A fusion polypeptide connected by non-peptide linker, preparation method and application thereof

Abstract

The application discloses a fusion polypeptide connected by a non-peptide linker, a preparation method and application, and belongs to the technical field of biological pharmacy. The application provides a fusion polypeptide connected by PEG, which is formed by connecting polypeptide A and polypeptide B through a non-peptide linker. n Polypeptide B or polypeptide B-(non-peptide linker) n Polypeptide A. The fusion polypeptide connected by PEG can improve solubility, reduce the synthesis cost of amino acids, prolong the survival time in an in-vitro environment, prolong the half-life in vivo, and provide sufficient spatial freedom for polypeptide A and polypeptide B.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a PEG-linked fusion polypeptide, its preparation method, and its application. Background Technology

[0002] Fusion peptides are complex molecules formed by linking two or more functional peptides together using a linker, and they are widely used in drug development, diagnostics, and therapy. For example, in targeted therapy, a fusion peptide can specifically recognize target cells through one peptide (such as a targeting peptide), while another peptide (such as an effector peptide) exerts a therapeutic effect. This design can significantly improve drug targeting and therapeutic efficacy while reducing toxic side effects on normal cells. However, the design and optimization of fusion peptides face many challenges, among which the selection of the linker is one of the key factors.

[0003] Linkers not only affect the stability, solubility, and functional synergy of peptides, but also determine their half-life and immunogenicity in vivo. Traditional fusion peptide design typically relies on amino acid linkers (such as (Gly-Ser)n), which suffer from significant problems in practical applications, including insufficient stability, high immunogenicity, steric hindrance, and poor solubility. To address these issues, researchers have begun exploring the application of non-peptide linkers, with polyethylene glycol (PEG) becoming a research hotspot due to its unique physicochemical properties. PEG linkers offer advantages such as high water solubility, prolonged half-life, reduced immunogenicity, and moderate flexibility, significantly improving the stability, solubility, and functional synergy of fusion peptides. PEG linkers can be linked to peptides through various chemical methods, such as NHS esterification, maleimide reaction, and click chemistry. Common PEG chain lengths include short-chain PEG (such as PEG2 and PEG4), medium-chain PEG (such as PEG8 and PEG12), and long-chain PEG (such as PEG24), with different PEG chain lengths having a significant impact on the function of the fusion peptide. Meanwhile, branched PEG modification technology enables multivalent linkage of peptide molecules, optimizes peptide ratio control, and better leverages the efficacy of fusion molecules. Examples of PEG linker applications in fusion peptide design include drug delivery, targeted therapy, and biosensors. For instance, using PEG linkers to connect targeting peptides and drug molecules improves drug targeting and therapeutic efficacy.

[0004] However, amino acid linkers, as one of the earliest technologies used in the biopharmaceutical industry, have some drawbacks. For example, insufficient stability: amino acid linkers are easily degraded by proteases in vivo, resulting in a short half-life for fusion peptides, which may limit their application in long-acting therapies; high immunogenicity: amino acid linkers may expose antigenic epitopes, triggering an immune response, which could lead to the fusion peptide being cleared by the immune system, reducing its therapeutic effect; high synthesis cost: the chemical composition of amino acid linkers is fundamentally different from small-molecule organic compounds, resulting in high synthesis costs for fusion peptides; steric hindrance effect: the limited length and flexibility of amino acid linkers may lead to steric hindrance between peptide A and peptide B, affecting the functional synergy of peptide A and peptide B; poor solubility: some amino acid linkers are highly hydrophobic, causing fusion peptides to easily aggregate or precipitate in aqueous environments, which can affect the bioavailability of the fusion peptides.

[0005] By using a different PEG linkage method, this invention aims to provide a fusion peptide with high stability, low immunogenicity, and good functional synergy, offering a new solution for its application in drug development, targeted therapy, and biosensors. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a PEG-linker-based fusion peptide that solves the problems of poor stability, high synthesis cost, and limited functionality associated with traditional amino acid linkers, while simultaneously improving the solubility, half-life, and functional synergy of the fusion peptide. Through comparative experimental tests of PEG-linker fusion peptides and amino acid linker fusion peptides, the PEG-linker fusion peptide demonstrates advantages in multiple parameter values.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] On one hand, the present invention provides a fusion polypeptide using PEG linkage, wherein the fusion polypeptide is formed by linking polypeptide A and polypeptide B through a non-peptide linker;

[0009] The general structural formula of the fusion polypeptide is: polypeptide A-(non-peptide linker) n - Peptide B or Peptide B- (non-peptide linker) n -Polypeptide A;

[0010] Among them, polypeptide A is an O-peptide, an O-peptide-derived peptide, or an O-peptide derivative; polypeptide B is a G-peptide, a G-peptide-derived peptide, or a G-peptide derivative.

