Novel coronavirus spike protein specific binding polypeptide and application thereof

Abstract

The invention provides a novel coronavirus spike protein specific binding polypeptide and application thereof. The amino acid sequence of the polypeptide is shown in a sequence table SEQ ID NO.1. The polypeptide is good in specific affinity, high in stability, capable of being efficiently prepared through an escherichia coli expression system, low in preparation cost and capable of being specifically bound with novel coronavirus spike protein. Further, the method is applied to detection and strain neutralization of 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 an RNA virus that emerged in recent years. This virus belongs to the β-CoV coronavirus family, the same as SARS-CoV. Its envelope has an average diameter of about 80 nm and approximately 30 spike S proteins on its surface. Research has found that, similar to the infection mechanism of SARS-CoV, SARS-CoV-2 uses the viral spike S protein to recognize the angiotensin-converting enzyme 2 (ACE2) receptor on human cells to enter the cells, and the expression level of ACE2 on the cell surface is positively correlated with the virus infection rate. That is, the key to SARS-CoV-2 infecting human cells lies in the binding of its spike S protein to the ACE2 protein.

[0003] The research team of Professor Zhou Qiang from Westlake University used cryo-electron microscopy technology to analyze the structure of the complex of the receptor-binding domain RBD (monomeric protein) of the SARS-CoV-2 spike S protein and the full-length human ACE2 receptor protein (Yan R, Zhang Y, Guo Y, Xia L, Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science, 2020, 367(6485):1444-1448), and clarified the interaction sites between the SARS-CoV-2 spike S protein and the extracellular domain of the cell receptor ACE2. Among them, 13 amino acid residues in the first α-helix of ACE2 participate in binding to the S protein, playing a key role in protein interaction.

[0004] SARS-CoV-2 is extremely prone to mutation. Currently, more than 1,000 mutant strains have been identified, and more than a dozen are widely spread. Analysis of research data has found that SARS-CoV-2 mutant strains with strong transmissibility and pathogenicity evade the immune response by reducing their affinity for neutralizing antibodies, and at the same time increase the infection efficiency by enhancing their specific binding to ACE2. As the key receptor for SARS-CoV-2 to infect cells, specific functional fragments in the extracellular domain where ACE2 specifically binds to the S protein have good specific affinity for both wild-type and mutant strains. That is, polypeptides with the same spatial structure in the extracellular domain of ACE2 will possess the general neutralizing activity against wild-type and mutant strains.

[0005] The extracellular domain of ACE2 can not only bind to the spike S protein of SARS-CoV-2, but also has ectopeptidase activity, and the binding of the S protein does not affect this enzyme activity. Using recombinant ACE2 extracellular domain with complete or low catalytic activity as a SARS-CoV-2 blocking drug may lead to the imbalance of the renin-angiotensin-aldosterone system (RAAS) homeostasis, and there are also problems of long preparation cycle and high cost. The affinity of the key fragment (amino acid residues 19-46) of ACE2 binding to the S protein is low, and the molecular stability is poor, which also has certain limitations in application.

[0006] Affibody is a new type of affinity ligand, which is derived from the B segment of the immunoglobulin-binding region of staphylococcal protein A (Nilsson B, Moks T, et al. A synthetic IgG-binding domain based on staphylococcal protein A. Protein Engineering, 1987, 1(2): 107-113). It is a single-chain structure composed of 58 amino acid residues and contains 3 α-helices in its structure. The receptor-binding site of the affibody contains 13 amino acid residues. Karin Nord replaced the codons of these 13 amino acids with degenerate codons respectively, and the mutants formed in this way constitute the affibody molecular library (Nord K, Nilsson J, et al. A combinatorial library of an alpha-helical bacterial receptor domain. Protein Engineering, 1995, 8(6): 601-608). Different affibodies recombinantly produced therefrom can respectively have specific binding activity and high affinity for many target protein molecules, and these target proteins include insulin, fibrin, transferrin (TRF), tumor necrosis factor α (TNF-α), etc. Affibody polypeptides are functionally similar to antibodies and have advantages that antibody molecules do not have at the same time, including small relative molecular weight, high stability, good affinity and specificity, flexible application, easy preparation, etc., and are widely used in many fields of biological science. Therefore, based on the principle of protein molecular design, through rational design combined with experimental verification guidance, on the basis of retaining its natural structure, obtaining an ACE2-affibody chimeric polypeptide with high affinity and high stability is expected to specifically bind to the spike proteins of wild-type and mutant strains of the novel coronavirus, and then be applied to the detection and strain neutralization of the novel coronavirus. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a polypeptide that specifically binds to the spike protein of the novel coronavirus.

