Protein-based biological material as well as preparation method and application thereof

Abstract

The embodiment of the invention discloses a protein-based biological material as well as a preparation method and application thereof. The method comprises the following steps: constructing a recombinant vector containing a coding collagen-like protein gene and a hedgehog protein gene, and performing expression and purification to obtain a recombinant protein; and reacting the recombinant protein with cholesterol in the presence of a TBS buffer solution to obtain the protein-based biological material. The mixed biological material with the functional cholesterol modified polypeptide shows programmable self-assembly on the nanoscale, has great market value potential in biomedical raw materials for treating diseases such as diabetes mellitus, tissue repair, tissue engineering scaffolds and treatment, drug delivery and the like, and has wide application prospects. And contributions are made to the development of biomedical materials in the future.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of biomaterials, and in particular to a protein-based biomaterial and a preparation method and application thereof. Background Art

[0002] As the most abundant protein in the extracellular matrix, collagen has a wide range of applications in food, cosmetics, pharmaceuticals, and biomedical engineering. Its ubiquity in human tissues and its unique properties (such as cell recognition signals, the ability to form 3D scaffolds, mechanical properties, and biodegradability) make it an ideal tissue engineering scaffold material for various clinical applications. Since collagen is considered an endogenous component of the body rather than a foreign substance, its acceptability as a biomaterial is high.

[0003] Collagen is a complex supramolecule that plays a key role in maintaining structural and biological integrity. These structures closely resemble natural supramolecular assemblies and have the ability to deliver bioactive molecules, making them important materials with challenges and opportunities in regenerative medicine. The development of protein-based materials with tunable assembly at the nanoscale is a hot research direction at the intersection of chemistry, biology and materials science. As an important component of nanomaterials, the composition, sequence and length of proteins can be precisely controlled at the genetic level. This genetic control allows proteins to seamlessly integrate many functional modes, such as enzymes or sequences that can bind to specific targets. In addition, proteins are easy to produce by Escherichia coli and are easily degraded into non-toxic building blocks. However, compared with synthetic materials, protein-based biomaterials have an important limitation, that is, the limited library of their constituent building blocks, which restricts the available design space and functionality of this important class of biomaterials.

[0004] Post-translational modifications (PTMs) are an orthogonal approach that can expand the chemical building block library of proteins and increase protein diversity. PTMs can alter protein structure and function by attaching non-protein moieties to polypeptides. Although hundreds of PTMs exist in natural proteins, they are currently limited to hydroxylation of tyrosine and proline in the construction of genetically encoded recombinant biomaterials, and are generally applied to biomaterials such as collagen. Summary of the invention

[0005] Protein-based materials are gaining increasing attention as sequence-defined polymers for various biomedical applications, such as tissue engineering scaffolds and therapeutics. Advances in gene synthesis and recombinant expression technologies have enabled us to create new genetically encoded sequence-defined peptide polymers more quickly and at lower cost. However, the precision and versatility of recombinant expression are limited by the available canonical amino acid repertoire, which constrains the diversity of peptide polymer sequences. Expanding the genetic code to include non-canonical amino acids is one solution to increase the chemical diversity of protein-based materials, and efforts have been made to achieve this goal.

[0006] Cholesterol is a lipid that can be synthesized in the human body. Since cholesterol is insoluble in water and cannot be directly dissolved in the blood, it must be combined with proteins to form lipoproteins in order to be transported in the body. There are two main types of cholesterol lipoproteins in the human body, namely high-density lipoproteins and low-density lipoproteins. Low-density lipoproteins are easily deposited on the blood vessel walls, leading to atherosclerosis and are the main cause of coronary heart disease. High-density lipoproteins have the function of clearing cholesterol deposited on the blood vessel walls, helping to maintain blood vessel patency and protect the heart. Cholesterol esters can be saponified, and the polar head of the cholesterol molecule is the hydroxyl group at the C-terminus, and the rest is a hydrophobic non-polar tail. Due to the rigidity of its ring structure, the presence of cholesterol reduces the fluidity of the membrane.

[0007] Hedgehog (Hh) protein contains two domains, an N-terminal signaling domain and a second domain fused to an autoprocessing C-terminal domain (HhC). HhC is homologous to intein-like proteins and contains an additional sterol binding site.

