Polypeptide and lysosome targeting chimera targeting IL17RA and application of polypeptide and lysosome targeting chimera in treatment of psoriasis

Abstract

The invention discloses polypeptide and lysosome targeted chimera targeting IL17RA and application of the polypeptide and the lysosome targeted chimera in treatment of psoriasis, and relates to the field of biological medicine, an amino acid sequence of the polypeptide targeting IL17RA has at least 80% identity with a sequence shown in SEQ ID NO: 1 or 2, and the polypeptide targeting IL17RA has high affinity for IL-17RA protein, can effectively block combination of the IL-17RA protein and IL-17A protein, and can be used for treating psoriasis. The lysosome targeting chimera constructed by using the polypeptide molecule has an excellent treatment effect and good biological safety on psoriasis, and has a good application prospect in treatment or adjuvant treatment of psoriasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide targeting IL17RA and a lysosome targeting chimera and applications thereof in the treatment of psoriasis. Background Art

[0002] Psoriasis is a common multi-gene inherited skin disease, which often manifests as localized or widespread scaly erythema or plaques. The pathogenesis of psoriasis is still unclear, but it is believed to be caused by the combined effects of many factors, including genetic factors and environmental factors.

[0003] There are many types of psoriasis, the most common of which is vulgaris, in addition to arthritis, pustular and erythrodermic types. The symptoms of psoriasis are diverse, mainly including red patches on the skin, usually covered with silvery white scales, itching, burning or pain, dry, cracked skin, and even bleeding. Some patients may also experience joint pain, swelling and abnormal nails.

[0004] Developing drugs that can effectively treat or improve psoriasis is one of the urgent issues that need to be addressed today.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a polypeptide targeting IL17RA and a lysosome targeting chimera and use thereof in the treatment of psoriasis.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a polypeptide, whose amino acid sequence has at least 80% identity with the sequence shown in SEQ ID NO: 1 or 2.

[0009] In a second aspect, an embodiment of the present invention provides a lysosome-targeting chimera, comprising: an aptamer targeting IL-17RA protein; the aptamer targeting IL-17RA protein comprises the polypeptide described in the aforementioned embodiment.

[0010] In a third aspect, an embodiment of the present invention provides a composition, which includes: raw materials for preparing the lysosome targeting chimera described in the above embodiments.

[0011] In a fourth aspect, an embodiment of the present invention provides a method for preparing the lysosome targeting chimera as described in the preceding embodiments, comprising: preparing the chimera using the composition described in the preceding embodiments.

[0012] In a fifth aspect, embodiments of the present invention provide use of the polypeptide described in the preceding embodiments or the lysosome targeting chimera described in the preceding embodiments in the preparation of a product for treating or assisting in the treatment of psoriasis or improving related clinical symptoms caused by psoriasis.

[0013] The present invention has the following beneficial effects:

[0014] The present invention provides a new polypeptide targeting IL-17RA, which has a high affinity for IL-17RA protein and can effectively block the binding of IL-17RA protein and IL-17A protein. The lysosome targeting chimera constructed using the polypeptide molecule has excellent therapeutic effect and good biosafety for psoriasis, and has good application prospects in the treatment or adjuvant treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 Schematic diagram of the phage screening process;

[0017] Figure 2 Schematic diagram of the structure of the lysosome-targeting chimera TdlTac-17RA;

[0018] Figure 3 The molecular docking results of CZG-7 and IL-17RA protein;

[0019] Figure 4 Western blot results showing that TdlTac-17RA targetedly degraded IL-17RA protein;

[0020] Figure 5 To verify the ELISA results of CZG-7 blocking ability;

[0021] Figure 6 The therapeutic effect of CZG-7 on psoriasis mice;

[0022] Figure 7 HE staining results of the back skin of psoriasis mice after CZG-7 treatment;

[0023] Figure 8 HE staining results of the liver of psoriasis mice after CZG-7 treatment;

[0024] Fig. 9HE staining results of kidneys of psoriasis mice after CZG-7 treatment;

