A protein-based biomaterial, its preparation method and application

Abstract

An embodiment of the present invention discloses a protein-based biomaterial, its preparation method and application. The method includes: constructing a recombinant vector containing a gene encoding collagen-like protein and a hedgehog protein gene, and after expression and purification, obtaining a recombinant protein; reacting the recombinant protein with cholesterol in the presence of TBS buffer to obtain the protein-based biomaterial. The present invention provides a hybrid biomaterial with functional cholesterol-modified polypeptides, which exhibits programmable self-assembly at the nanoscale and has great potential market value in biomedical raw materials such as treating diseases such as diabetes, tissue repair, tissue engineering scaffolds and treatment, and drug delivery, contributing to the development of future biomedical materials.

Description

Technical Field

[0001] Embodiments of the present invention relate to the technical field of biomaterials, and particularly to a protein-based biomaterial, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen, as the most abundant protein in the extracellular matrix, has a wide range of applications in the fields of food, cosmetics, pharmaceuticals, and biomedical engineering. Its widespread presence 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 raw material for tissue engineering scaffolds in various clinical applications. Since collagen is regarded as an endogenous component of the body rather than a foreign substance, its acceptability as a biomaterial is relatively high.

[0003] Collagen is a complex supramolecule that plays a key role in maintaining structural and biological integrity. These structures are closely similar to natural supramolecular assemblies and have the ability to deliver bioactive molecules, making them important materials with challenges and opportunities in regenerative medicine. Developing tunable assembled protein-based materials at the nanoscale is a hot research direction in the interdisciplinary field 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 gene level. This genetic control enables proteins to have the ability to seamlessly integrate many functional modes, such as enzymes or sequences that can bind to specific targets. In addition, proteins are easy to produce through 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 library of their constituent structural units is limited, which restricts the available design space and functions of this important class of biomaterials.

[0004] Using the orthogonal method of post-translational modification (PTM), the chemical building block library of proteins can be expanded, and the diversity of proteins can be increased. By attaching non-protein moieties to polypeptides, PTM can change the structure and function of proteins. Although there are hundreds of PTMs in natural proteins, in the construction of gene-encoded recombinant biomaterials, it is currently limited to the hydroxylation of tyrosine and proline, which is generally applied to biomaterials such as collagen. Summary of the Invention

[0005] Protein-based materials as sequence-defined polymers for various biomedical applications, such as tissue engineering scaffolds and therapeutics, are receiving increasing attention. With the advancement of gene synthesis and recombinant expression technologies, we are able to create new gene-encoded sequence-defined peptide polymers faster and at lower cost. However, the precision and versatility of recombinant expression are limited by the available canonical amino acid library, which restricts the diversity of peptide polymer sequences. Expanding the genetic code to include non-canonical amino acids is a 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 bound to proteins to form lipoproteins for transport in the body. There are two main cholesterol lipoproteins in the human body, namely high-density lipoprotein and low-density lipoprotein. Low-density lipoprotein is prone to deposition on the blood vessel wall, leading to atherosclerosis and being the main cause of coronary heart disease. On the other hand, high-density lipoprotein has the function of removing cholesterol deposited on the blood vessel wall, helping to maintain blood vessel patency and protecting 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 cyclic structure, the presence of cholesterol reduces the fluidity of the membrane.

[0007] Hedgehog protein (Hh) contains two domains, one is the N-terminal signal domain, and the other is the domain fused with the autocatalytic 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, the intein-like activity is blocked. When bound to cholesterol, the autocatalytic 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 of the bound cholesterol reacts with this intermediate, resulting in the cleavage of the C-terminal domain and the binding of the N-terminal signal domain to a part of cholesterol.

