Preparation method and application of protein-based artificial skin prepared by protein chain entanglement of eight tandem repeat polyproteins based on ferredoxin

Abstract

The invention relates to the technical field of polyacrylamide hydrogel, and particularly discloses a protein-based artificial skin prepared from eight tandem repeat polyproteins based on ferredoxin through protein chain entanglement, which is prepared by the following steps: step 1, modifying the eight tandem repeat polyproteins ((FI) 8) of ferredoxin; the purified (FI) 8 protein is concentrated and uniformly mixed with dimethyl sulfoxide (DMSO) containing NHS-PEG-AM in proportion, overnight modification is carried out at room temperature, a modification system is centrifuged, precipitates are collected, and freeze-drying is carried out. FI 8 protein is used as an artificial skin skeleton, NHS-PEG-AM is used for bridging the N terminal of the FI 8 protein and a polyacrylamide network, and a stable network structure is formed through entanglement of the C terminal of the protein. Wherein the (FI) 8 protein network provides high elasticity, the polyacrylamide network provides high toughness, and a network system with excellent and stable mechanical properties is formed. In addition, by adjusting the length of the protein chain, the mechanical property of the artificial skin can be adjusted so as to meet the requirements of different application scenes.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method and application of preparing protein-based artificial skin based on eight tandemly repeated polyproteins of ferredoxin-like proteins through protein chain entanglement. Background Art

[0002] In the field of artificial skin, we are pursuing a material that can adapt to the stretching and deformation of biological skin during movement, and has good biocompatibility and avoids inducing immune rejection. At present, technical research is mainly focused on materials such as hydrogels, organic gels and ionic liquid gels. Hydrogels, in particular, are widely used in biofunctional materials due to their high similarity to living tissues, high water content, excellent moisture retention, outstanding biocompatibility, and microstructure similar to the extracellular matrix. Hydrogels can be divided into two categories: natural hydrogels and synthetic hydrogels. Natural hydrogels include proteins, gelatin, hyaluronic acid, chitosan, etc.; while synthetic hydrogels cover polyacrylic acid and its derivatives, polyvinyl alcohol, polyacrylamide, etc. Among these synthetic materials, polyacrylamide hydrogels are widely used in the research and development of artificial skin due to their low synthesis cost, good biocompatibility, strong water retention and good stability.

[0003] Polyacrylamide hydrogel is a synthetic polymer material, which is formed by polymerization of acrylamide (AM) monomers to form a three-dimensional network structure and fixed by a cross-linking agent. Due to its excellent water absorption and good biocompatibility, polyacrylamide hydrogel plays an important role in the medical field, especially as a scaffold material for medical dressings, tissue engineering, as well as biomedical research and clinical applications. The pore structure of the three-dimensional network of the hydrogel gives it good mechanical properties and elasticity, and the size of the pores can be adjusted by changing the polymerization conditions, which makes it useful as a scaffold for cell growth or for the screening and separation of molecules. In addition, by changing the concentration of the cross-linking agent and the polymerization conditions, the mechanical strength, elastic modulus and toughness of the hydrogel can be adjusted to meet different application requirements. These characteristics of polyacrylamide hydrogel, combined with its potential for application in the biomedical field, highlight its important position as a multifunctional material in modern medicine and biotechnology.

[0004] In a Chinese patent with publication number CN109400818B, a method for preparing a polyacrylamide hydrogel is mentioned. It is difficult for traditional methods to achieve dynamic, reversible, and non-destructive design requirements. The method of the present invention uses the commercial product Pluronic F127 as a raw material, and prepares Pluronic F127DA modified with double bonds at both ends through a pyridine-catalyzed nucleophilic addition reaction. Pluronic F127DA is dissolved in deionized water containing dimethyl sulfoxide, and then the solution is mixed evenly with acrylamide monomer and an initiator, injected into a mold, and randomly copolymerized under ultraviolet light to obtain a polyacrylamide hydrogel with a specific shape. The method of the present invention can dynamically and reversibly adjust the modulus, elongation at break, etc. of the hydrogel while maintaining the strength of the polyacrylamide hydrogel by adjusting the ratio of dimethyl sulfoxide and deionized water in the solvent system;