[0025] Fig.10 HE staining results of the back skin of psoriasis mice after treatment with TdlTac-17RA. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0027] Current studies have shown that multiple immune signaling pathways are involved in the occurrence and deterioration of psoriasis. Helper T (Th) 1 cells and Th17 cells are believed to play an important role in the pathogenesis of psoriasis. Studies have found that the proliferation of Th1, Th17, and Th22 cells in patients with psoriasis leads to the production of a large number of proinflammatory mediators, including tumor necrosis factor-α (TNF-α), interferon and necrosis factor-α (TNF-α). IFN-c and interleukin (IL)-6, interleukin (IL)-17, interleukin (IL)-23, these proinflammatory mediators cause psoriasis lesions, among which the interleukin 17A (IL-17A) pathway is a key axis in the pathogenesis of psoriasis, which stimulates the production of antimicrobial peptides, attracts inflammatory cell infiltration, and aggravates the inflammatory response of psoriasis. At present, biological treatment of psoriasis has achieved certain clinical efficacy. The UK Psoriasis Guidelines 2020 covers biologics that are already available or about to be available in the UK, including TNF-α inhibitors, IL-12 / 23 (IL-12 / 23p40) inhibitors, IL-23 (IL-23p19) inhibitors, IL-17A inhibitors, IL-17RA inhibitors, etc. In psoriasis, the main receptor for IL-17A is IL-17RA. IL-17RA-mediated signaling molecules such as TNF receptor-associated factor 6 (TRAF6) activate the NF-κB signaling pathway by binding to IL-17RA, ultimately triggering immune cell activation and inflammatory response.

[0028] The embodiments of the present application provide a new polypeptide, which has a high affinity with the IL-17RA protein and can construct a lysosome-targeted chimera to target and degrade the IL-17RA protein to inhibit the activation of the IL-17 pathway, thereby inhibiting the occurrence of inflammatory response to treat psoriasis.

[0029] In one aspect, an embodiment of the present invention provides a polypeptide whose amino acid sequence is at least 80% identical to the sequence shown in SEQ ID NO: 1 or 2.

[0030] The at least 80% identity refers to a sequence identity of any one or any two of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100%. Identity refers to the proportion of sequences in which the nucleotides or amino acid residues in the same position of two sequences are exactly the same.

[0031] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1 or 2.

[0032] On the other hand, an embodiment of the present invention provides a lysosome targeting chimera, comprising: an aptamer targeting IL-17RA protein; the aptamer targeting IL-17RA protein comprises the polypeptide described in any of the aforementioned embodiments.

[0033] In some embodiments, the lysosome targeting chimera further comprises: an aptamer for a lysosome targeting receptor linked to the aptamer targeting the IL-17RA protein.

[0034] In some embodiments, the molar ratio of the aptamer targeting IL-17RA protein to the aptamer targeting lysosomal receptor is 1 to 6: 1 to 2. The molar ratio can be any one of 1: 1, 1: 2, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, or a range between any two of them.

[0035] In some embodiments, the aptamer for a lysosomal targeting receptor comprises an aptamer targeting IGFIIR.

[0036] In some embodiments, the aptamer of the lysosomal targeting receptor comprises retinoic acid. Optionally, the retinoic acid is ATRA.

[0037] In some embodiments, the aptamer targeting IL-17RA protein is directly linked to the aptamer targeting lysosomal receptor or is linked via a linker system.

[0038] In some embodiments, the linking system comprises a biotin-avidin linking system.

[0039] In some embodiments, the connection system includes biotin, a linker and avidin; the aptamer targeting IL-17RA protein and the aptamer targeting lysosomal receptor are respectively connected to biotin via the linker, and the biotin can non-covalently bind to the avidin.

[0040] In some embodiments, the avidin includes any one or more of avidin and streptavidin.

[0041] In some embodiments, the linker comprises PEG.

[0042] In some embodiments, the molecular weight of the PEG is 1k to 10k, specifically any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10k or a range between any two thereof.

[0043] On the other hand, an embodiment of the present invention provides a composition, which includes: a raw material for preparing the lysosome targeting chimera described in any of the above embodiments.

[0044] In some embodiments, the raw material includes: the aptamer targeting IL-17RA protein described in any of the preceding embodiments and the aptamer targeting lysosomal receptor described in any of the preceding embodiments.

