Affinity polypeptide of human serum albumin and design method thereof

Abstract

The invention relates to the technical field of biology, in particular to affinity polypeptide of human serum albumin and a design method of the affinity polypeptide. The molecular mechanism and microscopic details of interaction of HSA-Mgbc7HopQNbAlb1 compounds are analyzed through an MD simulation technology and an MM-PBSA free energy decomposition method, key binding sites between the compounds and spatial distribution of the key binding sites are determined, and an affinity model is established, so that a candidate affinity polypeptide library is constructed, and meanwhile, the peptide library is expanded through intelligent design. Then, screening and verifying the affinity polypeptide library through methods of molecular docking, conformation analysis, hydrophobicity analysis, MD simulation and the like, so as to obtain the high-affinity polypeptide aiming at the HSA target spot. The binding performance of the affinity polypeptide and the HSA is verified through a double-antibody one-step sandwich ELISA experiment, adsorption isotherm determination and an affinity chromatography experiment. The polypeptides such as FKITSGSLSR, DGALTPPSEY and the like obtained by screening have high affinity with the HSA (Human Serum Albumin).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to affinity peptides of human serum albumin and their design methods. Background Technology

[0002] Human serum albumin (HSA) has multiple functions, such as transporting endogenous and exogenous substances, regulating human physiological functions, antioxidation, and anticoagulation. HSA's binding affinity makes it a promising candidate in the biopharmaceutical field. It can be used as a standalone therapeutic agent or drug carrier for new drug development or to improve drug delivery, or it can be combined or fused with other proteins to develop novel therapeutic strategies. However, due to limitations in blood sources and pathogen safety concerns, the supply of HSA from human plasma is restricted. Therefore, obtaining HSA through genetic engineering expression has become an important approach, but the isolation and purification of HSA still presents many challenges. Developing high-affinity peptides targeting HSA is beneficial for improving its isolation efficiency and can be applied to the regulation of pharmacokinetic and other properties of adjuvant drugs, thus possessing significant research significance and practical value. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide an affinity polypeptide of human serum albumin and a method for designing it.

[0004] This invention provides an affinity polypeptide for human serum albumin, comprising any one of polypeptides A to C;

[0005] The polypeptide A is TIX1GSLSR, where X1 is any one of 20 common amino acids;

[0006] The polypeptide B is FX2ITX3GSLSR, wherein X2 or X3 is any one of 20 common amino acids;

[0007] The polypeptide C is DGALTPPSY1, where Y1 is absent or EY.

[0008] The polypeptide C is DGALTY1, where Y1 is PPS or PPSEY.

[0009] Furthermore, the affinity polypeptide of the present invention is characterized in that,

[0010] In the polypeptide A, X1 is selected from any one of D, E, Q, R, and P;

[0011] In the polypeptide B, X2 is selected from K, P, D or H; and X3 is selected from S, D, Q, H, C or K.

[0012] This invention provides a method for preparing the affinity polypeptide, which includes the following steps:

[0013] Step 1: Use HSA with PDB ID: 8oi2 and Mgb c7HopQ NbAlb1 A full-atom model was constructed based on the composite crystal structure, and then HSA and Mgb were determined using MD simulations and MM-PBSA. c7HopQ NbAlb1 Hotspot residues;

[0014] Step 2: Based on Mgb c7HopQ NbAlb1 The spatial distribution of hotspot residues was analyzed, and characteristic sequences were determined using amino acid localization to construct a biomimetic affinity peptide library.

[0015] Step 3: Using molecular docking technology, retain E in the biomimetic affinity peptide library. VINA A peptide library 1 was obtained by collecting peptides with a concentration of ≤-7.5 kcal / mol.

[0016] Step 4: Calculate Mgb c7HopQ NbAlb1 The root mean square deviation (RMSD) between the hotspot residues and the corresponding hotspot residues of the peptides in affinity peptide library 1 is calculated, and peptides with an RMSD ≤ 0.4 nm are retained to obtain affinity peptide library 2.

[0017] Step 5: Select peptides from the affinity peptide library 2 with a negative average hydrophilicity coefficient GRAVY value and a lipid-water partition coefficient log P ≤ -1.7, and obtain the affinity peptides of the present invention after verification by MD simulation;

[0018] Furthermore, in the preparation method described in this invention, the HSA with PDB ID: 8oi2 and Mgb c7HopQ NbAlb1 The conditions for constructing an all-atom model based on the composite crystal structure are as follows: molecular dynamics simulations were performed using the GROMOS96 43a1 force field; the solvent water molecules were modeled using the SPC model; and the simulation system of the composite crystal structure was placed in a space containing 372 Na⁺, 364 Cl⁻, and 123285 water molecules with a size of 19.0 × 11.8 × 17.5 nm. 3 Inside the cube-shaped box;

[0019] The conditions for the MD simulation were as follows: temperature 310.15 K; pressure 1 bar; bond length kept constant; long-range electrostatic force calculated; Coulomb potential energy calculation cutoff 1.2 nm; LJ potential energy calculation cutoff 1.2 nm; integration step size 2 fs; pre-equilibrium performed after energy minimization.