[0005] Compared with the above documents, in the above documents, the solution is mixed evenly with acrylamide monomer and initiator, injected into the mold, and randomly copolymerized under ultraviolet light to obtain a polyacrylamide hydrogel with a specific shape. This inventive method can dynamically and reversibly adjust the modulus, elongation at break, etc. of the polyacrylamide hydrogel while maintaining the strength of the polyacrylamide hydrogel by adjusting the ratio of dimethyl sulfoxide and deionized water in the solvent system. However, in the preparation process of polyacrylamide hydrogel, the selection of cross-linking agents is crucial. Common cross-linking agents include N,N-methylenebisacrylamide (MBAA), polyethyleneimine (PEI), hydroquinone (HQ), hexamethylenetetramine (HMTA), chromium ion (Cr 3+ ) and modified vesicles. N,N-methylenebisacrylamide may introduce unreacted residual monomers, which may be harmful to the human body; polyethyleneimine has high toxicity, which limits its application in the biomedical field; the instability of hydroquinone and hexamethylenetetramine under certain conditions may affect the overall performance of the gel; chromium ions as multivalent metal ions may not only pollute the environment, but also bring safety risks during their treatment. Therefore, a ferredoxin-like eight tandem repeat multiprotein-bound polyacrylamide is proposed, which is used to prepare artificial skin through a cross-linked network formed by entanglement between protein chains.

[0006] The present invention uses (FI)8 protein as the artificial skin skeleton, uses NHS-PEG-AM to bridge the N-terminus of the (FI)8 protein and the polyacrylamide network, and forms a stable network structure through the entanglement of the C-terminus of the protein. The (FI)8 protein network provides high elasticity and the polyacrylamide network provides high toughness, forming a network system with excellent and stable mechanical properties. The protein-based artificial skin has good biocompatibility and ensures the safety of the operation process. In addition, by adjusting the length of the protein chain, the mechanical properties of the artificial skin can be adjusted to meet the needs of different application scenarios. Summary of the invention

[0007] The object of the present invention is to provide a protein-based artificial skin prepared by entanglement of protein chains based on eight tandemly repeated polyproteins of ferredoxin-like proteins, so as to solve the problems raised in the above-mentioned background technology.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A preparation method and application of protein-based artificial skin prepared by entanglement of protein chains of eight tandemly repeated polyproteins of ferredoxin-like proteins, comprising:

[0010] Step 1: Modify the (FI)8 protein, concentrate the purified (FI)8 protein and mix it with dimethyl sulfoxide (DMSO) dissolved with NHS-PEG-AM in a certain proportion, modify it at room temperature overnight, collect the precipitate by centrifugation, and freeze-dry it;

[0011] Step 2: Dissolve the freeze-dried (FI)8-PEG-AM in 7M guanidine hydrochloride (GdHCl) to unfold the protein, add acrylamide and shake thoroughly to mix;

[0012] Step 3: Add ammonium persulfate (APS) and photoinitiator 2959 to the system after polymerization;

[0013] Step 4: Place the system described in the previous step under UV light for irradiation to obtain a polyacrylamide hydrogel using protein entanglement as a cross-linking method.

[0014] Preferably, the concentration of the purified (FI)8 protein is 14 mg / ml.

[0015] Preferably, the purified (FI)8 protein is mixed with dimethyl sulfoxide (DMSO) containing 4 mg / ml NHS-PEG-AM, and the mixing ratio of the purified (FI)8 protein and dimethyl sulfoxide (DMSO) containing 4 mg / ml NHS-PEG-AM is 9:1.

[0016] Through the setting of the above technical scheme, DMSO as a solvent can protect the purified protein from degradation and inactivation during the mixing process; NHS-PEG-AM PEGylates the protein to improve the stability and purity of the protein;

[0017] The strong permeability of DMSO helps NHS-PEG-AM to react with the amino groups of proteins to achieve PEGylation modification; the appropriate DMSO concentration can optimize the PEGylation reaction conditions and improve the reaction efficiency and yield;

[0018] In summary, the purified protein was mixed with dimethyl sulfoxide (DMSO) containing 4 mg / ml NHS-PEG-AM, which can fully utilize the solvent properties of DMSO and the modification function of NHS-PEG-AM, improve the stability and purity of the protein, promote the PEGylation reaction, and facilitate subsequent experimental operations.

[0019] Preferably, the centrifugation time of the modification system is 10 min, the centrifugation speed of the modification system is 12000 rpm, the concentration of the freeze-dried (FI)8-PEG-AM is 100 mg / ml, and the concentration of the acrylamide monomer is 150 mg / ml.