[0011] The O peptide is any one or more of the nucleic acid sequences SEQ ID NO.1-8;

[0012] G peptide is any one or more of the nucleic acid sequences SEQ ID NO. 9-17;

[0013] n represents the number of polymers aggregated, and n is an integer.

[0014] The aforementioned PEG-linked fusion peptide, wherein the non-peptide linker is a PEG linker, an alkyl chain linker, a click chemistry linker, or a polysaccharide linker; wherein, alkyl chain linkers (such as C6, C12) are suitable for hydrophobic environments, improving the stability of the fusion peptide; click chemistry linkers (such as DBCO-azide, TCO-tetraazine) are suitable for efficient linking, reducing reaction condition limitations; and polysaccharide linkers (such as hyaluronic acid, chitosan) are suitable for applications requiring high biocompatibility.

[0015] Preferably, the PEG Linker is a linear PEG or a branched PEG; (In this specific embodiment of the invention, the experiment was conducted using linear PEG as an example, but branched PEG can also achieve similar effects.)

[0016] More preferably, the branched PEG is a four-arm polyethylene glycol (4-arm PEG) or an eight-arm polyethylene glycol (8-arm PEG); using branched PEG (such as 4-arm PEG, 8-arm PEG) to link peptide A and peptide B achieves multivalent linkage and optimizes peptide ratio control. Specifically, 4-arm PEG can link 3 peptide A molecules and 1 peptide B molecule, optimizing peptide ratio control. 8-arm PEG can link 4 peptide A molecules and 4 peptide B molecules, suitable for multivalent ligand linkage.

[0017] Preferably, the number of PEGs in the PEG linker is 2 to 24. PEG linkers of different lengths (such as PEG2, PEG4, PEG8, PEG12, PEG24, etc.) are used to link peptide A and peptide B. Short-chain PEGs (such as PEG2 and PEG4) are suitable for scenarios requiring shorter linkers, reducing steric hindrance. Long-chain PEGs (such as PEG12 and PEG24) are suitable for scenarios requiring greater steric freedom, reducing interactions between peptide A and peptide B.

[0018] The PEG-linked fusion polypeptide, wherein the O-peptide-derived peptide is a polypeptide containing an O-peptide, or a sequence with sequence homology to the O-peptide higher than 50%, 80%, 90%, 95%, or 99%.

[0019] The O peptide derivative is a product obtained by conventional modification of the terminal or side chain of an O peptide or an O peptide-derived peptide, or a product obtained by attaching a tag for peptide or protein detection or purification, or a product obtained by isotope labeling modification.

[0020] The G peptide-derived peptide is a polypeptide containing a G peptide, or a sequence with a sequence homology to the G peptide higher than 50%, 80%, 90%, 95%, or 99%.

[0021] The G peptide derivative is a product obtained by conventionally modifying the terminal or side chain of a G peptide or G peptide-derived peptide, or a product obtained by attaching a tag for peptide or protein detection or purification, or a product obtained by isotope labeling modification.

[0022] The conventional modifications include fluorescent group modification, phosphorylation modification, disulfide bond cyclization modification, biotin labeling modification, photosensitizer, azide modification, PEG modification, methylation modification, fluorescence quencher group modification, protein coupling modification, small molecule compound modification, aminoation modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, nitration modification, esterification modification, or glycosylation modification;

[0023] Preferably, the fluorescent dye used in the fluorescent group modification is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, or ICG, and this modification can be used for fluorescence detection;

[0024] Preferably, the phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, and p-Tyr;

[0025] Preferably, the biotin in the biotin labeling modification is selected from D-biotin, biotinyl hydrazide, photosensitive biotin, or biotin-dUTP;

[0026] Preferably, the amino modification is an N-terminal acetylation modification of the polypeptide;

[0027] Preferably, the carboxylation modification is an amidation modification of the C-terminus of the polypeptide;

[0028] Preferably, the nitration modification is Tyr modification;

[0029] Preferably, the glycosylation modification is selected from one or more combinations of Ser, Asn, Thr, and Tyr;

[0030] Preferably, the conventional modification of the side chain is the modification of the R group of the amino acid side chain in the polypeptide;

[0031] The tags are His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityeXact;

[0032] The isotopes used in the isotope labeling modification are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, or 131I.

