A modified nanobody with pH-dependent binding properties to the original and variant strains of the novel coronavirus S-RBD and its potential applications.

Abstract

This invention discloses a modified nanobody with pH-dependent binding characteristics to the S-RBD of the original and mutant strains of SARS-CoV-2 and its potential applications, belonging to the field of biotechnology. The affinity purification packing material prepared based on any one of the nanobodies described in SEQ ID NO. 1-3 provided by this invention directly targets the S-RBD of wild-type and mutant SARS-CoV-2, achieving one-step purification of the target protein without a tag. The purified target protein has extremely high purity, binds to the target protein at pH 7.5, and is fully eluted at pH 5.0. The affinity purification packing material for the S-RBD nanobody can be regenerated at pH 4.5. Therefore, the nanobodies MNb-11, MNb-13, and MNb-14 provided by this invention have wide applications in the production, purification, binding, and / or dissociation of the S-RBD and S protein of wild-type and mutant SARS-CoV-2.

Description

Technical Field

[0001] This invention relates to the modification of nanobodies with pH-dependent binding properties to the original strain and variant strain S-RBD of the novel coronavirus and their potential applications, belonging to the field of biotechnology. Background Technology

[0002] Studies have shown that SARS-CoV-2 continues to generate new mutations, and previous vaccines are ineffective against the major circulating mutant strains that have emerged since, especially vaccines designed based on the original strain (Yang, H., Xie, Y. & Li, C. Understanding the mechanisms for COVID-19 vaccine's protection against infection and severe disease. Expert Rev Vaccines 22, 186–192 (2023)). Therefore, further research on SARS-CoV-2 and its variants is necessary, and effective vaccines must be developed against these strains to prevent breakthrough infections caused by new variants.

[0003] The spike protein (S) on the surface of SARS-CoV contains a receptor-binding domain (RBD) that specifically recognizes angiotensin-converting enzyme 2 (ACE2) to mediate coronavirus invasion of host cells (Shang, J. et al. Structural basis of receptor recognition by SARS-CoV-2. Nature 581, 221–224 (2020).; Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol 23, 3–20 (2022).). The S protein of coronaviruses, especially the RBD, can induce neutralizing antibodies (NAbs) and T-cell immune responses; therefore, the S protein and S-RBD are ideal antigen targets for SARS-CoV-2 vaccine development (Efficacy and Safety of COVID-19 Vaccines: A Systematic Review and Meta-Analysis of Randomized Clinical Trials. Vaccines (Basel) 9, 467 (2021).). Currently, the main components of recombinant protein vaccines against the novel coronavirus are S or S-RBDs. Combinations of multiple variant S-RBDs can provide broader-spectrum protection. Therefore, the preparation efficiency and purity of the original and variant S-RBDs of the novel coronavirus are crucial for vaccine development. There are currently two main types of affinity purification techniques for S-RBDs: one involves attaching purification tags such as His or Fc to the S-RBD for purification expression, enriching the target protein through the binding of the purification packing material to the tag, and then removing the purification tag from the S-RBD. This method is complex and may yield incompletely cleaved target protein. The other is a one-step purification method, where the purification packing material is directly bound to the S-RBD, allowing for direct elution with 100mM sodium acetate buffer (pH 3.5) to obtain high-purity S-RBD protein. However, the extremely acidic buffer significantly affects the biological activity and stability of both the S-RBD and the S protein.

[0004] Therefore, there is an urgent need for a method that can improve the purity of S-RBD proteins, has simple operation steps, and yields S-RBD proteins with high stability and broad spectrum. Summary of the Invention

[0005] Under different pH conditions, the high-purity nanobody protein to be screened was mixed with wild-type S-RBD. The binding state of the nanobody to S-RBD was analyzed using size exclusion chromatography, and its kinetic parameters were characterized by SPR. Furthermore, the nanobody protein was coupled to the surface of a blank solid-phase support (agarose purification packing material) using amino-coupling. The purification of wild-type S-RBD and its mutants was simulated under different elution conditions (pH, buffer components, etc.). The purification efficiency of the packing material was evaluated by SDS / PAGE and Coomassie brilliant blue staining. Finally, two nanobodies, MNb-11 and MNb-14, were screened and found to possess pH-dependent binding characteristics to S-RBD and S protein. These nanobodies were further immobilized onto agarose microspheres, showing stable binding to S-RBD (wild-type and mutant strains) under neutral pH conditions and complete elution under weakly acidic conditions. These nanobodies can be used for affinity purification of novel coronavirus S-RBD and S protein.

