Antibacterial material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of antibacterial materials, in particular to an antibacterial material and a preparation method and application thereof. The invention discloses an antibacterial master batch containing bioengineering protein as well as a preparation method and application of the antibacterial master batch, and belongs to the technical field of antibacterial materials. The antibacterial master batch is formed by compounding positively charged elastin-like protein (VPGRG) 72 and a matrix material, and has excellent broad-spectrum antibacterial performance and high temperature resistance. The material can be subjected to hot blending processing with a macromolecular matrix material, and other auxiliaries (such as an antioxidant and a dispersing agent) do not need to be added. The antibacterial master batch has good biocompatibility, has no irritation to skin and tissues, and can be used for various antibacterial medical instruments, such as antibacterial tooth socket membranes, in-vivo interventional catheters, abdominal repair patches and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial materials, and particularly to antibacterial materials, their preparation methods and applications. Background Art

[0002] Antibacterial materials are a new type of functional materials that can inhibit and kill bacteria in the external environment. With the increasing emphasis on health and environmental protection by people, the demand for antibacterial materials is continuously increasing.

[0003] Dental braces are a type of medical device used for oral care, mainly for tooth correction, tooth protection or treatment of periodontal diseases, etc. Traditional dental braces are mostly made of metal materials. With the development of technology, invisible dental braces have gradually become popular due to their advantages such as aesthetics and comfortable wearing. However, the oral environment is complex, and the growth of bacteria is likely to cause diseases such as dental caries and periodontitis. The demand for the antibacterial performance of dental braces is increasing day by day. On the one hand, if the dental braces themselves can be antibacterial, it can effectively inhibit the growth of bacteria and reduce the risk of oral diseases; on the other hand, antibacterial dental braces can extend the service life and reduce the maintenance cost. In addition to dental braces, the use of antibacterial materials in other medical devices is also becoming more and more common, which can not only reduce the risk of infection, but also reduce the cost of sterilization.

[0004] In nature, many substances themselves have good bactericidal or bacteriostatic functions, such as some organic compounds with specific groups, some inorganic metal materials and their compounds, some minerals and natural substances. Inorganic antibacterial agents utilize the antibacterial properties of metals or their ions such as silver, copper, zinc, etc., and through physical adsorption or ion exchange and other methods, fix these metal ions on the surface of porous materials, and achieve antibacterial by contact or controlling the release of metal ions. However, these metal ions usually have high toxicity and potential carcinogenicity. Organic antibacterial agents mainly include some quaternary ammonium salts, polypeptides, amino acids, etc. They have a fast bactericidal speed and strong bactericidal ability, but poor heat resistance, and are prone to decomposition during the thermoplastic processing with the matrix polymer material, resulting in the loss of antibacterial performance.

[0005] Therefore, the research and development of an antibacterial material with strong antibacterial ability, high temperature resistance and good biocompatibility, so as to prepare antibacterial medical devices, is still a research hotspot in this field. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide antibacterial materials, their preparation methods and applications.

[0007] The present invention provides the application of elastin-like protein (VPGRG)72 in the preparation of antibacterial materials.

[0008] In the present invention, the elastin-like protein (VPGRG)72 is abbreviated as R72 protein, and its amino acid sequence is MGQG[VG(VPGRG)9VPG]8LGAWH6, specifically as shown in SEQ ID NO.1. Among them, H6 is a purification tag set for convenient purification. It can be present or absent, and it can be a 6×His tag or other tags. The present invention does not limit this. As a feasible case, a 6×His tag is used for verification in the present invention.

[0009] Compared with the disadvantage that other antimicrobial peptides or antimicrobial proteins will deteriorate at high temperatures, the elastin-like protein described in the present invention not only has good antibacterial performance but also shows good high-temperature resistance in experiments. Therefore, this elastin-like protein can resist the high temperature generated by the melting of the matrix material, so that it can be well mixed with the matrix material and produce antibacterial performance. Experiments show that this protein still has good antibacterial effects on Gram-positive bacteria, Gram-negative bacteria and anaerobic bacteria after melt mixing.

[0010] In the present invention, the preparation of the elastin-like protein (VPGRG)72 as described above includes:

[0011] ① Synthesize the nucleic acid encoding the elastin-like protein (VPGRG)72 and construct it on an expression plasmid vector;

[0012] ② Transform or transfect the expression plasmid vector into a host cell;

[0013] ③ Culture the host cell and induce protein expression;

[0014] ④ Isolate the host cell expressing the bioengineered protein.

[0015] In the present invention, after the host cell expresses the elastin-like protein, it further includes a purification step, and the purification includes: disrupting the host cell and collecting the precipitate; further treating the precipitate with a protein purifier, and freeze-drying to obtain the bioengineered protein.

[0016] The antibacterial material provided by the present invention includes an elastin-like protein and a matrix material; the elastin-like protein is (VPGRG)72.

