WO2023108532A1 - 一种具有湿粘附性能的新型类细胞基质生物材料及其应用 - Google Patents

一种具有湿粘附性能的新型类细胞基质生物材料及其应用 Download PDF

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WO2023108532A1
WO2023108532A1 PCT/CN2021/138721 CN2021138721W WO2023108532A1 WO 2023108532 A1 WO2023108532 A1 WO 2023108532A1 CN 2021138721 W CN2021138721 W CN 2021138721W WO 2023108532 A1 WO2023108532 A1 WO 2023108532A1
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sbp9
seq
protein
cell
calcium
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PCT/CN2021/138721
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French (fr)
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刘伟治
王露露
薛波
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中国海洋大学
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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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D189/00—Coating compositions based on proteins; Coating compositions based on derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a novel cell-like matrix biological material with wet adhesion performance and its application.
  • Modern wound dressings include interactive wound dressings, calcium alginate dressings, silver dressings, foam dressings, and hydrogel dressings, among others. Modern wound dressings can not only avoid secondary infection, but also promote wound repair to a certain extent.
  • Chinese patent 201810344869.6 discloses a new type of bio-glue for wound covering, and the wound-covering product provided has the property of promoting healing, which is prepared by using polypeptide and hyaluronic acid derivatives; the results of use show that the new type of bio-glue has no obvious cytotoxicity , animal experiments prove that the wound covering product has a good effect of promoting healing.
  • Chinese patent 202010059780.2 discloses a protein fragment that can spontaneously form hydrogels for the preparation of hydrogels.
  • the hydrogel prepared from the protein provided by this patent has excellent properties such as controllable gelation time and mechanical properties, and good biocompatibility of the gel, so it has broad application prospects in the fields of biomedical materials and drug sustained release.
  • the present invention provides the combined application of Sbp9 protein and calcium ions in the preparation of cell-like matrix biomaterials.
  • the present invention provides an application of Sbp9 protein or its homologous protein in the preparation of cell-like matrix biomaterials.
  • the Sbp9 protein or its homologous protein is used together with Ca 2+ .
  • the homologous protein has more than 90% homology with Sbp9 protein.
  • the homologous protein has more than 95% homology with the Sbp9 protein; further preferably, the homologous protein has more than 99% homology with the Sbp9 protein.
  • the Sbp9 protein includes the combination of any number of sequences in SEQ ID NO.1-4.
  • any sequence can be selected from SEQ ID NO.1-4 for any number of repetitions, including but not limited to:
  • the Sbp9 protein includes in order: SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4 ;
  • the Sbp9 protein includes in order: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO. ID NO.2, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO .4;
  • the Sbp9 protein includes sequentially: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.3, SEQ ID NO.4;
  • the Sbp9 protein includes in order: SEQ ID NO.4, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.1;
  • the Sbp9 protein includes in order: SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.1;
  • the Sbp9 protein includes sequentially: SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4.
  • said Sbp9 protein consists of SEQ ID NO.1-4.
  • the Sbp9 protein includes in sequence: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4.
  • the Sbp9 protein is SEQ ID NO.5 or a sequence having more than 80% homology with SEQ ID NO.5.
  • the Sbp9 protein is SEQ ID NO.6 or a sequence having more than 80% homology with SEQ ID NO.6.
  • the Ca 2+ is used as a soluble salt.
  • the Ca2 + is calcium chloride, calcium nitrate, calcium gluconate, calcium dihydrogen phosphate, calcium bicarbonate, calcium bisulfate, calcium bisulfite, calcium hypochlorite, calcium bromide, iodide
  • the Ca2 + is calcium chloride, calcium nitrate, calcium gluconate, calcium dihydrogen phosphate, calcium bicarbonate, calcium bisulfate, calcium bisulfite, calcium hypochlorite, calcium bromide, iodide
  • the present invention provides a cell-like matrix biomaterial.
  • the cell-like matrix biological material includes: Sbp9 protein or its homologous protein and Ca 2+ .
  • the homologous protein has more than 90% homology with Sbp9 protein.
  • the homologous protein has more than 95% homology with the Sbp9 protein; further preferably, the homologous protein has more than 99% homology with the Sbp9 protein.
  • the Sbp9 protein includes the combination of any number of sequences in SEQ ID NO.1-4.
  • any sequence can be selected from SEQ ID NO.1-4 for any number of repetitions, including but not limited to:
  • the Sbp9 protein includes in order: SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4 ;
  • the Sbp9 protein includes in order: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO. ID NO.2, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO .4;
  • the Sbp9 protein includes sequentially: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.3, SEQ ID NO.4;
  • the Sbp9 protein includes in order: SEQ ID NO.4, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.1;
  • the Sbp9 protein includes in order: SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.1;
  • the Sbp9 protein includes sequentially: SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4, SEQ ID NO.4.
  • said Sbp9 protein consists of SEQ ID NO.1-4.
  • the Sbp9 protein includes in sequence: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4.
  • the Sbp9 protein is SEQ ID NO.5 or a sequence having more than 80% homology with SEQ ID NO.5.
  • the Sbp9 protein is SEQ ID NO.6 or a sequence having more than 80% homology with SEQ ID NO.6.
  • the concentration of the Sbp9 protein is ⁇ 0.2 mg/mL; further preferably, the concentration of the Sbp9 protein is 0.2-0.6 mg/mL; further, the concentration of the Sbp9 protein is 0.2-0.6 mg/mL.
  • the Ca 2+ is used as a soluble salt.
  • the Ca2 + is calcium chloride, calcium nitrate, calcium gluconate, calcium dihydrogen phosphate, calcium bicarbonate, calcium bisulfate, calcium bisulfite, calcium hypochlorite, calcium bromide, iodide
  • the Ca2 + is calcium chloride, calcium nitrate, calcium gluconate, calcium dihydrogen phosphate, calcium bicarbonate, calcium bisulfate, calcium bisulfite, calcium hypochlorite, calcium bromide, iodide
  • the Ca 2+ is calcium chloride.
