Active peptide with efficacy of repairing neuroinflammation after spinal cord injury as well as preparation method and application of active peptide

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an active peptide with a function of repairing neuroinflammation after spinal cord injury and a preparation method and application thereof. The invention firstly provides a recombinant active peptide. The structure of the recombinant active peptide at least comprises two groups of alpha spiral fragments; the amino acid sequence of the recombinant active peptide is as shown in SEQ ID NO. 2 or SEQ ID NO. 3. The bioactive peptide and the prepared nanofiber provided by the invention can effectively repair neuroinflammation after spinal cord injury, and have relatively high safety in a living body. And compared with the RK33A holoprotein, the RK33A holoprotein has a better effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an active peptide having the effect of repairing neuroinflammation after spinal cord injury, and a preparation method and application thereof. Background Art

[0002] Spinal cord injury (SCI) is a serious neurological disease characterized by significant neurological damage and persistent functional deficits, and its pathological process is highly regulated by neuroinflammation. Neuroinflammation is the main cause of secondary injury because microglia / macrophages rapidly infiltrate and secrete proinflammatory cytokines to promote nerve apoptosis and aggravate secondary injury. The prior art suggests that keratin materials have the function of regulating microglial polarization, and thus have potential therapeutic value in SCI repair. However, in actual clinical work, anti-inflammatory treatment of SCI remains a major challenge, and one of the problems that needs to be solved urgently is the demand for highly efficient anti-inflammatory materials.

[0003] The literature "Study on the Role of Recombinant Human Keratin in Regulating Neuroinflammation and Promoting Neural Regeneration after Spinal Cord Injury" publicly discusses the effects of recombinant human keratin (RKs) on microglia / macrophage polarization after SCI. Among them, recombinant human keratin 33A (RK33A) has the ability to regulate the M2 phenotype of microglia / macrophages. However, the existing technology for studying RK33A is based on its wild-type whole protein. The amino acids of the whole protein are too long, and the fragments that are truly active are not clear, which limits the potential for further development into small molecule composite materials. In addition, based on the three-dimensional conformation of the whole protein, some active sites may be encapsulated inside the protein. Although strategies for transforming wild-type whole proteins into small molecule peptides based on protein engineering have been reported, in actual operations, they often face problems such as loss of activity of the transformed small molecule peptides, inability to express smoothly, or cytotoxicity to organisms.

[0004] To sum up, it is necessary to propose new methods and strategies. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant RK33A active peptide and a preparation method and application thereof, so as to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0006] A recombinant active peptide having the effect of repairing neuroinflammation after spinal cord injury, wherein the recombinant active peptide is modified based on keratin RK33A, and the structure of the recombinant active peptide contains at least two groups of α-helical fragments (in RK33A); the amino acid sequence of the recombinant active peptide is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0007] Furthermore, the recombinant active peptide structure also includes a group of non-helical fragments (in RK33A); the amino acid sequence of the recombinant active peptide is shown in SEQ ID NO.3.

[0008] Furthermore, the recombinant active peptide shown in SEQ ID NO. 3 has almost equal numbers of hydrophilic amino acids and hydrophobic amino acids.

[0009] Furthermore, the number of glutamic acid in the recombinant active peptide shown in SEQ ID NO.3 is greater than 20.

[0010] A recombinant expression vector comprises a gene encoding the above recombinant active peptide.

[0011] In some preferred embodiments, the recombinant active peptide is the recombinant active peptide shown in SEQ ID NO.3.

[0012] Furthermore, the types of the vector include plasmid vectors, phage vectors or animal and plant virus vectors.

[0013] A recombinant bacterium expressing the above recombinant active peptide.

[0014] Furthermore, the cell types include Escherichia coli and / or Bacillus subtilis.

[0015] In some preferred embodiments, the recombinant active peptide is the recombinant active peptide shown in SEQ ID NO.3.

[0016] A nanofiber for repairing neuroinflammation after spinal cord injury, wherein the nanofiber is prepared from the above-mentioned recombinant active peptide and polycaprolactone.

[0017] In some preferred embodiments, the recombinant active peptide is the recombinant active peptide shown in SEQ ID NO.3.

[0018] Furthermore, the mass ratio of the recombinant active peptide to the polycaprolactone is 8-10:2.

[0019] In some preferred embodiments, the mass ratio of the recombinant active peptide to the polycaprolactone is 8:2.

[0020] The application of the above nanofibers in the preparation of medical materials for repairing neuroinflammation after spinal cord injury.

[0021] Furthermore, the medical material is in the form of an implantable stent.