[0033] The aforementioned PEG-linked fusion peptides utilize non-peptide linkers to link peptides A and B via the NHS ester method, maleimide method, and click chemistry method. This ensures that the structure and function of peptides A and B are not affected during the linking process. Specifically, the NHS ester reaction is suitable for linking to the amino groups of the peptides, forming stable amide bonds. The maleimide reaction is suitable for linking to the thiol groups of the peptides, forming stable thioether bonds. Click chemistry is suitable for efficient linking and reduces reaction condition limitations.

[0034] Secondly, the present invention provides a method for preparing a PEG-linked fusion polypeptide according to any one of the claims, comprising the following steps:

[0035] Peptide A and peptide B are synthesized using solid-phase synthesis or liquid-phase synthesis. Peptide A and peptide B are optionally activated and then linked sequentially using a non-peptide linker to obtain a fusion peptide.

[0036] Thirdly, the present invention provides a nucleic acid encoding any of the fusion polypeptides described in the present invention.

[0037] Fourthly, the present invention provides a carrier comprising the aforementioned nucleic acid.

[0038] Fifthly, the present invention provides a host cell transfected with the aforementioned vector.

[0039] In a sixth aspect, the present invention provides the use of any of the fusion peptides described in the preparation of medicaments, medical aesthetic products or cosmetics for treating and / or preventing hair loss, acne or dermatitis.

[0040] In the aforementioned application, the fusion polypeptide reduces androgen-induced apoptosis levels in human hair follicle papillary cells, maintains the growth level of supporting cells, restores the androgen-induced G0 / G1 cell ratio in human sebaceous gland cells, and inhibits cell proliferation.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. This invention utilizes the high water solubility of PEG chains to significantly improve the solubility of fusion peptides. In pharmaceutical formulations, the aggregation and precipitation of PEGylated fusion peptides are significantly reduced. This solves the problem in existing technologies where the strong hydrophobicity of amino acid linkers leads to the easy aggregation or precipitation of fusion peptides in aqueous environments.

[0043] 2. This invention uses PEG linkers instead of amino acid linkers, reducing the introduction of excess amino acids and lowering the synthesis cost of amino acids. Experimental verification shows that the PEGylated fusion peptides have a prolonged survival time in the in vitro environment. In animal models, the in vivo half-life of the PEGylated fusion peptides is also prolonged. This solves the problems in existing technologies where amino acid linkers are easily degraded by proteases in vivo, leading to a short half-life of the fusion peptides; and where peptide bonds are easily hydrolyzed under acidic, alkaline, or high-temperature conditions in the in vitro environment, resulting in unstable amino acid composition.

[0044] 3. This invention provides sufficient spatial freedom for peptide A and peptide B by selecting a PEG linker of appropriate length. Cellular function experiments have verified that the therapeutic effect of the PEGylated fusion peptide is significantly better than that of traditional amino acid linker fusion peptides. This solves the problem of steric hindrance between peptide A and peptide B caused by the limited length and flexibility of amino acid linkers in existing technologies. Attached Figure Description

[0045] Figure 1 This is a high-performance liquid chromatography (HPLC) chromatogram of the PZ1 fusion peptide.

[0046] Figure 2 This is a mass spectrometry (MS) detection image of the PZ1 fusion peptide;

[0047] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the PZ2 fusion peptide.

[0048] Figure 4 This is a mass spectrometry (MS) detection image of the PZ2 fusion peptide;

[0049] Figure 5 This is a high-performance liquid chromatography (HPLC) chromatogram of the PX1 fusion peptide.

[0050] Figure 6 This is a mass spectrometry (MS) detection image of the PX1 fusion peptide;

[0051] Figure 7 This is a high-performance liquid chromatography (HPLC) chromatogram of the PX2 fusion peptide.

