WO2022262509A1 - Leptin受体亲和肽及其应用 - Google Patents

Leptin受体亲和肽及其应用 Download PDF

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WO2022262509A1
WO2022262509A1 PCT/CN2022/093475 CN2022093475W WO2022262509A1 WO 2022262509 A1 WO2022262509 A1 WO 2022262509A1 CN 2022093475 W CN2022093475 W CN 2022093475W WO 2022262509 A1 WO2022262509 A1 WO 2022262509A1
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peptide
collagen
leptin receptor
affinity
affinity peptide
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PCT/CN2022/093475
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French (fr)
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戴建武
庄燕
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江苏独步生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • 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
    • 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
    • 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
    • A61L27/24—Collagen
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents

Definitions

  • the application belongs to the technical field of biomedicine, and specifically relates to a Leptin receptor affinity peptide, in particular to a Leptin receptor affinity peptide that can specifically bind to mesenchymal stem cells (MSC) and a screening method thereof, and is applied to tissue Engineering fixes.
  • a Leptin receptor affinity peptide in particular to a Leptin receptor affinity peptide that can specifically bind to mesenchymal stem cells (MSC) and a screening method thereof, and is applied to tissue Engineering fixes.
  • MSCs Mesenchymal stem cells
  • mesoderm mainly derived from mesoderm
  • MSCs are a type of adult stem cells with high self-renewal ability and differentiation potential, and are important members of the stem cell family.
  • MSCs were first discovered in the bone marrow, and later studies found that MSCs mainly exist in the connective tissue of the whole body and the interstitium of organs.
  • MSCs can be isolated and prepared from bone marrow, placenta, umbilical cord, fat and other tissues. Based on the multi-differentiation potential of MSC, it can be directed to differentiate into bone, cartilage, fat, cardiac muscle and other tissue cells in a specific induction environment in vivo and in vitro.
  • MSCs have the characteristics of low immunogenicity, so that they can avoid immune rejection in transplantation applications. These characteristics of MSC provide new treatment ideas and solutions for clinical treatment, and can be used as the most suitable seed cells in tissue engineering repair, and can be applied to the treatment of bone and muscle degenerative diseases, cerebrovascular diseases, autoimmune diseases, etc.
  • phage display technology provides a mature solution.
  • a large number of studies have confirmed that phage display technology plays an important role in the study of the properties, mutual recognition and interaction of polypeptides or proteins.
  • the foreign DNA fragment encoding the polypeptide is fused with the gene encoding the surface protein of the phage, it is presented on the surface of the phage in the form of a fusion protein, and the displayed polypeptide or protein can maintain the relative spatial structure and biological activity and be displayed on the surface of the phage .
  • Phage display random peptide library technology is developed on the basis of phage display technology.
  • a phage display library that can display a large number of different foreign peptides is formed.
  • the advantage of the phage display peptide library is that it provides a direct physical link between the protein and its genetic information, so it can effectively repeatedly screen the clones with the required functions, and then amplify them, and obtain these specific binding
  • the gene sequence corresponding to the exogenous peptide, and the biological function of these peptides can be studied in the later stage.
  • specific phage clones are continuously enriched due to their specific affinity for their ligands, so that relatively rare clones that can bind to ligands can be quickly and efficiently selected from a large library. filter out.
  • the application provides a Leptin receptor affinity peptide capable of specifically binding to mesenchymal stem cells (MSCs) and an application thereof.
  • MSCs mesenchymal stem cells
  • the present application provides a Leptin receptor affinity peptide, the sequence of the Leptin receptor affinity peptide is shown in SEQ ID No.1.
  • the Leptin receptor affinity peptide has a sequence more than 95% identical to the full-length sequence of SEQ ID No.1.
  • the Leptin receptor affinity peptide has a high affinity to the Leptin receptor and can specifically bind to mesenchymal stem cells.
  • the present application provides a method for screening Leptin receptor affinity peptides.
  • the method uses phage display technology to perform affinity screening with Leptin receptor recombinant proteins, and finally screens in a random 7-peptide library to have Leptin receptor has a high affinity polypeptide.
  • the present application provides an affinity peptide with collagen-binding ability, which includes the Leptin receptor affinity peptide and a short peptide with collagen-specific binding ability; the short peptide with collagen-specific binding ability passes A linker is connected to the N-terminus of the Leptin receptor affinity peptide, wherein the sequence of the Leptin receptor affinity peptide is shown in SEQ ID No.1, and the short peptide with collagen-specific binding ability is CBD , its sequence is shown in SEQ ID No.3, and the sequence of the linker is shown in SEQ ID No.4 or SEQ ID No.5.
  • the present application provides a functional material, which includes a collagen material and the affinity peptide with collagen binding ability, wherein the affinity peptide with collagen binding ability is bound to the surface of the collagen material and / or inside.
  • affinity peptide with collagen-binding ability specifically binds to the collagen material through a short peptide with collagen-specific binding ability.
  • the present application provides the application of the functional material described in the fourth aspect in the preparation of products with tissue damage repair function.
  • the present application provides a product with the function of repairing tissue damage, which includes the functional material described in the fourth aspect.
  • the product at least has the function of continuously adsorbing mesenchymal stem cells.
  • the Leptin receptor affinity peptide provided by this application has a high affinity with the Leptin receptor and can specifically bind to MSC.
  • the Leptin receptor affinity peptide can be connected to biological materials, and the The affinity peptides of short peptides can achieve specific binding to collagen scaffolds, thereby improving the recruitment of biomaterials to MSCs expressing Leptin receptors on the surface, and obtaining better tissue damage repair effects. Research provides experimental basis.
  • Fig. 1 is a schematic diagram of the binding and recovery of Leptin receptor recombinant protein by HY7 phage monoclonal and M13KE negative control phage in Example 1 of the present application.
  • Fig. 2A and Fig. 2B are schematic diagrams showing the binding recovery of HY7 phage monoclonal and M13KE negative control phage to human MSC and HS-5 cells in Example 2 of the present application.
  • Fig. 3 is a schematic diagram of the combination of FITC-HY7 and FITC-M7 with human MSCs taken by a confocal fluorescence microscope in Example 2 of the present application without adding the polypeptide group as a control.
  • Fig. 4 is a schematic diagram of flow cytometry detection of FITC-HY7 and FITC-M7 binding to rat MSCs in Example 2 of the present application.
  • Fig. 5 is a schematic diagram of the combination of CBD-HY7, sCBD-HY7, and CBD-sHY7 with collagen sponge taken by laser confocal fluorescence microscope in Example 3 of the present application.