[0022] A method for preparing nanofibers for repairing neuroinflammation after spinal cord injury comprises the following steps: S01: dissolving a recombinant active peptide with an amino acid sequence as shown in SEQ ID NO.2 or SEQ ID NO.3 and polycaprolactone in hexafluoroisopropanol, and fully stirring the mixed solution; the mass ratio of the recombinant active peptide to the polycaprolactone is 8-10:2; S02: The mixed solution after being fully stirred is placed in a syringe, connected to a power source, the voltage and rotation are set, and the nanofibers are obtained by injecting the solution into a drum collector covered with aluminum foil at room temperature through electrospinning technology; S03: (while at rest) separating the nanofibers from the aluminum foil and curling them longitudinally to form a cylindrical scaffold; S04: sterilizing the collected nanofibers using 60 Co γ rays.

[0023] In some preferred embodiments, the voltage is set to 15 kV, the rotation speed is set to 800-1000 rpm, and the flow rate is set to 1.5 mL h -1 .

[0024] Beneficial technical effects: The present invention is based on keratin RK33A and is truncated to provide a recombinant active peptide with the effect of repairing neuroinflammation after spinal cord injury. The recombinant active peptide structure contains at least two groups of α-helical fragments. In a most preferred case, the recombinant active peptide structure also contains a group of non-helical fragments. After experimental verification, the active peptides provided by the present invention and the prepared nanofibers can effectively repair neuroinflammation after spinal cord injury and have high safety in vivo. And compared with the whole RK33A protein, it has a better function of promoting nerve regeneration and a more significant effect on promoting the polarization of microglia M2 after SCI. However, due to the smaller molecular weight of the recombinant active peptide, it has the potential to be further developed into a small molecule composite material. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0026] Figure 1 Schematic diagram of the structure of RK33A whole protein, RK33A-1Aα peptide, RK33A-1Bα peptide and RK33A-2α peptide; Figure 2The physicochemical properties of RK33A whole protein, RK33A-1Aα peptide, RK33A-1Bα peptide and RK33A-2α peptide were analyzed; Figure 3 The biological effects of RK33A whole protein, RK33A-1Aα peptide, RK33A-1Bα peptide and RK33A-2α peptide on regulating microglial polarization are shown; Figure 4 To analyze the physical and chemical properties of RK33A nanofibers and RK33A-2α nanofibers; Figure 5 HE staining was used to observe the long-term biological toxicity of nanofibers in various tissues; Figure 6 To evaluate the biological effects and behavioral responses of RK33A nanofibers and RK33A-2α nanofibers in rats with spinal cord injury; Figure 7 The growth of nerve fibers after RK33A nanofibers and RK33A-2α nanofibers were implanted into rats with spinal cord injury. Figure 8 The effect of RK33A nanofibers and RK33A-2α nanofibers implanted into rats with spinal cord injury on the polarization of peripheral microglia toward the M2 anti-inflammatory phenotype; Fig. 9 The binding of RK33A-1Aα peptide, RK33A-1Bα peptide and RK33A-2α peptide to cell membrane surface integrin b2 was verified. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0029] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0030] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0031] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0032] Some specific drawings: Figure 2-1 The figure shows the SDS PAGE electrophoresis results of four recombinant proteins.

[0033] Figure 2-2 FT-IR results of four recombinant proteins.

[0034] Figure 2-3 The circular dichroism analysis results of four recombinant proteins are shown in Figure 2.

[0035] Figure 2-4 In vitro cytotoxicity test of four recombinant proteins.

[0036] Figure 3-1 This is a fluorescence staining image of CD206.

[0037] Figure 3-2 It is the percentage of CD206 in the statistical fluorescence staining results.

[0038] Figure 3-3 It is the percentage of CD206 in the statistical flow cytometry analysis results.

[0039] Figure 4-1 Schematic diagram of the process of constructing recombinant proteins into nanofibers that can be implanted into animal spinal cord injury models.

[0040] Figure 4-2 The morphology of nanofibers was observed using a scanning electron microscope.

[0041] Figure 4-3 To reflect the hydrophilicity and tissue compatibility of the material by detecting the water contact angle.

[0042] Figure 4-4 To observe the spatial distribution of recombinant proteins in nanofibers.

[0043] Figure 4-5 To detect the α-helical structure of the recombinant protein after it is constructed into nanofibers through Fourier transform infrared spectroscopy.

[0044] Figure 6-1 A real picture of nanofibers curling along the longitudinal direction of nanofiber orientation to form a cylindrical scaffold.

[0045] Figure 6-2 Schematic diagram for in vivo experimental design in animal models.

[0046] Figure 6-3 These are the motor images of the rat lower limbs at 1 week and 8 weeks after injury.

[0047] Figure 6-4 The BBB scores were 3 days before SCI and at 1, 3 dpi (days post-injury), 1, 2, 4 and 8 wpi (weeks post-injury).

