Affinity screening method and screened affinity

Abstract

The invention provides a method for screening an affinity body combined with a target protein. The method comprises the following steps: constructing a template polypeptide, preparing a protein structure, implementing molecular docking, optimizing docking conformation, establishing a virtual peptide library, verifying molecular dynamics, expressing the affinity body and measuring the affinity between the affinity body and the target protein. The invention also provides the affinity screened by the method, a nucleic acid molecule for coding the affinity, an expression vector containing the nucleic acid molecule, a pharmaceutical composition containing the affinity or the nucleic acid molecule or the expression vector and application of the pharmaceutical composition.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for screening an affibody and the screened affibody. Background Art

[0002] Prostate cancer is one of the most common malignant tumors of the male urinary reproductive system. The 2020 edition of the Global Cancer Statistics Report published by the WHO International Agency for Research on Cancer in February 2021 shows that in 2020, there were 1,414,259 new cases of prostate cancer worldwide, accounting for 7.3% of all malignant tumors in the body, and the incidence rate was second only to breast cancer and lung cancer, ranking third; there were 375,304 deaths from prostate cancer, accounting for 3.8% of all malignant tumors in the body, and the mortality rate ranked eighth. In January 2019, the National Cancer Center announced the latest incidence and mortality rates of malignant tumors in my country in 2015, including 72,000 new cases of prostate cancer, with an incidence rate of 10.23 / 100,000, ranking sixth among male malignant tumors; 31,000 deaths, with a mortality rate of 4.36 / 100,000, ranking tenth among male malignant tumors. From a global perspective, there are obvious geographical and racial differences in the incidence of prostate cancer. The incidence is high in Australia / New Zealand, North America and Europe, with an incidence of more than 85 / 100,000; the incidence is lowest in Asia, with an incidence of 4.5 / 100,000 to 10.5 / 100,000. Although the incidence of prostate cancer in my country is much lower than that in European and American countries, it has been increasing year by year in recent years. The main reasons for the increase in the incidence of prostate cancer in my country may be: the aging of the population, changes in people's lifestyles, and the popularization of prostate cancer screening methods such as prostate-specific antigen (PSA). Another feature of prostate cancer in my country is that the incidence in cities is significantly higher than in rural areas. In 2015, the incidence of prostate cancer in cities was 13.44 / 100,000, while that in rural areas was 6.17 / 100,000.

[0003] Carrying out early cancer screening and accurate cancer diagnosis is the key to preventing and treating cancer. Among them, prostate specific antigen (PSA) is a marker for early diagnosis and monitoring of prostate cancer, with an estimated sensitivity of up to 0.88. Prostate cancer markers including serum-bound PSA (Complex PSA, cPSA), free PSA (Free PSA, fPSA) and PSA isoform 2 (isoform [-2] proprostate-specific antigen, p2PSA) are increasingly receiving attention. Prostate specific antigen (PSA) is a single-chain polypeptide containing 237 amino acids, belonging to the family of serine proteases with tissue-specific chymotrypsin-like effects. PSA is tissue-specific and only exists in the cytoplasm of human prostate acinar and ductal epithelial cells, and is not expressed in other cells. Pathological conditions of the prostate can lead to elevated total PSA levels.

[0004] There are many chips and kits for detecting PSA on the market, most of which are based on antigen-antibody specific recognition. Antibodies are expensive and need to be stored at low temperatures, which results in high testing costs. At the same time, the amino acid sequences of cPSA, fPSA and p2PSA are highly homologous, so using these chips and kits for testing is prone to antibody cross-reactions.

[0005] Affibody is an artificial mutant derived from the B domain of Staphylococcal protein A (SPA). It is a new type of protein ligand with the ability to bind to other proteins. Affibody is composed of 58 amino acids, forming three α-helical structures, with a molecular weight of about 6.5kDa, which is only 1 / 20 of conventional antibodies and 1 / 2 of single-domain antibodies (sdAb). Due to its small relative molecular mass, it has strong permeability and tissue penetration. It can easily penetrate into the tissue gap in dense tissues such as solid tumors and specifically bind to cell surface or intracellular target proteins; unbound affibody in the blood can be quickly excreted from the body through glomerular filtration, and the background is lower when affibody labeled with fluorescent / radioactive isotopes is used as a tracer for in vivo imaging diagnosis; in addition, the immunogenicity of affibody is weak, and the probability of stimulating the body to produce an immune response is low. Conventional antibodies contain multiple domains. After the Fab segment binds to the antigen, the antibody conformation changes, which can activate complement and cause immune effects of the Fc segment such as opsonophagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading to unexpected immunotoxic side effects. Compared with monoclonal antibodies, the structure of the affibody molecule is very simple, with only three a helices and no Fc segment domain, thus avoiding the biological effects of the Fc end. Among them, the amino acid residues at 13 specific sites (9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32, 35) in the first and second helices have no obvious effect on the configuration of the affibody. These sites can be randomly mutated to form an affibody that can theoretically bind to any target molecule. The affibody has a high affinity for binding to the target protein and a stable structure, so it can be used for the detection, separation and purification of the target protein, and has a wide range of uses. Summary of the invention