  • FIG. 6A to FIG. 6F are respectively diagrams showing the proportion of MSC in the cells digested from the collagen sponge detected by flow cytometry in Example 3 of the present application.
  • Fig. 7 is a diagram of Masson staining results of lung tissue and collagen sponge area in Example 3 of the present application.
  • This application mainly provides an amino acid sequence, screening method and application of a Leptin receptor affinity peptide that can specifically bind to mesenchymal stem cells (MSC), and relates to the field of cell and polypeptide biotechnology.
  • This application uses phage display technology to perform affinity screening with the recombinant protein of Leptin receptor, and finally screens out a polypeptide with high affinity with Leptin receptor in a random 7-peptide library, and verifies that the Leptin receptor is Body affinity peptides can specifically bind MSCs.
  • MSC mesenchymal stem cells
  • Leptin receptor affinity peptide which can be named "HY7 peptide"
  • HY7 peptide which has the amino acid sequence shown in SEQ ID No.1, specifically SEQ ID No.1: HGGVRLY .
  • the Leptin receptor affinity peptide has a sequence more than 95% identical to the full-length sequence of SEQ ID No.1.
  • DNA sequence of the Leptin receptor affinity peptide is shown in SEQ ID NO.2: ATACAAACGAACCCCACCATG.
  • the Leptin receptor affinity peptide has a high affinity to the Leptin receptor and can specifically bind to mesenchymal stem cells (MSC).
  • the Leptin receptor affinity peptide is obtained through affinity screening with the Leptin receptor recombinant protein by using phage display technology, and then screening in a random 7-peptide library.
  • Another aspect of the embodiments of the present application also provides a method for screening Leptin receptor affinity peptides, the method utilizes a random seven-peptide library displayed on the surface of a phage to screen out a seven-peptide amino acid sequence that can specifically bind to the Leptin receptor, And identify its specific combination with MSC.
  • the screening method is to use phage surface display technology to perform affinity screening with the recombinant protein of the Leptin receptor, and finally screen a polypeptide with a high affinity with the Leptin receptor in a random 7-peptide library, And through a variety of detection methods to verify that the Leptin receptor affinity peptide can specifically bind to MSC.
  • the screening method utilizes a random 7-peptide library displayed on the surface of a phage, targeting the recombinant protein of Leptin receptor, and performing five rounds of biological screening.
  • 15 phage monoclonals were randomly selected, amplified, and then quickly purified by the sequencing module to generate sufficiently pure templates to be sent to biological companies for gene sequencing, and the corresponding phage monoclonals randomly selected were analyzed Display the peptide sequence, and analyze the proportion growth trend of the repeated peptide sequence, and select several phage monoclonals with a high proportion to detect their binding ability to the Leptin receptor recombinant protein.
  • Another aspect of the embodiment of the present application also provides an affinity peptide with collagen-binding ability, which includes the Leptin receptor affinity peptide and a short peptide with collagen-specific binding ability; the collagen-specific binding ability
  • the short peptide is connected to the N-terminus of the Leptin receptor affinity peptide through a linker.
  • the Leptin receptor affinity peptide has the amino acid sequence shown in SEQ ID No.1, specifically SEQ ID No.1: HGGVRLY.
  • the short peptide with collagen-specific binding ability is CBD, and its sequence is SEQ ID No. 3: TKKTLRT.
  • the present application introduced a short peptide CBD with collagen-specific binding ability, synthesized at the N-terminus of the Leptin receptor affinity peptide, and connected it with the Leptin receptor affinity peptide using a linker.
  • the linker when the linker is not fluorescent, the sequence uses SEQ ID No.4: GGGGS; when the linker is fluorescent, the sequence uses SEQ ID No.5: GGG-K-FITC.
  • Another aspect of the embodiment of the present application also provides a functional material, which includes a collagen material and the affinity peptide with collagen binding ability; the affinity peptide with collagen binding ability is bound to the surface of the collagen material and/or inside.
  • affinity peptide with collagen-binding ability specifically binds to the collagen material through a short peptide with collagen-specific binding ability.
  • Another aspect of the present application is the application and modification method of HY7 peptide modified collagen sponge scaffold implanted into damaged parts of the body.
  • the acquisition of the Leptin receptor affinity peptide makes it possible to specifically adsorb and recruit MSCs, but how to recruit MSCs to biological materials through the HY7 peptide is still a problem.
  • the simple adsorption method will cause the diffusion of peptides due to the infiltration and flow of body fluids, resulting in a decrease in the effective concentration of local affinity peptides in biomaterials, affecting the effect of adsorption and recruitment of MSCs.
  • the ratio of the amount of the collagen-binding affinity peptide to the collagen material is 1 ⁇ 10 -4 -2 ⁇ 10 -3 ⁇ mol:60mm 3 .
  • collagen material includes but not limited to collagen sponge scaffold.
  • the present application can link Leptin receptor affinity peptides to biological materials, thereby improving the recruitment of biological materials to MSCs, and can play an important role in the field of tissue engineering repair.
  • Another aspect of the present application is that the use of a Leptin receptor affinity peptide obtained through the screening of this application to connect to biomaterials can increase the recruitment of biomaterials to MSCs expressing Leptin receptors on the surface. And provide experimental basis for targeted therapy research.
  • Another aspect of the embodiments of the present application also provides the application of the aforementioned functional materials in the preparation of products with the function of repairing tissue damage (such as repairing lung damage).
  • tissue damage repair function which includes the functional material.
  • the product at least has the function of continuously adsorbing mesenchymal stem cells.
  • the Leptin receptor affinity peptide provided by the application has high affinity with the Leptin receptor, and can specifically bind to MSC, and the Leptin receptor affinity peptide can be connected to biological materials, and the Affinity peptides with short peptides with collagen-specific binding ability can achieve specific binding to collagen scaffolds, thereby improving the recruitment of biomaterials to MSCs expressing Leptin receptors on the surface, and obtaining better tissue damage repair effects.
  • reagents and raw materials used in the following examples are all commercially available, and the test methods for which specific conditions are not indicated are usually in accordance with conventional conditions or in accordance with the conditions suggested by each manufacturer. And, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this application all use conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, and recombinant DNA techniques in the technical field and conventional techniques in related fields.
  • This example uses phage display technology to screen for affinity polypeptides that specifically bind to the Leptin receptor
  • the phage random heptapeptide display library was purchased from NEB Company, 100 ⁇ L, with a titer of 1 ⁇ 10 13 pfu/mL. Stored in TBS buffer containing 50% glycerol (50mM Tris-HCl, 150mM NaCl [pH 7.5]), the storage capacity is 1.28 ⁇ 10 9 transformants. Escherichia coil ER2738 is the host strain of this peptide library.