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

[0009] The technical solution adopted by the present invention is: A polypeptide that specifically binds to the spike protein of the novel coronavirus, and its amino acid sequence is shown in Sequence Listing SEQ ID NO.1, SEQ ID NO.2, and / or SEQ ID NO.3.

[0010] Preferably, for the above-mentioned polypeptide that specifically binds to the spike protein of the novel coronavirus, its nucleotide sequence is shown in Sequence Listing SEQ ID NO.4, SEQ ID NO.5, and / or SEQ ID NO.6.

[0011] The above-mentioned polypeptide that specifically binds to the spike protein of the novel coronavirus is rationally designed based on the analysis of the primary structure and spatial structure of the amino acid residues at positions 19 - 46 of ACE2 and the affibody backbone sequence, and has high affinity activity for the spike protein of the novel coronavirus.

[0012] Preferably, for the above-mentioned polypeptide that specifically binds to the spike protein of the novel coronavirus, its amino acid sequence is shown in Sequence Listing SEQ ID NO.1.

[0013] Preferably, for the above-mentioned polypeptide that specifically binds to the spike protein of the novel coronavirus, its nucleotide sequence is shown in Sequence Listing SEQ ID NO.4.

[0014] A fusion protein, in which one or more protein tags are fused and expressed at the amino terminus or carboxyl terminus of the above-mentioned polypeptide.

[0015] Preferably, for the above-mentioned fusion protein, the protein tag is a histidine tag, myc tag, Flag tag, HA tag, or self-assembling short peptide.

[0016] Preferably, for the above-mentioned fusion protein, the protein tag is a self-assembling short peptide.

[0017] A fusion protein, in which other functional proteins are fused and expressed at the amino terminus or carboxyl terminus of the above-mentioned polypeptide, and the other functional proteins are enzyme proteins with catalytic functions, or proteins with fluorescence emission functions, or one or more of the above-mentioned polypeptides.

[0018] Preferably, for the above-mentioned fusion protein, it is covalently or non-covalently bound to a fluorescent group, biotin, radio group, or nanomaterial.

[0019] Use of the above-mentioned novel coronavirus spike protein-specific binding polypeptide and / or fusion protein in the preparation of a drug or reagent for neutralizing and blocking the novel coronavirus.

[0020] Use of the above-mentioned novel coronavirus spike protein-specific binding polypeptide and / or fusion protein in the preparation of a novel coronavirus detection reagent or drug.

[0021] Preferably, in the above application, when the detection reagent or drug is used to identify the novel coronavirus, the identification method is an enzyme-linked immunosorbent assay (ELISA) detection method.

[0022] The beneficial effects of the present invention are as follows: The above-mentioned novel coronavirus spike protein-specific binding polypeptide has good specific affinity and high stability. It can be efficiently prepared using an Escherichia coli expression system with low preparation cost and can specifically bind to the spike protein of the novel coronavirus (SARS-CoV-2). Based on the principle of protein molecular design, according to the analysis results of the primary structure and spatial structure of the amino acid residues at positions 19-46 of ACE2 and the Affibody backbone sequence, three novel coronavirus spike protein-specific binding polypeptides were rationally designed, and three polypeptides were prepared using the prokaryotic Escherichia coli expression system. The specific binding ability of the polypeptides to the spike protein of the novel coronavirus (SARS-CoV-2) was verified by ELISA experiments. The results show that the three designed polypeptides can specifically bind to the novel coronavirus spike protein, have no obvious affinity with BSA protein, and have significant advantages such as low preparation cost and good stability. They can be used as neutralizing blockers for the binding of the novel coronavirus spike protein to the ACE2 protein and can neutralize the binding of the novel coronavirus spike protein to the ACE2 protein. Description of the Drawings

[0023] Figure 1 It is a comparison result diagram of the Affibody backbone sequence and the amino acid residue sequence at positions 19-46 of ACE2. Among them, X represents any amino acid residue, which is the site where the Affibody molecule binds to the target.