[0045] In some embodiments, the raw material further comprises: a connection system as described in any of the preceding embodiments.

[0046] On the other hand, an embodiment of the present invention provides a method for preparing the lysosome targeting chimera as described in any of the aforementioned embodiments, comprising: preparing the chimera using the composition described in any of the aforementioned embodiments.

[0047] In some embodiments, the preparation comprises the following steps:

[0048] The aptamer targeting IL-17RA protein and the aptamer targeting lysosome receptor are respectively coupled to biotin through a linker to obtain the aptamer targeting IL-17RA protein coupled with biotin and the aptamer targeting lysosome receptor coupled with biotin;

[0049] Mixing the aptamer targeting IL-17RA protein coupled with biotin, the aptamer targeting lysosome receptor coupled with biotin, and avidin to obtain a lysosome targeting chimera;

[0050] The molar ratio of the aptamer targeting IL-17RA protein coupled with biotin, the aptamer targeting lysosomal receptor coupled with biotin and avidin is 1-2:1-6:1-2. The molar ratio can be any one of 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 2:3:1, 1:3:2 or a range between any two of them.

[0051] In addition, embodiments of the present invention also provide use of the polypeptide described in any of the foregoing embodiments or the lysosome targeting chimera described in any of the foregoing embodiments in the preparation of a product for treating or assisting in the treatment of psoriasis or improving related clinical symptoms caused by psoriasis.

[0052] In some embodiments, the improvement of relevant clinical symptoms caused by psoriasis includes: improving any one or more of liver cell damage, kidney cell damage, cell edema and skin damage caused by psoriasis.

[0053] In some embodiments, the product comprises a reagent or a kit.

[0054] As used herein, "treat" includes preventing or alleviating a condition, reducing the rate at which a condition develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0055] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0056] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0057] Example 1: Phage screening process to obtain targeting peptides

[0058] The process of obtaining the targeting peptide during phage screening is as follows, which can be referred to Figure 1 .

[0059] (1) Take two 96-well plates and set them as well plates 1 and 2. Add 135 μl of 5 mg / ml BSA solution and 15 μl of 1 μg / μl IL-17RA protein solution to each well of well plate 1. Then add 15 μL of 1 μg / μL IL-17RA protein solution to each well of well plate 2. Wrap the two well plates with a wet paper towel and mix overnight at 4°C on a 3D shaker.

[0060] (2) The next day, pour out the supernatant in well plate 1 and add 2 times the volume of IL-17A (solution concentration is 1 μg / μL). Wrap the well plate with a wet paper towel and mix on a 3D shaker in a refrigerator at 4°C for 2 h. Pour out the supernatant, add 200 μL TBST to each well, draw a cross and gently shake the well plate, pour out the TBST, and repeat three times.

[0061] (3) 1×10 11 Place the phages into the well with protein in well plate No. 1 and mix well on a 3D shaker at room temperature for 1 hour.

[0062] (4) Pour out the liquid in well plate No. 2, add 200 μL TBST to each well, draw a cross and gently shake the well plate to discard the TBST, repeat three times. Add the supernatant in well plate No. 1 to well plate No. 2. Mix and shake on a 3D shaker at room temperature for 1 hour. Pour out the supernatant, add 200 μL TBST to each well, draw a cross and gently shake the well plate to discard the TBST, repeat three times.

[0063] (5) Prepare two solutions of 0.2m clycine-HCL (pH = 2.2) and 1M Tris-HCL (pH = 9.1) in advance and filter them with a 0.22μm filter membrane. Add 150μL of 0.2m clycine-HCL (pH = 2.2) solution to well plate 2 and shake on a 3D shaker at room temperature for 30 minutes. Add 150μL of 1M Tris-HCL (pH = 9.1) solution for neutralization and resuspend and collect the phages at the bottom.

[0064] (6) Add the phage solution in (5) to the liquid culture medium of ER2738 Escherichia coli and incubate at 37°C, 200 rpm overnight.

[0065] (7) The next day, centrifuge the solution at 5000 rpm for 20 min to remove E. coli. Take the supernatant and transfer it to another conical flask. Add 90 mL of NaCl-TEG8000 solution, shake to mix, and leave at room temperature for 1 h.

