Interleukin 17 receptor IL-17RA targeted blocking peptide and application thereof

Abstract

The invention belongs to the technical field of biological medicine. In particular to an interleukin 17 receptor IL-17RA targeted blocking peptide and application thereof. The invention provides a targeting peptide AZG-1, and the amino acid sequence of the targeting peptide AZG-1 is shown as SEQ ID No.1. The targeting peptide has good tolerance, stereochemical characteristics of easy insertion and selective targets, and can generate effective interaction with the surface of a target protein; an embodiment verifies that the targeting peptide can realize specific binding with an IL-17RA / IL-17RC compound, has relatively high blocking capability, can effectively improve symptoms of liver and kidney cell injury, cell edema and skin injury caused by psoriasis after being applied to a psoriasis mouse model, and has a good application prospect in treatment and / or adjuvant treatment of psoriasis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an interleukin 17 receptor IL-17RA targeted blocking peptide and an application thereof. Background Art

[0002] Psoriasis is a chronic immune-mediated inflammatory skin disease. Under normal circumstances, the immune system protects the body from infection, but in patients with psoriasis, the innate immune pathway mediates the immune system to mistakenly attack its own skin cells, resulting in overgrowth and flourishing, forming plaques and scales. In genetically susceptible people, the actual onset of psoriasis can be triggered by stimuli such as mild mechanical trauma, upper respiratory tract streptococcal infection, stress, sunburn or certain systemic drugs (such as beta blockers, antimalarials, etc.). These stimuli induce the production of interleukin factors such as interleukin-17 (IL-17), IL-23, etc., which in turn change the function of keratinocytes and other skin tissue cells directly or through secondary mediators. Patients with psoriasis often have many comorbidities, including metabolic syndrome (diabetes, arterial hypertension, dyslipidemia and obesity), cardiovascular disease and depression. At present, the first-line treatments for psoriasis include topical treatment, phototherapy, and oral medications. However, clinical findings show that oral medications and immunosuppressants may cause more serious side effects, including immunosuppression, infection risk, liver damage, etc. The use of steroid drugs can easily cause skin thinning, bleeding, and other problems. Patients who use phototherapy for a long time may be at risk of skin cancer. Therefore, being able to effectively penetrate the lesion site, accurately block the specific innate immune pathways at the site of psoriasis inflammation, and inhibit the occurrence and development of inflammatory reactions has important scientific value and social significance in the treatment of psoriasis.

[0003] With the development of "precision medicine" and genomics, molecular targeted therapy has become an important option for the treatment of psoriasis. At present, targeted therapeutic drugs for psoriasis mainly include TGF-α inhibitors, IL-23 antibody inhibitors, IL-12 / 23 antibody inhibitors, IL-17 antibody inhibitors, etc., which improve the symptoms and course of psoriasis by regulating the immune system and reducing inflammatory responses. It is worth noting that the inhibition of inflammatory factor activity in inflammatory pathways is often accompanied by systemic immunosuppression, which in turn affects normal physiological activities. Existing drugs for the treatment of psoriasis often have poor targeting and large side effects; the effect is not good, the treatment is not thorough, and the quality of life of patients is reduced. Therefore, it is imperative to explore psoriasis-related inflammatory pathway receptor inhibitors, develop new targeted drugs, and directly act on skin functional cells to reduce inflammatory responses.

[0004] Interleukin 17 is a cytokine produced by the immune system that plays an important role in inflammatory responses and immunoregulation. The IL-17 family includes IL-17A to IL-17F, which play an important role in normal immune responses, especially against fungal and certain bacterial infections, as well as in tissue repair and protection of epithelial cells. However, IL-17 is associated with the occurrence of inflammatory and autoimmune diseases in some cases. In inflammatory diseases such as psoriasis, excessive production and signaling of IL-17 cause the immune system to attack its own tissues, causing excessive inflammatory responses and tissue damage. IL-17C and IL-17F show higher levels of expression in psoriasis, and experiments have shown that IL-17A is the most biologically active cytokine in the family (Blauvelt, A. & Chiricozzi, A. The immunologic role of IL-17 in psoriasis and psoriatic arthritis pathogenesis. Clin. Rev. Allergy Immunol. 55, 379–390 (2018).). IL-17A and IL-17F can function as homodimers or IL-17A / F heterodimers. IL-17A is mainly produced by Th17 cells. Meanwhile, innate immune cells, including ILC3 cells, γδT cells, neutrophils and mast cells, are also important sources of IL-17A in the development of psoriasis, and have received the most attention due to their proinflammatory effects in autoimmune diseases.