[0009] We hypothesized that replacing the N-terminal signal domain with a peptide polymer could reconstruct this post-translational modification in Escherichia coli. To test this concept, we used self-engineered collagen-like proteins as artificial peptide polymers to recombine with hedgehog protein (Hh), and utilized cholesterol to modify the expressed recombinant protein to form a functional protein-based biomaterial. This novel 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 hedgehog family proteins and collagen to achieve cholesterol modification through the natural post-translational modification process of cholesterol. Cholesterol cleavage is the only sterol-based protein modification process in natural lipids, and the hydrophobicity of cholesterol molecules is utilized to promote the self-assembly of polypeptides. Hedgehog protein (Hh) contains two domains, an N-terminal signal transduction domain and an autocatalytic 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, the intein-like activity is regulated. However, after binding to cholesterol, the autocatalytic 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-hydroxy group of the bound cholesterol reacts with this 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] Therefore, an embodiment of the present invention provides a protein-based biomaterial, its preparation method and application. The key point of the present invention is to recombine the gene-encoded collagen-like protein with the hedgehog protein (Hhc) sequence, and prepare the recombinant protein through biosynthesis. This recombinant protein is modified by cholesterol (in the absence of cholesterol, the intein-like activity is blocked. When binding to cholesterol, the autocatalytic 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-hydroxy group of the bound cholesterol reacts with this intermediate, resulting in the cleavage of the C-terminal domain and the cholesterol moiety in the N-terminal signal domain, forming a functional protein-based biomaterial, thus having biocompatibility.

[0012] In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

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

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

[0015] React the recombinant protein with cholesterol in the presence of TBS buffer to obtain the protein-based biomaterial.

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

[0017] Further, the TBS buffer includes TCEP, EDTA2 Na, Triton X-100, where TCEP and EDTA 2 The final concentrations of Na and Triton X-100 are 5 - 10 mM, 10 - 20 mM, and 1 - 5 mM in sequence.

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

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

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

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

[0022] According to the third aspect of the embodiments of the present invention, the present invention provides the application of the above-described protein-based biomaterial 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] In the present invention, a functional supramolecular protein material is prepared by recombining biosynthetic collagen-like protein with hedgehog protein. When the supramolecular protein material binds to cholesterol, the autocatalytic domain undergoes N→S acyl transfer, and then an intermediate with a thioester bond connecting the N-terminal and C-terminal domains is formed. Subsequently, the 3β-hydroxy group of cholesterol reacts with this intermediate, causing the cleavage of the C-terminal domain, and the N-terminal signal domain binds to the cholesterol moiety, ultimately achieving the construction of a functional protein-based biomaterial.

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

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

[0027] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained based on the provided drawings.

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

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

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

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

[0032] Figure 5 The electron micrographs of Chol-CLP1+Hhc and Chol-CLP2+Hhc observed by transmission electron microscopy (TEM), and Chol-CLP3+Hhc observed by cryo-transmission electron microscopy 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 The live / dead staining images of 3T3 cells after the action of Chol-CLP1+Hhc provided by the present invention;

[0036] Figure 8b The live / dead staining images of 3T3 cells after the action of Chol-CLP2+Hhc provided by the present invention;

[0037] Figure 8c The live / dead staining images of 3T3 cells after the action of Chol-CLP3+Hhc provided by the present invention;

[0038] Figure 9a The live / dead staining images of 3T3 cells after the action of CLP1+Hhc provided by the present invention;

[0039] Figure 9b This is the live / dead staining map of 3T3 cells after the action of CLP2+Hhc provided by the present invention;

[0040] Figure 9c This is the live / dead staining map of 3T3 cells after the action of CLP3+Hhc provided by the present invention. Detailed implementation manners

[0041] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0042] Example 1

[0043] This example provides a protein-based biomaterial, and its preparation method includes the following steps:

[0044] (1) Refer to Figure 2 , and design the basic sequence of a recombinant protein of an extracellular matrix recombinant supermolecular collagen-like protein and hedgehog protein.