[0020] More specifically, the conditions for the MD simulation are as follows: temperature is maintained at 310.15 K using the velocity-rescale algorithm; pressure is adjusted to 1 bar using the Parrinello-Rahman coupler; the bond length is kept constant using a linear constraint solver algorithm; the long-range electrostatic force is calculated using the particle-mesh Ewald algorithm; periodic boundary conditions are enabled in the x, y, and z directions; the cutoff for Coulomb potential energy calculation is set to 1.2 nm, and the cutoff for LJ potential energy calculation is set to 1.2 nm; the leapfrog integration method is used, with an integration step size of 2 fs; the steepest descent method is used for energy minimization; and a 100 ps NVT ensemble is used for pre-equilibrium.

[0021] In the preparation method described in this invention, after optimization and experimentation of the above parameters, the affinity polypeptide described in this invention is finally obtained;

[0022] In a specific embodiment of the present invention, in step 1, HSA and Mgb were determined. c7HopQ NbAlb1 Hotspot residues, specifically the Mgb c7HopQ NbAlb1 Hotspot residues include: SER-488, LYS-452, VAL-2, PHE-419, ARG-491, SER-490, LEU-489, ILE-485, THR-484, GLY-487, LYS-51, LYS-430, LYS-185;

[0023] The hotspot residues of the HSA include: LYS-313, ALA-320, ALA-362, LYS-372, ALA-364, CYS-361, LYS-378, and ASP-324.

[0024] In step 2, the characteristic sequence is determined using the amino acid localization method. The characteristic sequence is TIX1GSLSR, FX2ITX3GSLSR; where X1, X2, or X3 is any one of 20 common amino acids.

[0025] In step 4, the corresponding hotspot residues of the peptides in the affinity peptide library 1 are THR-484, ILE-485, PHE-419, GLY-487, SER-488, LEU-489, SER-490, and ARG-491.

[0026] The present invention provides a reagent containing at least one of the affinity peptides described in the present invention or the affinity peptides prepared by the preparation method described in the present invention, and an excipient for maintaining the activity of the affinity peptides.

[0027] Furthermore, the excipients for maintaining the activity of the affinity peptide include: buffers, stabilizers, preservatives, isotonic modifiers, lyophilization protectants, surfactants, antioxidants, excipients, cosolvents, and / or metal ion chelators.

[0028] Furthermore, the buffer is used to maintain the pH stability of the system and prevent the peptide from being degraded or aggregated due to acid-base changes. The buffer includes: phosphate, tris(hydroxymethyl)aminomethane, histidine and / or citric acid.

[0029] The stabilizers are used to maintain the spatial conformation of proteins / peptides and prevent denaturation, aggregation, or degradation; they also maintain activity under stress conditions such as lyophilization, high temperature, shaking, and acid-base fluctuations. The stabilizers include: sucrose, trehalose, glucose, lactose, mannitol, sorbitol, xylitol, EG 2000–8000, PVP, gelatin, dextran, glycine, arginine, proline, alanine, ammonium sulfate, sodium chloride, potassium phosphate, glycerol, Tween-20 / 80, and / or poloxamer 188.

[0030] The preservatives are used to inhibit the proliferation of microorganisms such as bacteria and fungi, ensuring the microbial safety of multi-dose or multiple-use formulations throughout their shelf life and usage period; the concentration must be below the protein toxicity threshold and not affect activity; the preservatives include: phenol, m-cresol, methyl ester, ethyl ester, propyl ester, butyl ester and their sodium salts, benzalkonium chloride, benzalkonium bromide, benzyl alcohol, chlorobutanol, sorbic acid, benzoic acid, imidazolidinyl urea and / or DMDM ​​hydantoin;

[0031] The isotonicity modifier is used to ensure that the formulation is isotonic with body fluids, reduce injection irritation and protein denaturation, and the isotonicity modifier includes: sodium chloride, glucose, mannitol and / or glycerol;

[0032] The freeze-drying protectant is used to prevent protein denaturation and aggregation during freezing and drying processes. The freeze-drying protectant includes sucrose, trehalose, mannitol and / or polyethylene glycol.

[0033] The surfactant is used to inhibit the adsorption and aggregation of proteins at the interface (gas-liquid, solid-liquid), and the surfactant includes: polysorbate 20, polysorbate 80 and / or poloxamer 188.

[0034] The antioxidant is used to prevent loss of peptide activity due to oxidation or reduction, and the antioxidant includes: methionine, ascorbic acid, glutathione and / or EDTA;

[0035] The excipients are used to provide a solid framework during freeze-drying to ensure appearance and reconstitution speed. The excipients include mannitol, glycine, lactose, and / or dextran.

[0036] The co-solvent is used to improve the solubility of the peptide and prevent precipitation. The co-solvent includes propylene glycol and / or cyclodextrin derivatives.