[0005] In psoriasis, the main receptor of IL-17A is IL-17RA, which activates IL-17RA-mediated signaling molecules such as TNF receptor-associated factor 6 (TRAF6) by binding to IL-17RA, activating the NF-κB signaling pathway, and ultimately triggering the activation of immune cells and inflammatory responses. IL-17RA is mainly expressed in a variety of skin cells, including keratinocytes, substratum corneum epidermal cells, fibroblasts, etc. The dimer formed by the binding of IL-17A / IL-17RA leads to excessive proliferation of the stratum corneum, forming the characteristic scales of psoriasis lesions, while promoting the stemness of keratinocytes, thereby promoting keratinocyte proliferation. Therefore, the development of IL-17RA inhibitors that precisely act on the receptors in the IL-17A / IL-17RA signaling pathway of psoriasis-related inflammation is expected to provide new candidate drugs for the clinical treatment of psoriasis. Summary of the invention

[0006] The purpose of the present invention is to provide a targeting peptide for blocking the binding of IL-17RA and IL-17A, and to provide a new candidate drug for the clinical treatment of psoriasis.

[0007] The present invention provides a targeting peptide AZG-1, and the amino acid sequence of the targeting peptide AZG-1 is shown as SEQ ID No.1.

[0008] The present invention also provides the use of the targeting peptide AZG-1 described in the above technical solution in blocking the binding between IL-17RA and IL-17A.

[0009] The present invention also provides the use of the targeting peptide AZG-1 described in the above technical solution in reducing liver and kidney cell damage caused by psoriasis.

[0010] The present invention also provides the use of the targeting peptide AZG-1 described in the above technical solution in improving cell edema caused by psoriasis.

[0011] The present invention also provides the use of the targeting peptide AZG-1 described in the above technical solution in alleviating skin damage caused by psoriasis.

[0012] The present invention also provides the use of the targeting peptide AZG-1 described in the above technical solution in the preparation of products for treating and / or assisting in the treatment of psoriasis.

[0013] The present invention also provides a product for treating and / or assisting in treating psoriasis, wherein the product comprises the targeting peptide AZG-1 described in the above technical solution.

[0014] Preferably, the product comprises a medicine.

[0015] Preferably, the concentration of the targeting peptide AZG-1 in the drug is 1-100 mg / kg.

[0016] Preferably, the dosage form of the drug includes injection and / or transdermal preparation.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides a targeting peptide AZG-1, the amino acid sequence of the targeting peptide AZG-1 is shown in SEQ ID No. 1, the targeting peptide has good tolerance, stereochemical properties that are easy to insert and selectable targets, and can effectively interact with the surface of a target protein; it is verified by examples that the targeting peptide can achieve specific binding to an IL-17RA / IL-17RC complex, has a high blocking ability, and after being applied to a psoriasis mouse model, can effectively improve the symptoms of liver and kidney cell damage, cell edema and skin damage caused by psoriasis, and has good application prospects in the treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 Schematic diagram of the process of screening targeting peptides for phage;

[0021] Figure 2 The 3D structure of the targeting peptide AZG-1;

[0022] Figure 3 This is the binding conformation of AZG-1 and IL-17RA / IL-17RC complex;

[0023] Figure 4 Western-blot validation results of the binding ability of AZG-1 to the IL-17RA / IL-17RC complex;

[0024] Figure 5 The confocal images of IL-17RA high and low cell lines MCF-7 and MCF10A after addition of FITC-AZG-1.

[0025] Figure 6 This is the Western blotting result of HACAT cells treated with AZG-1;

[0026] Figure 7 This is the validation result of the blocking ability of AZG-1 by ELISA method;

[0027] Figure 8 This is the HE staining result of the liver of the psoriasis mouse model after treatment;

[0028] Fig. 9 This is the HE staining result of the kidney of the psoriasis mouse model after treatment;

[0029] Fig.10 This is the HE staining result of the back skin of the psoriasis mouse model after treatment. DETAILED DESCRIPTION

[0030] The present invention provides a targeting peptide AZG-1, and the amino acid sequence of the targeting peptide AZG-1 is shown in SEQ ID No. 1, specifically: FYSSKRDWLQTN.

