Novel coronavirus spike protein-specific binding peptides and their applications

Abstract

The present invention provides a polypeptide that specifically binds to the novel coronavirus spike protein and its application. The amino acid sequence of the polypeptide is shown in the sequence listing SEQ ID NO.1. The polypeptide has good specific affinity and high stability. It can be efficiently prepared using an Escherichia coli expression system with low preparation cost. It can specifically bind to the novel coronavirus spike protein and thus be used in the detection and strain neutralization of the novel coronavirus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a novel coronavirus spike protein-specific binding polypeptide and its application. Background Art

[0002] The novel coronavirus (SARS-CoV-2) is a newly emerged RNA virus that belongs to the same β-CoV family as SARS-CoV. Its envelope has an average diameter of approximately 80 nm and features approximately 30 spike proteins on its surface. Studies have found that, similar to the SARS-CoV infection mechanism, SARS-CoV-2 utilizes its spike protein to recognize the angiotensin-converting enzyme 2 (ACE2) receptor on human cells to enter cells. Furthermore, the expression of ACE2 on the cell surface is positively correlated with the viral infection rate. This suggests that the key to SARS-CoV-2 infecting human cells lies in the binding of its spike protein to the ACE2 protein.

[0003] Professor Zhou Qiang's team at Westlake University used cryo-electron microscopy to determine the complex structure of the SARS-CoV-2 spike protein receptor domain RBD (monomeric protein) and the full-length human ACE2 receptor protein (Yan R, Zhang Y, Guo Y, XiaL, Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science, 2020, 367(6485):1444-1448). They identified the interaction sites between the SARS-CoV-2 spike protein and the extracellular domain of the cell receptor ACE2, and found that 13 amino acid residues in the first α-helix of ACE2 are involved in binding to the S protein, playing a key role in the protein-protein interaction.

[0004] SARS-CoV-2 is highly susceptible to mutation, with over 1,000 variants identified, including more than ten that are widely transmitted. Analysis of research data reveals that highly transmissible and pathogenic SARS-CoV-2 variants evade immune responses by reducing their affinity for neutralizing antibodies, while increasing their specific binding to ACE2 to increase infection efficiency. As a key receptor for SARS-CoV-2 infection of cells, specific functional fragments within the extracellular domain of ACE2 that specifically bind to the S protein have good specific affinity for both wild-type and variant strains. This means that peptides with the same spatial structure within the ACE2 extracellular domain will possess universal neutralizing activity against both wild-type and variant strains.

[0005] The ACE2 ectodomain not only binds to the SARS-CoV-2 spike protein but also exhibits exopeptidase activity, and S-protein binding does not affect this enzymatic activity. Using recombinant ACE2 ectodomains with full or reduced catalytic activity as SARS-CoV-2 blocking drugs could potentially disrupt the homeostasis of the renin-angiotensin-aldosterone system (RAAS), and is subject to long preparation times and high costs. The low affinity of ACE2 for the key S-protein binding fragment (amino acid residues 19-46) and poor molecular stability also limit its application.

[0006] Affibodies are a new type of affinity ligands derived from the immunoglobulin binding domain B of staphylococcal protein A (Nilsson B, Moks T, et al. A synthetic IgG-binding domainbased on staphylococcal protein A. Protein Engineering, 1987, 1(2): 107-113). They are single-chain structures composed of 58 amino acid residues and contain three α-helices. The receptor binding site of the affibody contains 13 amino acid residues. Karin Nord replaced the codons for these 13 amino acids with degenerate codons. The resulting mutants constitute the affibody library (Nord K, Nilsson J, et al. A combinatorial library of an alpha-helical bacterial receptor domain. Protein Engineering, 1995, 8(6): 601-608). The different affibodies produced by this recombinant method have specific binding activity and high affinity for many target protein molecules, including insulin, fibrin, transferrin (TRF), tumor necrosis factor α (TNF-α), etc. Affibody polypeptides are functionally similar to antibodies, and at the same time have advantages that antibodies do not have, including small relative molecular weight, high stability, good affinity and specificity, flexible application, and easy preparation. They are widely used in many fields of biological science. Therefore, based on the principles of protein molecular design, through rational design combined with experimental verification guidance, a high-affinity and high-stability ACE2-affibody chimeric polypeptide was obtained while retaining its natural structure. It is expected to specifically bind to the spike protein of the wild-type and mutant strains of the new coronavirus, and then be used for the detection and neutralization of the new coronavirus. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a novel coronavirus spike protein-specific binding polypeptide.