[0020] Through the setting of the above technical solution, oscillation mixing can ensure that the freeze-dried (FI)8-PEG-AM and acrylamide monomer are evenly distributed in the entire system, so that the polymerization process is fully and efficiently carried out, avoiding uneven or incomplete polymerization caused by excessively high or low local concentrations;

[0021] In summary, the lyophilized (FI)8-PEG-AM was dissolved in 7M guanidine hydrochloride to unfold the protein, and then fully oscillated and mixed. The unfolded protein can be used as a research model for subsequent experiments such as protein renaturation, folding mechanism, and drug screening. At the same time, in the process of drug development, understanding the relationship between the structure and function of the target protein is crucial for designing effective drugs. Through unfolding and renaturation experiments, small molecule drug candidates that interact with the target protein can be screened out.

[0022] Preferably, the final concentrations of the added ammonium persulfate (APS) and photoinitiator 2959 are 50 mM and 1% (w / v), respectively.

[0023] By setting the above technical solution, the benefits of adding ammonium persulfate (APS) are:

[0024] 1. Ammonium persulfate is a commonly used free radical initiator. It can decompose and produce free radicals under heating conditions. These free radicals can initiate the polymerization reaction of monomers. Adding an appropriate amount of APS to the system after polymerization can further promote the continued polymerization of monomers that have not reacted completely, thereby improving the conversion rate and yield of the polymer.

[0025] Second, APS generates free radicals, which rapidly connect monomers to form polymer chains. At the same time, the free radicals react and terminate, which helps to control the molecular weight and distribution of the polymer and prevent the unlimited growth of the polymer chain, thereby obtaining a polymer material with ideal properties.

[0026] 3. In the gel polymerization system, the redox property of APS helps promote the formation of gel. By interacting with other components in the reaction system (such as cross-linking agents, etc.), APS can enhance the stability of the polymer network and improve the mechanical properties and durability of the gel.

[0027] Benefits of adding photoinitiator 2959:

[0028] 1. Photoinitiator 2959 is a highly efficient UV initiator that can rapidly generate free radicals under UV light to initiate polymerization. Compared with APS, photoinitiator 2959 has a faster reaction speed and higher initiation efficiency, and is suitable for applications that require rapid curing.

[0029] Second, the hydroxyl functional group in the photoinitiator 2959 molecule improves its solubility in water-based coating formulations, so it is particularly suitable for water-based photocuring systems, which helps to expand the application range of polymerization reactions and meet the needs of more fields;

[0030] 3. Photoinitiator 2959 can initiate UV polymerization reaction of unsaturated prepolymer system to form polymer materials with excellent performance. By optimizing the dosage and reaction conditions of photoinitiator 2959, the curing effect can be further improved, so that the polymer materials have better mechanical properties, weather resistance and chemical corrosion resistance;

[0031] In summary, adding APS and photoinitiator 2959 to the system after polymerization can bring into play the synergistic effect of the two. APS, as a thermal initiator, can initiate polymerization under heating conditions; and photoinitiator 2959 can quickly generate free radicals under ultraviolet light irradiation, further promoting the polymerization reaction. This combination can improve the efficiency and yield of the polymerization reaction, while improving the properties of the polymer; according to different application requirements and experimental conditions, the amount and ratio of APS and photoinitiator 2959 can be flexibly adjusted to achieve the best polymerization effect.

[0032] Preferably, the irradiation time under UV light is 30 min.

[0033] Through the setting of the above technical solution, UV light irradiation can quickly trigger the photosensitive groups (such as photoinitiators) in the polyacrylamide molecules to produce free radicals or perform other chemical reactions, thereby promoting the cross-linking between the polyacrylamide molecular chains. This cross-linking process is usually faster than traditional thermal cross-linking or chemical cross-linking, which can significantly improve production efficiency;

[0034] The protein-based artificial skin prepared by entanglement of protein chains based on eight tandem repeat polyproteins of ferredoxin-like proteins has good compatibility in vivo. As a natural biological molecule, the presence of protein helps to reduce the stimulation and rejection reaction of hydrogels to biological tissues.

[0035] No toxic chemicals are introduced during UV light irradiation, thus avoiding the toxic residue problem that may be caused by traditional cross-linking methods, which makes the application of the hydrogel in the biomedical field safer and more reliable;

[0036] The polyacrylamide hydrogel obtained by UV light crosslinking has high physical and chemical stability, can resist erosion and damage from the external environment, and maintain long-term performance stability; at the same time, by changing parameters such as the concentration of polyacrylamide, the type and dosage of the crosslinker, and the irradiation conditions of UV light, hydrogels with different physical and chemical properties can be prepared to meet the application needs of different fields.