[0031] The examples show that the targeting peptide can achieve specific binding to the IL-17RA / IL-17RC complex, has a high blocking ability, and can effectively improve the symptoms of liver and kidney cell damage, cell edema and skin damage caused by psoriasis after being applied to the psoriasis mouse model. The concentration of the targeting peptide AZG-1 in the mouse psoriasis disease model is preferably 10 mg / kg.

[0032] Based on the above effects, the use of the targeting peptide AZG-1 in blocking the binding of IL-17RA and IL-17A, the use of the targeting peptide AZG-1 in reducing liver and kidney cell damage caused by psoriasis, the use of the targeting peptide AZG-1 in improving cell edema caused by psoriasis, and the use of the targeting peptide AZG-1 in alleviating skin damage caused by psoriasis should all fall within the protection scope of the present invention.

[0033] The present invention also provides the use of the targeting peptide AZG-1 described in the above technical solution in the preparation of products for treating and / or assisting in the treatment of psoriasis.

[0034] The present invention also provides a product for treating and / or assisting in treating psoriasis, wherein the product comprises the targeting peptide AZG-1 described in the above technical solution.

[0035] In the present invention, the product preferably includes a drug; the use concentration of the targeting peptide AZG-1 in the drug is preferably 1-100 mg / kg; the dosage form of the drug preferably includes an injection and / or a transdermal agent, and is more preferably an injection.

[0036] To further illustrate the present invention, the interleukin 17 receptor IL-17RA targeted blocking peptide and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] pass Figure 1 The phage screening process shown above yields targeting peptides

[0039] (1) Take two 96-well plates, named well plate 1 and well plate 2. Add 135 μL of 5 mg / mL BSA solution and 15 μL of 1 μg / μL IL-17RA&RC protein solution to each well of well plate 1. Add 15 μL of 1 μg / μL IL-17RA&RC protein solution to one well of well plate 2. Wrap the two well plates with a wet paper towel and mix overnight at 4°C for 3D.

[0040] (2) On the second day, discard the supernatant of well plate 1 and add 2 times the volume of 1 μg / μL IL-17A solution. Wrap the well plate with a wet paper towel and place it in a refrigerator at 4°C for 3D mixing for 2 h. Discard the supernatant, add 200 μL TBST to the well, make a cross and gently shake the well plate, discard the TBST, and repeat this operation three times.

[0041] (3) Add 1×10 11 Place the phages in a 3D shaker at room temperature and mix well for 1 hour.

[0042] (4) Discard the liquid in well plate No. 2, add 200 μL TBST to the well, and gently shake the well plate to discard the TBST by making a cross. Repeat this operation three times. Take the supernatant from well plate No. 1 and add it to well plate No. 2. Place it on a 3D shaker at room temperature and mix it for 1 hour. Discard the supernatant, add 200 μL TBST to the well, and gently shake the well plate to discard the TBST by making a cross. Repeat this operation three times.

[0043] (5) Prepare two solutions: 0.2m clycine-HCL pH=2.2 and 1M Tris-HCL pH=9.1, and filter them with a 0.22μm filter membrane. Add 150μL of the pH=2.2 solution to well plate 2, place it in a 3D shaker at room temperature, and mix and shake for 30 minutes. Add 150μL of the pH=9.1 solution to neutralize, and blow the bottom of the well plate to collect the phages bound to the bottom.

[0044] (6) Add the phage solution to the pre-shaken liquid culture medium of ER2738 Escherichia coli and incubate at 37°C, 200 rpm overnight.

[0045] (7) Centrifuge at 5000 rpm for 20 min to remove E. coli. After centrifugation, pour the supernatant into another conical flask, add 90 mL of NaCl-TEG8000 solution, shake the conical flask to mix, and leave at room temperature for 1 h.

[0046] (8) Centrifuge at 12000 rpm for 15 min to collect phages. Discard the supernatant, resuspend the phage precipitate with TBS solution, collect it in a 2 mL centrifuge tube, centrifuge at 8000 rpm for 10 min, remove E. coli again, transfer the supernatant to a new centrifuge tube, add 300 μL NaCl-TEG8000 solution, mix well, and incubate on ice for 1 h.