[0008] Studies have shown that in the absence of cholesterol, intein-like activity is blocked. When bound to cholesterol, the autoprocessing domain undergoes an N → S acyl transfer to form an intermediate with a thioester bond connecting the N-terminal and C-terminal domains. Subsequently, the 3β-hydroxyl group of the bound cholesterol reacts with this intermediate, resulting in cleavage of the C-terminal domain and binding of the N-terminal signaling domain to part of the cholesterol.

[0009] We hypothesized that this post-translational modification could be reconstructed in Escherichia coli by replacing the N-terminal signal domain with a peptide polymer. To verify this concept, we used a self-modified collagen-like protein as an artificial peptide polymer to reconstitute with hedgehog protein (Hh), and used cholesterol to modify the expressed recombinant protein to form a functional protein-based biomaterial. This new type of biomaterial can be applied in the biomedical field as a carrier for treating diseases and has certain practicality in the medical field.

[0010] The present invention utilizes the recombination of the hedgehog family proteins with collagen to achieve cholesterol modification through the natural cholesterol post-translational modification process. Cholesterol decomposition is the only sterol-based protein modification process in natural lipids, and the hydrophobicity of cholesterol molecules is used to promote the self-assembly of polypeptides. Hedgehog protein (Hh) contains two domains, an N-terminal signal transduction domain and an auto-processing C-terminal domain (HhC). HhC is homologous to intein-like proteins and contains an additional sterol binding site. Studies have shown that in the absence of cholesterol, intein-like activity is regulated. However, after binding to cholesterol, the auto-processing domain undergoes N→S acyl transfer to form an intermediate containing a thioester bond connecting the N-terminal and C-terminal domains. Subsequently, the 3β-hydroxyl group bound to cholesterol reacts with the intermediate, resulting in the cleavage of the C-terminal domain and the modification of cholesterol in the N-terminal signal transduction domain. This method provides a new strategy for protein-based biomaterials, overcomes the limitations of the currently available chemical design space, and opens up new avenues for the innovation of biomedical materials.

[0011] To this end, the embodiments of the present invention provide a protein-based biomaterial and a preparation method and application thereof. The key point of the present invention is to use genetically encoded collagen-like proteins and hedgehog protein (Hhc) sequences for recombination, and prepare recombinant proteins by biosynthesis. The recombinant protein is modified with cholesterol (in the absence of cholesterol, the intein-like activity is blocked. When combined with cholesterol, the autoprocessing domain undergoes N → S acyl transfer to form an intermediate with a thioester bond connecting the N-terminal and C-terminal domains. Subsequently, the 3β-hydroxyl group bound to cholesterol reacts with the intermediate, resulting in the cleavage of the C-terminal domain and the N-terminal signaling domain and the cholesterol portion, forming a functional protein-based biomaterial, thereby having biocompatibility.

[0012] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0013] According to a first aspect of an embodiment of the present invention, the present invention provides a method for preparing a protein-based biomaterial, the method comprising:

[0014] Constructing a recombinant vector containing a gene encoding a collagen-like protein and a hedgehog protein gene, and obtaining a recombinant protein after expression and purification;

[0015] The recombinant protein and cholesterol are reacted in the presence of TBS buffer to obtain the protein-based biomaterial.

[0016] Furthermore, the amino acid sequence of the recombinant protein is shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.

[0017] Furthermore, the TBS buffer comprises TCEP, EDTA2Na, and Triton X-100, wherein the final concentrations of TCEP, EDTA2Na, and TritonX-100 are 5-10 mM, 10-20 mM, and 1-5 mM, respectively.

[0018] Furthermore, the volume ratio of the recombinant protein to TBS buffer is 1:1.

[0019] Furthermore, the final concentration of cholesterol is 1-2 mM.

[0020] Furthermore, the reaction conditions are: stirring at room temperature for 12 hours.

[0021] According to a second aspect of the embodiments of the present invention, the present invention provides a protein-based biomaterial prepared by any of the methods described above.

[0022] According to a third aspect of an embodiment of the present invention, the present invention provides use of the protein-based biomaterial as described above in the preparation of products for treating diabetes, drug delivery, and bone repair.