[0048] Figure 6-5 and Figure 6-6 Representative images of the footprints of rats in different groups at 4 and 8 weeks after injury, respectively. The forelimb footprints are shown in red and the hindlimb footprints are shown in blue. The scale bar is 1 cm.

[0049] Figure 6-7 The stride length of the rat hind limb at 4 and 8 wpi according to footprint analysis.

[0050] Figure 7-1 and Figure 7-2 The lesion center ( Figure 7-1 ) β-tubulin III (green) and ( Figure 7-2 ) Representative images of immunofluorescence staining of Gfap (green). Scale bar is 100 µm.

[0051] Figure 7-3 and Figure 7-4 Based on the above fluorescence staining experiments, β-tubulin III ( Figure 7-3 ) and Gfap ( Figure 7-4 ) for quantitative analysis.

[0052] Figure 8-1 and Figure 8-3 To stain the lesion center of different treatment groups, CD206 + (Green) / Iba1+ Immunofluorescence staining and statistics. Scale bar is 100 μm.

[0053] Figure 8-2 and Figure 8-4 To stain the lesion center of different treatment groups, CD206 + (Green) / Iba1 + Immunofluorescence staining and statistics. Scale bar is 100 μm.

[0054] Figure 9-1 and Figure 9-2 This is the electrophoresis band diagram of RK33A-1Aα, RK33A-1Bα and RK33A-2α binding to integrin b2 on the cell membrane surface.

[0055] Example 1 First, the RK33A protein sequence was analyzed in UniProt. The RK33A protein consists of a central α-helical rod domain and two highly variable non-helical domains (head and tail) ( Figure 1 The central α-helical rod domain can be divided into three α-helical segments (1Aα, 1Bα and 2α) and two connecting regions (L1 and L12).

[0056] Furthermore, the team of the present invention used recombinant expression technology to synthesize three peptides, named RK33A-1Aα (1-91 Aa), RK33A-1Bα (92-203 Aa) and RK33A-2α (204-404 Aa). Among them, RK33A-1Aα is composed of head + 1Aα (non-helical fragment + helical fragment); RK33A-1Bα is composed of L1+1Bα (double helical fragment); RK33A-2α is composed of L12+2α+tail (double helical fragment + non-helical fragment).

[0057] The amino acid sequence information of the three peptides is shown in Table 1.

[0058] Table 1 The hydrophilicity analysis of the sequences shown in SEQ ID NO.1-3 showed that RK33A-1Aα contained 27 hydrophilic amino acids and 35 hydrophobic amino acids. RK33A-1Bα contained 33 hydrophilic amino acids and 38 hydrophobic amino acids. RK33A-2α contained 70 hydrophilic amino acids and 71 hydrophobic amino acids. The number of hydrophilic amino acids and hydrophobic amino acids contained in RK33A-2α was almost equal.

[0059] The RK33A and three peptides mentioned above are referred to as four recombinant proteins in the following experiments.

[0060] The above-mentioned RK33A and three peptides were expressed by an E. coli expression system and purified using a nickel column. The specific steps are as follows.

[0061] (1) The amino acid sequences of RK33A and the three peptides mentioned above were reverse translated into corresponding DNA sequences containing a 6×His tag, and the recombinant pET28a (+) plasmid was constructed, which was then transformed into Escherichia coli (BL21) strain for expression.

[0062] (2) Grow E. coli cells in Luria-Bertani medium (1 L) supplemented with 50 μg / mL ampicillin at 37°C until OD600 reaches 0.6-0.8. Add 1 mM isopropyl-β-D-thiogalactoside to induce enzyme expression at 37°C for 4 hours. Then, harvest the cells by centrifugation at 8000 rpm for 5 minutes at 4°C.

[0063] (3) The collected cell pellets were resuspended in buffer A (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 20 mM β-mercaptoethanol) and lysed twice at 700 °C using a high-pressure homogenizer and centrifuged at 10,000 rpm for 20 min at 4 °C.

[0064] (4) The precipitate was collected and resuspended in buffer B (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA; 20 mM β-mercaptoethanol, 1 M urea, and 0.5% Triton X-100) and buffer C (50 mM Tris, pH 8.0, 2.5 mM NaCl, 5 mM EDTA, and 20 mM β / mercaptoethanol), and centrifuged at 10,000 rpm for 20 min at 4°C. Afterwards, the precipitate was dissolved in buffer D (50 mM Tris, pH 8.0, 150 mM NaCl, 8 M urea, and 20 mM β-mercaptoethanol) for purification.

[0065] (5) The solution was allowed to bind to a Ni column (Ni Smart Beads 6FF) at 4 °C for 3 h in a vertical mixer. Then, the beads were washed with ddH2O to remove impurities, and the target protein was eluted by adding 10 or 250 mM imidazole to buffer D. Finally, the protein was dialyzed against ddH2O (molecular weight cutoff of 3 kDa) for 3 days, lyophilized, and sterilized by irradiation with 25 kGy of 60 Co γ-rays. The protein powder was collected and stored for subsequent analysis.