[0017] In the present invention, the matrix material is a polymer material, preferably a medical polymer material. The antibacterial material of the present invention has good plasticity and can be mixed with the aforementioned elastin-like protein. When the polymer material is in a molten state due to heat, it can fill the mold cavity through viscous flow according to common molding processes such as injection molding and extrusion molding, and obtain a product that meets the design expectations after cooling and solidification. It should be clear that the present invention does not limit the specific chemical category of the polymer matrix material, such as polyolefins, polyesters, polyamides, etc., and only requires that its performance can meet the relevant conditions for efficient mixing with antibacterial peptides and adapting to the melt molding process.

[0018] In the present invention, the matrix material includes at least one of polyurethane, polyethylene terephthalate-ethylene glycol copolyester, polycaprolactone, polylactic acid, polyvinyl chloride, polypropylene, and / or polyethylene. In some embodiments, the matrix material is polyurethane. In a specific embodiment, the polyurethane is a polyurethane with a Shore hardness of 60D to 82D. For example, it is a polyurethane with a Shore hardness of 60D, 75D, or 82D.

[0019] The present invention has explored and verified the content of elastin-like protein in the antibacterial material. Experiments show that adding the aforementioned elastin-like protein does not interfere with the mechanical properties of the matrix material. On the contrary, the addition of a certain amount of elastin-like protein can improve the tensile elastic modulus, yield stress, yield tensile strain, tensile strength, and elongation at break of the antibacterial material. In the present invention, the mass fraction of the elastin-like protein is 1% to 20%. In some specific embodiments, the mass fraction of elastin-like protein is 1%, 2%, 6%, 10%, or 20%.

[0020] The present invention also provides a preparation method of the aforementioned antibacterial material, which includes: mixing the elastin-like protein with the dried matrix material, heating to melt and then extruding, and obtaining the antibacterial material after cooling.

[0021] In the present invention, the antibacterial material can be directly melt-molded into the expected medical device. It can also be first prepared into masterbatch, and then remelted and / or added with matrix material to prepare the expected medical device. The present invention does not limit this. After the antibacterial material of the present invention is used to prepare a medical device, the mass fraction of elastin-like protein is 1% to 6%, and in the antibacterial material of the medical device, the elastin-like protein exists in a mass fraction of 1% to 6%. If a masterbatch is prepared, the elastin-like protein therein can be 2 to 100 times the mass fraction during use. For example, it is 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, or 100 times the mass fraction of elastin-like protein in the medical device material obtained.

[0022] In the embodiments of the present invention, the matrix material is polyurethane, and its drying step includes: the drying dew point is -30 to -40°C, the drying temperature is 90 to 120°C, and the drying time is 4 to 10 h.

[0023] The preparation method has a simple process, rich raw material sources, and does not require complex equipment, making it suitable for industrial production applications. In some embodiments: the matrix material is polyurethane, and its drying step includes: the drying dew point is -30°C, the drying temperature is 90 to 110°C, and the drying time is 6 to 10 h.

[0024] For example, in a specific embodiment, the drying step includes: the drying dew point is -30°C, the drying temperature is 100°C, and the drying time is 10 h. Or it includes: the drying dew point is -30°C, the drying temperature is 110°C, and the drying time is 10 h.

[0025] In the present invention, the extrusion temperature is 150 - 230°C. In some embodiments, the extrusion temperature is 210 - 220°C, and it is extruded into pellets or sheets.

[0026] In a specific embodiment, when the antibacterial material is prepared into an antibacterial masterbatch, the preparation method of the antibacterial material as described above includes:

[0027] S1. Take 10 - 20 parts by mass of the elastin-like protein (VPGRG) 72 and 90 - 80 parts by mass of the dried matrix material, and mix them in a drum mixer at 400 - 500 rpm for 1 - 3 min;

[0028] S2. Pass the material mixed in S1 through a pre-heated twin-screw extrusion device for melt blending to uniformly disperse the bioengineered protein into the matrix material to obtain an extruded material.

[0029] S3. Cut the extruded material into pellets by a pelletizer to obtain the antibacterial masterbatch.

[0030] In other embodiments, when the antibacterial material is directly melt-molded to prepare medical devices, taking the preparation of a dental appliance sheet as an example, the preparation method of the antibacterial material as described above includes:

[0031] s1. Take 1 - 6 parts by mass of the elastin-like protein (VPGRG) 72 and 94 - 99 parts by mass of the dried matrix material, and mix them in a drum mixer at 400 - 500 rpm for 1 - 3 min;

[0032] s2. Pass the material mixed in s1 through a pre-heated twin-screw extrusion device for melt blending to uniformly disperse the bioengineered protein into the matrix material to obtain an extruded material.

[0033] s3. Extrude and trim the extruded material to obtain an antibacterial dental appliance sheet containing bioengineered protein.