[0023] The embodiments of the present invention have the following advantages:

[0024] The present invention prepares a functional supramolecular protein material by recombining biosynthesized collagen-like proteins with hedgehog proteins. When the supramolecular protein material is combined with cholesterol, the auto-processing domain undergoes N→S acyl transfer, thereby forming an intermediate with a thioester bond connecting the N-terminal and C-terminal domains. Subsequently, the 3β-hydroxyl group of cholesterol reacts with the intermediate, causing the C-terminal domain to cleave, and the N-terminal signaling domain to combine with the cholesterol portion, ultimately achieving the construction of a functional protein-based biomaterial.

[0025] The material prepared by the present invention is based on a collagen-like molecule independently developed and designed. The collagen molecule has good cell compatibility and can provide support for cell growth. More importantly, it is prepared by biosynthesis, does not involve any chemical synthesis process, can achieve pollution-free preparation, and is a collagen-like molecule with excellent performance.

[0026] The present invention provides a functional cholesterol-modified polypeptide hybrid biomaterial, which exhibits programmable self-assembly properties at the nanoscale. As a raw material in the biomedical fields such as the treatment of diseases such as diabetes, tissue repair, tissue engineering scaffold construction, and drug delivery, it has great market value potential and is expected to contribute to the future development of biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0028] Figure 1 The production process of the protein-based biomaterial provided by the present invention;

[0029] Figure 2 The recombinant collagen CLP1-Hhc, CLP2-Hhc and CLP3-Hhc sequences provided by the present invention;

[0030] Figure 3 The SDS-PAGE diagram of the recombinant collagen CLP1-Hhc, CLP2-Hhc and CLP3-Hhc provided by the present invention;

[0031] Figure 4 Western blot identification results of the recombinant collagen CLP1-Hhc, CLP2-Hhc and CLP3-Hhc provided by the present invention;

[0032] Figure 5 Electron microscope images of Chol-CLP1+Hhc, Chol-CLP2+Hhc observed by transmission electron microscope (TEM), and Chol-CLP3+Hhc observed by cryo-transmission electron microscope provided by the present invention;

[0033] Figure 6 The CCK-8 assay results of NIH-3T3 cells provided by the present invention;

[0034] Figure 7 The CCK-8 assay results of Hacat cells provided by the present invention;

[0035] Figure 8a This is a live or dead staining image of 3T3 cells after Chol-CLP1+Hhc provided by the present invention acts on them;

[0036] Figure 8b This is a live or dead staining image of 3T3 cells after Chol-CLP2+Hhc provided by the present invention acts on them;

[0037] Figure 8c This is a live or dead staining image of 3T3 cells after Chol-CLP3+Hhc provided by the present invention acts on them;

[0038] Figure 9a This is a live or dead staining image of 3T3 cells after CLP1+Hhc provided by the present invention acts on them;

[0039] Figure 9b This is a live or dead staining image of 3T3 cells after CLP2+Hhc provided by the present invention acts on them;

[0040] Fig.9c This is a live or dead staining diagram after CLP3+Hhc provided by the present invention acts on 3T3 cells. DETAILED DESCRIPTION

[0041] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are 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.

[0042] Example 1

[0043] This embodiment provides a protein-based biomaterial, and the preparation method thereof comprises the following steps:

[0044] (1) See Figure 2 , design a basic sequence of a protein recombinant between extracellular matrix recombinant supramolecular collagen and hedgehog protein.

[0045] It mainly introduces V-domian at the N-terminus of the scl-2 gene from Streptococcus pyogenes and connects it with CPPC polypeptide. One, two, and three scl-2 genes are connected in the middle, and then the CPPC polypeptide and RGD are connected, and the Hhc hedgehog protein sequence is introduced at the N-terminus of the sequence.

[0046] The present invention designs the protein sequences of the recombinant proteins of three types of collagen (CLP1, CLP2, CLP3) and hedgehog protein:

[0047] The amino acid sequence of CLP1+Hhc is:

[0048] ADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDGGPCPPCGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGKDGEPGPVGPARGDGKGGSGGSTVHGCFTPESTALLESGVRKPLGELSIGDRVLSMTANGQAVYSEVILFMDRNLEQMQNFVQLHTDGGAVLTVTPAHLVSVWQPESQKLTFVFADRIEEKNQVLVRDVETGELRPQRVVKVGSVRSKGVVAPLTREGTIVVNSVAASCYAVINSQSLAHWGLAPMRLLSTLEAWLPAKEQLHSSPKVVSSAQQQNGIHWYANALYKVKDYVLPQSWRHDGHHHHHHHHG (SEQ ID No.1)