[0045] Mainly, a V-domian is introduced at the N-terminus of the scl-2 gene derived from Streptococcus pyogenes and connected with a CPPC polypeptide. One, two, or three scl-2 genes are respectively 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 three kinds of recombinant proteins of collagen-like proteins (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] Transfer the successfully constructed recombinant vector into expression-type (BL21) Escherichia coli. Pipette 1 μL of the recombinant vector into the BL21 competent cells, incubate on ice for 20 - 30 min, heat shock at 42 °C for 90 s, then incubate on ice for 5 min. Add 300 μL of antibiotic-free LB medium in a laminar flow hood, shake at 200 rpm / min on a shaker at 37 °C for 45 min, centrifuge for 1 min, pipette 20 μL and spread it on a culture plate with kanamycin resistance, incubate overnight in an incubator at 37 °C. Pick a single colony into 5 ml of medium with kanamycin resistance. When the medium becomes turbid, transfer all 5 mL of the bacterial solution into 500 mL of medium, shake the bacteria at 37 °C and 200 rpm / min. When the OD value is about 0.6 - 0.8, add 0.3 mM IPTG and incubate at 16 °C for 36 h. Collect the bacterial solution, discard the supernatant, add lysis buffer (composition: 300 mM NaCl, 50 mM Tris-HCl) to lyse, purify the protein through nickel affinity, and finally elute the protein to obtain the purified protein. Perform SDS-PAGE electrophoresis on the purified protein and stain with Coomassie Brilliant Blue, as Figure 3 shown in a: the sizes of CLP1+Hhc and CLP2+Hhc proteins are 44 kDa and 53 kDa respectively, and b: the size of CLP3+Hhc protein is 60 kDa. To further identify the correctness of the purified protein and perform western blot identification ( Figure 4 ).

[0055] (3)Cholesterol-modified recombinant protein

[0056] Mix the purified recombinant proteins CLP1+Hhc, CLP2+Hhc, and CLP3+Hhc with 1xTBS (including: 10 mM TCEP at final concentration, 15 mM EDTA 2 Na, 3 mM Triton X-100 at final concentration) at a volume ratio of 1:1. After mixing evenly, incubate at room temperature for 15 min, then add 30 mM cholesterol ethanol stock solution to control the final concentration of cholesterol to 1 mM, stir 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] Observe Chol-CLP1+Hhc and Chol-CLP2+Hhc using a transmission electron microscope (TEM), and observe Chol-CLP3+Hhc using a cryo-electron microscope. The results are as Figure 5Shown as follows: a, b, and c are the electron microscopy images of Chol-CLP1+Hhc, Chol-CLP2+Hhc, and Chol-CLP3+Hhc, respectively.

[0059] Characterization by transmission electron microscopy (TEM):

[0060] The TEM experiment was carried out on a HT7800 transmission electron microscope made in Japan with an accelerating voltage of 100 kV. The specific operation is as follows: Take 10 μL of the sample and place it on a carbon-coated copper grid (purchased from Electron Microscopy Sciences). After incubation for 1 min, the residual solution was removed by filter paper. Then, 5 μL of 1% (w / v) uranyl acetate was dropped onto the grid and incubated for 1 min, and the excess uranyl acetate solution was removed with filter paper. The sample was air-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 as Figure 5 shown.

[0061] Characterization by cryo-transmission electron microscopy: First, prepare the cryo-EM sample. Take 10 μL of the sample and place it on a carbon-coated copper grid, and then quickly freeze the copper grid in liquid ethane. After placing the frozen sample in the cryo-electron microscope, its structure was observed in the cryo-electron microscope mode under electron beam irradiation. The observation result of Chol-CLP3+Hhc is as 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 5 μL, 10 μL, 15 μL, 20 μL, and 25 μL, respectively. A cell experiment was carried out with 3T3 cells. The medium used was DMEM 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% CO 2 2.