[0066] (8) Centrifuge at 12,000 rpm for 15 min to collect phages, discard the supernatant, resuspend with TBS solution, and place in a 2-mL centrifuge tube. Centrifuge at 8,000 rpm for 10 min to 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.

[0067] (9) Centrifuge at 13,000 rpm for 10 min, discard the supernatant, and add TBS to resuspend.

[0068] 50% glycerol was added in a 1:1 volume ratio and finally stored in a -20°C refrigerator.

[0069] The above steps were repeated 3-5 times, and finally the phage solution was collected for sequencing to obtain its amino acid sequence QLRPLVR (SEQ ID NO: 1). Molecular docking was performed by computer simulation for sequence optimization, and the final selected sequence was named CZG-7, and its amino acid sequence was specifically: QLRPLFR (SEQ ID NO: 2).

[0070] Example 2: Preparation of lysosome-targeted chimera TdlTac-17RA

[0071] (1) Retinoic acid small molecules targeting IGFⅡR coupled with biotin:

[0072] 1mmol of retinoic acid small molecule targeting IGFⅡR was connected to 1mmol of PEG 5k coupled with biotin (Biotin-PEG 5k-NH3) through an amide bond through its own carboxyl group to synthesize an aptamer targeting IGFⅡR coupled with biotin (V, Biotin-PEG 5k-Va).

[0073] (2) Method for coupling peptide aptamers targeting membrane proteins with biotin:

[0074] 1mmol of the aptamer targeting IL-17RA protein (peptide CZG-7) was linked to 1mmol of PEG 5k coupled with biotin (Biotin-PEG 5k-COOH) through an amide bond via the amino group at the N-terminus to synthesize a peptide ligand targeting membrane protein coupled with biotin (P, Biotin-PEG 5k-CZG-7).

[0075] (3) The method for preparing a lysosome-targeted chimera targeting IL-17RA protein degradation comprises:

[0076] The aptamer targeting IGFⅡR coupled with biotin, the polypeptide ligand targeting membrane protein coupled with biotin and streptavidin were mixed at a molar ratio of 1:3:1, connected through an affinity-biotin system, and then synthesized into a lysosome-targeting chimera TdlTac-17RA.

[0077] The structure of the lysosomal targeting chimera TdlTac-17RA is shown in Figure 2 shown.

[0078] Molecular docking technology was used to predict the binding posture of TdlTac-17RA with the receptor and affinity analysis. AutoDock was used to perform molecular docking of TdlTac-17RA and IL-17RA / IL-17RC proteins in order to determine the binding site of TdlTac-17RA on the IL-17RA protein structure. The docking results were evaluated by analyzing the free functions of TdlTac-17RA and IL-17RA proteins to determine the most likely binding site of TdlTac-17RA. AutoDock is able to find the binding site of the ligand by combining the calculated affinity potential energy grid with the Lamarckian genetic algorithm for rapid energy evaluation. The docking mode of TdlTac-17RA and IL-17RA protein is shown in the figure. Figure 3As shown, the global docking of TdlTac-17RA and IL-17RA proteins was performed so that the receptor protein was contained in the grid box, with the goal of coinciding the center of the grid box with the center of the protein, so that the IL-17RA protein could remain rigid and the CZG-7 polypeptide could remain 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.

[0079] Example 3: Western analysis of the dual-targeted degradation ability of lysosome-targeted chimeras for targeted protein degradation on cell membrane proteins.

[0080] 1. Digest the HaCat cells cultured in a T25 culture flask with EDTA-trypsin, transfer to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody, and count using a cell counting plate. Take a six-well plate and place an appropriate amount of cell suspension in it, 2×10 cells per well. 6 The plate was gently shaken to make the cells evenly distributed, and then placed in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, the cells adhered to the wall and the drug was started.