[0034] Furthermore, the present invention also provides the use of the antibacterial material as described above or the antibacterial material prepared by the preparation method as described above in the preparation of antibacterial medical devices.

[0035] Even further, the present invention also provides an antibacterial medical device, the raw materials of which include the antibacterial material as described above or the antibacterial material prepared by the preparation method as described above.

[0036] In the present invention, the antibacterial medical device includes dental materials, specifically dental braces and / or antibacterial dental brace membranes. Alternatively, it may also be at least one of an in-vivo interventional catheter or an abdominal repair patch.

[0037] The antibacterial material provided by the present invention still has good antibacterial properties after being melted at high temperature. Therefore, it can be used as a dental material. For example, the dental brace membrane described in the present invention adds (VPGRG)72 as an antibacterial agent in the inner membrane close to the teeth, which has obvious antibacterial effects on pathogenic bacteria on the tooth surface such as Porphyromonas gingivalis and Streptococcus mutans, and has good biocompatibility. The inner antibacterial membrane only contacts the tooth surface, does not disrupt the microbial balance in the oral cavity, and can effectively ensure oral health.

[0038] In some embodiments, the dental material is a dental brace. More specifically, the dental brace includes an inner layer and an outer layer. The inner layer is the antibacterial material as described above (also referred to as the dental brace membrane in the present invention), and the hardness of the outer layer is higher than that of the inner layer, so that the patient can feel more comfortable during wearing and can ensure that it does not deform after repeated removal and wearing. In the present invention, the outer layer of the dental brace can be made of the same polymer material as the inner layer or different materials, and the present invention does not limit this. As a feasible case, the inner layer material of the antibacterial dental brace membrane is the antibacterial material as described above, and the outer layer material is polyethylene terephthalate-ethylene glycol copolyester (PETG).

[0039] Compared with the prior art, the bioengineered protein-containing antibacterial material provided by the present invention is composed of a positively charged elastin-like protein and a matrix material, and has: ① excellent broad-spectrum antibacterial properties, having certain antibacterial effects on Gram-positive bacteria, Gram-negative bacteria and anaerobic bacteria. ② High temperature resistance, and can be melt-processed by blending with medical polymer matrix materials, such as melt blending, extrusion and injection molding. ③ No other additives (such as antioxidants, dispersants, etc.) need to be added during the processing, and the composite material has good biocompatibility and no irritation to the skin and tissues. It can be applied to antibacterial medical devices, such as antibacterial dental brace membranes, in-vivo interventional catheters and abdominal repair patches, etc., and has good application prospects. Description of the Drawings

[0040] Figure 1Thermogravimetric (TG-DTA) curve of bioengineered protein (VPGRG) 72 (N2 atmosphere);

[0041] Figure 2 Thermogravimetric (TG-DTA) curve of organic antibacterial agent dodecyl dimethyl ammonium bromide (DDAB) (N2 atmosphere);

[0042] Figure 3 Thermogravimetric (TG-DTA) curve of antibacterial peptide lysozyme (N2 atmosphere). Detailed implementation manners

[0043] The present invention provides an antibacterial material, a preparation method thereof and applications. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate changes and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] Unless otherwise defined in the present invention, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0045] In addition, unless otherwise specified in this article, terms in the singular form in this article shall include the plural form, and terms in the plural form shall include the singular form. More specifically, as used in this specification and the appended claims, unless otherwise clearly indicated, the singular forms "a" and "the" include plural referents.

[0046] The terms "comprising", "including" and "having" can be used interchangeably in this article, and are intended to indicate the inclusiveness of the solution, meaning that the solution may contain other elements in addition to the listed elements. At the same time, it should be understood that when using "comprising", "including" and "having" to describe in this article, the solution of "consisting of..." is also provided.

[0047] When the term "and / or" is used in this article, it includes the meanings of "and", "or" and "any other combination of all or any of the elements linked by the term".

[0048] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single item (s) or plural item (s).

[0049] It should be understood that in various embodiments of the present application, the magnitudes of the serial numbers of the above processes do not imply the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined according to its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0050] In the embodiments, only some representative experimental results are presented. During the experiment, other similar elastin-like proteins stored in this laboratory or antibacterial peptides commonly used in the prior art could not exhibit the characteristic of high temperature resistance. The materials obtained after melt extrusion with polyurethane did not have antibacterial properties, and relevant experiments will not be elaborated here.

[0051] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0052] The elastin-like protein contains 72 repeated pentapeptide sequences (VPGRG), and a 6×His tag is also connected to the end of the elastin-like protein. The amino acid sequence thereof is as shown in SEQ ID NO.1, and the molecular weight is 38509.

[0053] The bioengineered protein (VPGRG)72 is expressed according to the following steps:

[0054] (1) Obtain a strain expressing the bioengineered protein (VPGRG)72. The nucleotide sequence of the bioengineered protein (VPGRG)72 is constructed on a Pet25b expression plasmid vector for transformation or transfection into the Escherichia coli prokaryotic expression competent cell BL21(DE3).