[0006] In order to improve the above technical problems, on the one hand, the present invention provides a method for screening an affinity body that binds to a target protein, the method comprising constructing a template polypeptide, preparing a target protein structure, performing molecular docking, optimizing the docking conformation, establishing a virtual peptide library, molecular dynamics verification, expressing the affinity body and determining the affinity between the affinity body and the target protein.

[0007] In one embodiment of the present invention, the affibody template polypeptide is the affibody template numbered 2B88 in the PDB structure database.

[0008] In one embodiment of the present invention, the preparation of the target protein structure is to download the protein crystal structure PDB format file of the PSA protein numbered 2ZCH from the PDB database.

[0009] In one embodiment of the present invention, the molecular docking is specifically: molecular docking the template polypeptide with the target protein, and outputting the top 2000 docking conformations with the best binding ability.

[0010] In a preferred embodiment of the present invention, the molecular docking is performed by using one or more of the following software: Discovery Studio TM , Rosetta, FoldIt, HADDOCK, HEX, and LightDock; preferably, Discovery Studio TM This can be done with the RDOCKR or ZDOCK tool in the example, preferably the ZDOCK tool.

[0011] In one embodiment of the present invention, the optimization of docking conformations is specifically to perform cluster analysis using the ZRANK tool, take the top 30 conformations with the highest ranking, re-optimize the docking conformations using the RDOCK method, calculate the binding free energy using E-Rdock, select conformations with low free energy and that meet the purpose of the transformation, and screen out the docking conformations with the strongest binding ability.

[0012] In one embodiment of the present invention, the establishment of a virtual peptide library is specifically to perform saturation mutation on 13 amino acid sites on the affinity body in the docking conformation to establish a virtual peptide library, and preliminarily evaluate the affinity and stability of the screened mutants to the target protein.

[0013] In one embodiment of the present invention, the molecular dynamics verification is specifically to re-molecularly dock the screened mutants with the target protein, and perform static simulation verification at a temperature of 300K and normal pressure (1 Bar).

[0014] In one embodiment of the present invention, the expressing of the affibody comprises directly synthesizing the polypeptide of the affibody and fusing the gene of the mutant to an expression vector for expression.

[0015] In a preferred embodiment of the present invention, the expression vector is selected from the group consisting of pET vector, pGEX vector, pmal-c and pFLAG vector, preferably PET-28 vector.

[0016] In a preferred embodiment of the present invention, the expressing the affibody further comprises purifying the expressed affibody, preferably the purification uses nickel ion resin, more preferably uses His60 Ni Superflow Resin resin.

[0017] In one embodiment of the present invention, the affinity between the affibody and the target protein is determined using microthermophoresis (MST), biomembrane interferometry (BLI), or surface plasmon resonance (SPR), preferably microthermophoresis (MST).

[0018] On the other hand, the present invention provides an affinity body screened by the aforementioned method, whose amino acid sequence has more than 98% identity with the amino acid sequence shown in any one of SEQ ID NOs: 2-4; preferably, its amino acid sequence is shown in any one of SEQ ID NOs: 2-4.

[0019] In another aspect, the present invention provides a nucleic acid molecule encoding the affibody.

[0020] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NOs: 5-7.

[0021] In another aspect, the present invention provides an expression vector comprising the nucleic acid molecule.

[0022] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned affibody or nucleic acid molecule or expression vector and a pharmaceutically acceptable carrier.

[0023] In one embodiment of the present invention, the present invention provides the use of the aforementioned affibodies, nucleic acid molecules, expression vectors and pharmaceutical compositions for treating or preventing prostate diseases in subjects in need thereof;

[0024] The prostate disease is selected from prostate cancer, prostate hyperplasia or prostatitis.

[0025] In another aspect, the present invention provides the use of the aforementioned affibodies, nucleic acid molecules, expression vectors and pharmaceutical compositions in the preparation of a medicament for treating or preventing a prostate disease in a subject in need thereof;

[0026] The prostate disease is selected from prostate cancer, prostate hyperplasia or prostatitis.

[0027] In another aspect, the present invention provides use of the aforementioned affibody, nucleic acid molecule, expression vector and pharmaceutical composition in preparing a chip or a kit for detecting the level of PSA antigen in a subject.