[0037] The metal ion chelating agent is used to inhibit metal-catalyzed oxidation or aggregation, and the metal ion chelating agent includes EDTA and / or citric acid.

[0038] This invention provides a conjugate comprising the affinity peptide and conjugation medium described herein;

[0039] The coupling media include protein carriers, magnetic microparticles, and / or chromatographic packing materials.

[0040] Furthermore, the protein carrier comprises keyhole hemocyanin; the chromatographic packing material comprises Sepharose 4Fast Flow (4FF) and / or EAH-Sepharose 4B.

[0041] This invention provides a kit comprising:

[0042] At least one of the following shown in A) to D) and an enzyme-linked immunosorbent assay (ELISA) reagent; or

[0043] At least one of the following shown in A) to D) and a protein purification reagent;

[0044] A) The affinity polypeptide described in this invention;

[0045] B) The affinity polypeptide prepared by the preparation method described in this invention;

[0046] C) The reagents described in this invention;

[0047] C) The coupling compound described in this invention.

[0048] This invention provides the use of at least one of the following (a) to (e) in the detection and / or purification of human serum albumin:

[0049] a) The affinity polypeptide described in this invention;

[0050] b) The affinity peptides prepared by the method described in this invention;

[0051] c) The reagents described in this invention;

[0052] d) The coupling described in this invention;

[0053] e) The reagent kit described in this invention.

[0054] The present invention provides a method for detecting and / or purifying human serum albumin, comprising using any one of the following I) to V) to achieve the detection and / or purification of human serum albumin;

[0055] I) The affinity polypeptide described in this invention;

[0056] II) The affinity peptides prepared by the method described in this invention;

[0057] III) The reagents described in this invention;

[0058] IV) The coupling compound described in this invention;

[0059] V), the reagent kit described in this invention.

[0060] The present invention also provides a nucleic acid encoding the said affinity polypeptide;

[0061] Furthermore, a heavy-load carrier containing the aforementioned affinity peptide is provided;

[0062] Furthermore, it provides host cells for transfection and / or transformation of the recombinant vector;

[0063] The present invention provides a method for preparing the aforementioned affinity peptide, which involves culturing host cells as described in the present invention to obtain a culture containing the aforementioned affinity peptide.

[0064] This invention obtains and verifies affinity peptides for human serum albumin through biomimetic and intelligent design, and its features include the following steps:

[0065] 1. Regarding HSA and Mgb c7HopQ NbAlb1 The crystal structure of the complex (PDB ID: 8oi2) was resolved, and HSA and Mgb were determined using MD simulation and MM-PBSA. c7HopQ NbAlb1 Hotspot residues;

[0066] The Mgb c7HopQ NbAlb1 Hotspot residues include: SER-488, LYS-452, VAL-2, PHE-419, ARG-491, SER-490, LEU-489, ILE-485, THR-484, GLY-487, LYS-51, LYS-430, LYS-185;

[0067] The hotspot residue sequences of the HSA are LYS-313, ALA-320, ALA-362, LYS-372, ALA-364, CYS-361, LYS-378, and ASP-324.

[0068] 2. Based on Mgb c7HopQ NbAlb1The spatial distribution of hotspot residues was analyzed, and a biomimetic affinity peptide library with characteristic sequences TIX1GSLSR and FX2ITX3GSLSR was determined using amino acid localization. Based on the hotspot residues of HSA, an intelligently designed peptide library containing affinity peptide sequences DGALTPPS (peptide 12, SEQ ID NO: 12) and DGALTPPSEY (peptide 13, SEQ ID NO: 13) was obtained using IDProMat (software copyright registration number 2023SR1603462) to expand the affinity peptides.

[0069] 3. 420 peptides from a biomimetic affinity peptide library were initially screened using Autodoc VINA molecular docking software. E0 was retained. VINA Affinity peptides with ≤-7.5 kcal / mol were identified, totaling 414. Mgb was calculated. c7HopQ NbAlb1 The conformational similarity between hotspot residues and corresponding hotspot residues in the aforementioned 414 affinity peptide sequences was analyzed. Among the decapeptide sequences, 25 peptides with RMSD ≤ 0.4 nm were selected. Due to the relatively small peptide library, 19 octapeptide sequences were retained for further analysis. The hydrophilicity and hydrophobicity of the peptides were assessed based on GRAVY (average hydrophilicity coefficient) and logP (oil-water partition coefficient), ultimately selecting 11 affinity peptides with good hydrophilicity for subsequent validation.

[0070] 4. Using the GROMACS software package, MD simulations were conducted to verify the binding of the above 13 affinity peptides (11 biomimetic design peptides + 2 intelligent design peptides) to HSA. Finally, four peptides, TIQGSLSR (peptide 1), FKITSGSLSR (peptide 6), DGALTPPS (peptide 12), and DGALTPPSEY (peptide 13), were selected for double-sandwich enzyme-linked immunosorbent assay.