[0066] (6) The protein solution was mixed with 5 μL of 5× SDS-PAGE loading buffer, and the mixture was heated at 100°C for 10 minutes to perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electrophoretically separated on a 10% SDS-polyacrylamide gel (RK33A) and a 15% SDS-polyacrylamide gel (RK33A fragment) system at 110 V for 90 minutes. The proteins were stained with Coomassie Brilliant Blue at room temperature for 30 minutes and destained with ethanol-acetic acid until visible.

[0067] The results of SDS PAGE showed that the molecular weights of RK33A, RK33A-1Aα, RK33A-1Bα and RK33A-2α were approximately 46, 11, 14 and 24 kDa, respectively. The final single band indicated that the recombinant protein was successfully purified. Figure 2-1 .

[0068] Furthermore, the physicochemical properties of the purified recombinant protein were analyzed as follows.

[0069] (1) Fourier transform infrared spectroscopy (FT-IR) was used to analyze the wavelength range from 500 to 4000 cm -1 Chemical structure of purified proteins in the spectral range. The samples were prepared by grinding the dried protein with KBr at a ratio of 1:100 and pressing it into thin sheets for testing.

[0070] (2) CD analysis of the purified recombinant protein solution was performed using a CD spectrophotometer at room temperature. -1 The spectra of all proteins were scanned from 190 to 260 nm at a rate of 1.5 and the measurement was repeated 3 times for each sample. A background spectrum was recorded before each sample and subsequently subtracted from the sample spectrum.

[0071] (3) The cytotoxicity of the above recombinant proteins was evaluated using the Cell Counting Kit-8 (CCK-8). BV2 cells were seeded into a 96-cell plate and incubated with the four recombinant proteins for 24 hours. Experimental and control groups were set up, and BV2 cells without any protein solution were set up as controls. The cells were then rinsed three times with PBS. Then, 10 μL of CCK-8 test solution was added to each well and incubated at 37°C for 2 hours. The optical density (OD) was measured at 450 nm using a microplate reader.

[0072] The results showed that the Fourier transform infrared (FT-IR) spectrum was between 500 and 4000 cm -1 Characteristic amide bands were detected in the range of 2.5 Å and 1.5 Å, which are spectrally related to peptide bonds ( Figure 2-2). The secondary structure of the purified protein was detected by circular dichroism (CD) analysis. All solutions showed a typical α-helical spectrum with two negative minima at about 208 and 222 nm, indicating that the recombinant protein fragment still had a secondary structure ( Figure 2-3 The in vitro cytotoxicity of the above recombinant proteins was evaluated by CCK-8 assay, which showed that there was no significant difference in the cell activity between RK33A-1Bα and RK33A-2α and RK33A, while the cell activity of RK33A-1Aα was relatively low.

[0073] Example 2 This example provides a comparison of the biological effects of RK33A-1Aα, RK33A-1Bα, RK33A-2α and RK33A full length synthesized in Example 1. Immunofluorescence staining and flow cytometry were used to detect the effects of RK33A-1Aα, RK33A-1Bα, RK33A-2α and RK33A full length on LPS (100 ng mL -1 ) and IFNγ (20 ng mL -1 ) (referred to as LPS)-induced polarization of M1 microglia.

[0074] Experimental methods: (1) BV2 microglia (Procell Life Science & Technology) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum and 1% streptomycin / penicillin (Gibco). When the cells reached approximately 60% confluence, they were incubated with LPS (100 ng mL -1 ) and IFN-γ (20 ng mL -1 , (referred to as LPS) were incubated together for 24 h, and 200 μg mL -1 The above recombinant protein fragments were processed.

[0075] (2) BV2 cells were seeded in 24-well culture dishes and treated with different treatments for 24 hours (same as step 1). The culture medium was then removed. BV2 cells were fixed with 4% PFA for 30 minutes at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. Then, the cells were permeabilized with Saponin for 20 minutes at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. Then, the cells were blocked with 5% BSA for 1 hour at room temperature, and the blocking solution was removed. The cells were incubated with the primary antibody CD206 (Proteintech, 1:500) at 4°C overnight, and then rinsed three times in PBS containing 0.05% Tween-20 for 5 minutes each. The cells were incubated with the appropriate secondary antibody purchased from Beyotime at a dilution of 1:500 for 2 hours at room temperature, and then rinsed three times in PBS containing 0.05% Tween-20 for 5 minutes each. The cell nuclei were stained with DAPI for 3 minutes. Images were captured using a fluorescence microscope.

