CN111939323B - Functional polypeptide hydrogel containing hyaluronic acid connecting peptide and connecting protein terminal peptide and application thereof - Google Patents
Functional polypeptide hydrogel containing hyaluronic acid connecting peptide and connecting protein terminal peptide and application thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
Abstract
The invention provides a functional polypeptide hydrogel containing hyaluronic acid connecting peptide and connecting protein terminal peptide, which comprises KLD12 polypeptide, KLD12-LPP polypeptide and KLD12-HLP polypeptide, wherein the mass ratio of the KLD12 polypeptide to the KLD12-LPP polypeptide to the KLD12-HLP polypeptide is 1:1: 1-3: 1: 1. The functional polypeptide hydrogel can be used as a scaffold material for treating articular cartilage damage caused by various diseases. The functional polypeptide hydrogel with the nanofiber network is prepared by intermolecular self-assembly of the KLD12-HLP polypeptide, the KLD12-LPP polypeptide and the KLD12 polypeptide, can be used as a scaffold of various stem cells and chondrocytes, has no obvious cytotoxicity, can promote migration of cells, and is beneficial to repair of damaged articular cartilage.
Description
Technical Field
The invention relates to the technical field of hydrogel, in particular to functional polypeptide hydrogel containing hyaluronic acid connecting peptide and connecting protein terminal peptide and application thereof.
Background
Osteoarthritis is one of the most common diseases in orthopedics, seriously affects the life quality of patients and brings great economic burden to patients, families and society. According to statistics, the prevalence rate of knee osteoarthritis in Chinese population is 9.56%, and 78.5% in people over 60 years old. In the united states, the cost of per-capita treatment prior to joint replacement is $ 3545.82. Early lesions in osteoarthritis are primarily manifested by damage to the articular cartilage. However, due to the lack of blood vessels, lymphatic vessels and nerves in articular cartilage, once damaged, it is extremely difficult to repair. It has been found that even very small articular cartilage lesions will become progressively larger and deeper if timely intervention is not taken, eventually leading to total joint degeneration. Therefore, how to repair the early and middle stage articular cartilage damage and further delay and even stop the progress of osteoarthritis is a problem which needs to be solved at present.
For patients with early and medium-term osteoarthritis, the existing treatment means such as medicines, physical therapy, glucocorticoid injection in joint cavities, hyaluronic acid injection in joints and the like can only temporarily relieve symptoms, but cannot influence the outcome of the disease, and finally severe osteoarthritis can be developed. In recent years, in situ tissue regeneration techniques by recruitment of endogenous stem cells have brought new ideas for the repair of articular cartilage. However, the lack of effective fusion between the new cartilage regenerated based on this strategy and the native cartilage, the new cartilage may prematurely degenerate, eventually leading to failure of articular cartilage repair. There is a large body of evidence that efficient chondrocyte migration favors interfacial integration of new cartilage with native cartilage. In addition, the migrating chondrocytes can further promote differentiation of the migrating stem cells into chondrocytes and prevent their hypertrophy. Therefore, a regenerative strategy that can induce both chondrocyte and stem cell migration would certainly facilitate interface integration and improve the quality of regenerated articular cartilage.
Disclosure of Invention
In view of the above, the present invention provides a method for preparing a functional polypeptide hydrogel having a lubricating function and capable of simultaneously inducing migration of chondrocytes and stem cells, and an application thereof, wherein the functional polypeptide hydrogel can be used as a scaffold material for treating osteoarthritis in early and middle stages.
The invention provides a functional polypeptide hydrogel containing hyaluronic acid connecting peptide and connecting protein terminal peptide, which comprises KLD12 polypeptide, KLD12-LPP polypeptide and KLD12-HLP polypeptide, wherein the mass ratio of the KLD12 polypeptide, KLD12-LPP polypeptide to the KLD12-HLP polypeptide is 1:1: 1-3: 1:1, the amino acid sequence of the KLD12 polypeptide is shown as SEQ ID No.1, the amino acid sequence of the KLD12-LPP polypeptide is shown as SEQ ID No.2, and the amino acid sequence of the KLD12-HLP polypeptide is shown as SEQ ID No. 3.
The invention also provides a preparation method of the functional polypeptide hydrogel containing the hyaluronic acid connecting peptide and the connecting protein terminal peptide, which comprises the following steps:
(1) weighing KLD12 polypeptide, KLD12-LPP polypeptide and KLD12-HLP polypeptide, respectively dissolving in sterile deionized water, performing ultrasonic treatment, and fully dissolving to obtain KLD12 polypeptide solution, KLD12-LPP polypeptide solution and KLD12-HLP polypeptide solution;
(2) and mixing the KLD12 polypeptide solution, the KLD12-LPP polypeptide solution and the KLD12-HLP polypeptide solution, placing the mixture in a microtube, and standing the mixture to ensure that the mixture is fully self-assembled to obtain the sticky functional polypeptide hydrogel.