[0071] 5. The results of the double-sandwich enzyme-linked immunosorbent assay (ELISA) showed that FKITSGSLSR (peptide 6) and DGALTPPSEY (peptide 13) exhibited a stronger binding affinity. Furthermore, adsorption isotherms and affinity chromatography experiments demonstrated that the peptides screened in this invention have good binding affinity with HSA, and can effectively adsorb and separate HSA.

[0072] This invention analyzes HSA-Mgb using MD simulation technology and MM-PBSA free energy decomposition method. c7HopQ NbAlb1The molecular mechanism and microscopic details of complex interactions were investigated, key binding sites and their spatial distribution between complexes were identified, and affinity models were established to construct a candidate affinity peptide library. This library was then expanded through intelligent design. Subsequently, the affinity peptide library was screened and validated using methods such as molecular docking, conformational analysis, hydrophobicity analysis, and MD simulation to obtain high-affinity peptides targeting HSA. The binding performance of the affinity peptides to HSA was verified using a one-step sandwich ELISA assay with double antibodies, adsorption isotherm determination, and affinity chromatography. Peptides such as FKITSGSLSR (peptide 6) and DGALTPPSEY (peptide 13) obtained through screening exhibited high affinity for HSA. Attached Figure Description

[0073] Figure 1 Mgb c7HopQ NbAlb1 The key binding sites are A for decapeptide ligand and B for octapeptide ligand.

[0074] Figure 2 ELISA results demonstrating the binding properties of affinity peptides;

[0075] Figure 3 The chromatographic purification chromatogram of HSA by EAH-FKITSGSLSR is shown.

[0076] Figure 4 The chromatographic chromatogram of HSA purification by 4FF-DGALTPPSEY is shown. Detailed Implementation

[0077] This invention provides affinity peptides for human serum albumin and their design methods. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0078] Mgb c7HopQ NbAlb1 It is an artificially designed chimeric protein, officially abbreviated as megabody (Mgb), which is composed of three parts assembled in sequence:

[0079] 1. Mgb: A common prefix for megabody

[0080] 2. c7HopQ: A “circular rearrangement” version of the adhesin HopQ from Helicobacter pylori (H. pylori), serving as a macromolecular rigid scaffold;

[0081] 3. NbAlb1: A nanobody (VHH) that can simultaneously bind to human and mouse serum albumin.

[0082] Mgb c7HopQ NbAlb1 It formed a stable complex with HSA, achieving a resolution of 2.3 Å;

[0083] EVINA is a scoring function used to assess the protein-ligand binding free energy, expressed in kcal / mol. A more negative value indicates a stronger binding affinity between the ligand and the protein.

[0084] RMSD (Root Mean Square Deviation) measures the average deviation of a protein structure from a reference structure (such as a crystal structure) during simulation or prediction, and is measured in nm. A smaller value indicates that the structure is closer to the reference conformation.

[0085] GRAVY (Grand Average of Hydropathy) is the average hydrophobicity value of all amino acids, used to assess the overall hydrophobicity of a protein. A more negative value indicates a more hydrophilic protein, while a more positive value indicates a more hydrophobic protein.

[0086] logP (partition coefficient) is the logarithm of the partition coefficient between octanol and water, used to measure the lipophilicity of a compound. A higher logP value indicates greater lipophilicity and a higher likelihood of crossing cell membranes.

[0087] Pbind (binding probability or binding constant) is usually used to represent the binding strength or binding constant (such as Kd or Ki) between a protein and a ligand. In some literature, it may also be the binding probability calculated through simulation.

[0088] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0089] Example 1 HSA and Mgb c7HopQ NbAlb1 Interaction Mechanism Analysis

[0090] This study uses HSA-Mgb c7HopQ NbAlb1 The complex crystal structure (PDB ID: 8oi2) was used as the initial template to construct an all-atom model. Molecular dynamics simulations were performed using the GROMOS96 43a1 force field, and the water solvent was represented using an SPC model. The system was placed in an area with dimensions of 19.0 × 11.8 × 17.5 nm. 3 372 Na+ molecules were introduced into the cube-shaped box. + 364 Cl -And 123,285 water molecules to simulate the saline environment.

[0091] MD simulations were performed using GROMACS. Temperature was maintained at 310.15 K using the velocity-rescale algorithm. Pressure was adjusted to 1 bar using a Parrinello-Rahman coupler. The Linear Constraint Solver (LINCS) algorithm was used to maintain bond length invariance. The Particle-Mesh Ewald (PME) algorithm was used to calculate long-range electrostatic forces. Periodic boundary conditions were enabled in the x, y, and z directions. The cutoff values ​​for both Coulomb and LJ potentials were set to 1.2 nm. Leap-frog integration was used with an integration step size of 2 fs. Steepest descent minimization was used for energy minimization. A 100 ps NVT ensemble was used for pre-equilibration. Three 100 ns MD simulations were performed using the NPT ensemble.