[0076] (3) BV2 cells were plated at 1×10 6 The cells were seeded at a density of 100 μg / mL in 6-well culture dishes and subjected to different treatments for 24 h (same as step 1). Afterwards, the cells were washed twice with PBS and then harvested into single cell suspensions containing 2% BSA. The suspensions were incubated with antibodies against APC-CD206 (1:100) at 4 °C in the dark for 1 h. Next, the cells were harvested by centrifugation at 2000 rpm for 5 min to remove unbound antibodies and washed twice with PBS. Subsequently, the suspensions were analyzed using flow cytometry.

[0077] The experimental results are shown in Figure 3 :CD206 after different treatments + Representative images of immunofluorescence stained BV2 cells are shown in Figure 3-1 , quantitative analysis results Figure 3-2 ; Scale bar, 100 µm. Figure 3-3 Representative flow cytometry histogram CD206 + Quantitative analysis of BV2 cells. The results showed that RK33A, RK33A-1Aα, RK33A-1 Bα and RK33A-2α could increase CD206 + However, compared with RK33A or other fragments, RK33A-2α was more effective in increasing CD206 expression. Based on this, RK33A-2α was selected for further verification.

[0078] Example 3 This example provides a validation of the effects of nanofibers prepared from full-length RK33A and RK33A-2α in an animal model. In this example, polycaprolactone (PCL) nanofibers and recombinant protein nanofibers (RKNF) were first prepared using an electrospinning method, and then the prepared recombinant protein nanofibers were implanted into a rat T9 spinal cord injury model to validate the effects.

[0079] Experimental methods: (1) PCL with or without recombinant protein (8 / 2 w / w, labeled with FITC) was dissolved in hexafluoroisopropanol at a final concentration of 10 wt% w / v and stirred continuously overnight. The solution was loaded into a 5 mL syringe with a 21-gauge stainless steel needle (positive electrode) and then injected into a drum collector covered with aluminum foil (negative electrode) by electrospinning at room temperature. The voltage was 15 kV, the receiving distance was controlled at 15-20 cm, the rotation speed was 1000 rpm, and the flow rate was 1.5 mL h -1 The nanofibers were separated from the aluminum foil and rolled longitudinally to form a cylindrical scaffold. The nanofibers were sterilized using 60 Co γ-ray irradiation (25 kGy) and kept dry for further experiments.

[0080] (2) Scanning electron microscopy (Nova 400) was used to characterize the surface morphology of PCL nanofibers and RKNFs. The samples were coated with metal under vacuum, examined for 15 s, and subsequently observed by SEM at an accelerating voltage of 5.0 kV.

[0081] (3) The diameter of the fibers was analyzed using SEM images and ImageJ software. Referring to the method of Example 1, the combination of PCL and RK in RKNF was analyzed using a Fourier transform infrared spectrometer. The hydrophilicity of PCL nanofibers and RKNF was evaluated using a contact angle meter (XG-CAMA). Distilled water was dropped onto the surface of the nanofibers and the shape of the droplet was immediately captured. The water contact angle was calculated based on the slope of the curve using ImageJ software. The spatial distribution of the embedded keratin fragments in the nanofibers was examined by fluorescence microscopy (XDS-2FL).

[0082] The experimental results are shown in Figure 4 . Figure 4-1 Schematic diagram of constructing the screened RK33A-2α active peptide segment and RK33A full-length into RK33A-2α nanofibers (RKNF33A-2α) and RK33A nanofibers (RKNF33A), respectively. Figure 4-2(Scale bar 1 μm) The morphology of nanofibers was observed by scanning electron microscopy (SEM). All nanofibers were arranged in an orderly manner, which is conducive to the adhesion and growth of axons. 100 nanofibers were randomly selected for analysis. The fiber diameters of PCL, RKNF33A, and RKNF33A-2α were 248.29 ± 60.35 nm, 198.35 ± 60.24 nm, and 210.94 ± 58.47 nm, respectively; the diameters of all nanofibers were within the range of 100-400 nm. Figure 4-3 The water contact angle reflects its hydrophilicity and tissue compatibility. The biocompatibility of biomaterials is related to their hydrophilicity, and hydrophilicity can be reflected by the water contact angle. RKNF33A (≈53°), RKNF33A-2α (≈40°), PCL nanofibers (≈73°), the water contact angle of RKNF33A-2α is significantly lower than that of PCL nanofibers (≈73°), and is also lower than that of RKNF33A, indicating that RKNF33A-2α can enhance the hydrophilicity of PCL and has the best tissue compatibility. Figure 4-4 (Scale bar 100 μm) In order to detect the spatial distribution of keratin fragments embedded in the nanofibers, RK33A and RK33A-2α were labeled with fluorescein isothiocyanate (FITC), and then the nanofibers were prepared and observed under a microscope, showing that RKNF33A and RKNF33A-2α were uniformly distributed in the nanofibers. Figure 4-5 Fourier transform infrared spectroscopy shows that RKNF33A and RKNF33A-2α still have the α-helical structure of the protein after being constructed into nanofibers. -1 and 1500 cm -1 To the left is the protein peak.

