in step S101, the preparation of the parthenocissus tricuspidata sucker polysaccharide includes:
s401, collecting the parthenocissus tricuspidata sucker, washing with deionized water, airing in the sun for 6-8 hours, then putting into an airflow crusher for crushing, and sieving with a 80-120-mesh sieve to obtain parthenocissus tricuspidata sucker powder;
s402, adding the parthenocissus tricuspidata sucker powder into an ethanol solution, heating in a water bath, carrying out reflux extraction for 2-4 h, and distilling at 60-70 ℃ to recover the ethanol solution to obtain degreased sucker powder;
s403, adding hot water with the temperature of 90-100 ℃ which is 10-20 times that of the degreased sucker powder, leaching for 3-5 hours, and performing centrifugal concentration to obtain the polysaccharide concentrated solution of the parthenocissus tricuspidata sucker.
The mass concentration of the ethanol solution provided by the embodiment of the invention is 95%.
As shown in fig. 5, in step S101 provided by the embodiment of the present invention, the preparation of maleylated hyaluronic acid includes:
s501, placing hyaluronic acid and dimethylformamide into deionized water, and stirring at room temperature for 8-24 hours to obtain a mixed solution A;
s502, adding maleic anhydride into the mixed solution A, uniformly stirring, and reacting for 12-48 hours at 35-80 ℃ to obtain a mixed solution B;
s503, adding 1mol/L NaHCO into the mixed solution B3Adjusting the pH value of the mixed solution to 7-8, dialyzing, and freeze-drying to obtain the horse extractTo acylate hyaluronic acid.
The freeze drying conditions provided by the embodiment of the invention are as follows: and (3) placing the mixed solution at-50 ℃ and under the pressure of 5-20 Pa, and freeze-drying for 48-72 h.
In step S105 provided in the embodiment of the present invention, the dialysis is: and carrying out deionized water dialysis on the reactant for 3-5 times by using a dialysis bag with a molecular weight cut-off value of 3500 Da.
In the description of the present invention, "a plurality" means two or more unless otherwise specified; the terms "upper", "lower", "left", "right", "inner", "outer", "front", "rear", "head", "tail", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are only for convenience in describing and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, should not be construed as limiting the invention. Furthermore, the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The biological adhesive based on the bioengineering protein is characterized by comprising, by mass, 30-50 parts of a solvent, 20-35 parts of the bioengineering protein, 15-20 parts of chitin whiskers, 10-15 parts of boston ivy sucker polysaccharide, 8-12 parts of tannic acid, 6-8 parts of maleylation hyaluronic acid and 2-4 parts of a catalyst.
2. The bioengineered protein-based bioadhesive of claim 1, wherein the solvent is any one of deionized water, water for injection, saline, gentamicin saline rinse, ringer's solution rinse, mannitol rinse, glucose rinse, chlorhexidine rinse, metronidazole rinse, glycine rinse or physiological equilibrating solution.