[0064] To evaluate cell viability, NIH-3T3 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. 1 μL, 5 μL, and 10 μL of the modified protein were mixed with 200 μL of the medium respectively for culturing cells, and the cytotoxicity of the cholesterol-modified protein on cells was detected by live / dead staining. Among them, green fluorescence represents live cells and red fluorescence represents dead cells. In addition, the CCK-8 assay was used to detect the effects of the cholesterol-modified protein on the viability of NIH-3T3 cells and HaCat cells. The specific operations are as follows: 1×10 4 cells were seeded in a 96-well plate, and the mixture of protein and medium was added for culturing for 20 h. The old medium was discarded and replaced with fresh medium, then CCK-8 reagent was added, and the plate was incubated in an incubator for 30 min. The optical density (OD) at 450 nm was measured and normalized with the medium control group. All tests were repeated three times to evaluate the cytotoxicity of the cholesterol-modified protein.

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

[0066] Based on the performance differences of the cholesterol-modified proteins Chol-CLP1+Hhc, Chol-CLP2+Hhc, and Chol-CLP3+Hhc, analysis was carried out according to the results of NIH-3T3 cell live / dead staining and the CCK-8 assay results of NIH-3T3 and HaCat cells.

[0067] Cholesterol-modified proteins usually show significant differences in cell uptake, stability, and biocompatibility. The following is a specific analysis of the performance differences of the 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 the immune response. However, due to the different lengths of the three types of collagen, CLP1, CLP2, and CLP3, the expressed proteins also differ in structure and function, resulting in different modification effects.

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

[0071] Chol-CLP2+Hhc: Has high stability, but low cell uptake rate.

[0072] Chol-CLP3+Hhc: Shows high biocompatibility and low cytotoxicity.

[0073] 2. Cell compatibility evaluation

[0074] Cell compatibility is a key indicator for evaluating cholesterol-modified proteins in biomedical applications. Based on the CCK-8 experimental results (see Figures 6 - 7 ), the cell compatibility analysis of the three proteins is as follows:

[0075] Chol-CLP1+Hhc: Has a high cell viability, and the cell metabolic activity decreases at high concentrations, indicating low cytotoxicity but may have a certain impact on cell metabolism.

[0076] Chol-CLP2+Hhc: Has a low cell viability, and the cell metabolic activity decreases significantly at high concentrations, indicating high cytotoxicity.

[0077] Chol-CLP3+Hhc: Has the highest cell viability, and no obvious cytotoxicity is observed at high concentrations, indicating good cell compatibility.

[0078] 3. Summary of performance differences:

[0079] Chol-CLP1+Hhc: Has a high cell uptake rate, and its cytotoxicity is slightly higher than that of the other two proteins.

[0080] Chol-CLP2+Hhc: Has a low cell uptake efficiency and high cytotoxicity.

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

[0082] In summary, based on the analysis of the CCK-8 experimental results and the cell viability staining results (Figures 8-9), Chol-CLP3+Hhc shows the best cell compatibility and low cytotoxicity, and has good application potential in the biomedical field.

[0083] Although the present invention has been described in detail above with general descriptions and specific examples, modifications or improvements can be made based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection of 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

[0099] Leu Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr 35 40 45

[0101] Tyr Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu 50 55 60

[0103] Leu Lys Gly Ile Gln Asp His Ala Leu Asp Gly Gly Pro Cys Pro Pro 65 70 75 80

[0105] Cys Gly Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp 85 90 95

[0107] Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly 100 105 110

[0109] Glu Gln Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly Asp 115 120 125

[0111] Arg Gly Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala 130 135 140

[0113] Gly Pro Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Arg 145 150 155 160

[0115] Gly Asp Gly Lys Gly Gly Ser Gly Gly Ser Thr Val His Gly Cys Phe 165 170 175

[0117] Thr Pro Glu Ser Thr Ala Leu Leu Glu Ser Gly Val Arg Lys Pro Leu 180 185 190

[0119] Gly Glu Leu Ser Ile Gly Asp Arg Val Leu Ser Met Thr Ala Asn Gly 195 200 205

[0121] Gln Ala Val Tyr Ser Glu Val Ile Leu Phe Met Asp Arg Asn Leu Glu 210 215 220