  • the concentration of calcium chloride is 0.1-10mM; more preferably, the concentration of calcium chloride is 5-10mM; furthermore, the concentration of calcium chloride is 5mM or 10mM.
  • the concentration of calcium chloride is 5 mM, and the concentration of Sbp9 protein is 0.6 mg/mL.
  • the concentration of calcium chloride is 10 mM, and the concentration of Sbp9 protein is 0.4 mg/mL.
  • the present invention provides the application of the aforementioned cell-like matrix biomaterial in the preparation of medical coating materials or medical devices.
  • the present invention provides a medical coating material.
  • the medical coating material includes the aforementioned cell-like matrix biological material.
  • the invention provides a medical device.
  • the medical device includes the aforementioned cell-like matrix biological material.
  • the present invention provides a method for preparing the aforementioned cell-like matrix biomaterial.
  • the preparation method includes expressing Sbp9 protein through cells.
  • Said cells include a vector expressing Sbp9 protein.
  • the vector includes the gene sequence for expressing Sbp9 protein.
  • the gene sequence includes SEQ ID NO.7 or SEQ ID NO.8.
  • the cell-like matrix biological material provided by the invention has waterproof performance and high adhesion performance, can promote cell growth, promote wound healing, prevent oxidative damage, and has both adhesion and biological activity of the material.
  • the cell-like matrix biomaterial provided by the present invention is different from the principle of cross-linking of collagen caused by calcium ions in the prior art, in which calcium ions interact with nitrogen atoms in amino acid residues of collagen to form a dense structure between protein molecules. network structure, thereby improving the mechanical properties of its derived materials.
  • the calcium ion provided by the present invention mainly cross-links the carboxylic acid groups of amino acid residues, and achieves the effect of depositing aggregates to form a coating by inducing protein particle size to increase.
  • FIG. 1 Amino acid sequence analysis of Sbp9.
  • Figure 2 SDS-PAGE image of Sbp9 after purification.
  • FIG. 3 Ca 2+ induces phase separation of Sbp9 protein.
  • Figure 4 Molecular dynamics analysis of Ca 2+ -induced Sbp9 protein assembly, where A is the turbidity change curve, and B is the protein particle size change curve.
  • FIG. 5 Dynamic topography (AFM) of Ca 2+ -induced coating of Sbp9 protein.
  • FIG. 6 Coat formation at different Ca2 + and Sbp9 protein concentrations.
  • FIG. 8 Surface morphology (SEM) of Sbp9 coating.
  • Figure 9 Stability analysis of Sbp9 coating, in which A is the topography of the coating formed at the interface of different materials placed in a water environment for 2 months, B is the analysis of the remaining coating area, and C is the coating in the Stability analysis in harsh environments.
  • Figure 11 Effect of Sbp9 coating on UVB irradiation-induced skin photoaging.
  • Figure 12 Effect of Sbp9 coating on wound healing in mice with diabetic full-thickness skin defects.
  • Figure 13 Wound healing rates of mice in different groups.
  • Sbp9 is a key component of scallop silk root, and its full-length sequence (SEQ ID NO.6) is characterized by highly repeated EGFL (epidermal growth factor-like protein) domains. Because the full-length sequence consists of 2033 amino acids, there are practical difficulties in recombinant expression in vitro. Therefore, in this example, a truncated sequence composed of four repeated EGFL domains is selected for substitution analysis.
  • the four repeated EGFL domains are E1- E4, the corresponding amino acid sequence is SEQ ID NO.1-4:
  • Sbp9 contains 136 amino acids and is arranged in tandem repeats of four EGFL domains, and its sequence is notably rich in cysteine (18%), and each EGFL domain contains six highly conserved Cysteine, this characteristic will have an important impact on the functionality of its derived materials, such as excellent antioxidant activity.
  • Sbp9 was obtained by constructing an expression system Sbp9-pET32-HisTT containing a hexahistidine tag, and performing recombinant expression in Escherichia coli.
  • the forward primer contains a restriction endonuclease BamH I enzyme cutting site (GGATCC), and a protective base CG is added upstream of the BamH I enzyme cutting site, thereby improving the restriction endonuclease digestion efficiency.
  • the reverse primer contains a restriction endonuclease Xho I enzyme cutting site (CTCGAG) and an expression guard base CCG is added upstream.
  • CTCGAG restriction endonuclease Xho I enzyme cutting site
  • Upstream primer SEQ ID NO.9
  • the purified PCR product and the pET32-HisTT vector were double-digested with restriction endonucleases BamH I and Xho I, respectively, and digested overnight at 30°C.
  • the purified gene fragment was ligated with the pET32-HisTT vector, and ligated at 16° C. for 4 h.
  • the constructed recombinant vector was transformed into Escherichia coli BL21 (DE3) competent cells, and the positive clones were sequenced. Sequencing results showed that the inserted target gene sequence was consistent with expectations.
  • the selected positive clones were inoculated into 5 mL of LB medium containing kanamycin resistance for culture, and the bacterial liquid cultured in the test tube was used as the seed liquid, inoculated in 1L of LB medium, and cultured at 37°C and 180 rpm with shaking.
  • 0.2 mM IPTG was added for protein induction, and culture was continued at 16° C. for 20 h.
  • Dialysis refolding use 7000Da dialysis bag, dialyze in 2.5L dialysate (20mM Tris-HCl, pH 8.5) for 24h.
  • the refolded protein solution was subjected to SDS-PAGE electrophoresis detection, and the detection results are shown in FIG. 2 .
  • the theoretical molecular weight of the Sbp9-pET32-HisTT fusion protein is 20kD, and the electrophoresis results show that the fusion protein is correctly expressed with high purity (>80%). Purified and refolded proteins can be used for subsequent property studies.
  • Embodiment 2Sbp9 self-assembly process research
  • the OD 340 turbidity change and dynamic light scattering (DLS) were used to further elucidate the self-assembly behavior of Sbp9 mediated by Ca 2+ .
  • DLS was used to detect the particle size change of the protein solution after the introduction of CaCl 2 within 30 min, and the data was read every 2 min.