[0008] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned novel coronavirus spike protein specific binding polypeptide.

[0009] The technical solution adopted in the present invention is:

[0010] A novel coronavirus spike protein-specific binding polypeptide, whose amino acid sequence is shown in the sequence listing SEQ ID NO.1, SEQ ID NO.2 and / or SEQ ID NO.3.

[0011] Preferably, the above-mentioned novel coronavirus spike protein specific binding polypeptide has a nucleotide sequence as shown in the sequence listing SEQ ID NO.4, SEQ ID NO.5 and / or SEQ ID NO.6.

[0012] The above-mentioned novel coronavirus spike protein-specific binding polypeptide was rationally designed based on the primary structure and spatial structure analysis of the 19-46 amino acid residues of ACE2 and the affibody backbone sequence, and has high affinity activity for the novel coronavirus spike protein.

[0013] Preferably, the above-mentioned novel coronavirus spike protein specific binding polypeptide has an amino acid sequence as shown in the sequence listing SEQ ID NO.1.

[0014] Preferably, the above-mentioned novel coronavirus spike protein specific binding polypeptide has a nucleotide sequence as shown in the sequence listing SEQID NO.4.

[0015] A fusion protein is expressed by fusing one or more protein tags to the amino terminus or carboxyl terminus of the above polypeptide.

[0016] Preferably, in the above-mentioned fusion protein, the protein tag is a histidine tag, a myc tag, a Flag tag, an HA tag or a self-assembling short peptide.

[0017] Preferably, in the above-mentioned fusion protein, the protein tag is a self-assembling short peptide.

[0018] A fusion protein is obtained by fusing other functional proteins to the amino or carboxyl terminus of the above polypeptide, wherein the other functional proteins are enzyme proteins with catalytic function, or proteins with fluorescent luminescence function, or one or more of the above polypeptides.

[0019] Preferably, the above fusion protein is covalently or non-covalently bound to a fluorescent light-emitting group, biotin, a radioactive group, or a nanomaterial.

[0020] The use of the above-mentioned novel coronavirus spike protein-specific binding polypeptides and / or fusion proteins in the preparation of drugs or reagents for neutralizing and blocking the novel coronavirus.

[0021] The use of the above-mentioned novel coronavirus spike protein-specific binding polypeptides and / or fusion proteins in the preparation of novel coronavirus detection reagents or drugs.

[0022] Preferably, in the above application, when the detection reagent or drug is used to identify the new coronavirus, the identification method is an enzyme-linked immunosorbent assay (ELISA) detection method.

[0023] The beneficial effects of the present invention are:

[0024] The novel coronavirus spike protein-specific binding peptides described above exhibit strong affinity and high stability. They can be efficiently and cost-effectively produced using an E. coli expression system and are capable of specifically binding to the novel coronavirus (SARS-CoV-2) spike protein. Based on the principles of protein molecular design and the analysis of the primary and spatial structures of amino acid residues 19-46 of ACE2 and the affibody backbone sequence, three novel coronavirus spike protein-specific binding peptides were rationally designed. These peptides were produced using a prokaryotic E. coli expression system and their specific binding ability to the novel coronavirus (SARS-CoV-2) spike protein was verified by ELISA. The results demonstrated that all three designed peptides specifically bound to the novel coronavirus spike protein, with no significant affinity for BSA. These peptides possess significant advantages, including low production cost and excellent stability. They are promising neutralizing agents for the binding of the novel coronavirus spike protein to the ACE2 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure shows the alignment results of the Affibody backbone sequence and the ACE2 amino acid residue sequence at positions 19-46, where X represents any amino acid residue and is the site where the Affibody molecule binds to the target.

[0026] Figure 2 Schematic diagram of the design of peptides that specifically bind to the spike protein of three novel coronaviruses (SARS-CoV-2).

[0027] Figure 3 This is a diagram showing the purification results of peptides specifically binding to the spike proteins of three novel coronaviruses (SARS-CoV-2).

[0028] Figure 4The results of the specific binding of three peptides to the novel coronavirus spike S protein are shown in Figure 1. a is the result of the Affibody-H5 experimental group, b is the result of the Affibody-A28 experimental group, and c is the result of the Affibody-A13 experimental group.

[0029] Figure 5 Schematic diagram of the expression vector for the polymerized novel coronavirus (SARS-CoV-2) spike protein-specific binding polypeptide.