[0052] Figure 8 This is a mass spectrometry (MS) detection image of the PX2 fusion peptide;

[0053] Figure 9 For testing the solubility of fusion peptides;

[0054] Figure 10 To detect apoptosis in human dermal papilla cells after DHT treatment using fusion peptides;

[0055] Figure 11 To detect the cell cycle of human sebaceous gland cells after DHT treatment with fusion peptides. Detailed Implementation

[0056] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0057] Example 1:

[0058] 1. Design of fusion peptides

[0059] The core of this invention is the connection between peptide A and peptide B via a PEG linker to form a fusion peptide. Its general structural formula is: Peptide A - (non-peptide linker) n - Peptide B or Peptide B- (non-peptide linker) n - Polypeptide A, where: Polypeptide A and Polypeptide B are polypeptide sequences with specific functions (A is an O peptide, an O peptide-derived peptide, or an O peptide derivative; B is a G peptide, a G peptide-derived peptide, or a G peptide derivative), the Linker part is PEG or other non-peptide Linkers (such as alkyl chains, click chemistry Linkers, polysaccharide Linkers, etc.), and n represents the number of polymers (n is an integer).

[0060] The above-mentioned O peptide is any one or more of the following sequences:

[0061] YLYQWLGAPVPYPDPLEPR(SEQ ID NO.1)

[0062] YLGASVPSPDPLEPT(SEQ ID NO.2)

[0063] LDPGLGAPAPYPDPLEPR(SEQ ID NO.3)

[0064] NYNRFYNARAAPTPDPLEPL(SEQ ID NO.4)

[0065] SNLRNAVFGTPVRDPLESK(SEQ ID NO.5)

[0066] AGAVTAADLSLTQLESL(SEQ ID NO.6)

[0067] YAQDSGVAGAPPNPLEAQ(SEQ ID NO.7)

[0068] SDFYER YFMHYKTPMEQM(SEQ ID NO.8)

[0069] The G peptide described above is any combination of one or more of the following sequences:

[0070] AWPYVTL (SEQ ID NO.9)

[0071] ADIRHIK (SEQ ID NO.10)

[0072] GPLIWFS (SEQ ID NO.11)

[0073] GFHYSLH (SEQ ID NO.12)

[0074] MPRLPPA (SEQ ID NO.13)

[0075] SILPVTR (SEQ ID NO.14)

[0076] TAKYLPMRPGPL (SEQ ID NO.15)

[0077] DYHDPSLPPLRK (SEQ ID NO.16)

[0078] GNNPLHVHHDKR(SEQ ID NO.17)

[0079] The O-peptide-derived peptide is a polypeptide containing an O-peptide, or a sequence with a sequence homology of 50%, 80%, 90%, 95%, or 99% higher than that of the O-peptide sequence; the G-peptide-derived peptide is a polypeptide containing a G-peptide, or a sequence with a sequence homology of 50%, 80%, 90%, 95%, or 99% higher than that of the G-peptide sequence; the O-peptide derivative is a product obtained by conventional modification of the O-peptide or O-peptide-derived peptide at the end or side chain, or a product obtained by attaching a tag for peptide or protein detection or purification, or a product obtained by isotope labeling modification; the G-peptide derivative is a product obtained by conventional modification of the G-peptide or G-peptide-derived peptide at the end or side chain, or a product obtained by attaching a tag for peptide or protein detection or purification, or a product obtained by isotope labeling modification.

[0080] 2. Preparation methods of fusion peptides

[0081] The fusion polypeptide PZ1 of the present invention (its nucleotide sequence is shown in SEQ ID NO.19):

[0082] YLYQWLGAPVPYPDPLEPR-PEG2-MPRLPPA (SEQ ID NO.19) was prepared by the following steps:

[0083] PZ1 monopeptides at both ends were synthesized using a solid-phase peptide synthesis method. The reaction proceeded from the C-terminus to the N-terminus, involving resin activation, amino acid linking, elution protection, and detection. Amino acids were linked one by one, followed by precipitation with excess diethyl ether and centrifugation. The crude peptides were then purified by HPLC, analyzed by mass spectrometry, and rapidly lyophilized in liquid nitrogen for later use. The specific process is as follows:

[0084] First, a matching PEG reagent is selected based on the characteristics of the terminal groups of the peptide. For peptides containing carboxyl groups, activation with EDC / NHS activator in pH 6.0 buffer for 30 minutes is required to convert -COOH to active NHS ester; peptides containing amino or thiol groups do not require activation. Then, a stepwise ligation reaction is performed: First, the activated peptide A is reacted with PEG in pH 7.4 PBS buffer at 4°C for 2 hours, maintaining a molar ratio of 1.2:1 to ensure single-end ligation by PEG. After the reaction, the intermediate is purified by ultrafiltration and centrifugation, and the ligation efficiency is monitored by RP-HPLC (typically >85%). Second, the PEG-peptide A intermediate is reacted with peptide B in pH 8.0 HEPES buffer at room temperature for 3 hours, with 1 eq of EDC added to promote the condensation reaction. Finally, the target product is purified by preparative HPLC (C18 column, acetonitrile / water gradient elution), and lyophilized to obtain a white powder. The product is then subjected to MALDI-TOFMS molecular weight verification (error <0.1%), analytical HPLC purity detection (>95%), and circular dichroism chromatography verification of secondary structure integrity to obtain the fusion peptide.

[0085] The results of high-performance liquid chromatography (HPLC) and mass spectrometry (MS) showed that... Figure 1 and 2 .

[0086] Example 2:

[0087] The fusion polypeptide PZ2 of the present invention (its nucleotide sequence is shown in SEQ ID NO.21):

[0088] MPRLPPA-PEG2-YLYQWLGAPVPYPDPLEPR was prepared by the following steps:

[0089] The above-mentioned fusion peptides were synthesized using conventional solid-phase or liquid-phase synthesis methods. The PZ2-terminal monopeptides were synthesized using a solid-phase peptide synthesis method, involving a reaction from the C-terminus to the N-terminus. This process included resin activation, amino acid linking, elution protection, and detection, with each amino acid linked individually. The peptides were then precipitated with excess diethyl ether and centrifuged. The crude peptides were purified by HPLC, analyzed by mass spectrometry, and rapidly lyophilized in liquid nitrogen for later use. The specific synthesis method was the same as in Example 1.

[0090] For PEG fusion peptides, after synthesizing the single peptide, it is necessary to activate the single peptide, sequentially link it with PEG, and then purify and characterize it to obtain the fusion peptide. The results of high-performance liquid chromatography (HPLC) and mass spectrometry (MS) show the following... Figure 3 and 4 .

[0091] Comparative Example 1:

[0092] The fusion polypeptide PX1 of the present invention (its nucleotide sequence is shown in SEQ ID NO.18):

[0093] YLYQWLGAPVPYPDPLEPR-GGGGS-MPRLPPA), prepared by the following steps:

[0094] PX1 monopeptides at both ends were synthesized using a solid-phase peptide synthesis method. The reaction proceeded from the C-terminus amino acid to the N-terminus. The amino acid linkage was completed one by one through steps such as resin activation, amino acid linking, elution protection, and detection. The crude peptide was then precipitated with excess diethyl ether and centrifuged. After purification by HPLC, mass spectrometry analysis was performed, and the peptide was rapidly freeze-dried in liquid nitrogen for later use.

[0095] The results of high-performance liquid chromatography (HPLC) and mass spectrometry (MS) showed that... Figure 5 and 6 .

[0096] Comparative Example 2:

[0097] The fusion polypeptide PX2 of the present invention (its nucleotide sequence is shown in SEQ ID NO.20):

[0098] MPRLPPA-GGGGS-YLYQWLGAPVPYPDPLEPR), prepared by the following steps:

[0099] PX2 monopeptides at both ends were synthesized using a solid-phase peptide synthesis method. The reaction proceeded from the C-terminus amino acid to the N-terminus. The amino acid linkage was completed one by one through steps such as resin activation, amino acid linking, elution protection, and detection. The crude peptide was then precipitated with excess diethyl ether and centrifuged. After purification by HPLC, the crude peptide was analyzed by mass spectrometry and then rapidly lyophilized in liquid nitrogen for later use.

[0100] The results of high-performance liquid chromatography (HPLC) and mass spectrometry (MS) showed that... Figure 7 and 8 .

[0101] According to the high performance liquid chromatography (HPLC) and mass spectrometry (MS) detection results of Examples 1-2 and Comparative Examples 1-2, the purity of amino acid linker fusion peptides PX1 and PX2 and the PEG linker fusion peptides PZ1 and PZ2 of the present invention are all greater than 98%, and the molecular weight is consistent with the predicted value.