  • the experimental results are shown in A in Figure 4.
  • Ca 2+ can cause a sudden increase in the turbidity of the Sbp9 protein solution. After 30 minutes, the turbidity basically reaches the plateau, and the assembly is basically completed.
  • the change curve of the particle size of the protein molecule shown in B in Figure 4 also shows that with the prolongation of time, the particle size of the Sbp9 protein molecule is continuously increased by Ca 2+ , and the protein particle size reaches 1600nm when it is assembled to 14min, and then The particle size monitoring is beyond the detection range of DLS equipment.
  • the dynamic morphology changes before and after Sbp9 assembly were monitored by atomic force microscopy (AFM).
  • AFM atomic force microscopy
  • Dilute 0.6 mg/mL protein 100 times before and after the introduction of CaCl 2 take 2.5 ⁇ L and drop it on the newly exfoliated mica sheet, let it stand for 30 min to dry, and then perform AFM image acquisition.
  • the scanning range of the image is 0.5-10 ⁇ m, and the scanning rate is 0.6-1 Hz.
  • the scanning results are shown in Figure 5.
  • the purified Sbp9 molecules are evenly distributed in the buffer, and Ca 2+ induces the aggregation and assembly of protein molecules to form larger particle groups.
  • a coating structure consisting of layer-by-layer assembly of protein particles was formed.
  • Ca 2+ induces the assembly of Sbp9 protein molecules, which is the basis for the formation of coating materials.
  • FTIR Fourier transform infrared spectroscopy
  • the results of FITR spectroscopy are shown in Figure 6.
  • the wave number of the protein after Ca 2+ binding is red-shifted, which means the stretching vibration of -NH 2 , and the amino nitrogen atom coordinates with Ca 2+ by providing electron pairs, which involves the sides of Arg and Lys chain group.
  • 1000-1100cm -1 is the deformation vibration of CO and -OH, involving the side chain groups of Ser and Thr.
  • 541cm -1 represents the antisymmetric stretching vibration of Glu's COO-.
  • calcium ions prefer to form complexes with negatively charged carboxylic acid groups than amino nitrogen atoms and water molecules. The above results clearly indicate the formation of coordination bonds between various functional groups in the protein and divalent calcium ions.
  • the formation of the coating requires a suitable concentration of Sbp9 and Ca 2+ .
  • concentration of Sbp9 purified in Example 2 was diluted to 0.2-1.2 mg/mL, 36 ⁇ L of Sbp9 of each concentration was mixed rapidly with 4 ⁇ L of CaCl 2 (final concentration was 0.1-10 mM), and the mixture was transferred to a On a 1cm circular glass slide, let it stand for 30 minutes and then remove the upper diluent phase solution to form protein coatings with various concentration ratios.
  • Coomassie Brilliant Blue staining of the coating adhered to the glass slide can be used to observe the structural differences of each group of coatings.
  • the surface morphology of the coating formed on the glass interface was observed by scanning electron microscopy. Mix 36 ⁇ L of 0.6mg/mL Sbp9 with 5mM CaCl 2 and drop it onto a glass slide, let it stand for 30min and then remove the upper diluted phase. Surface and longitudinal section observations were made after drying the coating adsorbed on the glass.
  • the morphology observation results are shown in Figure 8.
  • the Sbp9 coating formed on the glass interface has a regular and uniform structure, and a dense network structure of protein molecules can be observed, which is significantly different from the blank glass sheet.
  • the cross-section of the coating shows a clear coating portion, glass portion, and interface layer, and the coating material has completed adhesion to the glass flake.
  • the prepared coatings were exposed to turbulent and harsh environments to test their integrity.
  • Samples were taken at and 60 days for visualization and staining, and the porosity of the coating was calculated using Image J software to indicate the remaining coating area.
  • the coating was placed in an ultrasonic cleaner with a power of 180 W for 1 h to simulate a harsh water environment to show the resistance of the coating material.
  • the visual staining results of the coating in a wet environment are shown in A in Figure 9.
  • a single Sbp9 component cannot complete the adhesion to the medium, and the coating formed by Ca 2+ mediated Sbp9 assembly can be achieved on glass, plastic and aluminum sheets.
  • Good interfacial adhesion wherein the coating formed on each interface is substantially free from peeling off after being shaken in a water environment for 60 days.
  • the quantitative results shown in B in Figure 9 show that the integrity of the coating remains above 80% after 60 days
  • C in Figure 9 shows that the coating has no shedding after shaking in a harsh water environment for 1 hour, all indicating that The coating has excellent water resistance stability and harsh environment resistance, which is conducive to subsequent wet environment applications.
  • the cell attachment rate experiment was carried out.
  • the cells were divided into three groups: Control, Poly-lysine coating and Sbp9 coating.
  • the non-TC-treated 24-well cell culture plate was used for cell culture.
  • the well plates of each group were not treated, and 300 ⁇ L of Poly-lysine with a concentration of 100 ⁇ g/mL was spread. lysine and spread 300 ⁇ L of Sbp9 at a concentration of 25 ⁇ g/mL.
  • Example 5 Sbp9 coating antagonizes skin photoaging induced by oxidative stress
  • the Sbp9 purified in Example 2 was used to carry out coating antagonism skin photoaging experiment caused by UVB irradiation: the mice were divided into Control group, UVB group, UVB+Sbp9 coating group (UVB+Sbp9coating), UVB+Vitamin C group . Administration was carried out 10 minutes before irradiation, and the UVB+Sbp9 coating group was sprayed with Sbp9 coating (1 mg/mL protein); (2) the positive control group was smeared with Vitamin C (100 mg/kg); (3) Control group and UVB group Apply saline. Apply once every 2 days between dosing intervals.
  • UVB lamps were irradiated, and the radiation dose: a total of 5 days of irradiation, the daily radiation dose was 120mj ⁇ cm -2 in the first 3 days, and the daily radiation dose was 100mj ⁇ cm -2 in the next 2 days, and the distance from the light source to the irradiation area was 15- 20cm.