Further, the KLD12-LPP polypeptide consists of KLD12 polypeptide and LPP polypeptide, and is specifically synthesized by a solid phase polypeptide automatic synthesizer directly from a corresponding amino acid sequence, and the components and purity of the polypeptide are detected by a high performance liquid chromatograph and a mass spectrometer (3485.93 daltons, purity 97% -98%). The LPP polypeptide is connected to the amino terminal of the KLD12 polypeptide, the amino acid sequence of the LPP polypeptide is shown in SEQ ID NO.4, the LPP polypeptide and the KLD12 polypeptide are isolated by-Gly-and-Gly-can ensure that the rear active sequence has proper space, and the exposure of the active site after self-assembly is facilitated.
Further, the KLD12-HLP polypeptide consists of a KLD12 polypeptide and an HLP polypeptide, and is specifically synthesized by a solid phase polypeptide automatic synthesizer directly from a corresponding amino acid sequence, and the components and the purity of the KLD12-HLP polypeptide are detected by a high performance liquid chromatograph and a mass spectrometer (2925.35 daltons, the purity is 96.5% -98%). HLP polypeptide is connected to the amino terminal of the KLD12 polypeptide, the amino acid sequence of the HLP polypeptide is shown in SEQ ID NO.5, the HLP polypeptide and the KLD12 polypeptide are isolated by-Gly Gly-and-Gly-to ensure that the rear active sequence has proper space and is beneficial to the exposure of the active site after self-assembly.
Further, the amino-and carboxy-termini of KLD12 polypeptide, KLD12-LPP polypeptide and KLD12-HLP polypeptide were each acetylated and amidated, respectively, to avoid degradation of the polypeptide.
The invention also provides application of the functional polypeptide hydrogel containing the hyaluronic acid connecting peptide and the connecting protein terminal peptide in preparing a scaffold material for treating articular cartilage damage caused by various diseases.
Further, the disease includes, but is not limited to, osteoarthritis.
LPP polypeptides (connexin terminal peptides) are terminal peptides of connexins in the extracellular matrix of articular cartilage, and play an important role in the regulation of the biological behavior of chondrocytes and cartilage precursor cells. LPP polypeptides can promote synthesis of chondrocyte extracellular matrix proteoglycans and type ii collagen. In addition, it can promote the migration of cartilage precursor cells.
The HLP polypeptide (hyaluronic acid connecting peptide) is a specific amino acid sequence in articular cartilage extracellular matrix connecting protein, and can be specifically combined with hyaluronic acid.
Amphiphilic polypeptide KLD12-HLP containing hyaluronic acid connecting peptide, amphiphilic polypeptide KLD12-LPP containing connecting protein terminal peptide and amphiphilic polypeptide KLD12 can be subjected to intermolecular self-assembly to form hydrogel with certain strength; the pure KLD12-LPP polypeptide and the KLD12-HLP polypeptide can not generate intermolecular self-assembly to form hydrogel.
The technical scheme provided by the invention has the beneficial effects that: the functional polypeptide hydrogel with the nanofiber network is prepared by intermolecular self-assembly of the KLD12-HLP polypeptide, the KLD12-LPP polypeptide and the KLD12 polypeptide, can be used as a scaffold of various stem cells and chondrocytes, has no obvious cytological toxicity, can promote migration of cells, and is beneficial to repair of damaged articular cartilage; after the hydrogel containing the functional groups (the hyaluronic acid connecting peptide and the connexin terminal peptide) is injected into a joint cavity, the hyaluronic acid connecting peptide can target the hydrogel to hyaluronic acid exposed at a cartilage defect part, and the connexin terminal peptide further plays a good biological function. In addition, the hydrogel also has a good lubricating function, can obviously reduce the friction of joints and improve the symptoms of osteoarthritis.
Drawings
FIG. 1 is a diagram of the product of functional polypeptide hydrogel KHLP prepared in example 1 and example 3.
FIG. 2 is a graph showing the relationship between shear rate and viscosity of KHLP, which is a functional polypeptide hydrogel prepared in examples 1-3.
FIG. 3 is a graph showing the results of cell viability in KHLP coated on KLD12 polypeptide hydrogel and the functional polypeptide hydrogel prepared in example 3.