[0055] (2) Primary seed. The vector plasmid of the bioengineered protein (VPGRG)72 is transformed into the Escherichia coli BL21(DE3) expression strain and spread on an agar plate containing 100 μg / ml ampicillin. It is cultured at 37 °C for 12 - 16 h, and then a single colony is picked into 5 mL of LB liquid medium (the concentration of ampicillin is 100 μg / ml) and cultured overnight at 37 °C and 220 rpm. This is the primary seed.

[0056] (2) Secondary seed. The primary seed is transferred to 100 mL of LB liquid medium (the concentration of ampicillin is 100 μg / ml) at an inoculation amount of 10%, and cultured at 37 °C and 220 rpm for about 3 h. When the OD600 of the bacterial liquid reaches 0.6 - 0.8, 50 mL is taken out and added with an IPTG solution with a final concentration of 0.1 mM to induce protein expression. After 6 - 8 h, a sample is taken for SDS-PAGE electrophoresis to analyze the protein expression. After confirming protein expression, the uninduced bacterial liquid is transferred to 1 L of LB liquid medium (the concentration of ampicillin is 100 μg / ml) at an inoculation amount of 10%. This is the secondary seed.

[0057] (3) Fermentation. When the OD600 of the secondary seed culture reaches 5 - 10, it is transferred to the fermenter at an inoculation amount of 2%. The fermentation conditions are as follows: 37°C, stirring at 300 - 500 rpm with the dissolved oxygen linked and controlled at 30%, the pH value is controlled at 6.9 - 7.0 by ammonia water, the aeration rate is 1 vvm, and the tank pressure is controlled at 0.04 - 0.06 MPa. When the dissolved oxygen level rises, a glucose solution is supplemented through a peristaltic pump, and the sugar content is controlled within 2 g / L. When OD600 reaches 15 - 30, a solution of IPTG with a final concentration of 0.1 mM is added, and fermentation is stopped after induction for 5 - 6 h.

[0058] Preferably, the bioengineered protein (VPGRG)72 is purified according to the following steps: <{

[0059] (1) The fermented bacterial liquid is centrifuged at 8000 - 9000 rpm for 20 - 30 min, and the bacterial precipitate is collected.

[0060] (2) The wet bacteria are weighed, and for every gram of bacteria, 10 mL of 50 mM Tris-HCl (pH = 8.0) is added. 1 mg of lysozyme and 5 μg of DNase are added to each milliliter of the bacterial liquid and mixed well. Then, it is boiled at 37°C for 30 min to allow the enzymes to take effect, and boiled at 50°C for 60 min to inactivate the enzymes and precipitate the miscellaneous proteins. The bacterial cells are broken using a high-pressure homogenizer and centrifuged at 10000 - 12000 rpm for 20 - 30 min, and the supernatant is collected.

[0061] (3) The supernatant is passed through a 100 kD ultrafiltration column, and then decolorized through a hollow fiber column with a molecular weight cut-off of 30 kD. The pigment belongs to small molecules and is gradually reduced by dialysis with a buffer change (50 mM Tris-HCl, pH = 8.0). The buffer is continuously changed during the concentration process. When the color of the concentrated solution changes from brown to colorless, the protein solution in the hollow fiber column is discharged with 50 mM Tris-HCl, and then it can be loaded for purification.

[0062] (4) ① Wash the pump. Rinse the A1 and B1 pipelines of the protein purifier with deionized water through the membrane. After the UV absorption at 280 nm and the conductivity are constant, open the column position valve. ② Install the column. Connect the self-packed column filled with Q packing to the column position valve, and rinse the column with deionized water through the membrane. After the UV absorption and conductivity are constant, pause to prepare for liquid change. ③ Pre-equilibrate. Insert A1 into the 50 mM Tris-HCl (pH = 8.0) buffer solution, and insert B1 into the protein eluent 50 mM Tris-HCl, 1 M NaCl (pH = 8.0). Flush the pipeline at a flow rate of 10 mL / min with 100% A1. After the UV absorption at 280 nm and the conductivity are constant, prepare for sample loading. ④ Load the sample. Insert A1 into the protein solution and load the sample at a certain linear rate. When the UV absorption at 280 nm > 100, start to collect the flow-through liquid of the Q column until the sample is loaded completely, then pause and prepare for equilibration. ⑤ Equilibrate. Insert A1 into the 50 mM Tris-HCl (pH = 8.0) buffer solution and flush the column at a certain linear flow rate. Wait until the UV absorption at 280 nm and the conductivity are constant, then pause and prepare for elution. ⑥ Elute. Set the gradient elution program to increase B1 from 0% to 100% within 60 min. When the UV absorption at 280 nm > 60, collect the elution peak until B1 reaches 100% and then continue to wash for two column volumes. The elution is completed. Perform SDS-PAGE electrophoresis analysis on the received protein eluent. If the protein purity is low, it can be repeatedly loaded onto the Q column. <> <>