[0092] HSA-Mgb was calculated by free energy decomposition of MM-PBSA. c7HopQ NbAlb1 The binding free energy of the complex. Van der Waals interaction energy ΔG vdW = -75.32 kcal / mol, nonpolar solvation energy ΔG SASA =-7.73kcal / mol, Coulomb electrostatic interaction energy ΔG elec =-476.25kcal / mol, polarization solvation energy ΔG PB =498.73 kcal / mol. Based on the free energy calculation equation, ΔG polar =22.47 kcal / mol, ΔG nonpolar = -83.05 kcal / mol, final binding free energy ΔG bind = -60.57 kcal / mol. The results indicate that the binding between the two is mainly due to hydrophobic interactions.

[0093] HSA and Mgb were determined based on the combined free energy decomposition method. c7HopQ NbAlb1 The contribution of each amino acid residue to the binding free energy was determined. Ultimately, hydrophobic interactions were identified as the primary driving force for binding, with Mgb... c7HopQ NbAlb1 The key residues that make important contributions to the binding of the upper pair are SER-488, LYS-452, VAL-2, PHE-419, ARG-491, SER-490, LEU-489, ILE-485, THR-484, GLY-487, LYS-51, LYS-430, and LYS-185.

[0094] Example 2 Construction of affinity peptide library

[0095] I. Construction of a biomimetic affinity peptide library

[0096] Taking into account free energy contribution, interaction network, spatial orientation, and distance between key sites, the first sequence in the biomimetic design of affinity peptides selected THR-484, ILE-485, GLY-487, SER-488, LEU-489, SER-490, and ARG-491 as hotspot residues to construct the first affinity peptide library TIXGSLSR (where X represents 20 common amino acids, octapeptide ligands), comprising 20 sequences. The second sequence selected PHE-419, ILE-485, THR-484, GLY-487, SER-488, LEU-489, SER-490, and ARG-491 as hotspot residues to construct the second affinity peptide library FX2ITX3GSLSR (where X3 or X3 represents 20 common amino acids, decapeptide ligands), comprising 400 sequences, such as... Figure 1 As shown; the first affinity peptide library and the second affinity peptide library together constitute a biomimetic affinity peptide library.

[0097] II. Construction of Intelligent Design Peptide Libraries

[0098] Based on the hotspot residue sequences LYS-313, ALA-320, ALA-362, LYS-372, ALA-364, CYS-361, LYS-378, and ASP-324 on HSA, we constructed intelligently designed peptides using the protein ligand intelligent design model IDProMat (software copyright registration number 2023SR1603462) developed by our research group. These peptides consist of two peptides: DGALTPPS (peptide 12, SEQ ID NO:12) and DGALTPPSEY (peptide 13, SEQ ID NO:13).

[0099] Example 3: Screening of affinity peptides from a biomimetic affinity peptide library

[0100] The peptides in the biomimetic affinity peptide library were sequentially subjected to molecular docking screening, root mean square deviation comparison, and hydrophobic residue analysis. The selected affinity peptides were then used for the next step of MD simulation.

[0101] I. Molecular docking

[0102] The peptide was sequentially docked with HSA using Autodoc VINA. E VINA Using ≤-7.5kcal / mol as the standard, 414 peptides were obtained for further screening.

[0103] II. RMSD Calculation

[0104] Calculate Mgb c7HopQ NbAlb1 The conformational similarity between hotspot residues (SER-488, LYS-452, VAL-2, PHE-419, ARG-491, SER-490, LEU-489, ILE-485, THR-484, GLY-487, LYS-51, LYS-430, LYS-185) and corresponding hotspot residues in affinity peptide sequences was analyzed. Among the decapeptide sequences, 25 peptides with RMSD ≤ 0.4 nm were screened, and 19 octapeptide sequences were selected, all of which were retained.

[0105] III. Hydrophobicity Analysis

[0106] The hydrophilicity and hydrophobicity of affinity peptides were analyzed using GRAVY (average hydrophilicity coefficient) and logP (oil-water partition coefficient). Finally, 11 peptides (5 octapeptides and 6 decapeptides) were screened out, and two peptides from the intelligent design peptide library were selected for further MD simulation analysis.

[0107] Table 1. Properties of candidate affinity ligands

[0108]

[0109] Example 4 Molecular Dynamics Simulation

[0110] The binding of 13 initially screened affinity peptides (11 from a biomimetic affinity peptide library + 2 smart-designed peptides) to HSA was investigated using molecular dynamics (MD) simulations. A GROMOS96 43a1 force field was used. A cubic box was constructed using the editconf command, and the complex was placed at the center of the box. The simulation system containing the octapeptide and HSA was placed at a field size of 14.8 × 11.4 × 10.4 nm. 3 Inside the box; the simulated system containing decapeptide and HSA was placed at 15.7 × 11.4 × 10.4 nm. 3 Inside the box. Water molecules and Na+ needed to balance the charge are added to the box using the `solvate` and `genion` commands. + and Cl - The SPC model was selected as the water molecule model. The steepest descent method was then used to minimize the energy of the system. A 100 ps NVT ensemble simulation was performed using the mdrun command, followed by a 100 ns NPT ensemble MD simulation. The parameters for the MD simulation were the same as in Example 1.