[0024] Figure 2 It is a schematic diagram for the design of three novel coronavirus (SARS-CoV-2) spike protein-specific binding polypeptides.

[0025] Figure 3 It is a purification result diagram of three novel coronavirus (SARS-CoV-2) spike protein-specific binding polypeptides.

[0026] Figure 4 It is a specific binding result diagram of three polypeptides to the novel coronavirus spike S protein. Among them, 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.

[0027] Figure 5 Schematic diagram of an expression vector for a polypeptide specifically binding to the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for multimerization.

[0028] Figure 6 Purification result of a polypeptide specifically binding to the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for multimerization.

[0029] Figure 7 Result diagram of specific binding between a polypeptide specifically binding to the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for multimerization and the spike S protein of the severe acute respiratory syndrome coronavirus 2.

[0030] Figure 8 For the diploid tandem (Affibody-H5) 2 Result diagram of verification of blocking the binding between AEC2 and the spike protein of the severe acute respiratory syndrome coronavirus 2. Detailed implementation manners

[0031] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners.

[0032] The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used are all commercially available products unless otherwise specified.

[0033] Example 1 Design of a polypeptide specifically binding to the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The specific design idea is as follows: (1) The amino acid residue sequence of positions 19-46 of ACE2 was aligned with the amino acid sequence of the affibdoy molecular backbone (Nygren PA, Skerra A. Binding proteins from alternative scabfolds. Journal of Immunology Methods, 2004, 290(1-2): 3-28), and the homologous sequences were analyzed. (2) The spatial structure similarity between the amino acid residue sequence of positions 19-46 of ACE2 and the affibdoy molecular backbone was analyzed, and the binding site of the amino acid residue sequence of positions 19-46 of ACE2 to the spike protein of the severe acute respiratory syndrome coronavirus 2 was chimerized with the affibody backbone.

[0034] The alignment result of the amino acid sequence of the affibody molecular backbone and the amino acid residue sequence of positions 19-46 of ACE2 is shown in Figure 1. The results showed that the functional fragment of ACE2 was similar to the Affibody molecular backbone sequence, and the same or similar amino acid residues appeared at multiple sites (marked with a gray background); there were 9 overlaps (marked with a solid box with a bottom "△") between the binding site of the ACE2 functional fragment to the spike protein of SARS-CoV-2 (marked with a bottom "·") and the target binding site of the Affibody molecule (marked with the letter X). Based on the above analysis results, three ACE2-Affibody chimeric polypeptides were designed, and the design schematic diagram is shown in Figure 2 , where (a) Affibody-H5 (the amino acid sequence is shown in SEQ ID NO.1 of the sequence listing, and the nucleotide sequence is shown in SEQ ID NO.4 of the sequence listing) is to replace the first α-helix of Affibody ( Figure 1 Helix-1 in) with the amino acid residues at positions 19-46 of ACE2, and at the same time replace 6 binding sites (including 1 site before Helix-2) in the second α-helix of Affibody with Figure 1 the spike protein binding sites of the amino acid residues at positions 19-46 of ACE2 corresponding to the sequence alignment; (b) Affibody-A28 (the amino acid sequence is shown in SEQ ID NO.2 of the sequence listing, and the nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing) is to replace the Affibody fragment corresponding to the sequence alignment (Affibody residues 9-36) with the ACE2 functional fragment; (c) Affibody-A13 (the amino acid sequence is shown in SEQ ID NO.3 of the sequence listing, and the nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing) is to replace the target binding site in the Affibody backbone with the corresponding amino acid residues of the ACE2 functional fragment in the sequence alignment.