[0047] (9) Centrifuge at 13000 rpm for 10 min, discard the supernatant, add TBS to resuspend the phage precipitate, add 50% glycerol at a ratio of 1:1, and store in a -20°C refrigerator.

[0048] Repeat the above steps 3-5 times, and sequence the final concentrated phage solution to obtain its amino acid sequence. The final selected sequence is named AZG-1, and its amino acid sequence is specifically: FYSSKRDWLQTN (SEQ ID No. 1), and its three-dimensional structure is as follows Figure 2 shown.

[0049] Example 2

[0050] Computer simulation of docking

[0051] Molecular docking can predict the binding posture of ligand molecules and receptors and affinity analysis. Figure 2 The three-dimensional structure of the peptide constructed by Alphafold3 was used in the molecular docking of the peptide with IL-17RA and IL-17RC proteins (PDB: 7ZAN) using AutoDock4.2 to determine the binding site of the HA1 peptide on the IL-17RA or IL-17RC protein structure. In the molecular docking, the free function of the AZG-1 peptide and IL-17RA was used to evaluate the docking results to determine the most likely binding site of the AZG-1 peptide. AutoDock uses a fast energy evaluation method that combines a pre-calculated affinity potential grid with a Lamarckian genetic algorithm to quickly find the binding position of the ligand. First, the global docking of the AZG-1 peptide with IL-17RA and IL-17RC was performed. The entire receptor protein was contained in a grid frame so that the center of the grid frame coincided with the center of the protein, and IL-17RA and IL-17RC were kept rigid, and the AZG-1 peptide was kept flexible. The number of independent docking runs performed for each docking simulation was set to 100, and all other docking parameters were set to default values. Finally, the docking mode of AZG-1 and IL17RA was measured as follows Figure 3 shown.

[0052] Example 3

[0053] Western-blot to verify its binding ability

[0054] Biotin-AZG-1 was obtained by connecting Biotin-Mal and the polypeptide connected to the resin through a biotin-avidin system using a peptide synthesizer.

[0055] (1) Take out the frozen human immortalized keratinocytes (HACAT) from the -80℃ freezer. Thaw in a 37℃ water bath, transfer the cell suspension to a 15mL centrifuge tube filled with fresh DMEM medium, centrifuge at 1000rpm for 5min to remove the freezing solution, add fresh medium to resuspend the cell pellet, transfer to a T25 culture flask, gently shake the culture flask to evenly distribute the cells, and culture in a 37℃, 5% carbon dioxide constant temperature incubator.

[0056] (2) After the cells have grown to 95% of the bottom of the T25 cell culture flask after recovery, add 2 mL of trypsin containing 0.25% EDTA to digest the cells for 2 minutes, and then add 3 mL of fresh culture medium to stop digestion. Use a pipette to blow the cells attached to the bottom of the culture flask until all the cells are blown off from the bottom. Transfer the cell suspension to a 15 mL sterile centrifuge tube. Centrifuge at 1000 rpm for 5 minutes; discard the supernatant and add 3 mL of fresh culture medium. Take 20 μL of the cell suspension for counting. Plate 200,000 cells in each well of a six-well plate, and add fresh culture medium until the bottom of the well plate is covered. Gently shake the well plate in a cross shape to evenly distribute the cells on the bottom of the well plate. Place the well plate in an incubator and culture at 37°C, 5% CO2 for 24 hours. Transfer the remaining cells to a T25 culture flask and continue to culture for 24 hours.

[0057] (3) Take 0.5 mL sterile centrifuge tubes, add 25 μL Biotin-Va and 75 μL Biotin-AZG-1 to each centrifuge tube, vortex to mix, and leave at room temperature for 5 min. Add 25 μL streptavidin to each tube, vortex to mix quickly, and leave at room temperature for 30 min.

[0058] (4) Add the reagent in the centrifuge tube of step (3) to a centrifuge tube containing 875 μL of serum-free DMEM high-glucose medium. Take out the six-well plate laid out the day before from the incubator and mark CON and AZG-1 on the plate cover. Discard the medium in the wells and rinse with PBS to remove the serum. Add 1 mL of serum-free DMEM high-glucose medium to the wells marked CON and add the prepared drug to the wells marked AZG-1. Culture in a 37°C incubator. Extract the total protein of the cells after 24 hours.