  • the original library was diluted with TBST buffer, and 100 ⁇ L of the diluted library solution was added to the microwells of the microtiter plate pre-coated with Leptin receptor recombinant protein, and the amount of phage added was about 2 ⁇ 10 12 .
  • the collected phage supernatant was serially diluted 10 times with LB medium (suggested dilution range: unamplified screening eluate: 10 1 -10 4 , amplified phage culture supernatant: 10 8 -10 11 ).
  • LB medium suggested dilution range: unamplified screening eluate: 10 1 -10 4 , amplified phage culture supernatant: 10 8 -10 11 .
  • the output/input ratio of each round can be calculated to reflect the enrichment degree of specific phage (recovery rate Recovery). After five rounds of screening, it was found that the high-affinity phages specifically binding to the Leptin receptor recombinant protein were effectively enriched (see Table 1).
  • phage monoclonals were randomly selected from the titer plate, amplified, and then quickly purified by the sequencing module.
  • a phage monoclonal blue plaque was randomly selected and added to the above 1mL culture tube, and a total of 15 monoclonals were selected for amplification in each round.
  • Rapid purification of sequencing templates After phage monoclonal amplification according to the above method, take the phage-containing supernatant collected above (take 500 ⁇ L), add 200 ⁇ L PEG/NaCl, mix by inverting, place at room temperature for 10-20 minutes, and then store at 4°C , centrifuge at 14000rpm for 10 minutes, discard the supernatant, and perform centrifugation again, and thoroughly suck up the residual supernatant.
  • the precipitate was resuspended in 30 ⁇ L double-distilled water, which was the sequencing template, and 5 ⁇ L was taken and detected by 1% agarose gel electrophoresis, and the DNA that passed the test was sent to the company for sequencing with -96gIII sequencing primers.
  • the HY7 phage monoclonal obtained in this example was amplified according to the phage amplification method, and the control M13KE negative control phage (purchased from NEB Company, 40 ⁇ L, 1 ⁇ 10 13 pfu/mL) was simultaneously amplified.
  • the amplified HY7 phage monoclonal and the M13KE negative control phage were both titrated according to the aforementioned titration method to obtain the amplified phage concentration.
  • the target protein Leptin receptor recombinant protein
  • coating buffer 100 ⁇ g/mL
  • 100 ⁇ L was added to a well of the microtiter plate to ensure that the well plate was wet, and incubated overnight at 4°C in a humid box.
  • HY7 phage monoclonal and M13KE negative control phage were added to the target protein well for binding and elution, and the eluate of HY7 phage bound to the target protein and the eluate of M13KE negative control phage were respectively obtained. Titrate according to the phage titration method, and compare the binding of the two to the target molecule.
  • the HY7 phage monoclonal has more binding capacity to the Leptin receptor recombinant protein (as shown in Figure 1) , where the ordinate shows the recovery rate of binding of HY7 phage monoclonal and M13KE negative control phage to the Leptin receptor recombinant protein, that is, the ratio of the amount of phage recovered after binding to the amount of input phage.
  • Example 2 HY7 can specifically bind to human mesenchymal stem cells
  • HY7 phage monoclonal and M13KE negative control phage were added to human mesenchymal stem cell well plates pre-cultured to about 80% confluence, and an irrelevant human bone marrow stromal cell line HS-5 was set as a control. Bind and elute according to the phage screening steps to obtain the HY7 phage eluate bound to the target protein and the M13KE negative control phage eluate respectively, and titrate the two according to the phage titration method to compare the binding of the two to the target molecule.
  • FITC-HY7 labeled with fluorescein isothiocyanate (FITC)
  • FITC-M7 fluorescein isothiocyanate
  • human MSC and HS-5 cells were digested and planted in 6-well plate at a density of 5 ⁇ 10 5 cells/well, cultivated in a 37°C incubator for 24 hours, discarded the culture medium and washed with PBS After 3 times, the cells were pre-labeled with DiI dye for 15 minutes, washed 3 times with PBS, replaced with serum-free medium, and 5 ⁇ mol/L of the FITC-HY7 peptide and FITC-M7 peptide synthesized above were added to the cell well plate, and at the same time Use cells without peptide as blank control, incubate at 37°C for 30 minutes, wash the well plate 3 times with PBS
  • HY7 peptide In order to evaluate the species specificity of HY7 peptide and provide a basis for subsequent animal experiments, we specifically verified the binding of HY7 to rat mesenchymal stem cells. Rat bone marrow cells were obtained according to conventional methods, cultured and identified, and rat mesenchymal stem cells were obtained. The combination of FITC-HY7 peptide and FITC-M7 peptide with rat mesenchymal stem cells MSC was detected according to the aforementioned flow cytometry detection method. The results are shown in Figure 4, the binding ability of HY7 to rat MSC was significantly stronger than that of M7.
  • Example 3 CBD-affinity peptides can be bound to collagen materials for lung injury repair
  • CBD-affinity peptides can be bound to collagen materials
  • FITC-labeled CBD-HY7 was synthesized at the same time, and for the convenience of comparison, FITC-labeled CBD misconstructed control (sCBD-HY7) and hy7 misconstructed were also synthesized. Constructive control (CBD-sHY7).
  • the rat weighing about 150g was anesthetized, intubated and ventilated under the positive pressure of the ventilator, a partial resection of the right middle lobe was performed, and the lung tissue with a size of about 0.5cm*0.5cm*0.3cm was removed, and the CBD that had been evenly dripped in advance - Affinity peptides or control peptides, and collagen sponges incubated at 37°C for 2 hours were implanted into rat lung defect sites, respectively. After 12 hours, 24 hours or 48 hours, the rats were sacrificed, and the implanted collagen sponge was completely separated and taken out, and collagenase was used to dissolve the collagen sponge, and the cells adsorbed on the collagen sponge in vivo were separated and collected.
  • the present application obtains the Leptin receptor-specific binding polypeptide through phage display technology, and confirms that the polypeptide can be targeted to human mesenchymal stem cells with high affinity. During the process, it was found that the polypeptide has no obvious species specificity, so it is speculated that the affinity polypeptide has a wide range of applications.
  • the affinity peptide can be targeted to bind to the collagen material by using CBD, so as to achieve specific adsorption and recruitment of mesenchymal stem cells, so that the collagen material can be used as a scaffold When implanted in the body for lung injury repair, it can continuously adsorb mesenchymal stem cells, so as to obtain better repair effect.