  • Example 6 Sbp9 coating promotes wound healing in mice with diabetic full-thickness skin defect
  • the Sbp9 purified in Example 2 was used to carry out the wound healing experiment: the mice were divided into Control group, Sbp9 coating group (Sbp9coating), and rhEGF group, and a full-thickness skin injury wound was made with a trepan drill on the back of the mouse, with a diameter of 6 mm.
  • the wounds of the group were sprayed with Sbp9 coating with a protein concentration of 1 mg/mL once a day, the positive control group was smeared with rhEGF gel, and the negative control group was smeared with normal saline.
  • the wounds were photographed every day, and the wound area was analyzed by Image J software.
  • Figure 12-13 shows that at 3, 7, and 14 days, the wounds of the Sbp9 coating group and the rhEGF group healed faster, and compared with the Control group, the wound area decreased by 39.8% and 23.4% on the 3rd day; the wound area on the 7th day Reduced by 17.6%, 11.4%; on the 14th day, the wound area decreased by 30.9%, 12.7%.
  • the Sbp9 coating group reduced the wound area by 12.5% and 5.6% on the 3rd and 7th days; the wound area decreased by 16.3% on the 14th day. It shows that Sbp9 coating can promote the healing of diabetic chronic wounds, and it is slightly better than rhEGF in shrinking wounds.

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Abstract

提供了一种Sbp9蛋白或其同源蛋白在制备类细胞基质生物材料中的应用，其中Sbp9蛋白或其同源蛋白和Ca 2+共同应用。该类细胞基质生物材料具有防水性能和高粘附性能，能够促进细胞生长，促进伤口愈合，防止氧化损伤，兼具材料的粘附性和生物学活性。

Description

一种具有湿粘附性能的新型类细胞基质生物材料及其应用 技术领域
本发明属于生物技术领域，具体涉及一种具有湿粘附性能的新型类细胞基质生物材料及其应用。
背景技术
对于外部伤口的传统处理多采用纱布、绷带、创可贴等方式，但这些方式都存在较多的使用限制：一是药物需要额外操作敷用，步骤繁琐，且容易与血液组织液等发生黏连，如纱布、绷带之类；二是固定效果差，如创可贴之类；三是现有的伤口处理方式的防水效果都比较差，在不慎接触到液体时，无法避免感染的发生。
现代伤口敷料包括交互式伤口敷料、藻酸钙敷料、银敷料、泡沫敷料、和水凝胶敷料等。现代伤口敷料不仅能避免二次感染，并且对伤口具有一定的促进修复作用。
中国专利201810344869.6中公开了一种新型创面覆盖生物胶，所提供的具有促愈合性能的创面覆盖制品，是使用多肽和透明质酸衍生物制备成的；使用结果表明新型的生物胶无明显细胞毒性，动物实验证明该创面覆盖制品具有良好促愈合效果。
中国专利202010059780.2中公开了一种可自发形成水凝胶的蛋白片段用于制备水凝胶。该专利所提供的蛋白制备的水凝胶具有成胶时间和力学性能可控，胶的生物相容性好等优异的性能，因此在生物医用材料，药物缓释等领域具有广阔的应用前景。
技术问题
现有材料在成膜能力和防水能力上表现欠佳，有进一步优化的空间。
问题的解决方案
为了解决上述问题，本发明提供了Sbp9蛋白和钙离子在制备类细胞基质生物材料的联合应用。
一方面，本发明提供了一种Sbp9蛋白或其同源蛋白在制备类细胞基质生物材料中的应用。
所述的Sbp9蛋白或其同源蛋白和Ca 2+共同应用。
所述的同源蛋白与Sbp9蛋白具有90％以上的同源性。
优选地，所述的同源蛋白与Sbp9蛋白具有95％以上的同源性；进一步优选地，所述的同源蛋白与Sbp9蛋白具有99％以上的同源性。
所述的Sbp9蛋白包括SEQ ID NO.1-4中的任意数量的序列的组合。
所述的任意数量的序列的组合表示可以从SEQ ID NO.1-4选择任意序列进行任意数量的重复进行组合，包括但不限于：
所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.1、SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.1、SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.1、、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.3、SEQ ID NO.4；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.4、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.1；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.1；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4。