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

[0038] The present invention uses (FI)8 protein as the artificial skin skeleton, uses NHS-PEG-AM to bridge the N-terminus of the (FI)8 protein and the polyacrylamide network, and forms a stable network structure through the entanglement of the C-terminus of the protein. The (FI)8 protein network provides high elasticity and the polyacrylamide network provides high toughness, forming a network system with excellent and stable mechanical properties. The protein-based artificial skin has good biocompatibility and ensures the safety of the operation process. In addition, by adjusting the length of the protein chain, the mechanical properties of the artificial skin can be adjusted to meet the needs of different application scenarios.

[0039] The present invention provides a protein-based artificial skin prepared by entanglement of protein chains of eight tandemly repeated polyproteins based on ferredoxin, that is, (FI)8 protein is modified with NHS-PEG-AM, and the NHS on it undergoes a nucleophilic substitution reaction with the amino group on the protein molecule, releasing the NHS and generating a new amide bond at the same time, thereby realizing covalent connection between molecules;

[0040] PEG-AM was introduced into the amino terminus of (FI)8 protein to obtain (FI)8 protein functionalized with olefin double bonds. The modified protein and acrylamide monomer were polymerized in the same system. The N terminus of (FI)8 protein could polymerize with polyacrylamide, and the C terminus could be entangled with protein molecular chains and cross-linked into hydrogel under UV light initiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a flow chart of the steps of the present invention.

[0042] Figure 2 It is a mechanism diagram of the present invention.

[0043] Figure 3 It is a schematic diagram of the finished product of the present invention.

[0044] Figure 4This is a schematic diagram of the protein expression results in Example 1.

[0045] Figure 5 This is a schematic diagram of the formation of artificial skin in Example 2.

[0046] Figure 6 Schematic diagram of stress-strain of artificial skin in Example 3.

[0047] Figure 7 This is a schematic diagram of the results of culturing L929 and Hacat cell lines CCK8 with artificial skin extract in Example 4.

[0048] Figure 8 This is a schematic diagram of the live / dead staining results of the L929 cell line cultured with artificial skin extract in Example 4.

[0049] Fig. 9 This is a schematic diagram of the live / dead staining results of the Hacat cell line cultured in artificial skin leaching solution in Example 4. DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] like Figure 1 As shown, a preparation method and application of a protein-based artificial skin prepared by entanglement of protein chains of eight tandemly repeated polyproteins based on ferredoxin-like proteins, comprising:

[0052] Embodiment 1

[0053] Purification and modification of (FI)8 protein

[0054] (1) Protein shake flask fermentation production

[0055] Take 10 μl of glycerol bacteria and inoculate it into 5 ml of LB liquid medium containing kanamycin (100 μg / ml), and culture it at 37°C and 200 rpm overnight. Transfer 5 ml of overnight culture liquid into 1.5 L of LB liquid medium containing kanamycin (100 μg / ml), and culture it at 37°C and 200 rpm until OD600 is between 0.6-0.8, add 1M IPTG 450 μl, and induce protein expression at 16°C and 200 rpm for 48 hours.

[0056] (2) Protein separation and purification

[0057] Collect the bacterial solution by centrifugation at 4000rpm and 4℃ for 20min, add 100ml binding buffer to resuspend the bacterial solution, and ultrasonically disrupt the bacterial solution. After disruption, centrifuge the bacterial solution at 8000rpm for 100min and collect the supernatant. Use Binding buffer to balance the Ni-NTA affinity column, so that the collected supernatant passes through the Ni-NTA affinity column. Wash buffer is used to wash away non-specifically bound impurities. Elute buffer is used to elute the target protein and the eluate is collected. Take 20μl of the effluent from each step and add 5μl 5×SDS PAGE loading buffer. Heat it in a metal bath at 100℃ for 10min and perform SDS-PAGE detection. Figure 4 As shown, the protein was successfully expressed and purified.

[0058] (3) Protein concentration

[0059] The eluted protein was concentrated to a concentration of 14 mg / ml using an ultrafiltration tube at 3500 rpm.

[0060] (4) Protein modification

[0061] The concentrated (FI)8 protein was mixed with dimethyl sulfoxide (DMSO) containing 4 mg / ml NHS-PEG-AM at a ratio of 9:1 and modified overnight at room temperature. The modified system was lyophilized.