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Abstract

本申请公开了一种Leptin受体亲和肽及其应用。所述Leptin受体亲和肽的序列如SEQ ID No.1所示。本申请还公开了一种具有胶原结合能力的亲和肽，其包括所述Leptin受体亲和肽和具有胶原特异结合能力的短肽，该具有胶原特异结合能力的短肽结合在所述Leptin受体亲和肽的N端。本申请的亲和肽与Leptin受体具有高亲和性，且能够与MSC特异性结合，将该亲和肽连接到生物材料上，连接具有胶原特异结合能力的短肽的亲和肽可实现与胶原支架的特异结合，从而提高生物材料对于表面表达Leptin受体的MSC的募集，获得更好的组织损伤修复效果，为研究MSC在组织工程修复和靶向治疗研究方面提供实验依据。

Description

Leptin受体亲和肽及其应用 技术领域
本申请属于生物医药技术领域，具体涉及一种Leptin受体亲和肽，尤其涉及一种可特异性结合间充质干细胞(MSC)的Leptin受体亲和肽及其筛选方法，并应用于组织工程修复。
背景技术
间充质干细胞(mesenchymal stem cells,MSC)主要来源于中胚层，是一类具有高度自我更新能力和分化潜能的成体干细胞，是干细胞家族中的重要成员。MSC最初在骨髓中被发现，之后的研究发现MSC主要存在于全身结缔组织和器官间质中，目前已经可以从骨髓、胎盘、脐带、脂肪等组织中分离和制备间充质干细胞。基于MSC的多分化潜能，其在体内外特定的诱导环境中，可以定向分化成骨、软骨、脂肪、心肌等多种组织细胞。此外，MSC具有低免疫原性的特点，使其可以在移植应用中避免免疫排斥。MSC的这些特点给临床治疗提供了新的治疗思路和方案，可以作为组织工程修复中最适合的种子细胞，应用于骨与肌肉退行性疾病、脑血管疾病、自身免疫性疾病等的治疗。
在组织工程的发展过程中，利用支架材料吸附MSC应用于组织损伤修复再生已经被广泛认同。但由于体液的流动性，外源简单滴加到材料上的MSC和体内循环MSC均无法在支架材料上长时间停留，因此也无法发挥其分化和修复的作用。因此，如果可以对支架材料进行表面修饰，连接上与MSC具有强结合力的蛋白或多肽，从而提高MSC在材料表面的粘附率，必将更好地发挥支架材料上MSC的再生能力，提高支架材料的修复效果。在新近的研究中，研究人员发现，MSC中具有良好增殖分化能力的一小部分细胞表面表达Leptin受体，如果能找到能与Leptin受体具有强结合力的亲和肽，那就可以将之修饰到支架材料表面，使其作为桥梁作用特异性结合这一部分具有良好增殖分化能力的MSC。
如何去寻找Leptin受体亲和肽，噬菌体展示技术提供了成熟的解决方案。大量的研究已经证实，噬菌体展示技术在研究多肽或蛋白质的性质、相互识别和相互作用方面发挥了重要的作用。通过编码多肽的外源DNA片段与噬菌体表面蛋白的编码基因融合后，以融合蛋白的形式呈现在噬菌体的表面，被展示的 多肽或蛋白可保持相对的空间结构和生物活性，展示在噬菌体的表面。噬菌体展示随机肽库技术由噬菌体展示技术基础上发展而来，当导入了大量具有不同外源基因的噬菌体后，就构成一个可以展示大量不同外源肽的噬菌体展示库。噬菌体展示肽库的优势在于它将蛋白质与其遗传信息之间提供了直接的物理联系，因此可以有效的对所需功能的克隆进行反复筛选，并随之对其进行扩增，并获得这些特异结合的外源肽所对应的基因序列，并在后期可以对这些多肽进行生物学功能的研究。在文库筛选的过程中，特定的噬菌体克隆由于对其配体的特异亲和性而不断的得到富集，从而使相对稀少的可以结合配体的克隆能够快速、有效地从一个大文库中被筛选出来。
因此，如何获得一种可与Leptin受体具有高度亲和性，并与MSC特异性结合的多肽，以及将其应用于组织工程修复，已然成为业界研究人员长期以来一直努力的方向。
发明内容
本申请提供了一种可特异结合间充质干细胞(MSC)的Leptin受体亲和肽及其应用。
第一方面，本申请提供了一种Leptin受体亲和肽，所述Leptin受体亲和肽的序列如SEQ ID No.1所示。
进一步地，所述Leptin受体亲和肽具有与SEQ ID No.1的全长序列95％以上相同的序列。
进一步地，所述Leptin受体亲和肽与Leptin受体具有高度亲和性，且能够与间充质干细胞特异性结合。
第二方面，本申请提供了一种Leptin受体亲和肽的筛选方法，所述方法利用噬菌体展示技术通过与Leptin受体重组蛋白进行亲和筛选，最终在随机7肽库中筛选到具有与Leptin受体具有高度亲和性的多肽。
第三方面，本申请提供了一种具有胶原结合能力的亲和肽，其包括所述Leptin受体亲和肽和具有胶原特异结合能力的短肽；所述具有胶原特异结合能力的短肽通过一个linker连接结合在所述Leptin受体亲和肽的N端，其中，所述Leptin受体亲和肽的序列如SEQ ID No.1所示，所述具有胶原特异结合能力的短肽为CBD，其序列如SEQ ID No.3所示，且所述linker的序列如SEQ ID No.4 或SEQ ID No.5所示。
第四方面，本申请提供了一种功能性材料，其包括胶原材料和所述具有胶原结合能力的亲和肽，其中，所述具有胶原结合能力的亲和肽结合于所述胶原材料表面和/或内部。
进一步地，所述具有胶原结合能力的亲和肽通过具有胶原特异结合能力的短肽与所述胶原材料特异性结合。
第五方面，本申请提供了第四方面所述的功能性材料在制备具有组织损伤修复功能的产品中的应用。
第六方面，本申请提供了一种具有组织损伤修复功能的产品，其包括第四方面所述的功能性材料。
进一步地，所述产品至少具有能够持续性吸附间充质干细胞的功能。
与现有技术相比，本申请具有如下有益效果：