[0049] The amino acid sequence of CLP2+Hhc is:

[0050] MADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDGGPCPPCGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGKDGEPGPVGPARGDGKGGSGGSTVHGCFTPESTALLESGVRKPLGELSIGDRVLSMTANGQAVYSEVILFMDRNLEQMQNFVQLHTDGGAVLTVTPAHLVSVWQPESQKLTFVFADRIEEKNQVLVRDVETGELRPQRVVKVGSVRSKGVVAPLTREGTIVVNSVAASCYAVINSQSLAHWGLAPMRLLSTLEAWLPAKEQLHSSPKVVSSAQQQNGIHWYANALYKVKDYVLPQSWRHDGHHHHHHHHG (SEQ ID No.2)

[0051] The amino acid sequence of CLP3+Hhc is as follows:

[0052] ADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDGGPCPPCGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGKGGSGGSTVHGCFTPESTALLESGVRKPLGELSIGDRVLSMTANGQAVYSEVILFMDRNLEQMQNFVQLHTDGGAVLTVTPAHLVSVWQPESQKLTFVFADRIEEKNQVLVRDVETGELRPQRVVKVGSVRSKGVVAPLTREGTIVVNSVAASCYAVINSQSLAHWGLAPMRLLSTLEAWLPAKEQLHSSPKVVSSAQQQNGIHWYANALYKVKDYVLPQSWRHD (SEQ ID No.3).

[0053] (2)Express and purify the target protein

[0054] The successfully constructed recombinant vector was transferred into the expression type (BL21) Escherichia coli. 1 μL of the recombinant vector was taken into the BL21 competent medium, ice-bathed for 20-30 min, heat-shocked at 42°C for 90 s, and then ice-bathed for 5 min. 300 μL of non-resistant LB medium was added to the ultra-clean medium, shaken at 200 rpm / min on a 37°C shaker for 45 min, centrifuged for 1 min, and 20 μL was taken and spread on the kan-resistant culture plate, cultured overnight in a 37°C incubator, and a single clone was picked up in 5 ml of kan-resistant culture medium. When the culture medium became turbid, all 5 mL of bacterial solution was transferred to 500 mL of culture medium, and the culture was shaken at 37°C and 200 rpm / min. When the OD value was about 0.6-0.8, 0.3 mM IPTG was added, and cultured at 16°C for 36 h. Collect the bacterial suspension, discard the supernatant, add lysis buffer (composition: 300mM NaCl, 50mM Tris-HCl) for lysis, purify the protein by nickel ionization, and finally elute the protein to obtain the purified protein. The purified protein was subjected to SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue. Figure 3 In a, the sizes of CLP1+Hhc and CLP2+Hhc proteins are 44 kDa and 53 kDa respectively, and the size of CLP3+Hhc protein is 60 kDa. In order to further identify the correctness of the purified protein, western blot was performed ( Figure 4 ).

[0055] (3) Cholesterol-modified recombinant protein

[0056] The purified recombinant proteins CLP1+Hhc, CLP2+Hhc and CLP3+Hhc were mixed with 1xTBS (including: TCEP with a final concentration of 10 mM, EDTA2Na with a final concentration of 15 mM, and TritonX-100 with a final concentration of 3 mM) in a volume ratio of 1:1, respectively. After mixing, the mixture was incubated at room temperature for 15 min, and then 30 mM cholesterol ethanol stock solution was added to control the final concentration of cholesterol to 1 mM. The mixture was stirred at room temperature overnight (12 h) to form functional protein-based biomaterials, namely Chol-CLP1+Hhc, Chol-CLP2+Hhc, and Chol-CLP3+Hhc.

[0057] Test Example 1

[0058] Chol-CLP1+Hhc and Chol-CLP2+Hhc were observed using a transmission electron microscope (TEM), and Chol-CLP3+Hhc was observed using a cryo-electron microscope. The results are as follows: Figure 5Shown: a, b, c are electron micrographs of Chol-CLP1+Hhc, Chol-CLP2+Hhc, and Chol-CLP3+Hhc, respectively.