[0083] Example 4 This example provides biological toxicity testing of RK33A nanofibers and RK33A-2α nanofibers.

[0084] Experimental methods: (1) 2 × 2 cm PCL, RKNF33A, and RKNF33A-2α were incubated with 4 mL PBS (pH 7.4) in a shaking incubator at 37 °C for 14 days. The supernatant was measured by bicinchoninic acid assay (BCA) at 1, 2, 4, 12, 24 h, 3, 5, 7, 9, 11, and 14 days to analyze the degradation of the recombinant protein nanofibers.

[0085] (2) The degradation rate of RKNF was calculated using the RKs content in the supernatant and the RKs content in RKNF. After the sections were dewaxed and rehydrated, HE staining of important organs 8 weeks after nanofiber material implantation was performed according to the manufacturer's instructions (Solarbio), and the sections were observed under an optical microscope.

[0086] The experimental results are shown in Figure 5 (Scale bar 200 μm), HE staining observation showed that the nanofibers had almost no biological toxicity in various tissues (including heart, liver, spleen, lung tissue and kidney), indicating that RK33A-2α has high biological safety.

[0087] Example 5 This example provides BBB behavioral scoring and Footprint gait analysis to verify and compare the effects of RKNF33A and RKNF33A-2α in vivo. The animal experiments involved in this example have been approved by the Animal Care and Use Committee of Chongqing Medical University.

[0088] Experimental methods: (1) Six-week-old female Sprague-Dawley rats (200 ± 20 g) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China) and randomly divided into SCI (n = 6), PCL (n = 6), RKNF33A (n = 6), or RKNF33A-2α (n = 6) according to the nanofiber composites.

[0089] (2) A rat T9 lateral spinal cord hemisection model was used to evaluate nerve regeneration. The animals were allowed to acclimate to the environment for 1 week before the experiment. Pentobarbital (30 mg kg −1 ) The rats were anesthetized. The hair on the thoracic spine was shaved, and the surgical field was cleaned three times with iodine and ethanol. A midline skin incision (3 cm) was made on the back of the vertebrae T8−11. Under the operating microscope, the paraspinal muscles were bluntly separated and retracted from the surgical field to expose the spinous processes and vertebral lamina. The T9 spinous process was carefully cut, and the upper lamina was removed with biting forceps. The midline was determined by inserting a needle (30G) vertically through the midline into the spinal cord with the bevel facing the right side under the operating microscope. A 2-mm-long defect was created by removing the right spinal cord.

[0090] (3) A cylindrical scaffold (2 × 2 × 2 mm) was transplanted into the lesion site so that the orientation of the nanofibers was aligned with the long axis of the spinal cord. The muscle layer and skin layer were sutured separately. After surgery, each rat was subcutaneously injected with 0.9% sterile saline to maintain water and buprenorphine (2.0 mg kg) was administered twice daily. −1 ) to improve pain and continue for 2 days.

[0091] (4) All animals were housed in individual cages. Each rat received manual bladder compression twice a day until reflex bladder control was restored. At the same time, the rat urethra and perineum were disinfected with povidone-iodine to prevent urinary tract infection. Animal bedding was changed once a day to ensure a clean environment. At 1 and 8 wpi, high-dose sodium pentobarbital (100 mg / kg -1 The rats were randomly killed by intraperitoneal anesthesia.

[0092] (5) Two scorers were assigned to calculate the BBB score, ranging from 0 (no limb movement or weight support) to 21 (normal movement), 3 days before SCI and 1, 3 days, and 1, 2, 4, and 8 weeks after SCI to evaluate the hindlimb behavior of rats.

[0093] (6) Footprint analysis. The rats’ forelimbs and hindlimbs were dyed blue and red, respectively. Then, at 4 and 8 weeks after injury, the rats were allowed to walk on a homemade alley (80 × 40 cm) with a white floor, and footprint images were taken. ImageJ was used to measure stride length (the distance between the center pads of the forelimbs and hindlimbs) to assess limb coordination ability.