3. The bioengineered protein-based bioadhesive of claim 1, wherein the bioengineered protein is an elastin-like protein.
4. A preparation method of the biological engineering protein-based biological adhesive applying the biological engineering protein-based biological adhesive according to any one of claims 1 to 3, wherein the preparation method of the biological engineering protein-based biological adhesive comprises the following steps:
step one, preparing bioengineering protein: converting the ELP-like carrier plasmid into a pichia pastoris or escherichia coli expression strain, selecting a monoclonal colony, and carrying out overnight culture by using LB culture solution; adding the overnight activated expression seed liquid into a TB culture medium, adding an inducer isopropyl-beta-D-thiogalactopyranoside when the bacterial liquid reaches an OD600 value of 0.8-1.0, and cooling to 30 ℃ for overexpression; inducing for 10-12 h, collecting thalli, resuspending with lysis buffer, adding mixed microbial inoculum, crushing thalli, centrifuging at 10000-12000 rpm, collecting supernatant, and purifying by HPLC to obtain elastin-like protein (ELP) for later use;
step two, preparing the chitin whisker: weighing shrimp and crab shells, crushing the shrimp and crab shells into powder, dispersing the powder in 3M concentrated sulfuric acid, stirring the powder in a water bath at 85-95 ℃ for 10-12 hours, diluting the powder with deionized water, centrifuging the diluted solution to remove supernatant, and repeating the step for 3-5 times to obtain precipitate; resuspending the obtained precipitate with deionized water, transferring the precipitate into a dialysis bag with the molecular weight cutoff of 8000-14000 Da, and fully dialyzing until the pH value of the suspension in the dialysis bag is 6.0; performing ultrasonic treatment at the power of 600-800W for 10-30 min to completely suspend the precipitate, centrifuging the obtained suspension at 7000-7500 rpm, and freeze-drying for 5-20 min to obtain chitin whiskers for later use;
step three, preparing the parthenocissus tricuspidata sucker polysaccharide: collecting the parthenocissus tricuspidata sucker, washing with deionized water, airing in the sun for 6-8 h, then putting into an airflow crusher for crushing, and sieving with a 80-120-mesh sieve to obtain parthenocissus tricuspidata sucker powder; adding the parthenocissus tricuspidata sucker powder into an ethanol solution, heating in a water bath, performing reflux extraction for 2-4 h, and distilling at 60-70 ℃ to recover the ethanol solution to obtain degreased sucker powder; adding hot water with the temperature of 90-100 ℃ which is 10-20 times that of the degreased sucker powder, leaching for 3-5 hours, and performing centrifugal concentration to obtain a polysaccharide concentrated solution of the parthenocissus tricuspidata sucker for later use;
step four, preparation of maleylation hyaluronic acid is carried out: placing hyaluronic acid and dimethylformamide into deionized water, and stirring at room temperature for 8-24 hours to obtain a mixed solution A; adding maleic anhydride into the mixed solution A, uniformly stirring, and reacting for 12-48 h at 35-80 ℃ to obtain a mixed solution B; adding 1mol/L NaHCO into the mixed solution B3Adjusting the pH value of the mixed solution to 7-8, dialyzing, and freeze-drying to obtain maleylation hyaluronic acid for later use;
step five, weighing a solvent, a biological engineering protein, chitin whiskers, parthenocissus tricuspidata sucker polysaccharide, tannic acid, maleylation hyaluronic acid and a catalyst in sequence according to parts by weight for later use;
step six, adding 50-70 times of deionized water into the bioengineering protein, dissolving the protein under the condition of heating and stirring at 60 ℃, adding 12mol/L sodium hydroxide solution to adjust the pH value to 8.5, adding tannic acid, fully stirring and dissolving, and continuously introducing air;
and seventhly, sequentially adding the chitin whisker, the parthenocissus tricuspidata sucker polysaccharide, the maleylation hyaluronic acid and the catalyst, fully stirring and dissolving, adjusting the pH to 7.4 by using 0.5mol/L diluted hydrochloric acid after the reaction is finished, cooling the reactant to room temperature, dialyzing, and freeze-drying to obtain the biological adhesive based on the biological engineering protein.
5. The method of claim 4, wherein in step one, the molar ratio of the elastin-like protein to alkyl anionic surfactant is 1: (1.5 to 3).
6. The method for preparing biological adhesive based on bioengineered protein according to claim 4, wherein in the first step, the mixed microbial inoculum comprises protease inhibitor, DNase and lysozyme.
7. The method for preparing a biological adhesive based on bioengineering protein according to claim 4, wherein in the second step, the mixing ratio of the shrimp and crab shells to the concentrated sulfuric acid solution is 3-5% w/v.
8. The method for preparing a bio-adhesive based on bioengineered proteins according to claim 4, wherein the ethanol solution has a mass concentration of 95% in step three.
9. The method for preparing a bio-adhesive based on bio-engineered proteins according to claim 4, wherein in the fourth step, the conditions of freeze-drying are as follows: and (3) placing the mixed solution at-50 ℃ and under the pressure of 5-20 Pa, and freeze-drying for 48-72 h.
10. The method for preparing a bio-adhesive based on bioengineered proteins of claim 4, wherein in step seven, the dialysis is:
and carrying out deionized water dialysis on the reactant for 3-5 times by using a dialysis bag with a molecular weight cut-off value of 3500 Da.
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