[0006] The first objective of this invention is to provide a nanobody with an amino acid sequence as shown in any one of SEQ ID NO. 1 to 3, wherein the nanobody is capable of binding to the spike protein of the novel coronavirus and / or the receptor domain of the novel coronavirus spike protein.

[0007] In one embodiment, the novel coronavirus includes wild-type novel coronavirus strains and mutant novel coronavirus strains.

[0008] In one embodiment, the mutated novel coronavirus includes Beta, Delta, BA.4 / 5, XBB.1.5, CH.1.1, and BA.2.86.

[0009] In one embodiment, the nanobody sequence is linked to a tag protein.

[0010] In one embodiment, the tag protein includes 6×His.

[0011] A second objective of this invention is to provide a gene encoding the nanobody.

[0012] A third objective of this invention is to provide a vector carrying the gene.

[0013] A fourth object of the present invention is to provide a host cell that expresses the gene or carries the vector.

[0014] A fifth objective of this invention is to provide a purification packing material for purifying the novel coronavirus spike protein and / or the novel coronavirus spike protein receptor domain, the purification packing material comprising the nanobody.

[0015] In one embodiment, the nanobody is coupled to a solid-phase support.

[0016] In one embodiment, the solid support comprises an affinity chromatography medium.

[0017] In one embodiment, the affinity chromatography medium is based on styrene, acrylate, dextran, agarose, or polyacrylamide dextran.

[0018] A sixth object of the present invention is to provide a method for preparing the purification packing material, wherein the method comprises coupling the nanobody to a solid support.

[0019] In one embodiment, the solid support comprises an affinity chromatography medium.

[0020] In one embodiment, the affinity chromatography medium is based on styrene, acrylate, dextran, agarose, or polyacrylamide dextran.

[0021] A seventh objective of this invention is to provide the application of the above-described purification packing material in the purification of the novel coronavirus spike protein and / or the purification of the novel coronavirus spike protein receptor domain.

[0022] In one embodiment, the application includes the preparation of purification reagents or purification kits.

[0023] The present invention also provides a purification kit containing the purification packing material.

[0024] In one embodiment, the kit contains a buffer solution and a gravity column.

[0025] In one embodiment, the buffer solution comprises buffer I, buffer II, and buffer III; buffer I is 15–20 mM HEPES, 900 mM–1500 mM NaCl, pH 7.5; buffer II is 80–120 mM NaAC, 120–180 mM NaCl, pH 5.0; and buffer III is 80–120 mM NaAC, 120–180 mM NaCl, pH 4.5.

[0026] This invention provides a method for purifying the spike protein of the novel coronavirus and / or the receptor domain of the novel coronavirus spike protein. The method involves using the purification packing material or the purification kit to purify the spike protein of the novel coronavirus and / or the receptor domain of the novel coronavirus spike protein.

[0027] In one embodiment, the specific steps of the method are as follows:

[0028] S1. Combination: The sample to be purified is mixed with the purification packing material and incubated.

[0029] S2, Column packing: Add the incubated mixture from S1 into the gravity column to retain the purification packing material;

[0030] S3, Washing: Add pH-neutral high-salt buffer solution dropwise into a gravity column containing purification packing material, flow through, and repeat 3 times;

[0031] S4, Elution: Add a weakly acidic elution buffer to the gravity column after washing, collect the flow-through, and repeat 4 times.

[0032] In one embodiment, in S1, the mixture is incubated at 2–6°C for 50–70 minutes.

[0033] In one embodiment, in S3, the high-salt buffer solution comprises 15–20 mM HEPES and 900 mM–1500 mM NaCl.

[0034] In one embodiment, in S4, the elution buffer comprises 80–120 mM NaAC, 120–180 mM NaCl, and pH 5.0.

[0035] In one embodiment, after elution in step S4, the purified packing material is regenerated using a regeneration buffer at pH 4.5, the regeneration buffer comprising 80–120 mM NaAC and 120–180 mM NaCl.