[0094] (7) In order to detect the histopathological changes of the injury site after spinal cord injury, tissue immunofluorescence staining was used to detect and analyze the target protein. Paraffin sections were placed on a slide rack. Before staining, the slides were heated from all directions with a hair dryer to melt the paraffin. Then the slide rack was placed in xylene I and II treatment tanks for 25 minutes each. The sections were treated with gradient alcohol: 100%, 5 minutes; 95%, 5 minutes; 80%, 5 minutes; 75%, 5 minutes. Finally, the sections were washed with distilled water 3 times, 5 minutes each (on a shaker). The sections were placed on a plastic slide rack, sodium citrate buffer was added to the box of the plastic slide rack to submerge the sections, and the slide rack box was placed in a metal pot filled with pure water and heated at 100°C on an induction cooker for 30 minutes. After the sections returned to room temperature, they were washed with distilled water 3 times, 5 minutes each (on a shaker). Remove the slices from the slice rack, place them in a wet box, absorb excess moisture with filter paper, then add 0.5% TritonX-100 solution to cover the sample, permeabilize the membrane for 10 min at room temperature, wash with PBS three times, 5 min each time, and absorb excess moisture; add 3% BSA solution prepared with PBS to cover the sample, block at room temperature for 1.5 h, and gently absorb excess moisture with filter paper.

[0095] (8) Dilute the primary antibody in proportion, Iba-1 antibody (1:1000), CD206 antibody (1:500), GFAP antibody (CST1:500), β-tubulin III antibody (1:500), add about 50 μL of primary antibody to each sample, just covering the sample, and then place the wet box containing the slice sample in a 4℃ refrigerator overnight. Take out the slice sample from the refrigerator and warm it up at room temperature for 1 hour. Then put the slice sample into a slide staining jar containing PBST and shake it on a horizontal shaker for 3 times, 10 minutes each time, and then use filter paper to absorb the excess water on the slide. Dilute the corresponding secondary antibody, add an appropriate amount of secondary antibody diluent to each sample to ensure that the slice is completely covered, cover the wet box lid and incubate at room temperature in the dark for 2 hours; then put the slice sample into a slide staining jar containing PBST and wash it on a horizontal shaker for 3 times, 10 minutes each time. During all the slice processing, keep the slide dry and keep the wet box with water. The samples were mounted with anti-fluorescence quenching mounting medium containing DAPI, and images were collected using a fluorescence microscope.

[0096] The experimental results are shown in Figure 6 and Table 2 and Table 3.

[0097] Table 2 BBB scoring results Table 3 Stride length (cm) In summary, Figure 6 The results showed that the BBB score showed that the RKNF33A-2α group had better motor function recovery than the SCI, PCL, and RKNF33A groups at 2, 4, and 8 wpi, 3 days before injury and 1, 3 days, 1, 2, 4, and 8 weeks after injury (wpi). In addition, footprint analysis was performed at 4 wpi and 8 wpi to visually observe the recovery of the hindlimbs. The results showed that the RKNF33A-2α group had clearer right footprints and longer strides at 4 and 8 wpi compared with the SCI, PCL, and RKNF33A groups.

[0098] The growth of nerve fibers after RK33A nanofibers and RK33A-2α nanofibers were implanted into rats with spinal cord injury. Figure 7Results showed that immunofluorescence staining of the lesion center 8 weeks after SCI showed that the expression of glial fibrillary acidic protein (Gfap) was reduced in the RKNF33A-2α and RKNF33A groups compared with the other groups, indicating a reduction in glial scarring at 8 wpi. When the tissue samples were stained for β-tubulin III (newborn neurons), the RKNF33A-2α group had more β-tubulin III than the SCI, RKNF33A, and PCL groups. + cells, indicating that RKNF33A-2α has a better function in promoting nerve regeneration than RKNF33A.

[0099] Further research was conducted on the polarization of peripheral microglia toward the M2 anti-inflammatory phenotype after RK33A nanofibers and RK33A-2α nanofibers were implanted into rats with spinal cord injury. Figure 8 The results showed that RKNF33A-2α cells presented more CD206 than the other three groups. + / Iba1 + This suggests that RKNF33A-2α promotes M2 polarization of microglia after SCI better than RK33A.

[0100] Example 6 This example further studies the mechanism by which RK33A-1Aα, RK33A-1Bα and RK33A-2α promote microglial M2 polarization.

[0101] Example 1 analyzes the number of hydrophilic and hydrophobic amino acids in the sequences shown in SEQ ID NO. 1-3. This example continues to analyze the specific amino acid composition of the sequences shown in SEQ ID NO. 1-3, as shown in Table 4.

[0102] Table 4 The results in Table 4 show that RK33A-2α has the largest number of glutamic acid compared to the other two peptides.

[0103] Furthermore, the mechanisms by which several recombinant proteins promoted microglial M2 polarization were verified.

[0104] Experimental methods: (1) COIP: The interaction between the three RK33A peptides, Icam-1, and Itgb2 was evaluated using the BeaverBeadsTM Protein A (or A / G) Immunoprecipitation Kit (Beaver) according to the kit protocol. BV2 cells were co-cultured with the three RK33A peptides for 24 hours.