[0102] Example 3: Functional testing of fusion peptides

[0103] 1. Solubility test

[0104] The PEGylated fusion peptides prepared in Examples 1 and 2 and the amino acid linker fusion peptide solid powders prepared in Comparative Examples 1 and 2 were dissolved in PBS buffer (pH 7.4) to prepare solutions with a concentration of 10 mg / mL. These solutions were then diluted with PBS buffer to different concentrations (5 mg / mL, 1 mg / mL, 0.2 mg / mL). The diluted sample solutions were placed in a constant temperature shaker and incubated at 25°C for 1 hour to ensure complete dissolution. The incubated sample solutions were centrifuged at 10,000 rpm for 10 minutes to remove undissolved precipitates. The supernatant was collected for subsequent analysis. Standard curve plotting: A standard curve was plotted using fusion peptide solutions of known concentrations (0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, 25 mg / mL). The standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The solubility was calculated based on the concentration of the fusion peptide in the supernatant: solubility (mg / mL) = concentration of the fusion peptide in the supernatant. Chromatographic conditions: Agilent Advance Bio Peptide M, mobile phase: phase A is 0.1% TFA aqueous solution, phase B is 0.9% TFA acetonitrile solution, UV: 215nm, injection 10uL.

[0105] like Figure 9 As shown, the solubility test verified that the PEGylated fusion peptide has significantly better solubility in the aqueous environment than the traditional amino acid linker fusion peptide, providing experimental evidence for its application in subsequent drug formulations.

[0106] 2. Stability Test

[0107] The PEGylated fusion peptides prepared in Examples 1 and 2, and the conventional amino acid linker fusion peptides prepared in Comparative Examples 1 and 2, were dissolved in PBS (pH 7.4) at a concentration of 1 mg / mL. Under different environmental conditions, the content changes were tested at 2-8°C and 25°C / 60% RH for 0, 15, and 30 days under normal light / dark conditions. At each sampling, 10 μL of 10% trifluoroacetic acid (TFA) was added immediately to terminate the reaction. HPLC analysis was then performed. Chromatographic conditions: Agilent Advance Bio Peptide M, mobile phase: Phase A was 0.1% TFA aqueous solution, Phase B was 0.9% TFA acetonitrile solution, UV: 215 nm, injection 10 μL.

[0108] The content of peptide solutions under different storage conditions and at different times was determined by chromatography, and the results are shown in Table 1 below. Analysis revealed that the peptide content changed significantly under 25℃ / 60%RH conditions, but the PEG Linker fusion peptides PZ1 and PZ2 were more stable than the amino acid Linker fusion peptides PX1 and PX2. Normal light exposure had no significant effect on peptide stability, and the peptide solutions were stable when stored at 2-8℃.

[0109] Table 1. Results of stability tests on the fusion peptides

[0110]

[0111] 3. Half-life test

[0112] Before the experiment, SD rats were fasted for 12 hours but given free access to water. Six SD rats were randomly divided into two groups, A and B, and injected with PEG Linker fusion peptide and amino acid Linker fusion peptide solutions, respectively. Each rat was weighed before administration. The dosage was 2 mg / kg, administered via tail vein injection. Rats were restrained in a metal rat restraint device before tail vein administration. Blood samples (200 μl each) were collected from the orbital venous plexus of both groups at 0 hours (before administration), 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours. All blood samples were placed in centrifuge tubes treated with 0.5% heparin sodium and centrifuged at 3000 rpm for 15 min. 100 μl of the supernatant was transferred to a new centrifuge tube. Then, 100 μl of acetonitrile solution containing 0.1% TFA was added to precipitate impurities. The acetonitrile-treated plasma samples were vortexed for 30 s and centrifuged at 14000 rpm for 20 min. Finally, aspirate 30 μl of the supernatant and filter it through a 0.22 μm sterile filter, taking care to avoid aspirating the precipitate at the bottom. Then perform HPLC analysis. Chromatographic conditions: Agilent Advance Bio Peptide M, mobile phase: phase A is 0.1% TFA aqueous solution, phase B is 0.9% TFA acetonitrile solution, UV: 215 nm, injection 10 μL.