[0063] (5) Desalt the protein eluent. Desalt it with a G25 desalting column, and the buffer solution is 20 mM PB (9.5 mM NaH2PO4 + 10 mM Na2HPO4). Then pass through a multi-mode chromatography column. The difference from step (3) is that 50 mM PB is replaced with 20 mM PB, and the protein eluent is 20 mM PBS + 1 M NaCl. Finally, desalt again, and the buffer solution is 5 mM PB (2.7 mM NaH2PO4 + 2.3 mM Na2HPO4). <> <>

[0064] (6) Transfer the received solution to a freeze dryer for freeze-drying to obtain the bioengineered protein. <> <>

[0065] The matrix material is a medical polymer, including but not limited to medical polyurethane (TPU), polyethylene terephthalate-ethylene glycol copolyester (PETG), polycaprolactone (PCL), polylactic acid (PLA), polyvinyl chloride (PVC), polypropylene (PP), and polyethylene (PE), etc. <> <>

[0066] The following further elaborates the present invention in conjunction with embodiments: <> <>

[0067] Example 1 <> <>

[0068] An antibacterial masterbatch containing a bioengineered protein and its antibacterial dental appliance membrane sheet, comprising the following preparation steps:

[0069] S1. Preparation of bioengineered protein (VPGRG) 72

[0070] Expression of S1.1 and (VPGRG)72

[0071] (VPGRG)72 was expressed as follows:

[0072] S1.1.1. Obtain a strain expressing the bioengineered protein. The nucleotide sequence of (VPGRG)72 was synthesized by GenWeiZhi and constructed on the Pet25b expression plasmid vector for transformation or transfection into the prokaryotic expression-competent Escherichia coli BL21(DE3).

[0073] S1.1.2. Prepare primary seed: Transform the (VPGRG)72 vector plasmid into the Escherichia coli BL21(DE3) expression strain, spread on an agar plate containing 100 μg / ml ampicillin, and incubate at 37°C for 12-16 h. Then, pick a single colony and transfer it to 5 mL of LB liquid medium (with 100 μg / ml ampicillin) and incubate overnight at 37°C, 220 rpm. This is the primary seed;

[0074] S1.1.3. Preparation of secondary seeds: Transfer 10% of the primary seeds to 50 mL of LB liquid medium (with 100 μg / ml of ampicillin) and culture at 37°C and 220 rpm for approximately 3 h. When the OD600 of the bacterial solution reaches 0.6-0.8, add IPTG solution at a final concentration of 0.1 mM to induce protein expression. After 6-8 h, sample the culture and analyze protein expression by SDS-PAGE electrophoresis. After confirming protein expression, transfer 10% of the primary seeds to 1 L of LB liquid medium (with 100 μg / ml of ampicillin) to prepare the secondary seeds.

[0075] S1.1.4, Fermentation: When the OD600 of the secondary seed bacterial liquid reaches 5-10, transfer it to a 6L fermenter at a 10% inoculum volume. Fermentation conditions are 37°C, stirring at 300-500rpm, and the dissolved oxygen is controlled to 30%. The pH value is controlled at 6.9-7.0 with ammonia water, the ventilation rate is 1vvm, and the tank pressure is controlled at 0.04-0.06MPa. When the dissolved oxygen level rises, the glucose solution is supplemented by a peristaltic pump, and the sugar content is controlled within 2g / L. When the OD600 reaches 15-30, the flame is connected to a final concentration of 0.1mMIPTG solution, and the fermentation is stopped after induction for 5-6h.

[0076] S1.2 Purification of bioengineered protein (VPGRG) 72

[0077] Purify (VPGRG)72 according to the following steps:

[0078] S1.2.1. The fermented bacterial liquid is centrifuged at 8,000 - 9,000 rpm for 20 - 30 min to collect the bacterial precipitate.

[0079] S1.2.2. Weigh the wet bacteria. For every gram of bacteria, add 10 mL of 50 mM Tris - HCl (pH = 8.0). Add 1 mg of lysozyme and 5 μg of DNase to each milliliter of the bacterial liquid and mix well. Then, boil at 37°C for 30 min to allow the enzymes to act, and boil at 50°C for 60 min to inactivate the enzymes and precipitate the miscellaneous proteins. Use a high - pressure homogenizer to break the bacterial cells, centrifuge at 10,000 - 12,000 rpm for 20 - 30 min, and collect the supernatant.

[0080] S1.2.3. The supernatant is passed through a 100 kD ultrafiltration column, and then decolorized through a hollow fiber column with a molecular weight cut - off of 30 kD. The pigments are small molecules and are gradually reduced through dialysis by changing the solution (50 mM Tris - HCl, pH = 8.0). During the concentration process, the solution is continuously changed. When the color of the concentrated solution changes from brown to colorless, the protein solution in the hollow fiber column is discharged with 50 mM Tris - HCl, and then it can be loaded for purification.