[0062] Embodiment 2

[0063] Preparation of artificial skin

[0064] (1) The lyophilized (FI)8-PEG-AM was dissolved in 7M guanidine hydrochloride at a concentration of 100 mg / ml to unfold the protein. At the same time, the unmodified (FI)8 protein, BSA-PEG-AM and the three control groups without any crosslinking agent were dissolved in 7M guanidine hydrochloride at the same molar concentration. Acrylamide monomer was added to the above systems to a concentration of 150 mg / ml, and the mixture was thoroughly shaken and polymerized at room temperature for 30 min.

[0065] (2) adding ammonium persulfate (APS) and photoinitiator 2959 to the post-polymerization system to a final concentration of 50 mM and 1% (w / v), respectively;

[0066] (3) Add the system described in the previous step into a 2 ml EP tube and place it under UV light for 30 min. Invert the 2 ml EP tube. Figure 5 As shown, both modified and unmodified (FI)8 protein addition can form hydrogels.

[0067] Embodiment 3

[0068] Mechanical properties test of artificial skin

[0069] The cross-linked networks formed by (FI)8-PEG-AM, (FI)4-PEG-AM, (FI)8, Bis and polyacrylamide were prepared respectively, and the same molar concentration was required to ensure that the formed networks had the same theoretical cross-linking density, and the concentration of acrylamide was 150 mg / ml. Artificial skin was prepared as described in Example 2.

[0070] like Figure 6 As shown in the figure, the mechanical properties of artificial skin were tested using a tensile tester, and the stress-strain curve was obtained. Artificial skin prepared with (FI)8-PEG-AM, (FI)4-PEG-AM and polyacrylamide have significant differences in mechanical properties, so the mechanical properties can be adjusted by adjusting the length of the protein chain to meet the needs of different scenarios. Although the addition of unmodified (FI)8 can also form a cross-linked network, its mechanical properties are poor and have no great application value.

[0071] Embodiment 4

[0072] Cytocompatibility test

[0073] The artificial skin prepared in Example 2 was used to detect CCK8, and the cells used were Hacat and L929. Figure 7 As shown, from left to right are the CCK8 results of L929 and Hacat cells, indicating that 0.5% artificial skin extract has a certain effect of promoting cell proliferation.

[0074] The artificial skin prepared in Example 2 was used for cell death and viability staining, and the cells used were Hacat and L929. Figure 8 , 9 The figure shows the live / dead staining results of L929 and Hacat cells. Artificial skin extracts of different concentrations have good cell compatibility.

[0075] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0076]

[0077]

Claims

1. A method for preparing a protein-based artificial skin prepared by entanglement of protein chains of eight tandemly repeated polyproteins of ferredoxin-like proteins and its application, characterized in that: Using 150 mg / ml acrylamide as a raw material and 100 mg / ml double-bond modified (FI)8 protein as a cross-linking agent for cross-linking, a hydrogel is formed by entanglement between (Fl)8 protein chains. The nucleotide sequence of the (FI)8 protein is shown in SEQ ID No.

1.

2. The method for preparing protein-based artificial skin prepared by entanglement of protein chains of eight tandemly repeated polyproteins of ferredoxin-like proteins according to claim 1 and its application, characterized in that: The preparation steps are as follows: (1) The freeze-dried (FI)8-PEG-AM was dissolved in 7 M guanidine hydrochloride (GdHCl) at a concentration of 100 mg / ml to unfold the protein, and acrylamide monomer was added to make the concentration 150 mg / ml, and the mixture was thoroughly shaken and polymerized at room temperature for 30 min; (2) adding ammonium persulfate (APS) and photoinitiator 2959 to the post-polymerization system to a final concentration of 50 mM and 1% (w / v), respectively; (3) The system described in the previous step is placed under UV light for 30 minutes to obtain protein-based artificial skin.

3. The method for preparing and using a protein-based artificial skin prepared by entanglement of protein chains based on eight tandemly repeated polyproteins of ferredoxin-like proteins according to claim 1, characterized in that: The preparation method of the double-bond modified (FI)8 protein is as follows: the purified (FI)8 protein is concentrated to 14 mg / ml, the concentrated protein is mixed with dimethyl sulfoxide (DMSO) containing 4 mg / ml NHS-PEG-AM at a volume ratio of 9:1, the modification is carried out at room temperature overnight, the modified system is centrifuged to collect the precipitate and freeze-dried to obtain (FI)8-PEG-AM, i.e., the double-bond modified (FI)8 protein.

4. The method for preparing a protein-based artificial skin prepared by entanglement of protein chains of eight tandemly repeated polyproteins of ferredoxin-like proteins according to claim 1 and its application, characterized in that: The mechanical properties are adjusted by changing the length of the protein chain to meet the needs of different application scenarios.

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