优选地，所述的Sbp9蛋白由SEQ ID NO.1-4组成。
进一步优选地，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4。
优选地，所述的Sbp9蛋白为SEQ ID NO.5或与SEQ ID NO.5有80％以上同源性的序列。
优选地，所述的Sbp9蛋白为SEQ ID NO.6或与SEQ ID NO.6有80％以上同源性的序列。
具体地，所述的Ca 2+以可溶性盐的方式应用。
优选地，所述的Ca 2+为氯化钙、硝酸钙、葡萄糖酸钙、磷酸二氢钙、碳酸氢钙、硫酸氢钙、亚硫酸氢钙、次氯酸钙、溴化钙、碘化钙、氯酸钙、高氯酸钙、高锰酸钙中的一种或多种；进一步优选为氯化钙。
另一方面，本发明提供了一种类细胞基质生物材料。
具体地，所述的类细胞基质生物材料中包括：Sbp9蛋白或其同源蛋白和Ca 2+。
所述的同源蛋白与Sbp9蛋白具有90％以上的同源性。
优选地，所述的同源蛋白与Sbp9蛋白具有95％以上的同源性；进一步优选地，所述的同源蛋白与Sbp9蛋白具有99％以上的同源性。
所述的Sbp9蛋白包括SEQ ID NO.1-4中的任意数量的序列的组合。
所述的任意数量的序列的组合表示可以从SEQ ID NO.1-4选择任意序列进行任意数量的重复进行组合，包括但不限于：
所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.1、SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.1、SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.1、、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.2、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.3、SEQ ID NO.4；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.4、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.1；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.1；
或者，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4、SEQ ID NO.4。
优选地，所述的Sbp9蛋白由SEQ ID NO.1-4组成。
进一步优选地，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4。
优选地，所述的Sbp9蛋白为SEQ ID NO.5或与SEQ ID NO.5有80％以上同源性的序列。
优选地，所述的Sbp9蛋白为SEQ ID NO.6或与SEQ ID NO.6有80％以上同源性的序列。
优选地，所述的Sbp9蛋白的浓度≥0.2mg/mL；进一步优选地，所述的Sbp9蛋白的浓度为0.2-0.6mg/mL；更进一步地，所述的Sbp9蛋白的浓度为0.2-0.6mg/mL。
具体地，所述的Ca 2+以可溶性盐的方式应用。
优选地，所述的Ca 2+为氯化钙、硝酸钙、葡萄糖酸钙、磷酸二氢钙、碳酸氢钙、硫酸氢钙、亚硫酸氢钙、次氯酸钙、溴化钙、碘化钙、氯酸钙、高氯酸钙、高锰酸钙中的一种或多种。
进一步优选地，所述的Ca 2+为氯化钙。
优选地，所述的氯化钙的浓度为0.1-10mM；进一步优选地，所述的氯化钙的浓度为5-10mM；更进一步地，所述的氯化钙的浓度为5mM或10mM。
在一些实施例中，所述的氯化钙的浓度为5mM，所述的Sbp9蛋白的浓度为0.6mg/mL。
在一些实施例中，所述的氯化钙的浓度为10mM，所述的Sbp9蛋白的浓度为0.4mg/mL。
再一方面，本发明提供了前述的类细胞基质生物材料在制备医用涂层材料或医疗器械中的应用。
又一方面，本发明提供了一种医用涂层材料。
所述的医用涂层材料中包括前述的类细胞基质生物材料。
又一方面，本发明提供了一种医疗器械。
所述的医疗器械中包括前述的类细胞基质生物材料。
又一方面，本发明提供了前述类细胞基质生物材料的制备方法。
所述的制备方法中包括通过细胞表达Sbp9蛋白。
所述的细胞中包括表达Sbp9蛋白的载体。
所述的载体上包括表达Sbp9蛋白的基因序列。
优选地，所述的基因序列中包括SEQ ID NO.7或SEQ ID NO.8。
发明的有益效果
本发明提供的类细胞基质生物材料具有防水性能和高粘附性能，能够促进细胞生长，促进伤口愈合，防止氧化损伤，兼具材料的粘附性和生物学活性。
本发明的提供的类细胞基质生物材料与现有技术中钙离子引起胶原蛋白引起交联的作用原理不同，其中钙离子与胶原蛋白氨基酸残基中的氮原子相互作用致使蛋白分子间形成致密的网络结构，从而改善其衍生材料的机械性能。与胶原蛋白不同的是，本发明提供的钙离子主要交联氨基酸残基的羧酸基团，通过诱导蛋白粒径增大达到凝聚物沉积形成涂层的效果。
对附图的简要说明
图1：Sbp9的氨基酸序列分析。
图2：Sbp9纯化后的SDS-PAGE图。
图3：Ca 2+诱导Sbp9蛋白发生相分离。
图4：Ca 2+诱导Sbp9蛋白组装的分子动力学分析，其中A图是浊度变化曲线，B图是蛋白粒径变化曲线。
图5：Ca 2+诱导Sbp9蛋白形成涂层的动态形貌图(AFM)。
图6：在不同Ca 2+和Sbp9蛋白浓度下涂层的形成情况。
图7：Sbp9涂层可视化染色结果。
图8：Sbp9涂层表观形貌图(SEM)。
图9：Sbp9涂层的稳定性分析，其中A图是于不同材料界面形成的涂层在水环境中放置2月的形貌图，B图是剩余涂层面积分析，C图是涂层在恶劣环境下稳定性分析。
图10：Sbp9涂层对细胞贴壁的影响。
图11：Sbp9涂层对UVB辐照诱导的皮肤光老化的影响。
图12：Sbp9涂层对糖尿病全层皮肤缺损小鼠伤口愈合的影响。
图13：不同组小鼠伤口愈合率。
本发明的实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1Sbp9的重组表达与纯化
包括以下步骤：
1、Sbp9序列分析