[0111] The peptides exhibited good binding properties; for example, FKITSGSLSR (peptide 6) bound to HSA at 7.9 ns, and DGALTPPSEY (peptide 13) bound to HSA at 7.2 ns. C and E LJThis indicates that both electrostatic and hydrophobic interactions are beneficial for bonding (as shown in Table 2). The overall minimum distance (d) min ), centroid distance (d) com Lennard-Jones potential (E) LJ Coulomb potential energy (E) C ) and peptide conformation parameters (RMSD, R g The free energy decomposition of MM-PBSA was investigated. TIQGSLSR (peptide 1), FKITSGSLSR (peptide 6), DGALTPPS (peptide 12), and DGALTPPSEY (peptide 13) were selected for further validation.

[0112] Table 2. Binding properties of the 13 peptides initially screened

[0113]

[0114] Example 5: Combination Effect Validation Experiment - One-Step Sandwich ELISA Assay with Double Antibodies

[0115] I. Methods

[0116] 1. Sandwich enzyme-linked immunosorbent assay

[0117] A one-step sandwich enzyme-linked immunosorbent assay (ELISA) was used to verify the binding properties of TIQGSLSR (peptide 1), FKITSGSLSR (peptide 6), DGALTPPS (peptide 12), and DGALTPPSEY (peptide 13) to HSA. First, the affinity peptides were conjugated to keyhole hemocyanin (KLH) using an EDC. Then, the affinity peptides were coated onto 96-well ELISA plates using CBS. CBS served as a blank control, while the peptide sequence NFISLLG (SEQ ID NO: 14), which does not react with HSA, and KLH served as negative controls. The positive control was the albumin antibody coated in the human serum albumin ELISA kit. The specific steps are as follows:

[0118] Peptide conjugation: Affinity peptide, KLH, and EDC were dissolved in 50 mM MES buffer (pH=6) to final concentrations of 0.4 mg / mL, 0.2 mg / mL, and 100 mg / mL, respectively. 1 mL of affinity peptide solution was slowly injected into 2 mL of KLH solution. 200 μL of EDC solution was slowly added dropwise, and the mixture was magnetically stirred at room temperature for 2 h to promote the conjugation reaction between the peptide and KLH. After the reaction, the mixture was transferred to a 15 kDa molecular weight cutoff dialysis bag and dialyzed five times with 1 L PBS at 4 °C (changing the dialysate every 4–6 h) to completely remove residual EDC and byproducts, yielding the conjugated peptide.

[0119] Bottom adsorption: A 90 μg / mL affinity peptide and negative control solution were prepared using CBS. 100 μL of each solution was added to a 96-well microplate. 100 μL of CBS was also added as a blank control. The plate was incubated at 4°C for 24 h.

[0120] Well sealing: Discard the liquid in the wells and add 200 μL of 1% gelatin in PBS solution. Incubate at 37°C for 2 h.

[0121] Human serum albumin adsorption: Discard the liquid in the wells, add 50 μL of 1.5 mg / mL human serum albumin and 100 μL of horseradish peroxidase-labeled detection antibody to the wells (including the positive control wells), and incubate at 37°C for 1 h.

[0122] Discard the liquid in the well, add 350 μL of washing solution to each well, let stand for 1 min, shake off the washing solution, pat dry with absorbent paper, and repeat 3 times.

[0123] Add 50 μL each of urea peroxide and TMB to each well and incubate at 37°C in the dark for 15 min.

[0124] Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm.

[0125] 2. Determination of adsorption isotherms

[0126] Affinity peptide chromatography media were prepared by coupling FKITSGSLSR (peptide 6) to EAH-Sepharose 4B (coupling product: EAH-peptide 6) and coupling DGALTPPSEY (peptide 13) to Sepharose 4 fast flow (coupling product: 4FF-peptide 13).

[0127] Dissolve appropriate amounts of HSA in equilibration buffer (20 mmol / L PB, pH 5, 7, 9) and dilute to obtain HSA standard solutions of different concentrations. Take 10 mg of the affinity peptide chromatography medium, dry it, and place it in a 2 mL centrifuge tube. Add 1 mL of HSA standard solutions of different concentrations to the centrifuge tube and incubate at 25℃ and 100 rpm for 12 h. After the reaction, centrifuge at 3000 rpm for 2 min. Measure the absorbance of the supernatant at 280 nm and calculate the adsorption density of the affinity peptide chromatography medium.