FIG. 4 is a schematic representation of the effect of KLD12 polypeptide hydrogel and KHLP, the functional polypeptide hydrogel prepared in example 3, on the three-dimensional migration of chondrocytes and bone marrow stromal stem cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the accompanying drawings and examples.
In the following examples 1-3, the amino acid sequence of KLD12 polypeptide is shown as SEQ ID NO.1, the amino acid sequence of KLD12-LPP polypeptide is shown as SEQ ID NO.2, and the amino acid sequence of KLD12-HLP polypeptide is shown as SEQ ID NO. 3.
The KLD12-LPP polypeptide consists of KLD12 polypeptide and LPP polypeptide, is specifically synthesized by a solid phase polypeptide automatic synthesizer directly from corresponding amino acid sequences, and the components and the purity (3485.93 daltons, the purity is 97% -98%) of the polypeptide are detected by a high performance liquid chromatograph and a mass spectrometer. The LPP polypeptide is connected to the amino terminal of the KLD12 polypeptide, the amino acid sequence of the LPP polypeptide is shown in SEQ ID NO.4, the LPP polypeptide and the KLD12 polypeptide are isolated by-Gly-and-Gly-can ensure that the rear active sequence has proper space, and the exposure of the active site after self-assembly is facilitated.
The KLD12-HLP polypeptide consists of KLD12 polypeptide and HLP polypeptide, is specifically synthesized by a solid phase polypeptide automatic synthesizer directly from corresponding amino acid sequences, and the components and the purity (2925.35 daltons, the purity is 96.5% -98%) of the KLD12-HLP polypeptide are detected by a high performance liquid chromatograph and a mass spectrometer. The HLP polypeptide is connected to the amino terminal of the KLD12 polypeptide, the amino acid sequence of the HLP polypeptide is shown in SEQ ID NO.5, the HLP polypeptide and the KLD12 polypeptide are isolated by-Gly Gly-and-Gly Gly-can ensure that the rear active sequence has a proper space and is beneficial to exposing the active site after self-assembly.
The amino terminal and the carboxyl terminal of the KLD12 polypeptide, the KLD12-LPP polypeptide and the KLD12-HLP polypeptide are respectively acetylated and amidated to avoid the degradation of the polypeptides, and the structural formula of the KLD12 polypeptide is abbreviated as follows: AcN-KLDLKLDLKLDL-CONH2(ii) a The structural formula of the KLD12-LPP polypeptide is abbreviated as: AcN-KLDLKLDLKLDL-GG-DHLSDNYTLDHDRAIH-CONH2(ii) a The structural formula of the KLD12-HLP polypeptide is abbreviated as AcN-KLDLKLDLKLDL-GG-RYPISRPRKRC-CONH2。
Example 1:
example 1 provides a method for preparing a functional polypeptide hydrogel KHLP containing hyaluronic acid connecting peptide and connecting protein terminal peptide, comprising the following steps:
(1) weighing 10mg of KLD12 polypeptide, dissolving in 1mL of sterile deionized water, carrying out ultrasonic water bath treatment at 37 ℃ for 30 minutes, and fully dissolving to obtain a KLD12 polypeptide solution with the concentration of 10 mg/mL; weighing 10mg of KLD12-LPP polypeptide, dissolving in 1mL of sterile deionized water, carrying out ultrasonic water bath treatment at 37 ℃ for 30 minutes, and fully dissolving to obtain a 10mg/mL KLD12-LPP polypeptide solution; weighing 10mg of KLD12-HLP polypeptide, dissolving in 1mL of sterile deionized water, and carrying out ultrasonic water bath treatment at 37 ℃ for 30 minutes to fully dissolve the polypeptide to obtain a KLD12-HLP polypeptide solution with the concentration of 10 mg/mL;
(2) 200 mu L of KLD12 polypeptide solution, 200 mu L of KLD12-LPP polypeptide solution and 200 mu L of KLD12-HLP polypeptide solution are weighed and mixed, placed in a microtube and kept stand for 30 minutes to ensure that the functional polypeptide hydrogel KHLP is obtained through sufficient self-assembly.