Sbp9为扇贝足丝根部关键组成部分，其全长序列(SEQ ID NO.6)的主要特点是由高度重复的EGFL(表皮生长因子样蛋白)结构域组成。因全长序列由2033个氨基酸组成，在体外重组表达方面存在切实困难，因此本实施例选取截短的由四个重复EGFL结构域组成的序列进行替代分析，四个重复EGFL结构域为E1-E4，对应的氨基酸序列为SEQ ID NO.1-4：
Figure PCTCN2021138721-appb-000001
如图1所示，Sbp9含有136个氨基酸，由四个EGFL结构域串联重复排列，其序列的显著特点是富含半胱氨酸(18％)，每个EGFL结构域均含有六个高度保守的半胱氨酸，这一特性将对其衍生材料的功能性有重要影响，如优异的抗氧化活性。
2、构建重组表达系统
通过构建含有六聚组氨酸标签的表达系统Sbp9-pET32-HisTT，在大肠杆菌中进行重组表达获得Sbp9。
Sbp9-pET32-HisTT构建方法如下：
在编码Sbp9的基因序列SEQ ID NO.7，(对应的氨基酸序列为SEQ ID NO.5)3′末端加入终止密码子TAA后，从5′端和3′端分别设计正向引物和反向引物。正向引物中含有限制性内切酶BamH I酶切位点(GGATCC)，并且在BamH I酶切 位点的上游添加保护碱基CG，从而提高限制性内切酶的酶切效率。同样的，反向引物中含有限制性内切酶Xho I酶切位点(CTCGAG)以及在上游加入表达护碱基CCG。扩增所需引物信息如下所示：
上游引物(SEQ ID NO.9)：
5’-CGGGATCCGCACCGTGCGGCGGTAGTTG-3’；
下游引物(SEQ ID NO.10)：
5’-CCGCTCGAGTTAAACAACGCAAGAGCCTTTAAAC-3’；
将纯化后的PCR产物和pET32-HisTT载体分别利用限制性内切酶BamH I和Xho I进行双酶切，30℃酶切过夜。将纯化后的基因片段和pET32-HisTT载体进行连接，16℃连接4h。将构建好的重组载体转化入大肠杆菌BL21(DE3)感受态细胞中，并将阳性克隆进行测序。测序结果显示插入的目的基因序列与预期一致。
选择阳性克隆接种到含有卡那霉素抗性的5mL LB培养基中进行培养，将试管中培养的菌液作为种子液，接种于1L LB培养基中，37℃，180rpm振荡培养。当菌液浓度达到OD 600＝0.6-0.8时加入0.2mM IPTG进行蛋白诱导，16℃继续培养20h。在收集的菌体中加入1×PBS进行菌体重悬，利用超声波细胞破碎仪将菌体超声破碎，目标蛋白表达在包涵体中。
3、Sbp9蛋白的包涵体纯化
(1)上述菌体沉淀后加入20mL 1×PBS缓冲液重悬，冰水浴下超声破碎细胞，作用2s，间隔4s，功率50％，超声时间30min。12000g，4℃离心25min，弃上清，留沉淀。重复此步骤两次。
(2)用20mL洗涤液(1M尿素，1％甘油，1％TritonX-114，1mM EDTA)重悬沉淀，超声条件同(1)，弃上清，留沉淀。再重复洗涤两次。
(3)沉淀使用15mL(8M尿素，10mM DTT，1mM EDTA，20mM Tris-HCl，pH 8.5)变性缓冲液室温溶解。12000g，25℃离心15min，留取上清。最终将蛋白溶液稀释至1mg/mL。
(4)透析复性：使用7000Da透析袋，在2.5L透析液(20mM Tris-HCl，pH 8.5)中透析24h。将复性后的蛋白溶液进行SDS-PAGE电泳检测，检测结果如图2所示。Sbp9-pET32-HisTT融合蛋白的理论分子量为20kD，电泳结果显示融合蛋 白得到正确表达，且纯度较高(>80％)。纯化并复性后的蛋白可进行后续性质研究。
实施例2Sbp9自组装过程研究
1、Ca 2+介导Sbp9相分离的发生
将纯化后的蛋白溶液稀释至0.6mg/mL，此时放置在玻璃瓶中的样品呈现透明浅黄色，随后向蛋白溶液中引入5mM CaCl 2溶液，发现玻璃瓶中的样品浊度迅速增加，30min后即可观察到如图3所示的相分离现象。其中，吸附到玻璃瓶底部的称之为致密相，上层溶液为稀释相。Sbp9致密相的形成为制备新型涂层材料奠定了基础。
2、Sbp9自组装的动力学分析
利用OD 340浊度变化和动态光散射(DLS)来进一步阐述Sbp9在Ca 2+介导下的自组装行为。将180μL Sbp9溶液(0.6mg/mL)与20μL 50mM CaCl 2溶液(终浓度5mM)混匀后迅速放置96孔板中，在酶标仪下检测蛋白溶液在340nm处的吸光度变化，持续检测30min，每5min读取一次数据。此外，利用DLS检测30min内蛋白溶液在CaCl 2引入后的粒径变化，每2min读取一次数据。
实验结果如图4中的A所示，Ca 2+可引起Sbp9蛋白溶液浊度的骤然增加，30min后浊度基本达到平台期，组装基本完成。图4中的B所示的蛋白分子粒径变化曲线同样也表明随着时间的延长，Ca 2+致使Sbp9蛋白分子的粒径不断增大，在组装到14min时，蛋白粒径达到1600nm，随后的粒径大小监测超出了DLS设备的检测范围。
3、Sbp9自组装过程微观形貌变化
利用原子力显微镜(AFM)监测Sbp9组装前后的动态形貌变化。将0.6mg/mL的蛋白在引入CaCl 2前后各稀释100倍，取2.5μL滴到新剥落的云母片上，静置30min使其干燥，随后进行AFM图像采集。图像的扫描范围是0.5-10μm，扫描速率为0.6-1Hz。
扫描结果如图5所示，纯化后的Sbp9分子均匀分布在缓冲液中，Ca 2+诱导了蛋白分子聚集组装，形成较大的颗粒团体，蛋白分子抱团生长，最后观察到Sbp9沉积在云母片底部形成了由蛋白颗粒层层组装构成的涂层结构。Ca 2+诱导Sbp9蛋白分子组装是形成涂层材料的基础。
4、Sbp9与Ca 2+的结合机制
将纯化后的Sbp9稀释到0.6mg/mL，向溶液中添加5mM CaCl 2混匀后静置30min。分别将加钙前后的溶液进行液氮速冻，随后进行冷冻干燥。将冻干后的样品进行傅里叶变换红外光谱(FTIR)分析，在600-4000cm -1波长范围内进行扫描，步长为10μm。
FITR光谱结果如图6所示，Ca 2+结合后蛋白的波数红移，意味着-NH 2的伸缩振动，氨基氮原子通过提供电子对与Ca 2+配位，涉及到Arg和Lys的侧链基团。1000-1100cm -1是C-O和-OH的变形振动，涉及到Ser和Thr的侧链基团。541cm -1代表Glu的COO-的反对称拉伸振动。光谱中酰胺I带波数的减少代表C＝O的伸缩振动，涉及到Glu，Asp，Gln和Asn的侧链基团。一般情况下，与氨基氮原子和水分子相比，钙离子更倾向于与带负电荷的羧酸基团形成复合物。以上结果清晰地表明蛋白中各种官能团与二价钙离子间配位键的形成。