[0059] (5) Extraction of whole cell protein: First, pour out the culture medium in the six-well plate, and then wash the cells three times with 4°C PBS to remove the culture medium. Add trypsin solution to each well of the six-well plate to digest the cells, and remove the cells from the well wall by blowing up and down, and then collect the cells in a 1.5 mL EP tube. Next, centrifuge at medium speed (8000 rpm / min, for 2 min) at 4°C to sediment the cells, remove the liquid, add a mixture of RIPA and PMSF (ratio 100:1) to the precipitated cells, and vibrate with a vortexer, once every 5 min, for a total of 4-5 times to ensure that the cells are completely broken. Then centrifuge again at 4°C (12000 rpm / min, 10 min), carefully transfer the supernatant to a new EP tube, record the transferred volume, and then store it on ice.

[0060] (6) Protein concentration determination: The protein concentration was detected and calculated using a BSA protein concentration detection kit (purchased from MeilunBio, catalog number MA0082).

[0061] (7) Cooking protein samples: Mix the protein sample with a 5-fold diluted protein buffer at a volume ratio of 1:4. Heat the mixture into a 100°C metal solution and continue heating for 20 min. Then store the prepared protein sample at -20°C or -80°C, or use it for subsequent experiments.

[0062] (8) Western Blot detection of protein expression: First, when assembling the electrophoresis device, place two glass plates according to the rule of placing the short plate on the inside and the tall plate on the outside. After the device is ready, pour a little water to test for leaks and check whether the electrophoresis tank is leaking; after confirming that there is no leakage, drain the water and absorb the remaining water traces. According to the specific preparation method, we mixed about 7mL of separation gel and 3mL of concentrated gel. First, use a pipette to slowly add separation gel from one side, and then add 1mL of isopropanol to ensure that the liquid level is stable. Next, wait for about 20 minutes for the gel to solidify. After pouring off the isopropanol, use filter paper to absorb the residual liquid on the surface, then pour the pre-prepared concentrated gel and insert it into the comb. When the comb-like gel is completely solidified, apply a layer of glue film on the comb layer. After about 1 hour, place the gel plate in the electrophoresis tank, add sufficient Running Buffer, ensure that the liquid level completely covers the gel plate, and then remove the comb to load the sample.

[0063] For the loading and electrophoresis steps, the target protein and marker were injected into the loading wells in sequence according to the experimental grouping order. The marker required 3 μL and the loading amount of the target protein was 10 μg. The loading volume was calculated based on the standard curve. After the loading was completed, the lid was closed, the power supply was connected to the positive and negative poles, and the voltage parameter was set to 80V constant voltage operation. When the marker moved over the concentrated gel, the voltage was increased to 120V and continued to run until the bromophenol blue was close to the edge of the gel and the power was turned off.

[0064] When the transfer process begins, cut the PVDF membrane into a size that matches the gel and soak it in methanol for 5 minutes to activate it, so that it has positive ions so that it can bind to proteins more easily. Add transfer solution to the operating tray. After the electrophoresis is completed, take out the membrane and transfer it to the transfer solution. Open the transfer clip, with the black negative electrode facing down, and stack the asbestos mesh-filter paper-gel-PVDF membrane-filter paper-asbestos mesh on the transfer clip in sequence, ensuring that the PVDF membrane is tightly combined with the gel and releasing bubbles. Place the transfer clip in the transfer tank with an ice pack and adjust it to ensure that the black negative electrode faces the negative electrode in the tank and the white positive electrode faces the positive electrode in the tank. After adding the transfer solution, transfer the membrane with a constant voltage of 100V for 120 minutes and transfer on ice.

[0065] After the transfer process is completed, the PVDF membrane needs to be removed and carefully checked whether the marker has completely migrated to the surface of the membrane. Place the protein surface of the PVDF membrane upward, then soak it in the pre-prepared blocking solution and shake it slowly on a shaker at room temperature for 1 hour.

[0066] Then, according to the location of the target protein, the treated membrane was cut and placed in the corresponding primary antibody incubation box. At room temperature, it can be incubated by slow shaking for 2 hours, or placed on a shaker at 4°C overnight. After the primary antibody incubation, it was recovered, 1×TBST buffer was added, and it was placed on a shaker for 8 minutes, and it was performed 4 times in total.