[0028] In another aspect, the present invention provides the use of the aforementioned affibody, nucleic acid molecule, expression vector and pharmaceutical composition for screening PSA antibodies.

[0029] Beneficial Effects

[0030] The PSA-binding affibody provided by the present invention has good affinity and specificity with PSA, and can be applied to antigen-antibody detection of PSA to improve the detection efficiency of PSA. The present invention optimizes the coding sequence of the PSA-binding affibody provided to make it easier to express in bacteria.

[0031] The PSA-binding affibody provided by the present invention can be produced using bacterial fermentation technology, and its production capacity is much greater than that of PSA antibodies. It is easy to establish a platform system for the construction, expression, purification, and application of the affibody, effectively accelerating the establishment of an affibody industry platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the three-dimensional structure of the protein after PSA optimization with the PDB number 2ZCH.

[0033] Figure 2 This is a schematic diagram of the protein three-dimensional structure of the affinity body with the PDB number 2B88.

[0034] Figure 3 Schematic diagram of the main docking sites obtained by pre-processing the conformation of PSA and affibody protein using the ZDOCK tool.

[0035] Figure 4 Schematic diagram of the PSA-affine complex structure Pose35 screened using the ZRANK analysis tool.

[0036] Figure 5 The structural simulation diagram of the three affinity body mutants obtained by mutating the wild-type affinity body protein.

[0037] Figure 6 Figure 2 is the interaction diagram of three affimer mutants with PSA.

[0038] Figure 7 Schematic diagram showing the RMSD values ​​of the three affinity mutants and the PSA interaction model.

[0039] Figure 8 Schematic diagram showing the RMSF values ​​of the three affimer mutants and the PSA interaction model.

[0040] Fig. 9 Schematic diagram showing the changes in the number of hydrogen bonds between the three affinity mutants and the PSA interaction model during the kinetic simulation.

[0041] Fig.10 This is the 3D free energy topography diagram of the interaction system between three affinity mutants and PSA.

[0042] Fig.11 This is the alignment diagram of the sequencing results of mutant 1 and the designed sequence.

[0043] Fig.12 This is the alignment diagram of the sequencing results of mutant 2 and the designed sequence.

[0044] Fig.13 This is the alignment diagram of the sequencing results of mutant 3 and the designed sequence.

[0045] Fig.14 The figure shows the SDS-PAGE results of the purification of three affinity mutant proteins.

[0046] A is the result graph of mutant 1;

[0047] B is the result graph of mutant 2;

[0048] C is the result graph of mutant 3;

[0049] Lanes 1-4 are the uninduced total protein of the mutant, the induced total protein, the supernatant after centrifugal disruption, and the precipitate after centrifugal disruption, respectively;

[0050] Lane M is the marker.

[0051] Fig.15 The figure shows the SDS-PAGE results after gradient elution of three affinity mutant proteins.

[0052] A is the result graph of mutant 1;

[0053] B is the result graph of mutant 2;

[0054] C is the result graph of mutant 3;

[0055] Lanes 1 and 2 show the elution results with 100 mM imidazole;

[0056] Lanes 3, 4, and 5 are the results of elution with 200 mM imidazole;

[0057] Lanes 6, 7, and 8 are the results of elution with 300 mM imidazole;

[0058] Lanes 9, 10, and 11 are the results of elution with 500 mM imidazole;

[0059] Lane M is the marker.

[0060] Fig.16 The figure shows the SDS-PAGE results after the three affinity mutant proteins were concentrated.

[0061] A is the result graph of mutant 1;

[0062] B is the result graph of mutant 2;

[0063] C is the result graph of mutant 3;

[0064] Lane 1 is the concentrated protein;

[0065] Lane M is the marker.

[0066] Fig.17 The figure shows the MST detection results of the interaction between three affinity mutants and PSA antigen.

[0067] a, c, and e are negative controls BSA;

[0068] b is the result graph of mutant 1;

[0069] d is the result graph of mutant 2;

[0070] f is the result diagram of mutant 3. DETAILED DESCRIPTION

[0071] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0072] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0073] definition

[0074] As used herein, sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0075] As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0076] As used herein, "expression vector" includes a vector capable of expressing DNA that is operably linked to regulatory sequences such as promoter regions that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells as well as expression vectors that remain free or are integrated into the host cell genome.

[0077] As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment and / or cure. Prevention refers to preventing potential diseases and / or preventing symptoms from worsening or disease progression. Treatment also includes any pharmaceutical use of any nucleic acid molecules provided and the compositions provided herein.

[0078] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or disorder.