本申请提供的Leptin受体亲和肽与Leptin受体具有高亲和性，且能够与MSC特异性结合，可以将该Leptin受体亲和肽连接到生物材料上，连接具有胶原特异结合能力的短肽的亲和多肽可实现与胶原支架的特异结合，从而提高生物材料对于表面表达Leptin受体的MSC的募集，获得更良好的组织损伤修复效果，为研究MSC在组织工程修复和靶向治疗研究方面提供实验依据。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请中记载的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本申请实施例1中HY7噬菌体单克隆与M13KE阴性对照噬菌体对Leptin受体重组蛋白的结合回收情况示意图。
图2A和图2B是本申请实施例2中HY7噬菌体单克隆与M13KE阴性对照噬菌体分别对人MSC和HS-5细胞的结合回收情况示意图。
图3是本申请实施例2中以未加多肽组作为对照，共聚焦荧光显微镜拍摄FITC-HY7与FITC-M7与人MSC结合情况示意图。
图4是本申请实施例2中流式细胞术检测FITC-HY7与FITC-M7与大鼠 MSC结合情况示意图。
图5是本申请实施例3中激光共聚焦荧光显微镜拍摄CBD-HY7、sCBD-HY7、CBD-sHY7与胶原海绵结合情况示意图。
图6A至图6F分别是本申请实施例3中流式细胞术检测从胶原海绵上消化下来的细胞中MSC所占的比例图。
图7是本申请实施例3中肺组织及胶原海绵区域Masson染色结果图。
具体实施方式
本申请主要提供一种可特异结合间充质干细胞(MSC)的Leptin受体亲和肽的氨基酸序列、筛选方法及应用，涉及细胞、多肽生物技术领域。本申请利用噬菌体展示技术通过与Leptin受体重组蛋白进行亲和筛选，最终在随机7肽库中筛选到具有与Leptin受体具有高度亲和性的多肽，并通过多种检测方法验证该Leptin受体亲和肽可以特异性结合MSC。如下将对该技术方案、其实施过程及原理等作进一步的解释说明。
本申请实施例的一个方面提供了一种Leptin受体亲和肽(可命名为“HY7肽”)，它具有如SEQ ID No.1所示的氨基酸序列，具体为SEQ ID No.1：HGGVRLY。
进一步地，所述Leptin受体亲和肽具有与SEQ ID No.1的全长序列95％以上相同的序列。
进一步地，所述Leptin受体亲和肽的DNA序列为SEQ ID NO.2：ATACAAACGAACCCCACCATG所示。
进一步地，所述Leptin受体亲和肽与Leptin受体具有高度亲和性，且能够与间充质干细胞(MSC)特异性结合。
进一步地，所述Leptin受体亲和肽是采用噬菌体展示技术通过与Leptin受体重组蛋白进行亲和筛选，之后在随机7肽库中筛选得到的。
本申请实施例的另一个方面还提供了一种Leptin受体亲和肽的筛选方法，所述方法利用噬菌体表面展示随机7肽库筛选出能够与Leptin受体特异性结合的七肽氨基酸序列，并鉴定其与MSC的特异结合性。
在一些实施例中，所述筛选方法是利用噬菌体表面展示技术通过与Leptin受体重组蛋白进行亲和筛选，最终在随机7肽库中筛选到具有与Leptin受体具 有高度亲和性的多肽，并通过多种检测方法验证该Leptin受体亲和肽可以特异性结合MSC。
在一些更为优选的实施例中，所述的筛选方法利用噬菌体表面展示随机7肽库，以Leptin受体重组蛋白为靶标，进行五轮生物筛选。第三、四、五轮分别随机挑选15个噬菌体单克隆，对其进行扩增后进行测序模块的快速纯化以产生足够纯的模板送生物公司进行基因测序，分析随机挑选的噬菌体单克隆的相应展示肽序列，并分析重复出现的肽序列的比例增长趋势，挑选出出现比例较高的几个噬菌体单克隆通过检测其与Leptin受体重组蛋白的结合能力。并将这些噬菌体单克隆及合成的相应多肽与MSC进行结合能力的检测，得到一个与Leptin受体具有高亲和性，并与MSC特异性结合的七肽，具有SEQ ID NO.1所示的序列，命名为“HY7肽”。
本申请实施例的另一个方面还提供了一种具有胶原结合能力的亲和肽，其包括所述的Leptin受体亲和肽和具有胶原特异结合能力的短肽；所述具有胶原特异结合能力的短肽通过linker连接结合在所述Leptin受体亲和肽的N端。
其中，所述Leptin受体亲和肽具有如SEQ ID No.1所示的氨基酸序列，具体为SEQ ID No.1：HGGVRLY。
进一步地，所述具有胶原特异结合能力的短肽为CBD，其序列为SEQ ID No.3：TKKTLRT。
进一步地，本申请合成亲和多肽的同时，引入了具有胶原特异结合能力的短肽CBD，合成在Leptin受体亲和肽的N端，与Leptin受体亲和肽之间利用一个linker连接。其中，当linker不带荧光的时候，序列采用SEQ ID No.4：GGGGS；当linker带荧光的时候，序列采用SEQ ID No.5：GGG-K-FITC。
本申请实施例的另一个方面还提供了一种功能性材料，其包括胶原材料和所述具有胶原结合能力的亲和肽；所述具有胶原结合能力的亲和肽结合于所述胶原材料表面和/或内部。
进一步地，所述具有胶原结合能力的亲和肽通过具有胶原特异结合能力的短肽与所述胶原材料特异性结合。
本申请的另一方面是HY7肽修饰植入机体损伤部位胶原海绵支架的应用及其修饰方法。该Leptin受体亲和肽的获得使得特异性吸附募集MSC成为可能，但如何通过HY7肽募集MSC至生物材料上，依然是一个问题。简单吸附的方 法会由于体液的浸润和流动，造成多肽的扩散，导致生物材料局部亲和多肽的有效浓度降低，影响吸附募集MSC的效果。针对该技术问题，本申请中合成Leptin受体亲和肽的同时，引入了具有胶原特异结合能力的短肽collagen binding domain(CBD)，合成在亲和多肽的N端，与亲和多肽之间利用一个linker连接。连接有CBD的亲和多肽可实现与胶原支架的特异结合，从而提高胶原支架上HY7肽的分布浓度，加强了胶原支架在机体内对MSC的结合能力，获得了更良好的组织损伤修复效果。
进一步地，所述具有胶原结合能力的亲和肽与胶原材料的用量比例为1×10 -4-2×10 -3μmol：60mm 3。