[0123] Gln Met Gln Asn Phe Val Gln Leu His Thr Asp Gly Gly Ala Val Leu 225 230 235 240

[0125] Thr Val Thr Pro Ala His Leu Val Ser Val Trp Gln Pro Glu Ser Gln 245 250 255

[0127] Lys Leu Thr Phe Val Phe Ala Asp Arg Ile Glu Glu Lys Asn Gln Val 260 265 270

[0129] Leu Val Arg Asp Val Glu Thr Gly Glu Leu Arg Pro Gln Arg Val Val 275 280 285

[0131] Lys Val Gly Ser Val Arg Ser Lys Gly Val Val Ala Pro Leu Thr Arg 290 295 300

[0133] Glu Gly Thr Ile Val Val Asn Ser Val Ala Ala Ser Cys Tyr Ala Val 305 310 315 320

[0135] Ile Asn Ser Gln Ser Leu Ala His Trp Gly Leu Ala Pro Met Arg Leu 325 330 335

[0137] Leu Ser Thr Leu Glu Ala Trp Leu Pro Ala Lys Glu Gln Leu His Ser 340 345 350

[0139] Ser Pro Lys Val Val Ser Ser Ala Gln Gln Gln Asn Gly Ile His Trp 355 360 365

[0141] Tyr Ala Asn Ala Leu Tyr Lys Val Lys Asp Tyr Val Leu Pro Gln Ser 370 375 380

[0143] Trp Arg His Asp Gly His His His His His His His Gly 385 390 395

[0145] <210> 2

[0146] <211> 477

[0147] <212> PRT

[0148] <213> Synthetic Sequence

[0149] <400> 2

[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

[0154] Thr Leu Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu 35 40 45

[0156] Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg 50 55 60

[0158] Leu Leu Lys Gly Ile Gln Asp His Ala Leu Asp Gly Gly Pro Cys Pro 65 70 75 80

[0160] Pro Cys Gly Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys 85 90 95

[0162] Asp Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala 100 105 110

[0164] Gly Glu Gln Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly 115 120 125

[0166] Asp Arg Gly Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu 130 135 140

[0168] Ala Gly Pro Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala 145 150 155 160

[0170] Gly Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly 165 170 175

[0172] Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu 180 185 190

[0174] Gln Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly Asp Arg 195 200 205

[0176] Gly Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly 210 215 220

[0178] Pro Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Arg Gly 225 230 235 240

[0180] Asp Gly Lys Gly Gly Ser Gly Gly Ser Thr Val His Gly Cys Phe Thr 245 250 255

[0182] Pro Glu Ser Thr Ala Leu Leu Glu Ser Gly Val Arg Lys Pro Leu Gly 260 265 270

[0184] Glu Leu Ser Ile Gly Asp Arg Val Leu Ser Met Thr Ala Asn Gly Gln 275 280 285

[0186] Ala Val Tyr Ser Glu Val Ile Leu Phe Met Asp Arg Asn Leu Glu Gln 290 295 300

[0188] Met Gln Asn Phe Val Gln Leu His Thr Asp Gly Gly Ala Val Leu Thr 305 310 315 320

[0190] Val Thr Pro Ala His Leu Val Ser Val Trp Gln Pro Glu Ser Gln Lys 325 330 335

[0192] Leu Thr Phe Val Phe Ala Asp Arg Ile Glu Glu Lys Asn Gln Val Leu 340 345 350

[0194] Val Arg Asp Val Glu Thr Gly Glu Leu Arg Pro Gln Arg Val Val Lys 355 360 365

[0196] Val Gly Ser Val Arg Ser Lys Gly Val Val Ala Pro Leu Thr Arg Glu 370 375 380

[0198] Gly Thr Ile Val Val Asn Ser Val Ala Ala Ser Cys Tyr Ala Val Ile 385 390 395 400