[0067] Finally, after washing, the membrane was placed in an incubation box containing the secondary antibody and slowly shaken for 2 hours at room temperature. After the secondary antibody incubation was completed, we recovered the secondary antibody and used 1×TBST for a quick 8-minute membrane wash, completing a total of 4 operations. After washing, the working solution was prepared based on a 1:1 ratio of the developing substrate A solution to the B solution, and stored in a dark place. The exposure solution was evenly covered on the PVDF membrane, exposed using a gel imager, and photographed and recorded. The results are shown below. Figure 4 shown.

[0068] Depend on Figure 4It can be seen that compared with the CON group, the IL17RA protein on the surface of the three cell membranes was degraded by the biotin-avidin system after the addition of AZG-1, indicating that AZG-1 has the ability to bind to the IL-17RA / IL-17RC complex.

[0069] Example 4

[0070] Fluorescence photography verifies its binding ability

[0071] Human breast cancer cells (Michigan Cancer Foundation-7, MCF-7) and human normal breast cells (MCF10A) with high and low expression of IL-17RA were selected respectively. Cell recovery and plating were the same as in Example 3, FITC-AZG-1 was added, and laser confocal imaging was performed after incubation at 37°C for 2 hours. The results are shown in FIG. Figure 5 shown.

[0072] Figure 5 The two pictures in the middle are the results of confocal imaging. Figure 5 It can be seen that after adding FITC-AZG-1, it binds to the surface of MCF-7 cells that highly express IL-17RA, while there is no FITC-AZG-1 binding to the surface of MCF-10A cells that do not express IL-17RA, indicating that AZG-1 has binding ability.

[0073] Example 5

[0074] Western-blot verification of blocking ability

[0075] HACAT cells were selected for this experiment, and the cell treatment process was as follows Figure 6 As shown in the middle left picture, specifically:

[0076] IL-17A can activate the NF-κB pathway. After the pathway is activated, the P65 protein will undergo nuclear translocation. After the cells are treated, the nuclear protein and plasma protein are extracted to detect the P65 protein content to know the blocking effect of the polypeptide. The cell recovery and plating operations are the same as in Example 3. Three wells in the six-well plate are marked with con, and three wells are marked with AZG-1; the three wells marked with con are not treated, and 5 μL of AZG-1 at a concentration of 100 μM is added to each of the other three wells. Return to the incubator and continue to culture. After 2h, 10 μL of IL-17A solution at a concentration of 10 ng / μL is added to all wells. Return to the incubator and continue to culture for 24h. Extract nuclear protein and plasma protein.

[0077] Nuclear protein and plasma protein extraction steps:

[0078] 1. Prepare the solution: Dissolve the three reagents in the kit at room temperature, place on ice immediately after dissolution, and mix well. Take an appropriate amount of cytoplasmic protein extraction reagent A for use, add PMSF within a few minutes before use, so that the final concentration of PMSF is 1mM. Take an appropriate amount of nuclear protein extraction reagent for use, add PMSF within a few minutes before use, so that the final concentration of PMSF is 1mM.

[0079] 2. Cell treatment: Wash once with PBS, scrape off the cells with a cell scraper, or treat the cells with EDTA solution so that the cells are no longer tightly attached to the wall, and blow off the cells with a pipette. Collect the cells by centrifugation, and do your best to suck out the supernatant, leaving the cell pellet for later use. Try to avoid digesting the cells with trypsin to prevent the trypsin from degrading the target protein to be extracted.

[0080] 3. Add 200 μL of cytoplasmic protein extraction reagent A with PMSF added to every 20 μL of cell pellet. (For 2 million HeLa cells, the volume of the cell pellet is approximately 20 μL or 40 mg.)

[0081] 4. Vortex vigorously at the highest speed for 5 seconds to completely suspend and disperse the cell pellet. (If the cell pellet is not completely suspended and dispersed, the vortex time can be appropriately extended.)

[0082] 5. Ice bath for 10-15 minutes.

[0083] 6. Add 10 μL of cytoplasmic protein extraction reagent B. Vortex vigorously at the highest speed for 5 seconds and place on ice for 1 minute.

[0084] 7. Vortex vigorously at maximum speed for 5 seconds and centrifuge at 12000-16000g for 5 minutes at 4°C.