[0081] 2. Take 4 0.5ml centrifuge tubes, add 10μL of 20μM retinoic acid small molecules coupled with biotin to each centrifuge tube, add 30μL of 20μM CZG-7 coupled with biotin, place at room temperature for 5min, add 10μL of 20μM s-avidin, immediately vortex mix, and place at room temperature for 30min to obtain lysosome-targeted chimera TdlTac-17RA. Add culture medium (without serum and double antibody) to each centrifuge tube to a total volume of 1ml. Pour out the culture medium in the well plate, rinse with PBS, pour out PBS again, add the drug system in turn, directly add 1ml culture medium (without serum and double antibody) to the control group, and gently shake the well plate to make the culture medium evenly distributed. Place in an incubator for culture at 37℃, 5% CO2 for 10h, then add 1ml DMEM high-glucose culture medium (containing 10% FBS, 1% double antibody) to each well, and then culture for 24h, collect cells, and extract cell protein.

[0082] 3. Pour out the culture medium in the well plate, rinse with pre-cooled PBS, pour out the PBS, dissociate the cells from the bottom of the well plate with a cell scraper or a cell dissociation solution containing EDTA but not trypsin, transfer the cells to a 1.5ml centrifuge tube, centrifuge at 8000rpm for 2min to collect the cells, pour out the supernatant, centrifuge at 8000rpm for 30sec, gently aspirate the supernatant with a 200μL pipette, add cell lysis solution (RIPA:PMSF=100:1) according to the amount of cells, vortex the cell pellet with a vortexer, place it on ice for lysis, vortex once every ten minutes, repeat 3 times. Centrifuge at 12000rpm for 10min in a centrifuge. Then prepare the working solution, A solution: B solution=50:1, take 4 new 1.5ml centrifuge tubes, add 27μL water, 600μL working solution and 3μL supernatant (after centrifugation) to each centrifuge tube, vortex evenly, and bake at 37℃ for 30min. Take a new 96-well plate, take 180 μL of each sample and add it to the 96-well plate. Repeat three times for each sample, measure the absorbance at a wavelength of 562 nm, and substitute the absorbance into the standard curve prepared in advance to calculate the concentration and loading volume. After centrifugation, place the remaining supernatant in a new 1.5 ml centrifuge tube, add 5x protein loading buffer according to the supernatant volume, vortex to mix, and place in a 100 ° C metal bath for 10 minutes. Then perform protein electrophoresis.

[0083] 4. Prepare separation gel and stacking gel, load samples, perform electrophoresis, transfer to membrane, block, incubate primary and secondary antibodies, and expose bands.

[0084] Figure 4 The results show that the lysosome targeting chimera TdlTac-17RA achieves targeted degradation of the MCF-7 cell membrane protein IL-17RA.

[0085] Example 4: ELISA to verify blocking ability

[0086] The specific steps for quantitative analysis of the blocking ability of CZG-7 by ELISA experiment are as follows:

[0087] 1. Solution Preparation

[0088] Reagent buffer: 1% BSA; Washing buffer: PBS + 0.05% Tween 20; Blocking buffer: 2% BSA.

[0089] 2. Coating

[0090] First, prepare the IL-17RA working solution and dilute the IL-17RA mother solution (1μg / μL) to 1μg / mL with coating buffer. Add 100μL of working solution to each well and set up several control groups without coating. Seal the plate and shake it overnight at 4℃ on a small shaker.

[0091] 3. Cleaning

[0092] Pour out the liquid in the well plate, add 300 μL washing buffer to each well, shake on a 3D shaker for 1 min, then remove the supernatant, turn the well plate upside down on paper, tap and wait for it to dry, and repeat 3 times.

[0093] 4. Closure

[0094] Add 300 μL of bocking buffer to each well, seal with a vacuum film, and incubate in an incubator at 37°C for 1.5 h.

[0095] 5. Cleaning

[0096] Pour out the liquid in the well plate, add 300 μL washing buffer to each well, shake on a 3D shaker for 1 min, then remove the supernatant, turn the well plate upside down on paper, tap and wait for it to dry, and repeat 3 times.

[0097] 6. Loading

[0098] The monoclonal antibody and CZG-7 were diluted with 1% BSA to prepare a monoclonal antibody concentration of 1 μM and a CZG-7 concentration of 10 μM. 100 μL of sample solution was added to each well, Reagent buffer was added to the NO-coating wells, and the plate was sealed.