[0059] Transmission Electron Microscopy (TEM) Characterization:

[0060] TEM experiments were conducted on a Japanese-made HT7800 transmission electron microscope with an accelerating voltage of 100 kV. The specific operation is as follows: 10 μL of sample was placed on a carbon-coated copper grid (purchased from Electron Microscopy Science), incubated for 1 min, and then the residual solution was removed with filter paper; then 5 μL of 1% (w / v) uranyl acetate was dropped on the grid and incubated for 1 min, and then the excess uranyl acetate solution was removed with filter paper, and the sample was dried overnight. The prepared grid was observed on a HT7800 transmission electron microscope equipped with an 80 kV (FEI) cathode (LaB6, FEI), and the electron microscope (EM) images were recorded using an UltraScan 1000 charge-coupled device (CCD) camera (Gatan). The observation results of Chol-CLP1+Hhc and Chol-CLP2+Hhc are shown in Figure 2. Figure 5 shown.

[0061] Cryo-TEM characterization: First, prepare the cryo-TEM sample, take 10 μL of the sample and place it on a carbon-coated copper grid, then put the copper grid into liquid ethane for rapid freezing. After placing the frozen sample in the cryo-EM, observe its structure in cryo-EM mode under electron beam irradiation. The observation results of Chol-CLP3+Hhc are as follows: Figure 5 shown.

[0062] Test Example 2

[0063] The obtained cholesterol-modified Chol-CLP1+Hhc, Chol-CLP2+Hhc, and Chol-CLP3+Hhc proteins were prepared into solutions with the same concentration of 0.1 mol / mL. The volume gradients were set to 5μL, 10μL, 15μL, 20μL, and 25μL, respectively. Cell experiments were carried out with 3T3 cells. The culture medium used was DMEM culture medium containing 10% fetal bovine serum (FBS) and 1% double antibody (penicillin-streptomycin solution). The cells were cultured in a constant temperature incubator at 37°C and 5% CO2.

[0064] To evaluate cell viability, NIH-3T3 cells were cultured in DMEM containing 10% FBS and 1% double antibody. 1 μL, 5 μL, and 10 μL of the modified protein were mixed with 200 μL of culture medium and used to culture cells. The effect of cholesterol-modified protein on cytotoxicity was detected by live / dead staining. Green fluorescence represents live cells, and red fluorescence represents dead cells. In addition, the CCK-8 experiment was used to detect the effect of cholesterol-modified protein on the viability of NIH-3T3 cells and HaCat cells. The specific operation is as follows: 1×10 4 Cells were seeded in a 96-well plate, and a mixture of protein and culture medium was added for 20 h. The old culture medium was discarded and replaced with fresh culture medium. CCK-8 reagent was added and placed in an incubator for 30 min. The optical density (OD) at 450 nm was measured and standardized with the culture medium control group. All tests were repeated three times to evaluate the cytotoxicity of cholesterol-modified proteins.

[0065] The results of cell death and live staining are shown in Figure 8, indicating that Chol-CLP1+Hhc, Chol-CLP2+Hhc, and Chol-CLP3+Hhc cholesterol-modified proteins have good cell compatibility. NIH-3T3 cells ( Figure 6 )、HaCat cells( Figure 7 ) showed that in NIH-3T3 cells, Chol-CLP1+Hhc, Chol-CLP2+Hhc, and Chol-CLP3+Hhc modified proteins had a certain effect on promoting cell growth; in HaCat cells, when the volume of the modified protein was 10 μL, it had good cell compatibility and also had a certain promoting effect on cell growth (such as Figure 7 as shown).

[0066] The performance differences of cholesterol-modified proteins Chol-CLP1+Hhc, Chol-CLP2+Hhc, and Chol-CLP3+Hhc were analyzed based on the results of NIH-3T3 cell live and dead staining and the CCK-8 experimental results of NIH-3T3 and HaCat cells.

[0067] Cholesterol-modified proteins often show significant differences in cellular uptake, stability, and biocompatibility. The following is a detailed analysis of the performance differences of three cholesterol-modified proteins: Chol-CLP1+Hhc, Chol-CLP2+Hhc, and Chol-CLP3+Hhc:

[0068] 1. Biocompatibility analysis

[0069] Cholesterol modification usually enhances the biocompatibility of proteins and reduces immune responses. However, due to the different lengths of the three collagens CLP1, CLP2, and CLP3, the proteins expressed are also different in structure and function, resulting in different modification effects.

[0070] Chol-CLP1+Hhc: shows good cellular uptake effect, but may have a certain impact on cellular metabolism.