[0085] 8. Immediately pipette the supernatant into a pre-cooled plastic tube, which is the extracted cytoplasmic protein. It can be used immediately or frozen. (Do not touch the precipitate. You can keep a very small volume of supernatant above the precipitate to avoid touching the precipitate. The supernatant obtained after lysing 2 million cells with 200 μL of cytoplasmic protein extraction reagent A in this product has a cytoplasmic protein concentration of about 2-5 mg / mL, which varies from cell to cell.)

[0086] 9. For the precipitate, completely aspirate the remaining supernatant and add 50 μL of nuclear protein extraction reagent with PMSF added. (Failure to aspirate the supernatant will result in contamination of cytoplasmic proteins.)

[0087] 10. Vortex vigorously at the highest speed for 15-30 seconds to completely suspend and disperse the cell pellet. Then return to the ice bath and vortex vigorously at high speed for 15-30 seconds every 1-2 minutes for a total of 30 minutes.

[0088] Centrifuge at 12000-16000g for 10 min at 11.4°C.

[0089] 12. Immediately aspirate the supernatant into a pre-cooled plastic tube, which is the extracted nuclear protein.

[0090] The subsequent protein treatment and electrophoresis steps were the same as in Example 3. Figure 6 shown.

[0091] Figure 6 The middle right figure is the Western blotting result after treatment. It can be seen from the figure that compared with the control group (con), the degree of nuclear translocation of P65 protein is reduced after the addition of AZG-1, indicating that AZG-1 has a certain blocking effect.

[0092] Example 6

[0093] ELISA to verify blocking ability

[0094] The blocking ability of AZG-1 was quantitatively analyzed by building an ELISA blocking reagent platform. The specific steps are as follows:

[0095] 1. Solution Preparation

[0096] Washing buffer: PBS + 0.05% Tween 20;

[0097] Reagent buffer: 1% BSA;

[0098] Blocking buffer: 2% BSA.

[0099] 2. Coating

[0100] Dilute the Human IL-17RA stock solution (1μg / μL) to 1μg / mL with coating buffer to prepare the Human IL-RA working solution. Add 100μL of the working solution to each well, leaving a few wells uncoated as NO-coating controls. Seal the plate and shake overnight at 4℃.

[0101] 3. Wash

[0102] First pour out the liquid in the wells, then add 300μL washing buffer to each well, shake with 3D shaker for 1 min, discard the supernatant, then turn the well plate upside down on paper, tap and dry, and repeat 3 times.

[0103] 4. blocking

[0104] Add 300 μL bocking buffer to each well, seal with a membrane, and incubate at 37°C for 1.5 h.

[0105] 5. Wash

[0106] First pour out the liquid in the wells, then add 300μL washing buffer to each well, shake with 3D shaker for 1 min, discard the supernatant, then turn the well plate upside down on paper, tap and dry, and repeat 3 times.

[0107] 6. Sample loading (Brodalumab, AZG-1)

[0108] The monoclonal antibody and AZG-1 were diluted with 1% BSA, the working concentration of the monoclonal antibody was 1 μM, and the working concentration of AZG-1 was 10 μM. 100 μL of sample solution was added to each well, and Reagent buffer was added to the NO-coating wells, and the plate was sealed.

[0109] 7. Binding

[0110] Use Reagent buffer to dilute Biotin-IL-17A (50 μg / mL) to 0.1 μg / mL to make Biotin-IL-17A working solution. Add 50 μL Reagent buffer to the NO-coating wells and 50 μL Biotin-IL-17A working solution to other wells. Tap to mix, seal the plate, and incubate at 37°C for 1 h.

[0111] 8. Wash

[0112] First pour out the liquid in the wells, then add 300μL washing buffer to each well, shake with 3D shaker for 1min, discard the supernatant first, then turn the well plate upside down on paper, tap and dry, repeat 3 times.

[0113] 9. Add Streptavidin-HRP

[0114] Dilute the Streptavidin-HRP stock solution (50 μg / mL) to 0.1 μg / mL with substrate buffer to prepare the Streptavidin-HRP working solution. Add 100 μL to each well, seal the plate, and incubate at 37°C in the dark for 1 hour.

[0115] 10. Wash

[0116] First pour out the liquid in the wells, then add 300μL washing buffer to each well, shake with 3D shaker for 1min, discard the supernatant first, then turn the well plate upside down on paper, tap and dry, repeat 3 times.

[0117] 11. Substrate reaction

[0118] Add 100 μL TMB to each well, seal the plate, and incubate at 37°C in the dark for 20 min.