[0113] The results of the in vivo half-life detection of the fusion peptides in animals are shown in Table 2 below. By measuring the peptide content in rat blood using chromatography and plotting the relationship between peptide concentration and peak area, it was found that the PEG Linker fusion peptides PZ1 and PZ2 had a significantly longer terminal elimination half-life (1 / 2) and a significantly lower drug clearance rate in animals compared to the amino acid Linker fusion peptides PX1 and PX2.

[0114] Table 2 Results of in vivo animal half-life detection of fusion peptides

[0115]

[0116] Example 4: Detection of apoptosis in human hair follicle papillary cells (DHT treatment)

[0117] Human pilaris cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% CO2, with medium changed every other day. The same batch of human pilaris cells was cultured in 6-well plates and treated with 100 μM DHT to establish an androgen / seborrheic alopecia cell model. Comparative examples 1 and 2 included amino acid linker peptides, and examples 1 and 2 included 10 μM PEG linker peptides (DHT+ peptide group). Cell apoptosis was detected after 48 h of culture.

[0118] Aspirate the cell culture medium into centrifuge tubes and add an appropriate amount of trypsin cell digestion solution to digest the cells. Incubate at room temperature until the adherent cells are gently pipetted off. Aspirate the trypsin cell digestion solution and add it to the corresponding centrifuge tube (because DHT will induce apoptosis, the apoptotic adherent cells will float, so the culture medium should not be discarded directly). Centrifuge at 300g for 5 minutes, discard the supernatant, and collect the cells. Resuspend the cells in PBS, centrifuge at 300g for 5 minutes, discard the supernatant, and collect the cells again. Gently resuspend the cells in 100μL Annexin V-FITC binding solution, and add 5μL Annexin V-FITC and 10μL propidium iodide staining solution, and mix gently. Incubate at room temperature in the dark for 10 minutes, then place on ice, and then use flow cytometry to detect cell scattering and fluorescence.

[0119] Flow cytometry analysis results as follows Figure 10 As shown, the addition of DHT can effectively increase the proportion of apoptotic cells in dermal papilla cells, and DHT promotes the apoptosis process. Based on this, by adding two fusion peptides, the proportion of apoptotic cells decreased. The peptide drugs can alleviate apoptosis in human dermal papilla cells caused by high levels of androgen DHT. Among them, the PEG Linker fusion peptides PZ1 and PZ2 are more advantageous than the amino acid Linker fusion peptides PX1 and PX2.

[0120] Example 5: Human sebaceous gland cell cycle detection (DHT treatment)

[0121] Human sebaceous gland cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% CO2, with the medium changed every other day. The same batch of human sebaceous gland cells were cultured in 6-well plates and treated with 50 μM DHT to establish an androgen / seborrheic alopecia cell model. Comparative examples 1 and 2 included amino acid linker peptides, and examples 1 and 2 included 10 μM PEG linker peptides (DHT+peptide group). Cell cycle analysis was performed after 48 h of culture.

[0122] Carefully aspirate the cell culture medium, digest the cells with trypsin, and prepare a single-cell suspension. Centrifuge at 1000g for 5 min to pellet the cells, discard the supernatant, rinse the cells once with 1 mL of pre-chilled PBS, and centrifuge to collect the cells. Gently mix the cell pellet with 1 mL of pre-chilled 70% ethanol and fix at 4°C for at least 2 hours or overnight. Next, centrifuge at 1000g for 5 min to pellet the cells, and resuspend them in 1 mL of pre-chilled PBS. Then centrifuge at 1000g for 5 min again to pellet the cells. Add 10 μL of propidium iodide stock solution and 10 μL of RNase A solution to 0.5 mL of staining buffer and mix well. Add 0.5 mL of the prepared propidium iodide staining solution to each cell sample, gently mix, and resuspend the cells. Incubate at 37°C in the dark for 30 min, and then perform flow cytometry analysis.

[0123] Flow cytometry analysis results as follows Figure 11 As shown, adding DHT can effectively increase the number of sebaceous gland cells in the G2 / M phase and reduce the number in the G0 / G1 and S phases, thus blocking the normal progression of the cell cycle. On this basis, by adding two fusion peptides, the proportion of G0 / G1 cells has rebounded. Peptide drugs can alleviate the proliferation effect of human sebaceous gland cells caused by high androgen DHT. PEG Linker fusion peptides PZ1 and PZ2 are more advantageous than amino acid Linker fusion peptides PX1 and PX2.