[0079] As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom. A complete prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0080] As used herein, "pharmaceutical composition" refers to a variety of preparations. The preparation containing a therapeutically effective amount of the nucleic acid molecules provided by the present application is in the form of a sterile liquid solution, liquid suspension or lyophilized form, optionally containing a stabilizer or excipient.

[0081] It will be appreciated that the affibodies described above will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into formulations to provide improved transfer, delivery, tolerance, etc. A wide variety of suitable formulations can be found in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 of Blaug, Seymour therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TMSM102, DOPE, cholesterol, and PEG 1000-DMG), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsions of polyethylene glycol (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be suitable for use in treatments or therapies according to the present application, provided that the active ingredients in the formulation are not inactivated by the formulation and that the formulation is physiologically compatible and tolerated by the route of administration.

[0082] As used herein, the term "subject" refers to mammals, such as humans, cows, and dogs.

[0083] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0084] Example 1: Molecular design of PSA affibody

[0085] 1. Interaction between PSA and Affimer and Molecular Recognition Model

[0086] 1.1PSA protein structure

[0087] The three-dimensional structure (3D) of PSA used in the present invention is from the PDB structure database (PDB ID: 2ZCH) with a resolution of Using Simulation / Dynamics / Standard Dynamics Cascade software, the 3D structure of PSA was optimized through hydrogenation, elimination of unreasonable conformations between atoms, molecular mechanics optimization, and other steps. The results are as follows Figure 1 shown.

[0088] 1.2 Protein structure of the affibody

[0089] The present invention uses the affibody crystal structure in the PDB structure database (PDB ID: 2B88) as a template. Figure 2 As shown, the three-dimensional structure of PSA and affinity body was constructed and optimized.

[0090] 1.3 Molecular recognition model of PSA and Affibody

[0091] Using molecular simulation software Discovery Studio TM (DS for short) tool to study the interaction between PSA and affinity body and the molecular recognition model.

[0092] 1.3.1 Pre-process the protein conformations of PSA and affimer to reduce unnecessary conformations and improve prediction accuracy. The ZDOCK tool of the DSPotein Docking module is used to dock the pre-processed PSA and affimer. The ZDOCK tool can translate and rotate the affimer in the space around the PSA protein to calculate the mutual binding ability. Specific parameters: During the calculation process, the Euler angle of 6 degrees is used to sample the binding conformations. The final number of docking samples can reach 54,000 binding conformations, from which the top 2000 docking conformations with the best binding ability are output for further analysis. The main docking sites are Figure 3 The red ball indicates.

[0093] 1.3.2 Cluster analysis of ZDOCK docking results using ZRANK analysis tool

[0094] For the PSA and affinity body interaction models that meet the modification purpose screened out above, the top 30 conformations with the highest ranking were selected, and the docking conformations were re-optimized using the RDOCK method.

[0095] By calculating the binding free energy through E-Rdock, the conformations with low free energy and meeting the purpose of modification are selected, and the docking conformations with the strongest binding ability are screened out.

[0096] Apply the Analyze Protein Interface module and set the maximum hydrogen bond and salt bridge interaction distances, i.e. and The surface probe radius is The key amino acid residues in the binding interface are analyzed to infer the binding mechanism and make modifications.

[0097] The results were analyzed and summarized, and it was found that among the top 30 conformations, the docking conformation Pose35 ( Figure 4) has better binding force than other docking results and can produce a stable conformation with PSA protein (the relevant data are shown in Tables 1 and 2), and the amino acids PHE17, ASN18, LEU19, PRO20, LEU22, ASN23, GLY24, VAL25, VAL27, LYS28, ILE31, ARG35 and other amino acids that interact on the affibody are all outside the mutable sites. In theory, the binding ability can be further enhanced by modifying the amino acids at the mutable sites, which is more in line with the design purpose. Therefore, the docking conformation Pose35 was selected as the most suitable PSA and affibody complex structure for virtual mutation.

[0098] Table 1 Interaction types and distances between Affimer and PSA

[0099]

[0100] Table 2 Surface amino acid residues involved in the binding of Affimers to PSA

[0101]

[0102] Example 2: Virtual screening of PSA affibodies

[0103] 2.1 Amino acid screening and mutation strategy at key sites of affibody protein

[0104] The optimal docking conformation Pose35 was selected as the mutation model, and the Calculate MutationEnergy Binding module was used to perform single-point mutations and saturation mutations on 13 amino acid sites in the affinity body to construct a mutant library. The calculation conditions did not set the effects of system pH and temperature.

[0105] If one wants to establish an ideal virtual affinity database, the library capacity should be of appropriate size, and the affinity of the affinity bodies in the library should be as high as possible.