[0071] Chol-CLP2+Hhc: High stability, but low cellular uptake rate.

[0072] Chol-CLP3+Hhc: exhibits higher biocompatibility and lower cytotoxicity.

[0073] 2. Cytocompatibility assessment

[0074] Cytocompatibility is a key indicator for evaluating cholesterol-modified proteins in biomedical applications. Figure 6-7 ), the cell compatibility analysis of the three proteins is as follows:

[0075] Chol-CLP1+Hhc: The cell viability is relatively high, and the cell metabolic activity decreases at high concentrations, indicating that its cytotoxicity is relatively low, but it may have a certain effect on cell metabolism.

[0076] Chol-CLP2+Hhc: Cell viability was low, and at high concentrations, cell metabolic activity decreased significantly, indicating that its cytotoxicity was high.

[0077] Chol-CLP3+Hhc: The cell viability was the highest, and no obvious cytotoxicity was observed even at high concentrations, indicating that it has good cell compatibility.

[0078] 3. Summary of performance differences:

[0079] Chol-CLP1+Hhc: The cellular uptake rate is higher and the cytotoxicity is slightly higher than the other two proteins.

[0080] Chol-CLP2+Hhc: Low cellular uptake efficiency and high cytotoxicity.

[0081] Chol-CLP3+Hhc: has the best cell compatibility and low cytotoxicity, making it suitable for biomedical applications.

[0082] In summary, combined with the CCK-8 experimental results and the cell live and dead staining results (Figures 8-9), it can be seen that Chol-CLP3+Hhc exhibits the best cell compatibility and lower cytotoxicity, and has good application potential in the biomedical field.

[0083] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

[0084] Sequence Listing

[0085] <110> Inner Mongolia Hexun Biotechnology Co., Ltd.

[0086] <120> A protein-based biomaterial and its preparation method and application

[0087] <130> GG241436846A

[0088] <160> 3

[0089] <170> PatentIn version 3.5

[0090] <210> 1

[0091] <211> 398

[0092] <212> PRT

[0093] <213> Artificial sequence

[0094] <400> 1

[0095] Ala Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Glu Leu Ile Gln 1 5 10 15

[0097] Glu Leu Ala Gln Gly Leu Gly Gly Ile Glu Lys Lys Asn Phe Pro Thr 20 25 30 35

[0099] Leu Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr 40 45 50 55

[0101] Tyr Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu 60 65 70 75

[0103] Leu Lys Gly Ile Gln Asp His Ala Leu Asp Gly Gly Pro Cys Pro Pro 80 85 90 95

[0105] Cys Gly Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp 100 105 110 115

[0107] Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly 120 125 130 135

[0109] Glu Gln Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly Asp 140 145 150 155

[0111] Arg Gly Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala 160 165 170 175

[0113] Gly Pro Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Arg 180 185 190 195

[0115] Gly Asp Gly Lys Gly Gly Ser Gly Gly Ser Thr Val His Gly Cys Phe 200 205 210 215

[0117] Thr Pro Glu Ser Thr Ala Leu Leu Glu Ser Gly Val Arg Lys Pro Leu 220 225 230 235

[0119] Gly Glu Leu Ser Ile Gly Asp Arg Val Leu Ser Met Thr Ala Asn Gly 240 245 250 260

[0121] Gln Ala Val Tyr Ser Glu Val Ile Leu Phe Met Asp Arg Asn Leu Glu 265 270 275 280

[0123] Gln Met Gln Asn Phe Val Gln Leu His Thr Asp Gly Gly Ala Val Leu 285 290 295 300

[0125] Thr Val Thr Pro Ala His Leu Val Ser Val Trp Gln Pro Glu Ser Gln 305 310 315 320

[0127] Lys Leu Thr Phe Val Phe Ala Asp Arg Ile Glu Glu Lys Asn Gln Val 325 330 335 340

[0129] Leu Val Arg Asp Val Glu Thr Gly Glu Leu Arg Pro Gln Arg Val Val 345 350 355 360

[0131] Lys Val Gly Ser Val Arg Ser Lys Gly Val Val Ala Pro Leu Thr Arg 365 370 375 380

[0133] Glu Gly Thr Ile Val Val Asn Ser Val Ala Ala Ser Cys Tyr Ala Val 385 390 395 400