实施例3新型蛋白涂层材料的制备及稳定性表征
包括以下步骤：
1、Sbp9涂层的制备
涂层的形成要求Sbp9与Ca 2+具有合适的浓度搭配。将实施例2纯化后的Sbp9的浓度稀释到0.2-1.2mg/mL，将36μL各浓度的Sbp9分别与4μL的CaCl 2(终浓度为0.1-10mM)迅速混匀，将混合液转移到直径为1cm的圆形玻璃片上，静置30min后去除上层稀释相溶液，即可形成各个浓度配比的蛋白涂层。对粘附在玻璃片上的涂层进行考马斯亮蓝染色即可观察到各组涂层的结构差异。
可视化染色结果如图7所示，涂层的形成要求Sbp9的浓度高于0.2mg/mL。形成结构致密且均一的涂层需要合适的两者浓度搭配，比如0.4mg/mL的Sbp9需要10mM的CaCl 2。其中并非高浓度的蛋白和Ca 2+共混即可得到理想的涂层材料，比如10mM CaCl 2可导致1mg/mL Sbp9组装过度形成沉淀。从形成良好的涂层结构和样品的成本出发，随后的涂层表征工作均选择0.6mg/mL的Sbp9和5mM的CaCl 2。
2、Sbp9涂层的表面形貌观察
利用扫描电子显微镜观察于玻璃界面形成涂层的表面形貌。将36μL 0.6mg/mL Sbp9与5mM CaCl 2混匀后滴到玻璃片上，静置30min后去除上层稀释相。 将吸附到玻璃上的涂层干燥后进行表面和纵截面观察。
形貌观察结果如图8所示，于玻璃界面形成的Sbp9涂层结构规则均一，可观察到蛋白分子堆积的致密网络结构，与空白的玻璃片有显著区别。其次，涂层的横截面显示出清晰的涂层部分、玻璃部分和界面层，涂层材料已完成玻璃片的粘附。
3、Sbp9涂层在水环境下的稳定性表征
为了探索在不同界面上粘附的新型涂层材料在湿环境下的稳定性和耐受性，将制备的涂层暴露在动荡恶劣的环境下检测其完整性。分别在玻璃、塑料和铝片上制备大小均一的涂层，将涂层置于装有无菌水的烧杯中，放在100rpm的磁力搅拌器上模拟动荡的水环境，分别在10天、30天和60天时取样进行可视化染化，并利用Image J软件计算涂层的孔隙率以表明剩余的涂层面积。此外，将涂层置于功率为180W的超声波清洗机中1h以模拟恶劣的水环境，以表明涂层材料的耐受性。
涂层在湿环境下可视化染色结果如图9中的A所示，单一的Sbp9组份对介质不能完成粘附，Ca 2+介导Sbp9组装形成的涂层对玻璃、塑料和铝片均实现良好的界面粘附，其中在各个界面上形成的涂层在水环境中振荡60天后基本无脱落。图9中的B所示的定量结果显示涂层在60天后其完整性均保持在80％以上，且图9中的C显示涂层在恶劣的水环境中振荡1h后基本无脱落，均表明涂层具备优异的抗水稳定性和恶劣环境耐受性，有利于后续的湿环境应用。
实施例4 Sbp9涂层对细胞的贴附
为了Sbp9涂层促进细胞贴附的效果，进行细胞贴壁率实验。细胞分为Control，Poly-lysine涂层和Sbp9涂层3个组，采用非TC处理24孔细胞培养板进行细胞培养，各组孔板分别做不处理，铺300μL浓度为100μg/mL的Poly-lysine和铺300μL浓度为25μg/mL的Sbp9涂层。细胞以10 5/mL的密度接种，置于CO 2培养箱中培养1h，吸出未贴壁细胞，PBS清洗2遍并收集，血球计数板计数，统计未贴壁细胞数目，贴壁率＝(接种细胞总数-未贴壁数)/接种细胞总数。
图10结果表明Sbp9涂层促进L929和HUVEC细胞贴壁。与Poly-lysine组相比，Sbp9涂层组贴壁率显著提高。说明Sbp9涂层促进细胞的贴附，并且使用Sbp9蛋白的浓度是Poly-lysine蛋白浓度的1/4。
实施例5 Sbp9涂层拮抗氧化应激诱导的皮肤光老化
采用实施例2纯化后的Sbp9进行涂层拮抗UVB辐照造成的皮肤光老化实验：将小鼠分为Control组，UVB组，UVB+Sbp9涂层组(UVB+Sbp9coating)，UVB+Vitamin C组。辐照前10min进行给药，UVB+Sbp9涂层组进行Sbp9涂层喷涂(蛋白为1mg/mL)；(2)阳性对照组涂抹Vitamin C(100mg/kg)；(3)Control组和UVB组涂抹生理盐水。给药间隔每2天涂抹一次。UVB灯进行辐照，辐照剂量：共照射5天，前3天每日辐照剂量120mj·cm -2，后2天每日辐照剂量100mj·cm -2，光源距离辐照区域15-20cm。
图11结果表明UVB辐照造成小鼠背部皮肤光老化损伤，表现为皮肤干燥、起皮、红斑。应用Sbp9涂层涂抹预处理能减缓起皮、红斑、干燥的发生，从而对皮肤起到保护作用。
实施例6 Sbp9涂层促进糖尿病全层皮肤缺损小鼠伤口愈合
采用实施例2纯化后的Sbp9进行伤口愈合实验：将小鼠分为Control组，Sbp9涂层组(Sbp9coating)，rhEGF组，在小鼠背部用环钻制造全层皮肤损伤创面，直径6mm，实验组创面每天给予蛋白浓度为1mg/mL的Sbp9涂层喷雾1次，阳性对照组涂抹rhEGF凝胶，阴性对照组涂抹生理盐水，每天对创面拍照，利用Image J软件分析创面的面积。
图12-13结果表明在3、7、14天时，Sbp9涂层组和rhEGF组伤口愈合更快，与Control组相比，第3天创面面积减小39.8％，23.4％；第7天创面面积减小17.6％，11.4％；第14天创面面积减小30.9％,12.7％。Sbp9涂层组与rhEGF组相比，在第3、7天，创面面积减小12.5％，5.6％，；在第14天创面面积减小16.3％。表明Sbp9涂层能够促进糖尿病慢性伤口的愈合，在缩小创面效果上略优于rhEGF。

Claims (20)

  1. 一种Sbp9蛋白或其同源蛋白在制备类细胞基质生物材料中的应用，其特征在于，所述的Sbp9蛋白或其同源蛋白和Ca 2+共同应用。
  2. 根据权利要求1所述的应用，其特征在于，所述的Sbp9蛋白包括SEQ ID NO.1-4中的任意数量的序列的组合。
  3. 根据权利要求1所述的应用，其特征在于，所述的Sbp9蛋白为SEQ ID NO.5或与SEQ ID NO.5有80％以上同源性的序列或SEQ ID NO.6或与SEQ ID NO.6有80％以上同源性的序列。
  4. 一种类细胞基质生物材料，其特征在于，所述的类细胞基质生物材料中包括：Sbp9蛋白或其同源蛋白和Ca 2+。
  5. 根据权利要求4所述的类细胞基质生物材料，其特征在于，所述的Sbp9蛋白包括SEQ ID NO.1-4中的任意数量的序列的组合。