[0081] S1.2.4. ① Wash the pump. The A1 and B1 pipelines of the protein purifier are respectively rinsed with deionized water passing through the membrane. After the UV absorption at 280 nm and the conductivity are constant, open the column position valve; ② Install the column. Connect the self - packed column filled with Q packing to the column position valve, rinse the column with deionized water passing through the membrane. After the UV absorption and the conductivity are constant, pause and prepare to change the solution; ③ Pre - balance. Insert A1 into the 50 mM Tris - HCl (pH = 8.0) buffer solution, insert B1 into the protein elution solution 50 mM Tris - HCl, 1 M NaCl (pH = 8.0). Flush the pipeline with 100% A1 at a flow rate of 10 mL / min. After the UV absorption at 280 nm and the conductivity are constant, prepare to load the sample; ④ Load the sample. Insert A1 into the protein solution and load the sample at a certain linear rate. When the UV absorption at 280 nm > 100, start to collect the flow - through liquid of the Q column until the sample is loaded completely, then pause and prepare to balance; ⑤ Balance. Insert A1 into the 50 mM Tris - HCl (pH = 8.0) buffer solution and flush the column at a certain linear flow rate. Wait until the UV absorption at 280 nm and the conductivity are constant, then pause and prepare to elute; ⑥ Elute. Set the gradient elution program to increase B1 from 0% to 100% within 60 min. When the UV absorption at 280 nm > 60, collect the elution peak until B1 reaches 100% and then continue to wash for two column volumes. The elution is completed. Conduct SDS - PAGE electrophoresis analysis on the collected protein elution solution. For proteins with lower purity, they can be re - loaded onto the Q column.

[0082] S1.2.5. Protein eluate desalting. Desalt using a G25 desalting column, with the buffer being 20 mM PB (9.5 mM NaH2PO4 + 10 mM Na2HPO4). Then pass through a multi-mode chromatography column. The difference from step (3) is that 50 mM PB is replaced with 20 mM PB, and the protein eluate is 20 mM PBS + 1 M NaCl. Finally, desalt again, with the buffer being 5 mM PB (2.7 mM NaH2PO4 + 2.3 mM Na2HPO4).

[0083] S1.2.6. Transfer the receiving solution into a freeze dryer and freeze-dry to obtain the bioengineered protein (VPGRG) 72.

[0084] S2. Preparation of antibacterial masterbatch

[0085] S2.1. Weigh by weight parts: 10 parts of bioengineered protein and 90 parts of medical polyurethane (TPU, 75D). The drying dew point of medical polyurethane (TPU) is -30°C, the drying temperature is 100°C, and the drying time is 8 h. Then mix (VPGRG) 72 and medical polyurethane (TPU) in a roller mixer at 500 rpm for 10 min.

[0086] S2.2. Melt and blend the above mixed materials through a co-rotating twin-screw extruder, cool with water, pelletize, dry, and package. Obtain a TPU antibacterial masterbatch containing 10% bioengineered protein (VPGRG) 72.

[0087] S3. Preparation of antibacterial dental appliance membrane sheet containing bioengineered protein (VPGRG) 72

[0088] S3.1. The inner layer membrane sheet material is composed of the antibacterial masterbatch obtained in S2 and medical polyurethane (TPU). Weigh by weight parts 10:90 (10 parts of 10% antibacterial masterbatch, and then mix with 90 parts of the matrix material. The protein content in the obtained antibacterial material is 1%). Obtain an inner layer membrane sheet material containing 1% bioengineered protein (VPGRG) 72. Dry the inner layer membrane sheet material and the outer layer membrane sheet material polyethylene terephthalate-ethylene glycol copolyester (PETG). The drying dew point is -30°C, the drying temperature is 100°C, and the drying time is 10 h.

[0089] S3.2. Load the dried inner and outer layer membrane sheet materials into a multi-layer co-extrusion device. The outer layer extrusion temperature is 220°C, and the inner layer extrusion temperature is 210°C. The thickness of the membrane sheet body is 0.8 mm, and the thickness of the inner layer membrane sheet is 0.2 mm. The thickness of the membrane sheet can be adjusted by the extrusion device during the production process. Trim after extrusion molding to obtain the antibacterial dental appliance membrane sheet containing bioengineered protein (VPGRG) 72.

[0090] Example 2

[0091] An antibacterial masterbatch containing bioengineered protein and its antibacterial dental appliance membrane sheet, comprising the following preparation steps:

[0092] S1. The specific conditions for preparing the bioengineered protein are the same as those in Example 1.

[0093] S2. Prepare an antibacterial masterbatch containing bioengineered protein

[0094] S2.1. Weigh by weight, 20 parts of bioengineered protein and 80 parts of medical polyurethane (TPU, 60D). The drying dew point of medical polyurethane (TPU) is -30°C, the drying temperature is 90°C, and the drying time is 10h. Then mix (VPGRG)72 with medical polyurethane (TPU) in a roller mixer at 600 rpm for 20 min.