Example 2:
example 2 provides a method for preparing a functional polypeptide hydrogel KHLP containing hyaluronic acid connecting peptide and connecting protein terminal peptide, comprising the following steps:
(1) weighing 10mg of KLD12 polypeptide, dissolving in 1mL of sterile deionized water, carrying out ultrasonic water bath treatment at 37 ℃ for 30 minutes, and fully dissolving to obtain a KLD12 polypeptide solution with the concentration of 10 mg/mL; weighing 10mg of KLD12-LPP polypeptide, dissolving in 1mL of sterile deionized water, carrying out ultrasonic water bath treatment at 37 ℃ for 30 minutes, and fully dissolving to obtain a 10mg/mL KLD12-LPP polypeptide solution; weighing 10mg of KLD12-HLP polypeptide, dissolving in 1mL of sterile deionized water, carrying out ultrasonic water bath treatment at 37 ℃ for 30 minutes, and fully dissolving to obtain a KLD12-HLP polypeptide solution with the concentration of 10 mg/mL;
(2) weighing 400 mu L of KLD12 polypeptide solution, 200 mu L of KLD12-LPP polypeptide solution and 200 mu L of KLD12-HLP polypeptide solution, mixing, placing in a microtube, standing for 30 minutes, and fully self-assembling to obtain the functional polypeptide hydrogel KHLP.
Example 3:
example 3 provides a method for preparing a functional polypeptide hydrogel KHLP containing hyaluronic acid connecting peptide and connecting protein terminal peptide, comprising the following steps:
(1) weighing 10mg of KLD12 polypeptide, dissolving in 1mL of sterile deionized water, carrying out ultrasonic water bath treatment at 37 ℃ for 30 minutes, and fully dissolving to obtain a KLD12 polypeptide solution with the concentration of 10 mg/mL; weighing 10mg of KLD12-LPP polypeptide, dissolving in 1mL of sterile deionized water, carrying out ultrasonic water bath treatment at 37 ℃ for 30 minutes, and fully dissolving to obtain a 10mg/mL KLD12-LPP polypeptide solution; weighing 10mg of KLD12-HLP polypeptide, dissolving in 1mL of sterile deionized water, carrying out ultrasonic water bath treatment at 37 ℃ for 30 minutes, and fully dissolving to obtain a KLD12-HLP polypeptide solution with the concentration of 10 mg/mL;
(2) weighing 600 mu L of KLD12 polypeptide solution, 200 mu L of KLD12-LPP polypeptide solution and 200 mu L of KLD12-HLP polypeptide solution, mixing, placing in a microtube, standing for 30 minutes, and fully self-assembling to obtain the functional polypeptide hydrogel KHLP.
The functional polypeptide hydrogel KHLP products prepared in examples 1 and 3 are shown in fig. 1, fig. 1A and 1B show Hyaluronic Acid (HA) products in fig. 1, fig. 1C shows KLD12 polypeptide hydrogel products in fig. 1D shows the functional polypeptide hydrogel KHLP product prepared in example 3, fig. 1E shows the functional polypeptide hydrogel KHLP product prepared in example 1, and fig. 1E shows the KLD12 polypeptide itself forms hydrogel with certain strength, and KLD12 polypeptide, KLD12-LPP polypeptide and KLD12-HLP polypeptide form hydrogel with different strength when mixed in different proportions, as can be seen from fig. 1.
FIG. 2 is a graph showing the relationship between shear rate and viscosity of KHLP, which is a functional polypeptide hydrogel prepared in examples 1-3, and FIG. 2 also shows the relationship between shear rate and viscosity of a hydrogel obtained by self-assembly of Hyaluronic Acid (HA) and KLD12 polypeptide (i.e., KLD12 polypeptide hydrogel). In fig. 2, curve a shows the relationship between shear rate and viscosity of Hyaluronic Acid (HA), curve B shows the relationship between shear rate and viscosity of KLD12 polypeptide hydrogel, curve C shows the relationship between shear rate and viscosity of functional polypeptide hydrogel KHLP prepared in example 1, curve D shows the relationship between shear rate and viscosity of functional polypeptide hydrogel KHLP prepared in example 2, and curve E shows the relationship between shear rate and viscosity of functional polypeptide hydrogel KHLP prepared in example 3, as shown in fig. 2: as the shear rate increases, the viscosities of the functional polypeptide hydrogel KHLP, KLD12 polypeptide hydrogel and HA become thinner gradually, which indicates that the functional polypeptide hydrogel KHLP prepared in examples 1-3 HAs shear thinning characteristics similar to hyaluronic acid, and is a good lubricant.
In order to evaluate the cytotoxicity of KHLP, chondrocytes were co-cultured with KLD12 polypeptide hydrogel and KHLP obtained from example 3 in three dimensions, respectively, and after co-culturing for 3D and 7D, the survival rate of the cells coated with KLD12 polypeptide hydrogel and KHLP obtained from example 3 was more than 90%, as shown in FIG. 3, bar A shows the survival rate of the cells coated with KLD12 polypeptide hydrogel after co-culturing for 3D, bar B shows the survival rate of the cells coated with KHLP obtained from example 3, bar C shows the survival rate of the cells coated with KHLP obtained from example 12 polypeptide after co-culturing for 7D, and bar D shows the survival rate of the cells coated with KHLP obtained from example 3 after co-culturing for 7D, the above results show that: the newly synthesized functional polypeptide hydrogel KHLP and KLD12 polypeptide hydrogel have no obvious cytotoxicity.