[0030] Figure 6 This is the purification result of the peptide that specifically binds to the polymerized novel coronavirus (SARS-CoV-2) spike protein.

[0031] Figure 7 This is a diagram showing the specific binding results of the polymerized novel coronavirus (SARS-CoV-2) spike protein-specific binding peptide to the novel coronavirus spike S protein.

[0032] Figure 8 This is the verification result of the diplicate concatemer (Affibody-H5) 2 blocking the binding of AEC2 to the new coronavirus S protein. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used are commercially available products unless otherwise specified.

[0035] Example 1

[0036] The design of peptides that specifically bind to the spike protein of the novel coronavirus (SARS-CoV-2) is as follows:

[0037] (1) The amino acid residue sequence of ACE2 at positions 19-46 was compared with the amino acid sequence of the affibdoy molecular skeleton (Nygren PA, Skerra A. Binding proteins from alternative scabfolds. Journal of Immunology Methods, 2004, 290(1-2): 3-28) to analyze homologous sequences;

[0038] (2) Analyze the similarity between the amino acid residue sequence of ACE2 at positions 19-46 and the spatial structure of the affibdoy molecular skeleton, and chimera the binding site of the SARS-CoV-2 spike protein in the amino acid residues of ACE2 at positions 19-46 with the affibody skeleton.

[0039] The amino acid sequence of the Affibody molecular backbone is aligned with the ACE2 amino acid residue sequence at positions 19-46. Figure 1 The results showed that the ACE2 functional fragment was similar to the Affibody molecular backbone sequence, with identical or similar amino acid residues appearing at multiple sites (marked with a gray background); the ACE2 functional fragment overlapped with the SARS-CoV-2 spike protein binding site (marked with "·" at the bottom) and the Affibody molecular target binding site (marked with the letter X) at 9 locations (marked with "△" combined with a solid line box at the bottom). Based on the above analysis results, three ACE2-Affibody chimeric peptides were designed. The design schematic is shown in the figure. Figure 2 , wherein (a) Affibody-H5 (amino acid sequence as shown in SEQ ID NO.1 of the sequence listing, nucleotide sequence as shown in SEQ ID NO.4 of the sequence listing) is a molecule in which the amino acid residues 19-46 of ACE2 are substituted for the first α-helix of Affibody ( Figure 1 At the same time, the six binding sites in the second α-helix of Affibody (including the one before Helix-2) were replaced by Figure 1 The polypeptide contains the spike protein binding site of the ACE2 amino acid residues 19-46 corresponding to the sequence alignment; (b) Affibody-A28 (the amino acid sequence is shown in SEQ ID NO.2 in the sequence listing, and the nucleotide sequence is shown in SEQ ID NO.5 in the sequence listing) is obtained by replacing the ACE2 functional fragment with the corresponding Affibody fragment (Affibody residues 9-36) in the sequence alignment; (c) Affibody-A13 (the amino acid sequence is shown in SEQ ID NO.3 in the sequence listing, and the nucleotide sequence is shown in SEQ ID NO.6 in the sequence listing) is obtained by replacing the target binding site in the Affibody backbone with the corresponding amino acid residues of the ACE2 functional fragment in the sequence alignment.

[0040] Example 2

[0041] Expression and purification of novel coronavirus (SARS-CoV-2) spike protein-specific binding peptides.

[0042] The three designed polypeptide coding genes were obtained by chemical synthesis, and the target protein genes were cloned into the pET21b prokaryotic expression vector (with artificial His-HA-tag tag added). The recombinant expression vector with correct sequencing was transformed into E. coli For BL21(DE3), single colonies were picked and transferred to 5 mL of LB / A medium (1 g tryptone, 0.5 g yeast extract, 1 g sodium chloride, dilute to 100 mL, autoclave; ampicillin was added to a final concentration of 100 μg / mL) and cultured overnight at 37°C, 180 rpm. The culture was inoculated into 200 mL of LB / A medium at a ratio of 1:50 and grown to logarithmic phase at 37°C, 180 rpm. Induction was then carried out overnight at 20°C, 180 rpm, with the addition of IPTG to a final concentration of 0.1 mM. The culture was harvested by centrifugation at 4400 rpm at 4°C. The cells were resuspended in 20 mL of pre-chilled PBS (8 g NaCl, Na2HPO4•12H2O, 0.2 g KCl, 0.24 g NaH2PO42•H2O), disrupted by sonication (3 s supersonication, 10 s off-time), and centrifuged at 12000 rpm for 15 min. The supernatant was collected.