[0128] 3. Affinity Chromatography Experiment

[0129] The dynamic adsorption properties of the affinity peptide chromatographic medium were determined using a liquid chromatography system. The specific steps are as follows:

[0130] Column packing: Wash the affinity peptide chromatography medium (EAH-peptide 6 or 4FF-peptide 13) stored in 20% ethanol with PB buffer, dry it, and equilibrate it thoroughly with 2 mL of PB buffer. Vertically fix the column and add the packing suspension (i.e., affinity peptide chromatography medium) in portions, compacting the packing at a linear velocity of 0.9 mL / min. Adjust the adapter to ensure a stable and sealed column bed.

[0131] Column equilibration: The column was equilibrated with 20 mM PB solution under optimal adsorption conditions at a linear velocity of 1 mL / min. The UV absorbance of the detector was automatically zeroed and the wavelength was set to 280 nm.

[0132] Sample injection: Take an appropriate concentration of human serum albumin and inject it at a flow rate of 0.4 mL / min.

[0133] Column washing: Wash the column with equilibration buffer until the baseline returns to a stable state.

[0134] Elution: Human serum albumin adsorbed on the column was eluted using a gradient elution under optimal elution conditions. The linear velocity was 0.8 mL / min, and the eluent concentration increased linearly from 0% to 100% within 10 min, with elution continuing for a period of time.

[0135] Regeneration: The column was washed with 0.1 mol / L Gly-HCl (pH 2.4) at a linear flow rate of 1 mL / min for 5 CVs. The column was then reequilibrated with 10 CV equilibration buffer.

[0136] II. Results

[0137] 1. Results of double-sandwich enzyme-linked immunosorbent assay

[0138] ELISA experiments were performed on TIQGSLSR, FKITSGSLSR, DGALTPPS, and DGALTPPSEY, and the results are as follows: Figure 2 As shown ( Figure 2 In the sample, CBS served as a blank control, NFISLLG (SEQ ID NO:14) as a negative control, KLH as a negative control, and Antibody as a positive control. The affinity peptide was immobilized on a well plate, and HSA and HRP-labeled detection antibodies were added. The substrate TMB was used for color development; TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity was positively correlated with the HSA concentration in the sample. (The blank control group OD...) 450 =0.0644±0.0019, OD of NFISLIG in the negative control group 450 =0.0725±0.0025 (p=0.098, >0.05), OD of KLH in the negative control group 450=0.0714±0.0065 (p=0.127,>0.05), OD of albumin antibody in the positive control group 450 =0.1449±0.0021 (p<0.001), OD of TIQGSLSR 450 =0.0764±0.0055 (p=0.012, <0.05), OD of FKITSGSLSR 450 =0.0835±0.0035 (p<0.001), OD of DGALTPPS 450 =0.0809±0.0057 (p=0.001), OD of DGALTPPSEY 450 =0.0848±0.0102 (p<0.001). No significant differences were observed in either of the two negative control groups, and all four tested peptides were significantly higher than the blank control. Therefore, all the above-mentioned affinity peptides can bind effectively, with FKITSGSLSR (peptide 6) and DGALTPPSEY (peptide 13) showing stronger binding affinity.

[0139] 2. Results of adsorption isotherm measurements

[0140] FKITSGSLSR and DGALTPPSEY were coupled to EAH-Sepharose 4B and Sepharose 4 fastflow, respectively, to prepare affinity peptide chromatography media. Adsorption isotherms of HSA were determined at different pH and NaCl concentrations. Both media exhibited a relatively balanced adsorption capacity and affinity at pH=9. The qa of FKITSGSLSR-EAH was [not specified in the original text]. m =139.7 mg / mL, K d =2.04 μmol / L; q of 4FF-DGALTPPSEY m =59.10953 mg / mL, K d =4.16 μmol / L.

[0141] 3. Affinity chromatography results

[0142] Performance evaluation of affinity peptide chromatographic media was conducted using a liquid chromatography system. Results are as follows: Figure 3 and 4As shown. The adsorption conditions for the affinity peptide chromatographic medium EAH-FKITSGSLSR (EAH-peptide 6) were 20 mmol / L PB (pH=9), and the elution conditions were 20 mmol / L PB (pH=5, 500 mmol / L NaCl); the adsorption conditions for the affinity peptide chromatographic medium 4FF-DGALTPPSEY (4FF-peptide 13) were 20 mmol / L PB (pH=9), and the elution conditions were 20 mmol / L PB (pH=5, 200 mmol / L NaCl). The results indicate that HSA can be effectively separated and purified.

[0143] III. Summary

[0144] This invention proposes an affinity peptide for HSA and its design method. HSA-Mgb is analyzed using MD simulation technology and the MM-PBSA free energy decomposition method. c7HopQ NbAlb1 The molecular mechanism and microscopic details of complex interactions were analyzed to identify key binding sites and their spatial distribution, establishing affinity models to construct a candidate affinity peptide library. This library was then expanded through intelligent design. The affinity peptide library was screened and validated using methods such as molecular docking, conformational analysis, hydrophobicity analysis, and MD simulation to obtain high-affinity peptides targeting HSA. The binding performance of the affinity peptides to HSA was verified using a one-step sandwich ELISA assay with dual antibodies, adsorption isotherm determination, and affinity chromatography. The screened peptides, such as FKITSGSLSR and DGALRTPPSEY, were validated as effective affinity peptides for HSA, demonstrating the effectiveness of the design method.