[0135] Ile Asn Ser Gln Ser Leu Ala His Trp Gly Leu Ala Pro Met Arg Leu 405 410 415 420

[0137] Leu Ser Thr Leu Glu Ala Trp Leu Pro Ala Lys Glu Gln Leu His Ser 425 430 435 440

[0139] Ser Pro Lys Val Val Ser Ser Ala Gln Gln Gln Asn Gly Ile His Trp 445 450 455 460

[0141] Tyr Ala Asn Ala Leu Tyr Lys Val Lys Asp Tyr Val Leu Pro Gln Ser 465 470 475 480

[0143] Trp Arg His Asp Gly His His His His His His His His Gly 485 490 495

[0145] <210> 2

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[0148] <213> Artificial Sequence

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[0150] Met Ala Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Glu Leu Ile 1 5 10 15

[0152] Gln Glu Leu Ala Gln Gly Leu Gly Gly Ile Glu Lys Lys Asn Phe Pro 20 25 30 35

[0154] Thr Leu Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu 40 45 50 55

[0156] Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg 60 65 70 75

[0158] Leu Leu Lys Gly Ile Gln Asp His Ala Leu Asp Gly Gly Pro Cys Pro 80 85 90 95

[0160] Pro Cys Gly Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys 100 105 110 115

[0162] Asp Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala 120 125 130 135

[0164] Gly Glu Gln Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly 140 145 150 155

[0166] Asp Arg Gly Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu 160 165 170 175

[0168] Ala Gly Pro Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala 180 185 190 195

[0170] Gly Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly 200 205 210 215

[0172] Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu 220 225 230 235

[0174] Gln Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly Asp Arg 240 245 250 255

[0176] Gly Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly 260 265 270 275

[0178] Pro Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Arg Gly 280 285 290 295

[0180] Asp Gly Lys Gly Gly Ser Gly Gly Ser Thr Val His Gly Cys Phe Thr 300 305 310 315

[0182] Pro Glu Ser Thr Ala Leu Leu Glu Ser Gly Val Arg Lys Pro Leu Gly 320 325 330 335

[0184] Glu Leu Ser Ile Gly Asp Arg Val Leu Ser Met Thr Ala Asn Gly Gln 340 345 350 355

[0186] Ala Val Tyr Ser Glu Val Ile Leu Phe Met Asp Arg Asn Leu Glu Gln 360 365 370 375

[0188] Met Gln Asn Phe Val Gln Leu His Thr Asp Gly Gly Ala Val Leu Thr 380 385 390 395

[0190] Val Thr Pro Ala His Leu Val Ser Val Trp Gln Pro Glu Ser Gln Lys 400 405 410 415

[0192] Leu Thr Phe Val Phe Ala Asp Arg Ile Glu Glu Lys Asn Gln Val Leu 420 425 430 435

[0194] Val Arg Asp Val Glu Thr Gly Glu Leu Arg Pro Gln Arg Val Val Lys 440 445 450 455

[0196] Val Gly Ser Val Arg Ser Lys Gly Val Val Ala Pro Leu Thr Arg Glu 460 465 470 475

[0198] Gly Thr Ile Val Val Asn Ser Val Ala Ala Ser Cys Tyr Ala Val Ile 480 485 490 495

[0200] Asn Ser Gln Ser Leu Ala His Trp Gly Leu Ala Pro Met Arg Leu Leu 500 505 510 515

[0202] Ser Thr Leu Glu Ala Trp Leu Pro Ala Lys Glu Gln Leu His Ser Ser 520 525 530 535

[0204] Pro Lys Val Val Ser Ser Ala Gln Gln Gln Asn Gly Ile His Trp Tyr 540 545 550 555

[0206] Ala Asn Ala Leu Tyr Lys Val Lys Asp Tyr Val Leu Pro Gln Ser Trp 560 565 570 575

[0208] Arg His Asp Gly His His His His His His His His Gly 580 585 590

[0210] <210> 3

[0211] <211> 541

[0212] <212> PRT

[0213] <213> Artificial Sequence

[0214] <400> 3

[0215] Ala Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Glu Leu Ile Gln 1 5 10 15

[0217] Glu Leu Ala Gln Gly Leu Gly Gly Ile Glu Lys Lys Asn Phe Pro Thr 20 25 30 35

[0219] Leu Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr 40 45 50 55

[0221] Tyr Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu 60 65 70 75

[0223] Leu Lys Gly Ile Gln Asp His Ala Leu Asp Gly Gly Pro Cys Pro Pro 80 85 90 95