[0043] Ni-TED column (purchased from Sangon Biotech Co., Ltd., catalog number C600803) was selected for the purification of recombinant polypeptides. First, the Ni-TED column was equilibrated with 10 column volumes of phosphate buffer; the collected protein supernatant was filtered through a 0.45μm filter membrane and loaded, flowed through 4 times, and the flow-through was retained for SDS-PAGE analysis; the column was washed with phosphate buffer containing 20mM imidazole (20 column volumes) to wash away impurities; phosphate buffer containing 50mM, 100mM, 250mM, and 400mM imidazole was prepared, and the target protein was eluted with 2 column volumes respectively. The protein samples collected with different concentrations of imidazole were analyzed by SDS-PAGE, and the purification results of Affibody-H5, Affibody-A28, and Affibody-A13 are shown in Figure 2. Figure 3 a. Figure 3 b and Figure 3 c, All three affibody peptides were purified successfully.

[0044] Example 3

[0045] Analysis of the binding characteristics of novel coronavirus (SARS-CoV-2) spike protein-specific binding peptides.

[0046] The three polypeptides expressed and purified in Example 2 were used as further research objects, and their affinity properties for the novel coronavirus (SARS-CoV-2) spike protein were detected by ELISA. At the same time, BSA was selected as an irrelevant antigen negative control. The specific operation is as follows:

[0047] The novel coronavirus (SARS-CoV-2) spike protein / BSA was diluted to 10 μg / mL in carbonate buffer (pH 9.6) and coated on a 96-well microplate at 4°C overnight. The plate was blocked with 3% skim milk powder-PBS and incubated at 37°C for 2 h. Three recombinant proteins, Affibody-H5, Affibody-A28, and Affibody-A13, with HA tags were added and incubated at 37°C for 2 h. Diluted HA antibody (purchased from Sangon Biotech Co., Ltd., Catalog No. D191044) was added and incubated at 37°C for 2 h. Diluted HRP-labeled rabbit anti-mouse secondary antibody (purchased from Sangon Biotech Co., Ltd., Catalog No. D110098) was added and incubated at 37°C for 45 min. TMB substrate was added for color development for 10 min. 2 M H2SO4 was added to terminate the reaction, and the OD was read on a microplate reader. 450 During the ELISA process, the plate was washed 4 times with PBST (PBS + 0.5% Tween-20) at each step. Figure 4 , it can be seen that Affibody-H5 has the best specific binding ability.

[0048] Example 4

[0049] Preparation and affinity analysis of novel coronavirus (SARS-CoV-2) spike protein-specific binding peptide polymers.

[0050] The peptide Affibody-H5 with high affinity and good specificity for the novel coronavirus (SARS-CoV-2) spike protein in Example 3 was used as a further research object to prepare a two-fold concatemer of Affibody-H5 (Affibody-H5) 2, a homotrimer based on a Foldon tag Affibody-H5-Foldon, and a homotrimer of the two-fold concatemer of Affibody-H5 based on a Foldon tag (Affibody-H5) 2-Foldon as peptides for capturing the novel coronavirus (SARS-CoV-2) spike protein in ELISA experiments.

[0051] References to the above preparation methods: (1) LiuJ, JiangY, et al. Development of activeaffibody aggregates induced by a self-assembling peptide for high sensitive detection of alpha-fetoprotein. Chemical Engineering Journal, 2022, 456:13508; (2) Meier S, Sarah Güthe, et al. Foldon, the natural trimerization domain of T4 fibritin, dissociates into a monomeric A-state form containing a stablebeta-hairpin: atomic details of trimer dissociation and local beta-hairpinstability from residual dipolar couplings. Journal of Molecular Biology, 2004,344(4):1051-1069.

[0052] Expression host selection E. coli BL21 (DE3) (Beijing Quanshijin, Catalog No. CD601-02), the expression vector used was pET-21b (Novagen, Catalog No. 69741-3), the schematic diagram of the diploid concatemer recombination vector is shown in Figure 5 a, Schematic diagram of homotrimeric recombination vector Figure 5 b, Schematic diagram of the homotrimeric recombination vector of the diploid concatemer. Figure 5 c, where G4SG4S represents the linker GGGGSGGGGS. The protein expression and purification process was referred to Example 2, where the purification results of (Affibody-H5)2, Affibody-H5-Foldon and (Affibody-H5)2-Foldon are shown in Figure 6 a. Figure 6 b and Figure 6 c.