Claims

1. A fusion polypeptide linked by PEG, characterized in that, The fusion polypeptide is formed by linking polypeptide A and polypeptide B through a non-peptide linker. The general structural formula of the fusion polypeptide is: polypeptide A-(non-peptide linker) n - Peptide B or Peptide B- (non-peptide linker) n -Polypeptide A; Among them, polypeptide A is an O-peptide, an O-peptide-derived peptide, or an O-peptide derivative; polypeptide B is a G-peptide, a G-peptide-derived peptide, or a G-peptide derivative. The O peptide is any one or more of the nucleic acid sequences SEQ ID NO.1-8; G peptide is any one or more of the nucleic acid sequences SEQ ID NO. 9-17; n represents the number of polymers aggregated, and n is an integer.

2. The PEG-linked fusion polypeptide as described in claim 1, characterized in that, The non-peptide linker is a PEG linker, an alkyl chain linker, a click chemistry linker, or a polysaccharide linker. Preferably, the PEG Linker is a linear PEG or a branched PEG; More preferably, the branched PEG is a four-armed polyethylene glycol or an eight-armed polyethylene glycol.

3. The PEG-linked fusion polypeptide as described in claim 1, characterized in that, The O-peptide-derived peptide is a polypeptide containing an O-peptide, or a sequence with a sequence homology to the O-peptide sequence greater than 50%, 80%, 90%, 95%, or 99%. The O peptide derivative is a product obtained by conventional modification of the terminal or side chain of an O peptide or an O peptide-derived peptide, or a product obtained by attaching a tag for peptide or protein detection or purification, or a product obtained by isotope labeling modification. The G peptide-derived peptide is a polypeptide containing a G peptide, or a sequence with a sequence homology to the G peptide higher than 50%, 80%, 90%, 95%, or 99%. The G peptide derivative is a product obtained by conventionally modifying the terminal or side chain of a G peptide or G peptide-derived peptide, or a product obtained by attaching a tag for peptide or protein detection or purification, or a product obtained by isotope labeling modification.

4. The PEG-linked fusion polypeptide as described in claim 3, characterized in that, The conventional modifications include fluorescent group modification, phosphorylation modification, disulfide bond cyclization modification, biotin labeling modification, photosensitizer, azide modification, PEG modification, methylation modification, fluorescence quencher group modification, protein coupling modification, small molecule compound modification, aminoation modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, nitration modification, esterification modification, or glycosylation modification; Preferably, the fluorescent dye used in the fluorescent group modification is selected from at least one of AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, or ICG; Preferably, the phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, and p-Tyr; Preferably, the biotin in the biotin labeling modification is selected from D-biotin, biotinylate, photosensitive biotin, or biotin-dUTP; Preferably, the amino modification is an N-terminal acetylation modification of the peptide; Preferably, the carboxylation modification is an amidation modification of the C-terminus of the peptide; Preferably, the nitration modification is Tyr modification; Preferably, the glycosylation modification is selected from one or more combinations of Ser, Asn, Thr, and Tyr; Preferably, the conventional modification of the side chain is the modification of the R group of the amino acid side chain in the polypeptide; The tags are His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityeXact; The isotopes used in the isotope labeling modification are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, or 131I.

5. The PEG-linked fusion polypeptide as described in claim 1, characterized in that, The non-peptide linker connects peptide A and peptide B using the NHS ester method, maleimide method, and click chemistry method.

6. A method for preparing a PEG-linked fusion polypeptide according to any one of claims 1-5, characterized in that, Includes the following steps: Peptide A and peptide B are synthesized using solid-phase synthesis or liquid-phase synthesis. Peptide A and peptide B are optionally activated and then linked sequentially using a non-peptide linker to obtain a fusion peptide.

7. A nucleic acid, characterized in that, Its encoding is the fusion polypeptide as described in any one of claims 1-5.

8. A carrier, characterized in that, It contains the nucleic acid as described in claim 7.

9. A host cell, characterized in that, The vector as described in claim 8 was transfected.

10. The use of the fusion polypeptide according to any one of claims 1-5 in the preparation of medicaments, medical aesthetic products or cosmetics for treating and / or preventing hair loss, acne or dermatitis; Preferably, the fusion polypeptide reduces the level of androgen-induced apoptosis in human hair follicle papillary cells and restores the androgen-induced G0 / G1 cell ratio in human sebaceous gland cells.

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