[0145] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An affinity polypeptide of human serum albumin, characterized in that, comprising any one of polypeptides A-C; said polypeptide A is TIX1GSLSR, wherein X1 is any one of 20 common amino acids; said polypeptide B is FX2ITX3GSLSR, wherein X2 or X3 is any one of 20 common amino acids; said polypeptide C is DGALTPPSY1, wherein Y1 is null or EY.

2. The affinity polypeptide of claim 1, wherein, in said polypeptide A, X1 is selected from any one of D, E, Q, R, P; in said polypeptide B, X2 is selected from K, P, D or H; and X3 is selected from S, D, Q, H, C or K.

3. Process for the preparation of the affinity polypeptide according to claim 1 or 2, characterized in that, comprising the following steps: Step 1: HSA with Mgb of PDB ID: 8oi2 c7HopQ NbAlb1 Complex crystal structure is the basis for building full-atom model, and then with the help of MD simulation and MM-PBSA to determine the hot spot residues of HSA and Mgb c7HopQ NbAlb1 ​ Step 2: The characteristic sequence is determined by amino acid positioning method according to the spatial distribution of hotspot residues of Mgb c7HopQ NbAlb1 The biomimetic affinity polypeptide library is constructed. Step 3: Using molecular docking techniques, polypeptides from the biomimetic phage display library that bind E VINA polypeptides with a binding energy of < -7.5 kcal / mol are obtained. Step 4: Calculate Mgb c7HopQ NbAlb1 the root mean square deviation between the hot spot residues of said affinity polypeptide library 1 and the corresponding hot spot residues of the polypeptides of said affinity polypeptide library 2, retaining the polypeptides with a root mean square deviation < 0.4 nm, obtaining an affinity polypeptide library 3: Step 5: selecting polypeptides with negative GRAVY value and log P≤-1.7 from the affinity polypeptide library 2, and obtaining the affinity polypeptide of the present application after MD simulation verification; The HSA with Mgb of PDB ID: 8oi2 c7HopQ NbAlb1 The conditions for constructing the all-atom model based on the complex crystal structure are: the GROMOS96 43a1 force field is used for molecular dynamics simulation, the SPC model is used for solvent water molecules, the simulation system of the complex crystal structure is placed in a cubic box with a size of 19.0*11.8*17.5nm containing 372 Na⁺, 364 Cl⁻ and 123285 water molecules; 3 ​ said MD simulation is under the following conditions: temperature is 310.15 K; pressure is 1 bar; bond length is kept constant; long-range electrostatic force is calculated; Coulomb potential energy calculation cutoff is 1.2 nm, and LJ potential energy calculation cutoff is 1.2 nm; integral step is 2 fs; and pre-equilibrium is performed after energy minimization.

4. An agent characterized in that, at least one of the affinity polypeptide of claim 1 or 2 or the affinity polypeptide prepared by the preparation method of claim 3 and an excipient for maintaining the activity of the affinity polypeptide.

5. The agent of claim 4, wherein the excipient for maintaining the activity of the affinity polypeptide comprises a buffer, a stabilizer, a preservative, an isotonicity adjusting agent, a lyophilization protective agent, a surfactant, an antioxidant, an excipient, a co-solvent and / or a metal ion chelating agent.

6. Conjugate characterized in that, comprising: at least one of the affinity polypeptide of claim 1 or 2 or the affinity polypeptide prepared by the preparation method of claim 3 and a coupling medium; said coupling medium comprises a protein carrier, a magnetic microparticle and / or a chromatographic filler.

7. The conjugate of claim 6, wherein, said protein carrier comprises keyhole limpet hemocyanin.

8. A kit, characterized in that, comprising: at least one of A) to D) shown below and an enzyme-linked immunosorbent reagent; or at least one of A) to D) shown below and a protein purification reagent; A), the affinity polypeptide of claim 1 or 2; B), the affinity polypeptide prepared by the preparation method of claim 3; C), the reagent of claim 4 or 5; C), the conjugate of claim 6 or 7.

9. Application of at least one of a) to e) shown below in human serum albumin detection and / or purification: a), the affinity polypeptide of claim 1 or 2; b), the affinity polypeptide prepared by the preparation method of claim 3; c), the reagent of claim 4 or 5; d), the conjugate of claim 6 or 7; e), the kit of claim 8.

10. A method for the detection and / or purification of human serum albumin, characterized in that, comprising using any one of I) to V) to achieve human serum albumin detection and / or purification; I), the affinity polypeptide of claim 1 or 2; II), the affinity polypeptide prepared by the preparation method of claim 3; III), the reagent of claim 4 or 5; IV), the conjugate of claim 6 or 7; V), the kit of claim 8.

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