[0053] The affinity and specificity of Affibody-H5 and its two-fold concatemer (Affibody-H5) 2, homotrimer Affibody-H5-Foldon, and homotrimer (Affibody-H5) 2-Foldon of Affibody-H5 two-fold concatemer to the spike protein of the new coronavirus (SARS-CoV-2) were analyzed by referring to Example 3. The results are shown in Figure 7 , OD measured after (Affibody-H5)2 binds to the spike protein 450 The value was much higher than that of the negative control group.

[0054] Example 5

[0055] (Affibody-H5)2 blocking experiment on ACE2 binding to the novel coronavirus (SARS-CoV-2) spike protein.

[0056] The peptide (Affibody-H5) 2 in Example 4 was used as a further research object to verify that (Affibody-H5) 2 blocks ACE2 binding to the novel coronavirus (SARS-CoV-2) spike protein. The specific method is as follows:

[0057] The novel coronavirus (SARS-CoV-2) spike protein was diluted to 10 μg / mL in carbonate buffer (pH 9.6) and coated on a 96-well microplate at 4°C overnight. The plate was blocked with 3% skim milk powder-PBS and incubated at 37°C for 2 h. ACE2 protein and ACE2 protein monoclonal antibody were added to the control group, and (Affibody-H5)2, ACE2 protein, and ACE2 protein monoclonal antibody were added to the test group, and the plates were incubated at 37°C for 2 h. Diluted HRP-labeled rabbit anti-mouse secondary antibody (purchased from Sangon Biotech Co., Ltd., catalog number D110098) was added and incubated at 37°C for 45 min. TMB substrate was added for color development for 10 min. 2 M H2SO4 was added to terminate the reaction, and the OD was read on a microplate reader. 450 During the ELISA process, the plate was washed 4 times with PBST (PBS + 0.5% Tween-20) at each step. Figure 8 .Depend on Figure 8 It can be seen that after adding (Affibody-H5)2 (50μg / mL) to the test group, OD 450 The significant decrease indicated that (Affibody-H5)2 blocked the binding of ACE2 to the spike protein of the new coronavirus (SARS-CoV-2).

[0058] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements to the technical solutions of the present invention made by ordinary engineers and technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A novel coronavirus spike protein-specific binding polypeptide, characterized by: Its amino acid sequence is shown in the sequence listing SEQ ID NO.

1.

2. A fusion protein, characterized in that: The invention comprises the polypeptide according to claim 1 and one or more protein tags fused to the amino terminus or carboxyl terminus of the polypeptide according to claim 1.

3. The fusion protein according to claim 2, characterized in that: The protein tag is a histidine tag, a myc tag, a Flag tag, an HA tag or a self-assembling short peptide.

4. A fusion protein, characterized in that: The invention comprises the polypeptide according to claim 1 and other functional proteins fused with the amino terminus or carboxyl terminus of the polypeptide according to claim 1, wherein the other functional proteins are enzyme proteins with catalytic function or proteins with fluorescent luminescence function.

5. A novel coronavirus spike protein-specific binding polypeptide, characterized by: The polypeptide is in one of the following forms: (a) a diploid formed by connecting two polypeptides according to claim 1 in series via a connecting peptide having the amino acid sequence GGGGSGGGGS; (b) a homotrimer formed by linking three polypeptides of claim 1 to a Foldon trimerization tag; (c) A complex multimer formed by connecting two polypeptides according to claim 1 in series via a connecting peptide having the amino acid sequence of GGGGSGGGGS to form a diploid, and then connecting them with a Foldon trimerization tag.

6. The polypeptide according to claim 5, characterized in that: The Foldon trimerization tags in formats (b) and (c) are derived from the T4 bacteriophage fiber protein Foldon domain.

7. The fusion protein according to claim 2 or 4, characterized in that: The fusion protein is covalently or non-covalently combined with a fluorescent luminescent group, biotin, a radioactive group, or a nanomaterial.

8. Use of the polypeptide according to claim 1, 5 or 6 or the fusion protein according to one of claims 2 to 4 or 7 in the preparation of a drug for neutralizing and blocking the new coronavirus.

9. Use of the polypeptide of claim 1, 5 or 6 or the fusion protein of one of claims 2 to 4 or 7 in the preparation of an agent for neutralizing and blocking the new coronavirus.

10. Use of the polypeptide according to claim 1, 5 or 6 or the fusion protein according to one of claims 2 to 4 or 7 in the preparation of a novel coronavirus detection reagent, wherein the detection method is enzyme-linked immunosorbent assay.

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