[0106] If all 13 specific sites are saturatedly mutated, that is, each amino acid residue at the 13 sites is randomly mutated into another 19 amino acids, 2013 affibodies will be formed, which cannot be calculated by current calculation software, and the affinity of most molecules is not high.

[0107] Therefore, in order to find the desired high-affinity affibody from the established virtual combinatorial affibody library, the determination of the affibody mutation site and the selection of the mutated residues become the key to establishing the virtual library.

[0108] The present invention adopts the following strategies:

[0109] First, using the optimal binding model Pose35 as a template, single-point mutations were performed on 13 specific amino acid sites of the affinity body to ALA. Due to the small molecule and low steric hindrance of ALA, the effect of the mutation on the stability of the binding complex can reflect the magnitude of the binding effect of this site.

[0110] According to the calculation results of five groups of parameters, namely mutation free energy, effect of mutation, van der Waals term, electrostatic term and entropy term, the mutation free energy is used as the basis for screening. The calculation formula is as follows:

[0111] ΔGmut=ΔGfold(mut)-ΔGfold(wild);

[0112] Among them, ΔGmut (kcal / mol) is the folding free energy difference between the binding energy after mutation ΔGfold(mutant) and the binding energy before mutation ΔGfold(wild type).

[0113] The mutation results are divided into three states: stable, medium, and unstable. When the mutation free energy is less than -0.5, it is a stable state, that is, the affinity body has a stronger binding ability to PSA after mutation; when the mutation free energy is between -0.5 and 0.5, it is a medium state, that is, the affinity body has a similar binding ability to PSA after mutation; when the mutation free energy is greater than 0.5, it is an unstable state, that is, the binding ability becomes weaker after mutation.

[0114] Then, based on the mutation results of 13 amino acid sites, the key residues for the interaction between the affinity body and PSA were analyzed, the mutation sites and residue types of new affinity bodies were designed, the affinity body virtual mutant library was constructed, and the affinity body with the best binding ability was screened.

[0115] The calculation results of the single point mutation ALA show that mutations at the 13 amino acid sites have more or less effects on the thermal stability of binding, but the sites that can make the binding more stable are mainly 9, 11, 13, 17, 18, 25, 27, 28, 32, and 35. The effects of mutations at the remaining sites that have produced strong interaction on the binding energy are all neutral.

[0116] Finally, considering the above calculation results, the key amino acids involved in the affinity binding surface, and the capacity of the virtual affinity library, the mutation sites and mutation residue types of the following affinity library were designed. Sites 9, 11, 13, 17, 18, 25, 27, 28, 32, and 35 were mutated in the direction of more stable binding, that is, LEU9 mutated to ARG, LYS; TRP11 mutated to PRO; THR13 mutated to VAL; PHE17 mutated to TRP, ARG; ASN18 mutated to TYR, HIS; VAL25 mutated to TRP, PHE; VAL27 mutated to ARG, TRP; LYS2 8 mutated to ARG, PRO; ASP32 mutated to GLY; ARG35 mutated to TRP, PHE. As shown in Table 3, the binding ability was improved to varying degrees.

[0117] Table 3 Mutation screening of key amino acid sites

[0118]

[0119] According to the above mutation direction, saturation mutations were performed on amino acid sites 9, 11, 13, 17, 18, 25, 27, 32, and 35, and the generated mutant library contained a total of 128 mutants. Three mutants with the strongest ability to bind to PSA were screened out based on mutation free energy, and the mutation sites and mutated amino acids of the three mutants are shown in Table 4. The screened mutants have strong ability to bind to PSA and stable overall structure, so the three affinity mutants were modeled and simulated for verification.

[0120] Table 4 Key amino acid mutation scheme

[0121]

[0122] 2.2 Key amino acid replacement scheme and verification

[0123] According to the saturation mutation results, three affinity mutant sequences were determined, as shown in Table 5. The three mutant sequences selected were used to construct virtual mutants using Swiss-model, as shown in Table 5. Figure 5 As shown, the three mutants are similar to the original affinity structure ( Figure 2 )Comparative analysis can preliminarily determine that the amino acid mutation has not changed the secondary structure of the affinity body.

[0124] Table 5 Amino acid sequences of three mutants

[0125]

[0126] The three affimer mutants were re-docked with PSA using the ZDOCK module of DS software, the optimal conformations were screened out, and the interaction patterns between the affimer mutants and PSA were analyzed. Figure 6 The ribbon diagrams of the three Affimer mutants and PSA are shown respectively. Combined with the results of the interaction type and distance between the mutant Affimer and PSA (Table 6), it can be seen that the specific sites on the first and second α helices of the mutants have more interactions and larger contact areas after modification. The ZDOCK and ZRANK results also show that the interaction between the mutants and PAS is significantly enhanced. Therefore, the design of the three mutants is consistent with expectations.