[0095] S2.2. Melt and blend the above mixed materials through a co-rotating twin-screw extruder, cool with water, pelletize, dry, and package. Obtain a TPU antibacterial masterbatch containing 20% bioengineered protein (VPGRG)72.

[0096] S3. Prepare an antibacterial dental appliance membrane sheet containing bioengineered protein

[0097] S3.1. The inner layer membrane sheet material is composed of the antibacterial masterbatch obtained in S2 and medical polyurethane (TPU). Weigh by weight 5:95 to obtain an inner layer membrane sheet material containing 1% bioengineered protein (VPGRG)72, and weigh an appropriate amount of the outer layer membrane sheet material polyethylene terephthalate-ethylene glycol copolyester (PETG). Dry the inner and outer layer membrane sheet materials, with a drying dew point of -30°C, a drying temperature of 100°C, and a drying time of 10h.

[0098] S3.2. Load the dried inner and outer layer membrane sheet materials into a multi-layer co-extrusion device. The outer layer extrusion temperature is 220°C, and the inner layer extrusion temperature is 170°C. The thickness of the membrane sheet body is 0.8 mm, and the thickness of the inner layer membrane sheet is 0.3 mm. Trim after extrusion molding to obtain an antibacterial dental appliance membrane sheet containing bioengineered protein.

[0099] Example 3

[0100] An antibacterial masterbatch containing bioengineered protein and its antibacterial dental appliance membrane sheet, comprising the following preparation steps:

[0101] S1. The specific conditions for preparing the bioengineered protein are the same as those in Example 1.

[0102] S2. Prepare an antibacterial masterbatch containing bioengineered protein

[0103] S2.1. Weigh by parts by weight, 15 parts of bioengineered protein and 85 parts of medical polyurethane (TPU, 82D). The drying dew point of medical polyurethane (TPU) is -30°C, the drying temperature is 110°C, and the drying time is 6 h. Then mix (VPGRG)72 with medical polyurethane (TPU) in a roller mixer at 800 rpm for 10 min.

[0104] S2.2. Melt and blend the above mixed materials through a co-rotating twin-screw extruder, cool with water, pelletize, dry, and package. Obtain a PTU antibacterial masterbatch containing 15% bioengineered protein (VPGRG)72.

[0105] S3. Prepare an antibacterial dental guard film sheet containing bioengineered protein

[0106] S3.1. The film sheet material is composed of the antibacterial masterbatch obtained in S2 and medical polyurethane (TPU). Weigh by parts by weight 40:60 and then dry. The drying dew point is -30°C, the drying temperature is 100°C, and the drying time is 10 h. The obtained film sheet material contains 6% bioengineered protein (VPGRG)72.

[0107] S3.2. Load the dried film sheet material into an extrusion casting device, and the extrusion temperature is 220°C. The thickness of the film sheet is 0.8 mm. Extrusion molding and trimming yield an antibacterial dental guard film sheet containing bioengineered protein.

[0108] Comparative Example 1

[0109] The difference from Example 3 is that the parts by weight of the antibacterial masterbatch containing bioengineered protein is 0, the hardness of the medical polyurethane is 82D, and other conditions and steps are the same as those in Example 3. What is obtained in Comparative Example 1 is an antibacterial dental guard film sheet without bioengineered protein (VPGRG)72.

[0110] Comparative Example 2

[0111] The difference from Example 1 is that the inorganic antibacterial agent IONPURE is used to replace the bioengineered protein. IONPURE is a heat-resistant glass material combined with silver ions or zinc ions and copper ions with antibacterial activity. Other conditions and steps are the same as those in Example 1. What is obtained in Comparative Example 2 is an antibacterial dental guard film sheet containing the inorganic antibacterial agent IONPURE.

[0112] Comparative Example 3

[0113] The difference from Example 1 is that the organic antibacterial agent dodecyl dimethyl ammonium bromide (DDAB) is used to replace the bioengineered protein. Other conditions and steps are the same as those in Example 1. What is obtained in Comparative Example 3 is an antibacterial dental guard film sheet containing the inorganic antibacterial agent IONPURE.

[0114] Comparative Example 4

[0115] The difference from Example 1 is that the antimicrobial peptide lysozyme (purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.) is used to replace the bioengineered protein (VPGRG)72. Other conditions and steps are the same as those in Example 1. The obtained antimicrobial dental retainer membrane of Comparative Example 4 contains lysozyme.

[0116] Performance test

[0117] Antibacterial performance test: The antimicrobial dental retainer membranes obtained in Examples 1 to 3 and Comparative Examples 1 to 4 were subjected to antibacterial performance tests. The test standard was based on GB / T 31402-2015 "Test method for antibacterial properties of plastics on the surface of plastics". The control sample was a standard PE film. The test strains were: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Porphyromonas gingivalis ATCC33277, Streptococcus mutans ATCC 25175. The relevant test results are shown in Table 1.