[0225] Cys Gly Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp 100 105 110 115

[0227] Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly 120 125 130 135

[0229] Glu Gln Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp 140 145 150 155

[0231] Arg Gly Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala 160 165 170 175

[0233] Gly Pro Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly 180 185 190 195

[0235] Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu 200 205 210 215

[0237] Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln 220 225 230 235

[0239] Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly 240 245 250 255

[0241] Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro 260 265 270 275

[0243] Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly Pro Lys 280 285 290 295

[0245] Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu Ala Gly 300 305 310 315

[0247] Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln Gly Glu 320 325 330 335

[0249] Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly Glu Thr 340 345 350 355

[0251] Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly 360 365 370 375

[0253] Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly Lys Gly Gly Ser 380 385 390 395

[0255] Gly Gly Ser Thr Val His Gly Cys Phe Thr Pro Glu Ser Thr Ala Leu 400 405 410 415

[0257] Leu Glu Ser Gly Val Arg Lys Pro Leu Gly Glu Leu Ser Ile Gly Asp 420 425 430 435

[0259] Arg Val Leu Ser Met Thr Ala Asn Gly Gln Ala Val Tyr Ser Glu Val 440 445 450 455

[0261] Ile Leu Phe Met Asp Arg Asn Leu Glu Gln Met Gln Asn Phe Val Gln 460 465 470 475

[0263] Leu His Thr Asp Gly Gly Ala Val Leu Thr Val Thr Pro Ala His Leu 480 485 490 495

[0265] Val Ser Val Trp Gln Pro Glu Ser Gln Lys Leu Thr Phe Val Phe Ala 500 505 510 515

[0267] Asp Arg Ile Glu Glu Lys Asn Gln Val Leu Val Arg Asp Val Glu Thr 520 525 530 535

[0269] Gly Glu Leu Arg Pro Gln Arg Val Val Lys Val Gly Ser Val Arg Ser 540 545 550 555

[0271] Lys Gly Val Val Ala Pro Leu Thr Arg Glu Gly Thr Ile Val Val Asn 560 565 570 575

[0273] Ser Val Ala Ala Ser Cys Tyr Ala Val Ile Asn Ser Gln Ser Leu Ala 580 585 590 595

[0275] His Trp Gly Leu Ala Pro Met Arg Leu Leu Ser Thr Leu Glu Ala Trp 600 605 610 615

[0277] Leu Pro Ala Lys Glu Gln Leu His Ser Ser Pro Lys Val Val Ser Ser 620 625 630 635

[0279] Ala Gln Gln Gln Asn Gly Ile His Trp Tyr Ala Asn Ala Leu Tyr Lys 640 645 650 655

[0281] Val Lys Asp Tyr Val Leu Pro Gln Ser Trp Arg His Asp 660 665 670 675

Claims

1. A method for preparing a protein-based biomaterial, characterized in that: The method comprises: Constructing a recombinant vector containing a gene encoding a collagen-like protein and a hedgehog protein gene, and obtaining a recombinant protein after expression and purification; The recombinant protein and cholesterol are reacted in the presence of TBS buffer to obtain the protein-based biomaterial.

2. The method for preparing a protein-based biomaterial according to claim 1, characterized in that: The amino acid sequence of the recombinant protein is shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.

3.

3. The method for preparing a protein-based biomaterial according to claim 1, characterized in that: The TBS buffer comprises TCEP, EDTA2Na and Triton X-100, wherein the final concentrations of TCEP, EDTA2Na and TritonX-100 are 5-10 mM, 10-20 mM and 1-5 mM respectively.

4. The method for preparing a protein-based biomaterial according to claim 1, characterized in that: The volume ratio of the recombinant protein to TBS buffer was 1:

1.

5. The method for preparing a protein-based biomaterial according to claim 1, characterized in that: The final concentration of cholesterol was 1-2 mM.

6. The method for preparing a protein-based biomaterial according to claim 1, characterized in that: The reaction conditions are: stirring at room temperature for 12 hours.

7. A protein-based biomaterial, characterized in that: The invention is prepared by the method according to any one of claims 1 to 6.

8. Use of the protein-based biomaterial according to claim 7 in the preparation of products for treating diabetes, drug delivery, and bone repair.

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