In order to evaluate the effect of the prepared functional polypeptide hydrogel KHLP on the three-dimensional migration of chondrocytes and bone marrow stromal stem cells, cells are planted on the surface of the hydrogel, and after co-culture for 7D, both chondrocytes and bone marrow stromal stem cells planted on the surface of the functional polypeptide hydrogel KHLP prepared in example 3 can spontaneously migrate into the interior of the polypeptide hydrogel, as shown in FIG. 4, FIG. 4A shows that chondrocytes are planted on the surface of KLD12 polypeptide hydrogel, FIG. 4B shows that chondrocytes are planted on the surface of the functional polypeptide hydrogel KHLP, FIG. 4C shows that bone marrow stromal stem cells are planted on the surface of KLD12 polypeptide hydrogel, and FIG. 4D shows that bone marrow stromal stem cells are planted on the surface of the functional polypeptide KHLP. The above results show that: the functional polypeptide hydrogel KHLP has the biological function of the functional group LPP carried by the functional polypeptide hydrogel KHLP.
The features of the embodiments and embodiments described herein above may be combined with each other without conflict.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
<110> Wuhan Supu Biotechnology GmbH
<120> functional polypeptide hydrogel containing hyaluronic acid connecting peptide and connecting protein terminal peptide and application thereof
<160> 5
<210> 1
<211> 12
<212> polypeptide
<213> Artificial Synthesis (Artificial)
<400> 1
Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu
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<210> 2
<211> 30
<212> polypeptide
<213> Artificial Synthesis (Artificial)
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Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu Gly Gly Asp
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His Leu Ser Asp Asn Tyr Thr Leu Asp His Asp Arg Ala Ile His
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<210> 3
<211> 25
<212> polypeptide
<213> Artificial Synthesis (Artificial)
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Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu Gly Gly Arg
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Tyr Pro Ile Ser Arg Pro Arg Lys Arg Cys
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<210> 4
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<212> polypeptide
<213> Artificial Synthesis (Artificial)
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Asp His Leu Ser Asp Asn Tyr Thr Leu Asp His Asp Arg Ala Ile His
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<213> Artificial Synthesis (Artificial)
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Arg Tyr Pro Ile Ser Arg Pro Arg Lys Arg Cys
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Claims (4)
1. A method for preparing a functional polypeptide hydrogel containing a hyaluronic acid connecting peptide and a connecting protein terminal peptide, which is characterized by comprising the following steps:
(1) weighing KLD12 polypeptide, KLD12-LPP polypeptide and KLD12-HLP polypeptide, respectively dissolving in sterile deionized water, performing ultrasonic treatment, and fully dissolving to obtain KLD12 polypeptide solution, KLD12-LPP polypeptide solution and KLD12-HLP polypeptide solution; the mass ratio of the KLD12 polypeptide to the KLD12-LPP polypeptide to the KLD12-HLP polypeptide is 1:1: 1-3: 1:1, the amino acid sequence of the KLD12 polypeptide is shown as SEQ ID NO.1, the amino acid sequence of the KLD12-LPP polypeptide is shown as SEQ ID NO.2, and the amino acid sequence of the KLD12-HLP polypeptide is shown as SEQ ID NO. 3;
(2) and mixing the KLD12 polypeptide solution, the KLD12-LPP polypeptide solution and the KLD12-HLP polypeptide solution, standing, and fully self-assembling to obtain the functional polypeptide hydrogel.
2. The method for preparing the functional polypeptide hydrogel containing the hyaluronic acid connecting peptide and the connecting protein terminal peptide according to claim 1, wherein the KLD12-LPP polypeptide consists of KLD12 polypeptide and LPP polypeptide, and the amino acid sequence of the LPP polypeptide is shown in SEQ ID No. 4.
3. The method for preparing the functional polypeptide hydrogel containing the hyaluronic acid connecting peptide and the connecting protein terminal peptide according to claim 1, wherein the KLD12-HLP polypeptide consists of a KLD12 polypeptide and an HLP polypeptide, and the amino acid sequence of the HLP polypeptide is shown as SEQ ID No. 5.
4. Use of the functional polypeptide hydrogel prepared by the preparation method of claim 1 in preparing a scaffold material for treating articular cartilage damage caused by diseases.
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| CN111939323A (en) | 2020-11-17 |
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