[0118] Biocompatibility test

[0119] The antimicrobial dental retainer membranes obtained in Examples 1 to 3 and Comparative Examples 1 to 4 were subjected to biocompatibility evaluation. The test standards were based on GB / T 16886.5-2017 In vitro cytotoxicity test and GB / T 16886.10-2017 Tests for irritation and skin sensitization, and the samples were prepared according to ISO 10993-12 Biological evaluation of medical devices—Part 12: Sample preparation and reference materials.

[0120] The relevant test results are shown in Table 1.

[0121] Table 1 Antibacterial and biocompatibility test results

[0122]

[0123] As can be seen from Table 1, the antibacterial dental appliance membrane added with the bioengineered protein (VPGRG) 72 and the inorganic antibacterial agent IONPURE has good antibacterial performance. The dental appliance membranes without added antibacterial agent and those added with the organic antibacterial agent quaternary ammonium salt DDAB and the antibacterial peptide lysozyme have almost no antibacterial performance. This is because the organic antibacterial agent and the antibacterial peptide have poor heat resistance and decompose during the thermoplastic processing with the matrix polymer material. Moreover, the cell survival rate of the antibacterial dental appliance membrane added with the bioengineered protein (VPGRG) 72 is above 95%, and the intradermal reaction score is all <1, indicating good biocompatibility. While for the control dental appliance membrane added with the inorganic antibacterial agent IONPURE, the relative cell growth rate is 26%, showing cytotoxicity; the intradermal reaction score is 1.6, indicating an intradermal irritation reaction, suggesting that the dental appliance membrane added with the inorganic antibacterial agent IONPURE has poor biocompatibility. The reason may be the precipitation of silver ions, zinc ions and copper ions in the inorganic antibacterial agent IONPURE, which produces toxic side effects on cells and causes an intradermal irritation reaction.

[0124] Mechanical property test

[0125] The antibacterial dental appliance membrane obtained in Example 3 was subjected to a mechanical property test, and the test standard was based on GB / T1040.1 - 2006 Test Method for Tensile Properties of Plastics. The results obtained are shown in Table 2.

[0126] Table 2 Results of mechanical property test

[0127] Project Comparative Example 1 Example 3 Tensile Modulus of Elasticity (MPa) 1261 1632 Yield Stress (MPa) 36.5 36.8 Yield Tensile Strain (%) 5.82 5.94 Tensile Strength (MPa) 42 44.1 Elongation at Break (%) 132 150

[0128] As can be seen from Table 2, the tensile elastic modulus, yield stress, yield tensile strain, tensile strength and elongation at break of the antibacterial dental appliance membrane added with 6% of the bioengineered protein (VPGRG) 72 are all increased compared with the blank dental appliance membrane without added antibacterial agent. The main reason is that the bioengineered protein reacts with the matrix polymer material and further reacts with each other during the hot processing mixing, increasing the mechanical properties of the dental appliance membrane.

[0129] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An antibacterial material, comprising elastin-like protein and a matrix material; the elastin-like protein is (VPGRG)72.

2. The antibacterial material according to claim 1, characterized in that, The matrix material is a medical polymer, including at least one of polyurethane, polyethylene terephthalate-ethylene glycol copolyester, polycaprolactone, polylactic acid (PLA), polyvinyl chloride, polypropylene, and / or polyethylene.

3. The antibacterial material according to claim 1, wherein The mass fraction of the elastin-like protein is 1% to 20%.

4. The antibacterial material according to claim 1, characterized in that, The matrix material is polyurethane, and the mass fraction of the elastin-like protein is 1%, 2%, 6%, 10%, or 20%.

5. The preparation method of the antibacterial material according to any one of claims 1 to 4, comprising: The elastin-like protein is mixed with the dried matrix material, heated to melting and then extruded, and cooled to obtain the antibacterial material.

6. The preparation method according to claim 5, characterized in that, The matrix material is polyurethane, and its drying step includes: a drying dew point of -30°C, a drying temperature of 90 to 110°C, and a drying time of 6 to 10 h.

7. The preparation method according to claim 5, characterized in that, The extrusion temperature is 210 to 220°C, and it is extruded into particles or sheets.

8. Use of the antibacterial material according to any one of claims 1 to 4 or the antibacterial material prepared by the preparation method according to any one of claims 5 to 7 in the preparation of antibacterial medical device materials.

9. An antibacterial medical device, the raw material of which includes the antibacterial material according to any one of claims 1 to 4 or the antibacterial material prepared by the preparation method according to any one of claims 5 to 7.

10. The antibacterial medical device according to claim 9, characterized in that, It is an antibacterial dental tray sheet, an in-vivo interventional catheter, an abdominal repair patch, etc.

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