进一步地，所述胶原材料包括但不限于胶原海绵支架。
本申请可以将Leptin受体亲和肽连接到生物材料上，从而提高生物材料对于MSC的募集，在组织工程修复领域可以发挥重要的作用。
本申请的另一方面在于，利用本申请筛选获得的一种Leptin受体亲和肽连接到生物材料上，可以提高生物材料对于表面表达Leptin受体的MSC的募集，为研究MSC在组织工程修复和靶向治疗研究方面提供实验依据。
本申请实施例的另一个方面还提供了前述的功能性材料于制备具有组织损伤修复功能(如肺损伤修复)的产品中的应用。
相应的，本申请实施例的另一个方面还提供了一种具有组织损伤修复功能的产品，其包括所述功能性材料。
进一步地，所述产品至少具有能够持续性吸附间充质干细胞的功能。
藉由上述技术方案，本申请提供的Leptin受体亲和肽与Leptin受体具有高亲和性，且能够与MSC特异性结合，可以将该Leptin受体亲和肽连接到生物材料上，连接有具有胶原特异结合能力的短肽的亲和多肽可实现与胶原支架的特异结合，从而提高生物材料对于表面表达Leptin受体的MSC的募集，获得更良好的组织损伤修复效果，为研究MSC在组织工程修复和靶向治疗研究方面提供实验依据。
为了使本申请技术方案及优点更加清楚明白，以下结合附图及若干较佳实施例对本申请的技术方案做进一步详细说明。显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于 本申请保护的范围。下列实施例中未注明具体条件的试验方法，实施例中的试验方法均按照常规条件进行。此外，下面所描述的本申请各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
下列实施例中所用试剂和原料均市售可得，而其中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。又及，除非另外说明，本申请中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
实施例1
本实施例利用噬菌体展示技术筛选与Leptin受体特异性结合的亲和多肽
噬菌体随机七肽展示文库购自NEB公司，100μL，滴度为1×10 13pfu/mL。贮存于含50％甘油的TBS缓冲液(50mM Tris-HCl，150mM NaCl[pH值为7.5])中，库容量1.28×10 9个转化子。Escherichia coil ER2738是此肽库的宿主菌。
1.靶蛋白包被
将Leptin受体重组蛋白用包被缓冲液(0.1M碳酸氢钠缓冲液[pH值为8.6])稀释至终浓度100μg/mL，取100μL加至酶标板一孔中，保证孔板湿润，在湿盒中4℃孵育过夜。
2.亲和筛选
第二天倒掉酶标板中的包被液，在干净的吸水纸上拍净残余液体，在每孔中加入200μL封闭液(0.1M NaHCO 3，5mg/mL牛血清白蛋白(BSA)[pH值为8.6])，置湿盒中4℃孵育至少1小时。弃去封闭液，每孔加满TBST缓冲液(TBS+体积比0.1％[v/v]聚山梨酯-20(Tween-20))，于脱色摇床上温和摇动洗涤6次，每次洗涤5分钟，倾去缓冲液，在干净的吸水纸上拍干以除去残余的液体，要求该实验步骤应快速进行，以避免过程中孔板干燥。接着用TBST缓冲液稀释原始文库，取稀释后的文库液100μL加入到预先包被Leptin受体重组蛋白的酶标板微孔内，加入的噬菌体数量约为2×10 12。随后，室温放置于脱色摇床上温和摇动孵育1-2小时，弃去孔内液体，将酶标板干净的吸水纸上拍干除去残余溶液，并用TBST缓冲液洗板10次，操作同前(洗去未结合的噬菌体)。最后一遍洗涤后，将孔板内液体在干净的吸水纸上拍干，向酶标板微孔中加入100μL非特异性缓冲液(0.2M甘氨酸-盐酸(Glycine-HCl)缓冲液[pH值为2.2]， 1mg/mL BSA)来洗脱已结合的噬菌体，室温放置于脱色摇床上温和摇动10分钟以充分洗脱，收集洗脱液至一个无菌EP管中，用15μL中和液(1M Tris-HCl[pH值为9.1])中和洗脱液。按常规M13滴定方法取5μL洗脱液用来测定洗脱物的滴度，剩余洗脱液均加入到当天提前进行扩增并已处于对数前期的20mL ER2738菌液中，进行第一轮噬菌体洗脱物的扩增，于37℃，220rpm摇床振荡孵育4.5小时，收集扩增菌液，转移到无菌离心管中，经4℃，12000g离心10分钟后，取约80％的噬菌体上清，转移到另一无菌离心管中，加入1/6体积的聚乙二醇(PEG)/NaCl(20％[w/v]PEG-8000，2.5M NaCl)，4℃放置至少2小时或过夜处理，进行噬菌体沉淀。沉淀完成后，4℃，12000g离心15分钟，用微量移液器弃去残余上清，可再进行短暂离心以彻底吸弃上清。沉淀物重悬于1mLTBS缓冲液中，再次离心(4℃，14000rpm，5分钟)后收集上清，转移到无菌离心管中，加入1/6体积的PEG/NaCl(20％[w/v]PEG-8000，2.5M NaCl)，冰上孵育15-60分钟，4℃，14000rpm，离心10分钟，弃上清，再次离心，用200μL TBS缓冲液重悬沉淀，微离心1分钟除去不溶物质，即获得扩增后的洗脱物。取5μL扩增后的洗脱物再次进行噬菌体滴度的测定，留取部分用于第二轮亲和筛选，其余部分按与甘油1:1比例冻存于-20℃备用。重复以上步骤共进行五轮筛选。逐轮筛选增加洗涤步骤中TBST的洗涤次数。
3.噬菌体滴度的测定