[0127] Table 6 Comparison of the interaction between PSA protein and wild type and three mutants

[0128]

[0129] The molecular dynamics verification of the interaction model between the three mutants and the affinity body was carried out, and static simulation verification was carried out at a temperature of 300K and normal pressure (1Bar). The force field was Amber99sb-ildn, and the solvent was water molecules (Tip3p water model). By adding an appropriate amount of Na + ions to neutralize the total charge of the simulated system.

[0130] The molecular dynamics simulation system first uses the steepest descent method to minimize the energy, and then performs 100,000 steps of isothermal and isochoric ensemble (NVT) equilibrium and isothermal and isobaric ensemble (NPT) equilibrium, with a coupling constant of 0.1 ps and a duration of 100 ps. Finally, a free molecular dynamics simulation is run, which consists of 5,000,000 steps, a step size of 2 fs, and a total duration of 100 ns.

[0131] After completing the simulation, the software's built-in tools were used to analyze the trajectory, calculate the root mean square deviation (RMSD), root mean square fluctuation (RMSF) and protein gyration radius of each amino acid trajectory, and analyze and evaluate the docking effect based on data such as the free energy (MMGBSA) free energy profile.

[0132] From the RMSD values ​​of the three mutants ( Figure 7 ), the overall RMSD of the complex of mutant 1 and mutant 2 with PSA is less than 1.5nm, which has good structural stability. Among them, the complex system is blue with a lower RMSD, indicating that the addition of the affinity is conducive to the further stabilization of the PSA structure. The average RMSD of mutant 1 is 0.34nm, and the variance is 0.057nm, the average RMSD of mutant 2 is 0.32nm, and the variance is 0.052nm, and the average RMSD of mutant 3 is 0.35nm, and the variance is 0.0360nm. The average RMSD and variance of the overall compound are relatively low, and the fluctuation is small.

[0133] In the interaction model between different mutants and PSA, the residue freedom of PSA in mutant 1 fluctuates more at 60 and 75 than in mutant 2, and that in mutant 3 fluctuates more around amino acid 95. The overall trends of the three systems are basically the same, such as Figure 8 shown.

[0134] Mutant 1 has a large fluctuation in the number of hydrogen bonds at 18000ps and 58000ps, while the number of hydrogen bonds in the system of mutant 2 is stable between 5-10. For mutant 3, the number of hydrogen bonds is stable at about 10 in the first 40000ns, and increases to about 15-20 after 43000ns ( Fig. 9 ).

[0135] Table 7 MMPBSA binding free energy scores for the three systems

[0136]

[0137] From the overall table 7, mutant 1, mutant 2 and mutant 3 all have lower binding free energy, and mutant 3 has a binding free energy of -75.59 kcal / mol, which has better binding force. Mutant 3 is significantly superior to the other two mutants in the van der Waals force level.

[0138] The data was further visualized and the MMPBSA analysis results were plotted, such as Fig.10 shown.

[0139] From the above kinetic simulation results, the two systems have similar effects, relatively stable structures, and low binding energy. Among them, mutant 3 is more stable than mutant 1 and mutant 2 in terms of RMSD, RMSF, and the number of hydrogen bonds. Its binding free energy score is -75.59 kcal / mol, which is better than mutant 1 and mutant 2, which have binding free energy scores of -44.41 and -47.62 kcal / mol, respectively.

[0140] Example 3: Prokaryotic expression and affinity verification of affinity mutants

[0141] 3.1 Construction of expression vector for affinity mutants

[0142] According to the affinity mutant sequences obtained based on the molecular docking screening, Wuhan Jinkairui Bioengineering Co., Ltd. was commissioned to synthesize affinity mutant genes. The synthesized genes (including the pET-28 vector) were named pET-28-2a76 (mutant 1), pET-28-2a77 (mutant 2), and pET-28-2a78 (mutant 3). The NdeI cloning site on the pET-28 vector was selected, and the synthesis was CAT with NdeI retained at the N-terminus and CATATG with NdeI retained at the C-terminus. After sequencing verification, the nucleotide sequences of affinity mutant 1 (2a76), affinity mutant 2 (2a77) and affinity mutant 3 (2a78) are shown in SEQ ID NO: 5-7, respectively. The sequencing results of mutants 1-mutant 3 were aligned with the designed sequences as shown in Figure 11-13 shown.

[0143] 3.2 Induction and expression of affibody mutants

[0144] Taking the protein expression and purification of mutant 1 as an example, the specific operation steps are described. The expression and purification operation steps of mutant 2 and mutant 3 are the same.