[0035] Example 2 Expression and purification of a polypeptide specifically binding to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0036] The coding genes of the three designed polypeptides were obtained by chemical synthesis. The target protein genes were respectively cloned into the pET21b prokaryotic expression vector (artificially added His-HA-tag), and the correctly sequenced recombinant expression vectors were transformed E.coliBL21(DE3), pick a monoclonal colony and inoculate it into 5 mL of LB / A medium (1 g tryptone, 0.5 g yeast extract, 1 g sodium chloride, made up to 100 mL and autoclaved; then add ampicillin with a final concentration of 100 μg / mL), culture overnight at 37°C and 180 rpm; inoculate the culture into 200 mL of LB / A medium at a ratio of 1:50, culture at 37°C and 180 rpm until the logarithmic phase, add IPTG with a final concentration of 0.1 mM, and induce overnight at 20°C and 180 rpm. Centrifuge the culture at 4°C and 4400 rpm to collect the bacterial cells; resuspend the cells with 20 mL of pre-cooled PBS (8 g NaCl, 2 Na 4 2 2 HPO 2 4 4 •12H 2 2O, 0.2 g KCl, 0.24 g NaH

[0037] For the purification of the recombinant polypeptide, a Ni-TED column (purchased from Sangon Biotech Co., Ltd., product number C600803) was selected. First, equilibrate the Ni-TED column with 10 column volumes of phosphate buffer; filter the collected protein cell lysate supernatant through a 0.45 μm filter membrane and then load the sample, wash through 4 times, and retain the flow-through for SDS-PAGE analysis; wash the column with phosphate buffer containing 20 mM imidazole (20 column volumes) to remove the contaminating proteins; prepare phosphate buffers containing 50 mM, 100 mM, 250 mM, and 400 mM imidazole, and elute the target protein with 2 column volumes respectively. Perform SDS-PAGE analysis on the protein samples collected with different concentrations of imidazole. The purification results of Affibody-H5, Affibody-A28, and Affibody-A13 are shown in Figure 3 a, Figure 3 b, and Figure 3 c respectively, and all three Affibody polypeptides were successfully purified.

[0038] Example 3 Analysis of the binding characteristics of the polypeptide specifically binding to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0039] Using the 3 polypeptides expressed and purified in Example 2 as the further research objects, the ELISA method was used to detect their affinity characteristics with the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the same time, BSA was selected as an irrelevant antigen negative control. The specific operation is as follows: Dilute the spike protein / BSA of the novel coronavirus (SARS-CoV-2) to 10 μg / mL with carbonate buffer at pH 9.6, coat a 96-well ELISA plate, and incubate overnight at 4°C; block with 3% skim milk-PBS at 37°C for 2 h; add three recombinant proteins, Affibody-H5, Affibody-A28, and Affibody-A13, with HA tags, and incubate at 37°C for 2 h; add the diluted HA antibody (purchased from Sangon Biotech Co., Ltd., product number D191044), and incubate at 37°C for 2 h; add the diluted HRP-labeled rabbit anti-mouse secondary antibody (purchased from Sangon Biotech Co., Ltd., product number D110098), and incubate at 37°C for 45 min; add TMB substrate for color development for 10 min; add 2M H 2 SO 4 to terminate the reaction, and read the OD on an ELISA reader 450 . During the ELISA process, wash the plate 4 times with PBST (PBS + 0.5% Tween-20) at each step. The results are shown in Figure 4 , and it can be seen that Affibody-H5 has the best specific binding ability.

[0040] Example 4 Preparation and affinity characterization of a polypeptide multimer specifically binding to the spike protein of the novel coronavirus (SARS-CoV-2).

[0041] Using the polypeptide Affibody-H5 with high affinity and good specificity for the spike protein of the novel coronavirus (SARS-CoV-2) in Example 3 as the further research object, prepare the tandem dimer of Affibody-H5 (Affibody-H5) 2 , the homotrimer Affibody-H5-Foldon based on the Foldon tag, and the homotrimer of the tandem dimer of Affibody-H5 based on the Foldon tag (Affibody-H5) 2 -Foldon as the polypeptide for capturing the spike protein of the novel coronavirus (SARS-CoV-2) in the ELISA experiment.