[0119] 12. Termination

[0120] Add 50 μL of stop solution to each well and gently shake the plate to mix thoroughly (blue → yellow).

[0121] 13.Measure absorbance

[0122] The absorbance at 450nm-630nm of the ELISA reader is the final result. Figure 7 shown.

[0123] Figure 7 In the figure, PBS was the negative control group and the monoclonal antibody was the positive control group. "****" in the figure means p < 0.0001. By comparing the two, it was found that AZG-1 had a good blocking effect.

[0124] Example 7

[0125] In vivo experiments to verify blocking ability

[0126] Construction of mouse psoriasis disease model

[0127] Six-week-old female Balb / c mice were selected. After one week of breeding, the mice were adapted to the growth environment and randomly divided into four groups, namely, CON, AZG-1, and MTX (methotrexate), with 6 mice in each group, and treated as follows:

[0128] Normal group: normal feeding, no treatment;

[0129] PBS group: The backs of the mice were depilated, 5% imiquimod cream was applied every day, the backs of the mice were photographed every day, and when wrinkles appeared on the back skin of the mice, PBS was injected intraperitoneally into the mice, 100 μL per mouse each time;

[0130] AZG-1 group: The backs of the mice were depilated, 5% imiquimod cream was applied daily, and the backs of the mice were photographed daily. When wrinkles appeared on the back skin of the mice, the mice were given medication via intraperitoneal injection at a concentration of 10 mg / kg of AZG-1. Subsequently, psoriasis was induced and medication was given once a day for one week.

[0131] MTX group: The backs were depilated, 5% imiquimod cream was applied daily, the backs of the mice were photographed daily, and when wrinkles appeared on the back skin of the mice, the mice were given medication by intraperitoneal injection. The MTX concentration was 1 mg / kg, and the injection was performed once every two days. Subsequently, psoriasis was induced every day for one week.

[0132] After the administration, the mice were killed, and their liver, kidney and back skin were taken for HE staining to detect the biological safety of AZG-1 and the therapeutic effect of psoriasis. The results are as follows: Figure 8-Figure 10 shown.

[0133] Figure 8-Figure 10 The following are the HE staining results of the liver, kidney, and back skin of psoriasis mice after treatment. Figure 8 and Fig. 9 It can be seen that the liver and kidney cells of the mice in the PBS control group were fragmented and necrotic, with unclear structures and edema. The cells in the AZG-1 and MTX treatment groups were neatly arranged, with uniform cytoplasm coloring and clear nuclei, indicating that AZG-1 has no cytotoxicity and has extremely high biosafety. Fig.10 It can be seen that the stratum corneum of the back skin in the PBS group was obviously keratinized, the granular layer in the epidermis was thinned, and the granular layer was thinned, which showed obvious symptoms of psoriasis. After treatment with AZG-1 and MTX, various indicators were significantly improved.

[0134] It can be seen from the above examples that the polypeptide AZG-1 provided by the present invention can effectively block the binding of IL-17RA and IL-17A and has good biosafety, and provides a new candidate drug for the clinical treatment of psoriasis.

[0135] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A targeting peptide AZG-1, characterized in that: The amino acid sequence of the targeting peptide AZG-1 is shown in SEQ ID No.

1.

2. Use of the targeting peptide AZG-1 according to claim 1 in blocking the binding between IL-17RA and IL-17A.

3. Use of the targeting peptide AZG-1 according to claim 1 in reducing liver and kidney cell damage caused by psoriasis.

4. Use of the targeting peptide AZG-1 according to claim 1 in improving cell edema caused by psoriasis.

5. Use of the targeting peptide AZG-1 according to claim 1 in alleviating skin damage caused by psoriasis.

6. Use of the targeting peptide AZG-1 according to claim 1 in the preparation of products for treating and / or assisting in the treatment of psoriasis.

7. A product for treating and / or assisting in the treatment of psoriasis, characterized in that: The product comprises the targeting peptide AZG-1 according to claim 1.

8. The product according to claim 7, characterized in that The products include pharmaceuticals.

9. The product according to claim 8, characterized in that The use concentration of the targeting peptide AZG-1 in the medicine is 1-100 mg / kg.

10. The product according to claim 9, characterized in that The dosage forms of the drug include injections and / or transdermal preparations.

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