[0099] 7. Combine

[0100] First prepare Biotin-IL-17A working solution, dilute Biotin-IL-17A (50 μg / mL) to 0.1 μg / mL with Reagent buffer, set up experimental group and NO-coating group, add 50 μL Biotin-IL-17A working solution to each well of the experimental group, add 50 μL Reagent buffer to each well of the NO-coating group, tap to mix, seal the plate, and incubate in an incubator at 37°C for 1 h.

[0101] 8. Cleaning

[0102] Pour out the liquid in the well plate, add 300 μL washing buffer to each well, shake on a 3D shaker for 1 min, then remove the supernatant, turn the well plate upside down on paper, tap and wait for it to dry, and repeat 3 times.

[0103] 9. Add Streptavidin-HRP

[0104] Prepare the Streptavidin-HRP working solution first, dilute the Streptavidin-HRP stock solution (50 μg / mL) to 0.1 μg / mL with a substrate, add 100 μL to each well, seal the plate, and incubate in a 37°C incubator away from light for 1 hour.

[0105] 10. Cleaning

[0106] Pour out the liquid in the well plate, add 300 μL washing buffer to each well, shake on a 3D shaker for 1 min, then remove the supernatant, turn the well plate upside down on paper, tap and wait for it to dry, and repeat 3 times.

[0107] 11. Substrate reaction

[0108] 100 μL TMB was added to each well, the plate was sealed, and incubated in an incubator at 37°C in the dark for 20 min.

[0109] 12. Termination

[0110] Add 50 μL of stop solution to each well and gently shake the plate to mix thoroughly (blue → yellow).

[0111] 13.Measure absorbance

[0112] The absorbance at 450nm-630nm of the microplate reader is the final result.

[0113] Figure 5 In the experiment, PBS was used as the negative control group and the monoclonal antibody was used as the positive control group. By comparing the two, it was found that CZG-7 had a good blocking effect.

[0114] Example 5: In vivo experiments to verify therapeutic ability

[0115] Experimental Procedure

[0116] 1. Construction of mouse psoriasis disease model

[0117] Six-week-old female Balb / c mice were purchased and fed for one week to adapt to the growth environment. The mice were divided into four groups, with 6 mice in each group, namely PBS group, CON group, CZG-7 group, and MTX (methotrexate) group. The experimental operation was as follows:

[0118] Normal group: normal feeding, no treatment;

[0119] PBS group: The backs of mice in this group were depilated, 5% imiquimod cream was applied daily, and the backs were photographed. When wrinkles appeared on the back skin of mice, 100 μL PBS was injected intraperitoneally into each mouse for multiple times.

[0120] CZG-7 group: The backs of mice in this group were depilated, 5% imiquimod cream was applied daily and the backs were photographed. When wrinkles appeared on the back skin of mice, the mice were given a dose of 10 mg / kg, 125 μl by intraperitoneal injection, and then psoriasis was induced and treated once a day for one week.

[0121] MTX group: The backs of mice in this group were depilated, 5% imiquimod cream was applied daily and their backs were photographed. When wrinkles appeared on the back skin of mice, the drug was administered at a dose of 1 mg / kg by intraperitoneal injection of 100 μl. Thereafter, the injections were given every two days for one week, and psoriasis was induced every day.

[0122] After the administration, the mice were killed, and their livers, kidneys and back skin were taken for HE staining to verify the biological safety of CZG-7 and the therapeutic effect of psoriasis. The results are as follows Figure 6-9 shown.

[0123] Figure 6-Figure 9 The following are the HE staining results of the back skin, liver, and kidney of psoriasis mice after treatment. Figure 6 and Figure 7 As shown in the figure, the stratum corneum of the back skin in the PBS group was obviously keratinized, the granular layer in the epidermis was thinned, and obvious psoriasis symptoms could be seen; Figure 8 and Fig. 9 As shown, the liver and kidney cells of mice in the PBS control group were fragmented and necrotic, with unclear structures and edema. The cells in the CZG-7 and MTX experimental groups were neatly arranged, with uniform cytoplasm coloring and clear nuclei, which verified that CZG-7 had no cytotoxicity and had extremely high biosafety. After treatment with CZG-7 and MTX, various indicators were significantly improved.

[0124] On this basis, we purchased 6-week-old female Balb / c mice, raised them for one week to adapt them to the growth environment, and divided the mice into two groups, 6 mice in each group, namely the PBS group and the TdlTac-17RA group.