[0105] (2) Collect the cells into 1.5 mL EP tubes. 5 Add binding buffer at a ratio of 20-30 μL per cell, and immediately add 1 mM PMSF. After mixing, place the EP tube on ice for 10 minutes. Collect the supernatant by centrifugation (4°C, 14,000 × g, 10 minutes) and place on ice.

[0106] (3) Place 50 μL of magnetic bead suspension into a 1.5 mL EP tube and vortex the immunoprecipitated magnetic beads for 1 minute to completely resuspend them. Wash the cells three times with 200 μL of binding buffer and separate them on a magnetic separator (Beyotime).

[0107] (4) Add 100 μL of antibody working solution (final concentration is 50 μg / mL anti-His antibody or 50 μg / mL Icam-1 antibody or non-specific IgG), quickly suspend, and place on a vertical mixer at room temperature. After 30 minutes, magnetically separate the cells for subsequent detection.

[0108] (5) Lysates were prepared before gently pipetting through the antigen and bead-antibody complexes until they were evenly dispersed. The mixture was placed in a stand mixer at 4°C overnight to allow complete binding of the antibody. The beads were washed three times with IP buffer. Finally, the immunoprecipitates were eluted by boiling the beads in 1× SDS-PAGE loading buffer, followed by SDS-PAGE and western blotting. To avoid the effect of RKs addition on Itgb2 expression, the relative reduction of Itgb2 was calculated and analyzed using the housekeeping gene β-tubulin.

[0109] The experimental results are shown in Fig. 9 . Fig. 9 It was shown that RK33A-1Aα, RK33A-1Bα and RK33A-2α can all bind to integrin b2 (Itgb2) on the cell membrane surface, thereby hindering the binding of Itgb2 and its natural receptor Icam 1, inhibiting the expression of the downstream Jak3 / Stat5 signaling pathway, promoting microglial M2 polarization, reducing inflammatory response, and promoting repair after spinal cord injury.

[0110] It is further speculated that the difference in effect between different α-helical segments may be attributed to glutamate (GLU). GLU is a key feature of integrin ligand binding motion. Since RK33A-2α has the largest number of GLU, it interacts with the amino acid sites of Itgb2, forming more diverse hydrogen bonds and salt bonds than other α-helical segments. Therefore, RK33A-2α has a greater affinity for Itgb2 than other segments, and has a more significant inhibitory effect on downstream Jak3 / Stat5.

[0111] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0112] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A recombinant active peptide having the effect of repairing neuroinflammation after spinal cord injury, characterized in that: The recombinant active peptide is modified based on keratin RK33A, and the structure of the recombinant active peptide contains at least two groups of α-helical fragments; the amino acid sequence of the recombinant active peptide is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. The recombinant active peptide according to claim 1, characterized in that: The recombinant active peptide structure also includes a group of non-helical fragments; the amino acid sequence of the recombinant active peptide is shown in SEQ ID NO.

3.

3. A recombinant expression vector, characterized in that: Comprising a gene encoding the recombinant active peptide according to claim 1 or 2.

4. A recombinant bacterium, characterized in that Expressing the recombinant active peptide according to claim 1 or 2.

5. A nanofiber for repairing neuroinflammation after spinal cord injury, characterized in that: The nanofiber is prepared from the recombinant active peptide according to claim 1 or 2 and polycaprolactone.

6. The nanofiber according to claim 5, characterized in that The mass ratio of the recombinant active peptide to the polycaprolactone is 8-10:

2.

7. Use of the nanofibers according to claim 5 in the preparation of medical materials for repairing neuroinflammation after spinal cord injury.

8. The use according to claim 7, characterized in that The medical material is in the form of an implantable stent.

9. A method for preparing nanofibers for repairing neuroinflammation after spinal cord injury, characterized in that: The following steps are involved: S01: dissolving a recombinant active peptide with an amino acid sequence as shown in SEQ ID NO.2 or SEQ ID NO.3 and polycaprolactone in hexafluoroisopropanol, and fully stirring the mixed solution; the mass ratio of the recombinant active peptide to the polycaprolactone is 8-10:2; S02: The mixed solution after being fully stirred is placed in a syringe, connected to a power source, the voltage and rotation are set, and the nanofibers are obtained by injecting the solution into a drum collector covered with aluminum foil at room temperature through electrospinning technology; S03: separating the nanofibers from the aluminum foil and curling them longitudinally to form a cylindrical stent; S04: sterilizing the collected nanofibers using 60 Co γ rays.

10. The preparation method according to claim 9, characterized in that Set the voltage to 15 kV, the rotation speed to 800-1000 rpm, and the flow rate to 1.5 mL h -1 .

Images