[0200] Asn Ser Gln Ser Leu Ala His Trp Gly Leu Ala Pro Met Arg Leu Leu 405 410 415

[0202] Ser Thr Leu Glu Ala Trp Leu Pro Ala Lys Glu Gln Leu His Ser Ser 420 425 430

[0204] Pro Lys Val Val Ser Ser Ala Gln Gln Gln Asn Gly Ile His Trp Tyr 435 440 445

[0206] Ala Asn Ala Leu Tyr Lys Val Lys Asp Tyr Val Leu Pro Gln Ser Trp 450 455 460

[0208] Arg His Asp Gly His His His His His His His His Gly 465 470 475

[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

[0219] Leu Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr 35 40 45

[0221] Tyr Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu 50 55 60

[0223] Leu Lys Gly Ile Gln Asp His Ala Leu Asp Gly Gly Pro Cys Pro Pro 65 70 75 80

[0225] Cys Gly Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp 85 90 95

[0227] Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly 100 105 110

[0229] Glu Gln Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp 115 120 125

[0231] Arg Gly Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala 130 135 140

[0233] Gly Pro Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly 145 150 155 160

[0235] Pro Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu 165 170 175

[0237] Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln 180 185 190

[0239] Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly 195 200 205

[0241] Glu Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro 210 215 220

[0243] Ala Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly Pro Lys 225 230 235 240

[0245] Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu Ala Gly 245 250 255

[0247] Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln Gly Glu 260 265 270

[0249] Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly Glu Thr 275 280 285

[0251] Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly 290 295 300

[0253] Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly Lys Gly Gly Ser 305 310 315 320

[0255] Gly Gly Ser Thr Val His Gly Cys Phe Thr Pro Glu Ser Thr Ala Leu 325 330 335

[0257] Leu Glu Ser Gly Val Arg Lys Pro Leu Gly Glu Leu Ser Ile Gly Asp 340 345 350

[0259] Arg Val Leu Ser Met Thr Ala Asn Gly Gln Ala Val Tyr Ser Glu Val 355 360 365

[0261] Ile Leu Phe Met Asp Arg Asn Leu Glu Gln Met Gln Asn Phe Val Gln 370 375 380

[0263] Leu His Thr Asp Gly Gly Ala Val Leu Thr Val Thr Pro Ala His Leu 385 390 395 400

[0265] Val Ser Val Trp Gln Pro Glu Ser Gln Lys Leu Thr Phe Val Phe Ala 405 410 415

[0267] Asp Arg Ile Glu Glu Lys Asn Gln Val Leu Val Arg Asp Val Glu Thr 420 425 430

[0269] Gly Glu Leu Arg Pro Gln Arg Val Val Lys Val Gly Ser Val Arg Ser 435 440 445

[0271] Lys Gly Val Val Ala Pro Leu Thr Arg Glu Gly Thr Ile Val Val Asn 450 455 460

[0273] Ser Val Ala Ala Ser Cys Tyr Ala Val Ile Asn Ser Gln Ser Leu Ala 465 470 475 480

[0275] His Trp Gly Leu Ala Pro Met Arg Leu Leu Ser Thr Leu Glu Ala Trp 485 490 495

[0277] Leu Pro Ala Lys Glu Gln Leu His Ser Ser Pro Lys Val Val Ser Ser 500 505 510

[0279] Ala Gln Gln Gln Asn Gly Ile His Trp Tyr Ala Asn Ala Leu Tyr Lys 515 520 525

[0281] Val Lys Asp Tyr Val Leu Pro Gln Ser Trp Arg His Asp 530 535 540

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; reacting the recombinant protein with cholesterol in the presence of TBS buffer to obtain the protein-based biomaterial; The amino acid sequence of the recombinant protein is shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.

3.

2. 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.

3. 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.

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

5. 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.

6. A protein-based biomaterial, characterized in that: It is prepared by the method according to any one of claims 1 to 5.

7. Use of the protein-based biomaterial according to claim 6 in preparing a product for drug delivery.

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