接种ER2738单菌落于5mL LB培养基中，37℃，200rpm摇床孵育4.5-5小时至对数中期(OD600在0.5左右)。期间将顶层琼脂于微波炉中加热溶解，分成3mL/份分装到5mL灭菌EP管中，管数根据噬菌体稀释梯度数量而定，每个稀释梯度一管即可。分装的顶层琼脂置于45℃水浴锅备用；同时准备LB/IPTG/Xgal培养平板，提前一小时置于37℃培养箱备用。对收集的噬菌体上清用LB培养基进行10倍梯度稀释(建议稀释范围：未扩增的筛选洗脱物：10 1-10 4，扩增的噬菌体培养上清：10 8-10 11)。稀释完成后将处于对数中期的ER2738菌液分成200μL/管至1.5mL灭菌EP管中，每个噬菌体稀释度准备一管。将稀释好的各个梯度的噬菌体溶液各取10μL立即加到含ER2738菌液的EP管中，快速振荡混匀后于室温孵育1-5分钟。随后将45℃放置的顶层琼脂取出，将已加入噬菌体的ER2738菌液加入45℃预温的顶层琼脂中，每次一管，快速混匀，立即倾注于37℃预温的LB/IPTG/Xgal培养平板上，适当倾斜平板使顶层琼脂均匀 分布。待冷却凝固约5分钟后，37℃孵箱倒置培养过夜。选取噬菌斑总数在100个左右的平板，计数平板上长出来的噬菌斑数目并计算出噬菌体效价(pfu)。根据每轮投入筛选的噬菌体量(投入滴度Input)与洗脱得到的噬菌体量(回收滴度Output)，可计算每轮的产出/投入比，反映特异性噬菌体的富集程度(回收率Recovery)。经过五轮筛选发现，与Leptin受体重组蛋白特异性结合的高亲和力的噬菌体得到了有效富集(见表1)。
表1.每轮投入滴度、回收滴度与回收率的情况
Figure PCTCN2022093475-appb-000001
4.噬菌体单克隆DNA的制备与测序
筛选至第三、四、五轮，对每轮获得的噬菌体扩增无进行滴定后，从滴定板上分别随机挑选15个噬菌体单克隆，对其进行扩增后再进行测序模块的快速纯化以产生足够纯的模板送生物公司进行基因测序：将过夜培养的ER2738扩增菌液(OD值约0.5)按照1：100接种于LB培养基中振摇到对数期后，1mL/管分装至培养管中。随机挑选一个噬菌体单克隆蓝色菌斑加入到上述1mL培养管中，每一轮共挑选15个单克隆进行扩增。37℃摇床250rpm培养4.5-5小时后，将孵育后的噬菌体菌液转入微量离心管中，10000rpm离心30秒，上清转入一个新离心管中，再次离心，吸取80％上清液转入新离心管中，此即为扩增噬菌体贮液，若不立即进行接下去的实验，可暂存于4℃，需长期贮存应用甘油1:1稀释后，-20℃贮存。
测序模板的快速纯化：按上述方法进行噬菌体单克隆扩增后，取上述收集的含噬菌体上清(取500μL)，加入200μL PEG/NaCl，颠倒混匀，室温放置10-20分钟后，4℃，14000rpm离心10分钟，弃上清，可再进行离心操作，彻底吸弃残余上清。沉淀物彻底重悬于100μL碘化物缓冲液(10mM Tris-HCl，1mM EDTA，4M NaI[pH值为8.0])，再加入250μL乙醇，室温孵育10-20分钟(使DNA沉淀)；4℃，14000rpm离心10分钟，弃上清。用500μL预冷的70％乙 醇清洗沉淀一次后再次离心，弃上清后开盖风干，使乙醇充分挥发。最后将沉淀重悬于30μL双蒸水中，此即为测序模板，取5μL用1％琼脂糖凝胶电泳检测，检测合格后的DNA选用-96gIII测序引物送到公司进行测序。
通过比对，本案发明人获得了一条在几轮筛选随机选送测序的单克隆中出现频率最高的一条短肽，命名为“HY7肽”，其氨基酸序列为SEQ ID No.1：HGGVRLY。
5.检测HY7噬菌体单克隆对靶分子的结合能力
将本实施例中获得的HY7噬菌体单克隆按照噬菌体扩增方法进行扩增，同时扩增对照M13KE阴性对照噬菌体(购于NEB公司，40μL，1×10 13pfu/mL)。对扩增后的HY7噬菌体单克隆和M13KE阴性对照噬菌体均根据前述滴定方法进行滴定，获得扩增后的噬菌体浓度。提前一晚将靶蛋白即Leptin受体重组蛋白用包被缓冲液稀释至终浓度100μg/mL，取100μL加至酶标板一孔中，保证孔板湿润，在湿盒中4℃孵育过夜。根据噬菌体筛选步骤将HY7噬菌体单克隆和M13KE阴性对照噬菌体加入至靶蛋白孔中进行结合并洗脱，分别获得与靶蛋白结合的HY7噬菌体洗脱液及M13KE阴性对照噬菌体洗脱液，对两者按照噬菌体滴定方法进行滴定，比较两者与靶分子的结合情况，可以看到与M13KE阴性对照噬菌体对比，HY7噬菌体单克隆对Leptin受体重组蛋白有更多的结合量(如图1所示)，其中纵坐标显示的是HY7噬菌体单克隆与M13KE阴性对照噬菌体对Leptin受体重组蛋白的结合回收率，即结合后洗脱回收的噬菌体量与投入的噬菌体量的比例。
实施例2 HY7可以特异性结合人间充质干细胞
1.检测HY7噬菌体单克隆对人间充质干细胞的结合能力
参照实施例1中的方法扩增HY7噬菌体单克隆和M13KE阴性对照噬菌体，并进行滴定。将HY7噬菌体单克隆和M13KE阴性对照噬菌体加入至预先培养至80％左右汇合度的人间充质干细胞孔板中，并设置无关的人骨髓基质细胞系HS-5作为对照。根据噬菌体筛选步骤进行结合并洗脱分别获得与靶蛋白结合的HY7噬菌体洗脱液及M13KE阴性对照噬菌体洗脱液，对两者按照噬菌体滴定方法进行滴定，比较两者与靶分子的结合情况。结果可见，HY7可以特异性结合至MSC细胞表面，而不结合至对照细胞HS-5，图2A和图2B是HY7噬菌体单克隆与M13KE阴性对照噬菌体分别对人MSC和HS-5细胞的结合回收情况示 意图。
2.荧光显微镜检测HY7与人间充质干细胞的结合情况
委托公司进行体外合成带有异硫氰酸荧光素(FITC)标记的多肽(FITC-HY7)，并选用文献报道不与MSC结合的的一个七肽，也同时合成带有FITC标记的阴性参照多肽(FITC-M7)，将人MSC和HS-5细胞分别消化后以5×10 5个/孔的密度种于6孔板中，37℃孵箱培养24小时后，弃去培养液，PBS洗涤3遍后，细胞预先用DiI染料标记15分钟后，PBS洗涤3遍，换成无血清培养基，在细胞孔板中分别加入5μmol/L上述合成的FITC-HY7肽和FITC-M7肽，同时用不在肽的细胞作为空白对照，37℃共孵育30分钟，PBS洗孔板3遍，用4％多聚甲醛固定20min后，PBS洗3遍，加入DAPI(1:10000稀释)，孵育10min，PBS洗板3遍后，以未加多肽组作为对照，共聚焦荧光显微镜下观察荧光多肽细胞FITC-HY7与FITC-M7与人MSC的结合情况，结果如图3所示，FITC-HY7肽与人MSC结合量明显高于阴性对照肽。