[0042] The above preparation method references: (1) Liu J, Jiang Y, et al. Development of active affibody 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 stable beta-hairpin: atomic details of trimer dissociation and local beta-hairpin stability from residual dipolar couplings. Journal of Molecular Biology, 2004, 344(4): 1051-1069. Expression host selection E.coli BL21(DE3) (TransGen Biotech, product number CD601-02) was selected as the expression host, and pET-21b (Novagen, product number 69741-3) was selected as the expression vector. The schematic diagrams of the recombinant vectors of the double tandem body are shown in Figure 5 a, and the schematic diagram of the recombinant vector of the homotrimer is shown in Figure 5 b, and the schematic diagram of the recombinant vector of the homotrimer of the double tandem body is shown in Figure 5 c, where G 4 SG 4 S represents the linker GGGGSGGGGS. The protein expression and purification process was referred to Example 2, in which (Affibody-H5) 2 、Affibody-H5-Foldon and (Affibody-H5) 2 -Foldon purification results are shown in Figure 6 a、 Figure 6 b and Figure 6 c.

[0043] Affibody-H5 and its double tandem body (Affibody-H5) 2, Homotrimeric Affibody-H5-Foldon, homotrimer of Affibody-H5 tandem dimer (Affibody-H5) 2 - Analysis of the affinity and specificity of Foldon with the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The method was referred to Example 3, and the results are shown in Figure 7 , (Affibody-H5) 2 The OD 450 value measured after binding to the spike protein was much higher than that of the negative control group.

[0044] Example 5 (Affibody-H5) 2 Blocking experiment of ACE2 binding to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0045] Using the polypeptide (Affibody-H5) 2 in Example 4 as the further research object to verify the blocking of ACE2 binding to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by (Affibody-H5). The specific method is as follows: 2 Dilute the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to 10 μg / mL with carbonate buffer at pH 9.6, coat a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and incubate overnight at 4 °C; block with 3% skim milk-PBS at 37 °C for 2 h; add ACE2 protein and ACE2 protein monoclonal antibody to the control group, and add (Affibody-H5) , ACE2 protein and ACE2 protein monoclonal antibody to the test group, and incubate at 37 °C for 2 h; add diluted HRP-labeled rabbit anti-mouse secondary antibody (purchased from Sangon Biotech Co., Ltd., product number D110098), and incubate at 37 °C for 45 min; add TMB substrate for color development for 10 min; add 2M H 2 SO 2 to terminate the reaction, and read the OD 4 in an enzyme-linked immunosorbent assay reader. During the ELISA process, wash the plate 4 times with PBST (PBS + 0.5% Tween-20) at each step. The results are shown in 450 . From Figure 8 . It can be seen that after adding (Affibody-H5) Figure 8 (50 μg / mL) to the test group, the OD 2 decreased significantly, indicating that (Affibody-H5) 450 blocked the binding of ACE2 to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 2

[0046] ​The above-described embodiments are merely descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

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

1.

2. The novel coronavirus spike protein specific binding polypeptide according to claim 1, characterized in that: Its nucleotide sequence is shown in the sequence listing SEQ ID NO.

4.

3. A fusion protein, characterized in that: One or more protein tags are fused and expressed at the amino or carboxyl terminus of the polypeptide of claim 1 or 2.

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

5. The fusion protein according to claim 3, characterized in that: Covalently or non-covalently bind to fluorescent light-emitting groups, biotin, radioactive groups, and nanomaterials.

6. A fusion protein, characterized in that: Other functional proteins are fused and expressed at the amino terminus or carboxyl terminus of the polypeptide described in claim 1 or 2, and the other functional proteins are enzyme proteins with catalytic function, or proteins with fluorescent luminescence function, or one or more polypeptides described in claim 1 or 2.

7. The fusion protein according to claim 6, characterized in that: Covalently or non-covalently bind to fluorescent light-emitting groups, biotin, radioactive groups, and nanomaterials.

8. Use of the novel coronavirus spike protein-specific binding polypeptide described in claim 1 or 2 and / or the fusion protein described in any one of claims 3-7 in the preparation of drugs or reagents for neutralizing and blocking the novel coronavirus.

9. Use of the novel coronavirus spike protein-specific binding polypeptide described in claim 1 or 2 and / or the fusion protein described in any one of claims 3-7 in the preparation of novel coronavirus detection reagents or drugs.

10. The use according to claim 9, characterized in that: When the detection reagent or drug is used to identify the new coronavirus, the identification method is an enzyme-linked immunosorbent assay.

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