  6. 根据权利要求5所述的类细胞基质生物材料，其特征在于，所述的Sbp9蛋白中按顺序依次包括：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4。
  7. 根据权利要求6所述的类细胞基质生物材料，其特征在于，所述的Sbp9蛋白的浓度≥0.2mg/mL。
  8. 根据权利要求7所述的类细胞基质生物材料，其特征在于，所述的Sbp9蛋白的浓度为0.2-1.2mg/mL。
  9. 根据权利要求8所述的类细胞基质生物材料，其特征在于，所述的Sbp9蛋白的浓度为0.2-0.6mg/mL。
  10. 根据权利要求4所述的类细胞基质生物材料，其特征在于，所述的Ca 2+为氯化钙、硝酸钙、葡萄糖酸钙、磷酸二氢钙、碳酸氢钙、硫酸氢钙、亚硫酸氢钙、次氯酸钙、溴化钙、碘化钙、氯酸钙、高氯酸钙、高锰酸钙中的一种或多种。
  11. 根据权利要求10所述的类细胞基质生物材料，其特征在于，所述的Ca 2+为氯化钙。
  12. 根据权利要求11所述的类细胞基质生物材料，其特征在于，所述的氯化钙的浓度为0.1-10mM。
  13. 根据权利要求12所述的类细胞基质生物材料，其特征在于，所述的氯化钙的浓度为5-10mM。
  14. 根据权利要求13所述的类细胞基质生物材料，其特征在于，所述的氯化钙 的浓度为5mM或10mM。
  15. 根据权利要求14所述的类细胞基质生物材料，其特征在于，所述的氯化钙的浓度为5mM，所述的Sbp9蛋白的浓度为0.6mg/mL。
  16. 根据权利要求14所述的类细胞基质生物材料，其特征在于，所述的氯化钙的浓度为10mM，所述的Sbp9蛋白的浓度为0.4mg/mL。
  17. 权利要求4-16任一项所述的类细胞基质生物材料在制备医用涂层材料或医疗器械中的应用。
  18. 一种医用涂层材料，其特征在于，所述的医用涂层材料中包括权利要求4-16任一项所述的类细胞基质生物材料。
  19. 一种医疗器械，其特征在于，所述的医疗器械中包括权利要求4-16任一项所述的类细胞基质生物材料。
  20. 一种类细胞基质生物材料的制备方法，其特征在于，所述的制备方法中包括通过细胞表达Sbp9蛋白，再将Sbp9蛋白与Ca 2+结合。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105647937A (zh) * 2016-02-29 2016-06-08 中国海洋大学 一种扇贝足丝蛋白体外表达方法及应用
CN107474128A (zh) * 2017-09-15 2017-12-15 中国海洋大学 一种仿生止血生物胶
CN108379650A (zh) * 2018-04-17 2018-08-10 中国海洋大学 一种新型促创面愈合生物胶及其应用
CN111253481A (zh) * 2020-01-19 2020-06-09 中国海洋大学 一种仿生智能水凝胶的制备及应用
CN114213521A (zh) * 2021-12-16 2022-03-22 中国海洋大学 一种具有湿粘附性能的新型类细胞基质生物材料及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105647937A (zh) * 2016-02-29 2016-06-08 中国海洋大学 一种扇贝足丝蛋白体外表达方法及应用
CN107474128A (zh) * 2017-09-15 2017-12-15 中国海洋大学 一种仿生止血生物胶
CN108379650A (zh) * 2018-04-17 2018-08-10 中国海洋大学 一种新型促创面愈合生物胶及其应用
CN111253481A (zh) * 2020-01-19 2020-06-09 中国海洋大学 一种仿生智能水凝胶的制备及应用
CN114213521A (zh) * 2021-12-16 2022-03-22 中国海洋大学 一种具有湿粘附性能的新型类细胞基质生物材料及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Study on the Composition and Adhesion Mechanism of Scallop Foot Filament Protein", BAIDU BAIKE, 26 August 2021 (2021-08-26), XP009547205 *
DATABASE Nucleotide 5 December 2018 (2018-12-05), ANONYMOUS : "Azumapecten farreri scallop byssal protein 9 (Sbp9) mRNA, complete cds ", XP093072966, retrieved from NCBI Database accession no. MK050192.1 *
MIAO, YAN ET AL.: "Marine Bioadhesin Study Based on Omics Technology", THE 6TH PAN-BOHAI SEA SOCIETY OF BIOCHEMISTRY AND MOLECULAR BIOLOGY ACADEMIC EXCHANGE MEETINGS PROCEEDINGS SHANDONG SOCIETY OF BIOCHEMISTRY AND MOLECULAR BIOLOGY CONFERENCE PROCEEDINGS, 14 August 2016 (2016-08-14) *
XU PINGPING, DAI XIAOTING, WANG DANDAN, MIAO YAN, ZHANG XIAOKANG, WANG SHUOSHUO, TENG LUYAO, DONG BO, BAO ZHENMIN, WANG SHI, LYU Q: "The discovered chimeric protein plays the cohesive role to maintain scallop byssal root structural integrity", SCIENTIFIC REPORTS, vol. 8, no. 1, XP093072960, DOI: 10.1038/s41598-018-35265-y *

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