3.流式细胞术检测HY7与大鼠间充质干细胞的结合情况
为评价HY7肽的种属特异性，也为后续的动物实验提供依据，我们特地验证了HY7与大鼠间充质干细胞的结合情况。根据常规方法获取大鼠骨髓细胞并进行培养和鉴定，取得大鼠间充质干细胞。按前述流式细胞术检测方法检测FITC-HY7肽和FITC-M7肽分别与大鼠间充质干细胞MSC的结合情况。结果如图4所示，HY7与大鼠MSC结合能力明显强于M7。
实施例3 CBD-亲和肽可以结合到胶原材料上，用于肺损伤修复
1.CBD-亲和肽可以结合到胶原材料上
为了避免简单吸附方法所导致的亲和肽无法有效地驻留在生物材料上保持有效浓度，影响亲和肽发挥作用。本申请中合成亲和多肽的同时，引入了具有胶原特异结合能力的短肽CBD(序列具体为SEQ ID No.3：TKKTLRT)，合成在亲和多肽的N端，与亲和多肽之间利用一个linker连接，序列具体为SEQ ID No.5：GGG-K-FITC。为评价CBD-亲和肽是否可以有效结合到胶原材料上，同时合成了FITC标记的CBD-HY7，并为方便对照，也分别合成了FITC标记的CBD错构对照(sCBD-HY7)和hy7错构对照(CBD-sHY7)。具体实验方法：将直径5mm的胶原海绵片先用PBS充分润湿，用无菌滤纸吸去多余液体，将溶解好的FITC标记的亲和肽和对照肽分别均匀滴加至相应的胶原海绵片上(浓 度为5-100μmol/L)，所述FITC标记的亲和肽与胶原海绵片的用量比例为1×10 -4-2×10 -3μmol：60mm 3，保证胶原海绵充分吸收，液体不溢出。放置于37℃孵育2小时后，PBS洗涤3-5遍，共聚焦荧光显微镜拍摄CBD-HY7、sCBD-HY7、CBD-sHY7与胶原海绵结合情况。结果如图5所示，表达CBD的肽均可以特异性结合至胶原海绵上，而错构CBD对照则失去了结合胶原海绵的能力。
2.胶原海绵结合CBD-亲和肽植入大鼠肺损伤部位可以在短时间内募集内源性MSC
将体重约150g大鼠麻醉后，插管并在呼吸机正压通气情况下，行右肺中叶部分切除术，切除约0.5cm*0.5cm*0.3cm大小肺组织，将预先已经均匀滴加CBD-亲和肽或对照肽，并在37℃孵育2小时的胶原海绵分别植入大鼠肺缺损部位。分别于12小时、24小时或48小时后，处死大鼠后，完整分离取出植入的胶原海绵，利用胶原酶溶解胶原海绵，将在体吸附于胶原海绵上的细胞分离收集下来。通过鉴定细胞表面CD45、CD44、CD71、CD90的表达，分析在植入部位吸附至胶原海绵上的MSC的数量比例。流式细胞术检测从胶原海绵上消化下来的细胞中MSC所占的比例，结果如图6A至图6F所示，与对照组相比，CBD-HY7可以在短时间内，吸附更多的MSC至胶原海绵上。
3.胶原海绵结合CBD-亲和肽植入大鼠肺损伤部位可以修复肺损伤
按前述方法，将CBD-亲和肽及对照肽植入大鼠肺损伤部位后，35天后取出肺组织，并进行病理学鉴定，可以发现，植入CBD-亲和肽的组织部位与对照组相比，有更好的修复效果。肺组织及胶原海绵区域Masson染色结果见图7。
综上所述，本申请通过噬菌体展示技术获得Leptin受体特异性结合多肽，并证实该多肽可以高度亲和靶向至人间充质干细胞，同时在对该亲和多肽进行种属特异性鉴定的过程中发现该多肽并无明显的种属特异性，所以推测该亲和多肽具有广泛的应用范围。接着，通过将CBD合成至筛选获得的Leptin受体亲和肽上，利用CBD使得亲和肽可以靶向结合到胶原材料上，从而实现特异性吸附募集间充质干细胞，使得该胶原材料作为支架植入体内进行肺损伤修复的时候，能够持续性吸附间充质干细胞，从而获得更好的修复效果。
尽管已参考说明性实施例描述了本申请，但所属领域的技术人员将理解，在不背离本申请的精神及范围的情况下可做出各种其它改变、省略和/或添加且可用实质等效物替代所述实施例的元件。另外，可在不背离本申请的范围的情 况下做出许多修改以使特定情形或材料适应本申请的教示。因此，本文并不打算将本申请限制于用于执行本申请的所揭示特定实施例，而是打算使本申请将包含归属于所附权利要求书的范围内的所有实施例。

Claims (8)

  1. 一种具有胶原结合能力的亲和肽，其包括：Leptin受体亲和肽和具有胶原特异结合能力的短肽；所述具有胶原特异结合能力的短肽通过linker连接结合在所述Leptin受体亲和肽的N端，其中，所述Leptin受体亲和肽的序列如SEQ ID No.1所示，所述具有胶原特异结合能力的短肽为CBD，它的序列如SEQ ID No.3所示，所述linker的序列如SEQ ID No.4或SEQ ID No.5所示。
  2. 根据权利要求1所述的具有胶原结合能力的亲和肽，其中，所述Leptin受体亲和肽是采用噬菌体展示技术通过与Leptin受体重组蛋白进行亲和筛选，之后在随机7肽库中筛选得到的。
  3. 一种功能性材料，其包括：胶原材料和权利要求1-2中任一项所述的具有胶原结合能力的亲和肽；其中，所述具有胶原结合能力的亲和肽结合于所述胶原材料表面和/或内部。
  4. 根据权利要求3所述的功能性材料，其中，所述具有胶原结合能力的亲和肽与胶原材料的用量比例为1×10 -4-2×10 -3μmol：60mm 3。
  5. 根据权利要求3所述的功能性材料，其中，所述胶原材料包括胶原海绵支架。
  6. 权利要求3-5中任一项所述的功能性材料在制备具有组织损伤修复功能的产品中的应用。
  7. 一种具有组织损伤修复功能的产品，其包括权利要求3-5中任一项所述的功能性材料。
  8. 根据权利要求7所述的产品，其中，所述产品至少具有能够持续性吸附间充质干细胞的功能。
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