[0145] The correctly sequenced recombinant plasmid of mutant 1 was transferred into E. coli Rosetta (DE3) competent cells by chemical transformation, spread on LB solid plates (kanamycin resistance), and cultured for about 12 hours. A single colony was picked and inoculated into LB liquid culture medium (kanamycin + thiamphenicol resistance), and cultured overnight at 37°C and 180rpm. Then, it was transferred (1%) to a 1.5L Erlenmeyer flask (the liquid volume in the bottle was 500mL LB liquid culture medium) and cultured at 37°C and 180rpm until the OD600 absorbance of the bacterial solution was between 0.6 and 0.8. IPTG (final concentration was 0.2mM / L) was added, induced overnight at 18°C ​​and 150rpm, and the bacteria were collected by centrifugation at 4000rpm. Add 100 μL of protease inhibitors, lysozyme with a final concentration of 200 μg / mL, and 10 μL of DNase I to the bacterial suspension, place on ice, let stand for 30 minutes, ultrasonically disrupt for 15 minutes, and then centrifuge at 4°C, 15,000rpm for 30 minutes. Transfer the supernatant to a 13mL filter column containing 1mL His60 NiSuperflow Resin, centrifuge at 4°C, 10rpm, incubate for 2 hours, allow the target protein to bind to the filler, then wash the nickel column 5 times with wash buffer (20mM imidazole), then wash 10 times with wash buffer (50mM imidazole), and finally elute with 4mL of eluent for each gradient (100mM imidazole, 200mM imidazole, 300mM imidazole, 500mM imidazole), collect the eluent, and use 15% SDS-PAGE electrophoresis to detect the protein purification effect.

[0146] To test whether the affinity mutant protein can be induced to express, SDS-PAGE was performed after a small amount of induced expression. The results are as follows Fig.14 As shown. The molecular weight of the known mutant proteins is 9kDa. Fig.14 There is no band between 6.5KDa and 14.4KDa in lane 1, but there is a band at the corresponding position in lane 2, indicating that the protein is successfully expressed. There are also bands at the corresponding positions of lanes 3 and 4, indicating that the target protein is soluble and expressed.

[0147] The results of SDS-PAGE detection of the mutant 1, mutant 2 and mutant 3 protein after nickel column purification are as follows Fig.15 Lanes 4, 5, 6, 7, 8, 9, 10, and 11 all have bands between 6.5 and 14.4 kDa, which is consistent with the molecular weight of the target protein. After gradient elution, the impurity proteins gradually decrease.

[0148] According to the imidazole elution result chart ( Fig.15 ), the target bands were collected and combined, desalted and concentrated by ultrafiltration tube, and the concentration of the target protein after concentration was mutant 1: 2.5 mg / mL; mutant 2: 0.6 mg / mL; mutant 3: 5 mg / mL. The SDS-PAGE detection results after concentration are shown in Fig.16 .

[0149] In general, during the affinity chromatography stage, mutant 1 protein was stable and had a relatively high yield; mutant 2 protein was less stable and had a relatively low yield; and mutant 3 protein had the best stability and the highest yield.

[0150] 3.3 Purification of affinity mutants and affinity testing

[0151] The binding of PSA protein to the affinity mutant was detected by microscale thermophoresis (MST). Take mutant 1 protein as an example, and the negative control operation steps are the same. First, mutant 1 protein (2a76 protein) was labeled with RED-cy5 dye, and the specific steps are as follows. 10μL 100μM mutant 1 protein (2a76 protein) and 2.5μL 1200μM RED-cy5 dye were added to 87.5μL buffer (a mixture of 10μM 2a76 protein and 30nM RED-cy5 dye at a final concentration), incubated on ice in the dark for 30min, and then the excess dye was removed using column B. Before determining the Kd value, the fluorescent labeled protein was diluted to a final concentration of 20nM, that is, 297μL buffer was added to the centrifuge tube, and then 3μL 2μM RED-cy5 labeled 2a76 protein was added, and mixed. This operation was performed on ice. Secondly, the ligand (PSA protein) protein is gradiently diluted as follows. Prepare 16 PCR tubes, and add 10 μL of MST buffer to each tube 2-16, and add 10 μL of ligand (PSA) protein to the first and second PCR tubes respectively. After mixing the MST buffer and ligand protein in the second PCR tube, pipette 10 μL of the mixture of MST buffer and ligand protein into the third PCR tube, and dilute stepwise to the 16th tube in the above manner. After gradient dilution, add 10 μL of fluorescently labeled mutant 1 protein (2a76 protein) to each of the above PCR tubes, mix thoroughly and let stand for 10 minutes. The sample was drawn up using a capillary (NT.115.k002) and placed into the MST instrument (Monolith NT.115 instrument, Nanotemper Technologies) in descending order of ligand concentration. The instrument parameters selected the Nano-Red red light channel, the affinity determination option, the labeled fluorescent molecule concentration of 30 nM, and the initial ligand concentration of 10 μM. After the instrument was run, data analysis was performed using the instrument's own software, Nanotemper Analysis software, v 2.3.