[0125] Normal group: normal feeding, no treatment;

[0126] PBS group: The backs of mice in this group were depilated, 5% imiquimod cream was applied daily, and the backs were photographed. When wrinkles appeared on the back skin of mice, 100 μL PBS was injected intraperitoneally into each mouse for multiple times.

[0127] TdlTac-17RA group: The backs of mice in this group were depilated, 5% imiquimod cream was applied daily and the backs were photographed. When wrinkles appeared on the back skin of mice, treatment was given by intraperitoneal injection at a concentration of 10 mg / kg, 125 μl each time. Psoriasis was induced and drug treatment was performed once a day for one week.

[0128] Fig.10 This figure shows the effect of TdlTac-17RA treatment on the back skin of psoriasis mice. As shown in the figure, after a period of TdlTac-17RA treatment, the psoriasis symptoms of the mice were significantly reduced, verifying that TdlTac-17RA has a therapeutic effect on psoriasis mice.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that Its amino acid sequence has at least 80% identity with the sequence shown in SEQ ID NO: 1 or 2.

2. The polypeptide according to claim 1, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

3. A lysosome targeting chimera, characterized in that It includes: An aptamer targeting IL-17RA protein; the aptamer targeting IL-17RA protein comprises the polypeptide of claim 1 or 2.

4. The lysosomal targeting chimera according to claim 3, characterized in that The lysosome targeting chimera further comprises: an aptamer of a lysosome targeting receptor connected to the aptamer targeting the IL-17RA protein; Optionally, the molar ratio of the aptamer targeting IL-17RA protein to the aptamer targeting lysosome receptor is 1-6:1-2; Optionally, the aptamer for the lysosomal targeting receptor comprises an aptamer targeting IGFIIR; Optionally, the aptamer for the lysosomal targeting receptor comprises retinoic acid.

5. The lysosomal targeting chimera according to claim 4, characterized in that The aptamer targeting IL-17RA protein is directly connected to the aptamer targeting lysosome receptor or is connected via a connection system; Optionally, the linking system comprises a biotin-avidin linking system; Optionally, the avidin includes any one or more of ovalbumin and streptavidin; Optionally, the connection system comprises: biotin, a linker and avidin; the aptamer targeting IL-17RA protein and the aptamer targeting lysosome receptor are respectively connected to biotin via a linker, and the biotin can be non-covalently bound to the avidin; Optionally, the linker comprises PEG; Optionally, the molecular weight of the PEG is 1k to 10k.

6. A composition, characterized in that It comprises: a raw material for preparing the lysosome targeting chimera according to any one of claims 3 to 5; Optionally, the raw materials include: the aptamer targeting IL-17RA protein as described in claim 3 and the aptamer targeting lysosome receptor as described in claim 4; Optionally, the raw material further comprises: the connection system described in claim 5.

7. The method for preparing a lysosome targeting chimera according to any one of claims 3 to 5, characterized in that: It includes: The composition according to claim 6 is used for preparation.

8. The preparation method according to claim 7, characterized in that: The preparation comprises: The aptamer targeting IL-17RA protein and the aptamer targeting lysosome receptor are respectively coupled to biotin through a linker to obtain the aptamer targeting IL-17RA protein coupled with biotin and the aptamer targeting lysosome receptor coupled with biotin; Mixing the aptamer targeting IL-17RA protein coupled with biotin, the aptamer targeting lysosome receptor coupled with biotin, and avidin to obtain a lysosome targeting chimera; The mixing molar ratio of the biotin-coupled aptamer targeting IL-17RA protein, the biotin-coupled aptamer targeting lysosome receptor and avidin is 1-2:1-6:1-2.

9. Use of the polypeptide according to claim 1 or 2 or the lysosome targeting chimera according to any one of claims 3 to 5 in the preparation of a product for treating or assisting in the treatment of psoriasis or improving relevant clinical symptoms caused by psoriasis.

10. The use according to claim 9, characterized in that: The improvement of the relevant clinical symptoms caused by psoriasis includes: improving any one or more of liver cell damage, kidney cell damage, cell edema and skin damage caused by psoriasis.

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