[0152] The MST test results of negative control BSA and PSA are as follows Fig.17 (Figure a, c, e), its uniformity is good, there is no protein aggregation, but the affinity is poor, there is no obvious binding within the given BSA concentration range. The MST test results of mutant 1, mutant 2, mutant 3 and PSA are shown in Fig.17(Figure b, d and f), among which mutant 1 has the smallest Kd value with PSA, about 0.835μM; followed by mutant 2, with a Kd value of 2.701μM; mutant 3 has the largest Kd value, which is estimated by the software to be 28.366μM, but the actual binding is not obvious from the figure. It is similar to the negative control BSA. In summary, mutant 1 has the strongest affinity with PSA and has good specificity for PSA.

[0153] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. 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 method for screening an affinity body that binds to a target protein, the method comprising constructing a template polypeptide, preparing a protein structure, performing molecular docking, optimizing a docking conformation, establishing a virtual peptide library, molecular dynamics verification, expressing the affinity body, and determining the affinity between the affinity body and the target protein.

2. The screening method according to claim 1, wherein the template polypeptide is an affibody template numbered 2B88 in the PDB structure database; The molecular dynamics verification is specifically to re-dock the selected mutants with the target protein, and perform static simulation verification at a temperature of 300K and normal pressure (1 Bar).

3. The screening method according to claim 1 or 2, wherein the target protein structure is prepared by downloading a protein crystal structure PDB format file of the PSA protein numbered 2ZCH from the PDB database; Preferably, the molecular docking is specifically: molecular docking the template polypeptide with the target protein, and outputting the top 2000 docking conformations with the best binding ability; More preferably, the molecular docking is performed by using one or more of the following software: DiscoveryStudio TM , Rosetta, FoldIt, HADDOCK, HEX, and LightDock; preferably, Discovery Studio TM The RDOCKR or ZDOCK tool in the embodiment is used, preferably the ZDOCK tool; Preferably, the optimization of docking conformations is specifically to use the ZRANK tool to perform cluster analysis, take the top 30 conformations with the highest ranking, re-optimize the docking conformations using the RDOCK method, calculate the binding free energy through E-Rdock, select conformations with low free energy and meeting the purpose of transformation, and screen out the docking conformations with the strongest binding ability; Preferably, the virtual peptide library is established by performing saturation mutation on 13 amino acid sites on the affinity body in the docking conformation to establish a virtual peptide library, and preliminarily evaluating the affinity and stability of the screened mutants to the target protein; Preferably, the expressing of the affibody comprises directly synthesizing the polypeptide of the affibody and fusing the gene of the mutant to an expression vector for expression; Preferably, the affinity between the affibody and the target protein is determined using microthermophoresis (MST), biomembrane interferometry (BLI), or surface plasmon resonance (SPR), preferably microthermophoresis (MST).

4. The screening method according to any one of claims 1 to 3, wherein the expression vector is selected from the group consisting of pET vector, pGEX vector, pmal-c and pFLAG vector, preferably PET-28 vector; Preferably, the expressing the affibody further comprises purifying the expressed affibody, and preferably the purification uses nickel ion resin, more preferably uses His60 Ni Superflow Resin resin.

5. The affibody screened out by the screening method according to any one of claims 1 to 4, wherein the amino acid sequence thereof has more than 98% identity with the amino acid sequence shown in any one of SEQ ID NOs: 2 to 4; preferably, the amino acid sequence thereof is shown in any one of SEQ ID NOs: 2 to 4.

6. A nucleic acid molecule encoding the Affibody according to claim 5; Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in any one of SEQ ID NOs: 5-7.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A pharmaceutical composition comprising the affibody according to claim 5 or the nucleic acid molecule according to claim 6 or the expression vector according to claim 7 and a pharmaceutically acceptable carrier.

9. Use of the affibody according to claim 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7 and the pharmaceutical composition according to claim 8 in the preparation of a drug for treating or preventing a prostate disease in a subject in need thereof; The prostate disease is selected from prostate cancer, prostate hyperplasia or prostatitis.

10. Use of the affibody according to claim 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7 and the pharmaceutical composition according to claim 8 in preparing a chip or a kit for detecting the level of PSA antigen in a subject.

11. Use of the affinity body according to claim 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7 and the pharmaceutical composition according to claim 8 for screening PSA antibodies.

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