[0036] Beneficial effects:

[0037] 1. The affinity purification packing material prepared based on the S-RBD nanobody described in any one of SEQ ID NO. 1 to 3 provided by this invention is currently the affinity purification packing material with the broadest binding range to the novel coronavirus S protein and S-RBD protein and the mildest elution conditions. This packing material directly targets S-RBD, achieving one-step purification of the tagless target protein. The purified target protein has extremely high purity, and the elution conditions are mild (binding to the target protein at pH 7.5 and sufficient elution at pH 5.0), effectively protecting the conformational stability and biological activity of the target protein while improving purification efficiency.

[0038] 2. Because nanobodies target the conserved region of the S-RBD, they broadly bind to the S-RBD protein of the novel coronavirus mutant. This makes it the most effective affinity purification packing material for the novel coronavirus mutant S-RBD, offering the broadest range and best purification efficiency. Due to the conserved nature of its binding site, no currently circulating mutant strains have mutated at this site. It can be confirmed that it can purify the S-RBD and S protein of all currently circulating mutant strains (Beta, Delta, BA.4 / 5, XBB.1.5, CH.1.1, BA.2.86). Regarding the sustainable use of the packing material, the modified nanobodies still possess extremely strong stability and high yield. Regeneration of the nanobodies' S-RBD affinity purification packing material can be achieved under pH 4.5 conditions, ensuring the durability of the packing material while saving production time, greatly improving production efficiency, and reducing production and usage costs.

[0039] The nanobodies MNb-11, MNb-13, and MNb-14 provided by this invention have broad application value and can be used in the production, purification, binding / dissociation of all S-RBDs and S proteins, such as general experiments related to the novel coronavirus, including specific binding / dissociation and protein preparation processes. They are especially suitable for the production of high-purity, unlabeled target proteins, such as vaccine production and immune antigen preparation. Attached Figure Description

[0040] Figure 1 Nanobody purification molecular sieve and SDS / PAGE plot. The line in the molecular sieve plot represents the absorbance at 280 nm. The SDS / PAGE plot shows the protein corresponding to the highest absorbance.

[0041] Figure 2 The nanobodies bound to S-RBDs in vitro at neutral (pH 7.5) and slightly acidic (pH 6.0, pH 5.5, pH 5.0) pH levels. The interaction between the nanobodies and S-RBDs was characterized by gel filtration chromatography and analyzed by SDS / PAGE and Coomassie Brilliant Blue staining.

[0042] Figure 3 Characterizing the stability of Nb-WT, MNb-11, MNb-13, and MNb-14 proteins. A. DSF analysis was used to characterize the thermal stability of Nb-WT, MNb-11, MNb-13, and MNb-14 at pH 7.5 and 4.5 for 0 hours, 6 hours, 24 hours, and 48 hours, respectively, and the corresponding fluorescence unit curves were displayed. B. The calculated melting temperatures (Tm) were summarized.

[0043] Figure 4Surface plasmon resonance (SPR) spectroscopy was used to characterize the real-time binding kinetics of S-RBD and nanobodies. SPR response curves and binding affinity values ​​of S-RBD with Nb-WT, MNb-11, MNb-13, and MNb-14 were obtained at pH 7.5, 6.0, 5.5, and 5.0. Slow binding / slow dissociation kinetics data were analyzed using a 1:1 binding model, while fast binding / fast dissociation kinetics data were analyzed using a steady-state method.

[0044] Figure 5 The purification efficiency of S-RBD protein using S-RBD affinity purification packing material with MNb-11 and MNb-14 as ligands under different pH elution conditions. The left figure shows the SDS-PAGE analysis of S-RBD bound to NHS-Activated LA Beads 4FF (blank resin) with MNb-11 and MNb-14 (blank resin). The right figure shows the SDS-PAGE analysis of the purified S-RBD protein, with elution conditions of pH 6.0, pH 5.5, pH 5.0, and pH 4.5. Numbers 1 / 2 / 3 / 4 indicate the elution order of S-RBD protein, and Resin indicates the affinity purification packing material after elution.

[0045] Figure 6 Six mutant S-RBD proteins were purified using affinity purification packing material. MNb-11 and MNb-14 were coupled to NHS-Activated LA Beads 4FF (blank resin) and bound to the six mutant S-RBD proteins. SDS-PAGE analysis of the eluted mutant S-RBD proteins is shown in the right figure. Numbers 1 / 2 / 3 / 4 indicate the elution order of the S-RBD proteins, and Resin indicates the affinity purification packing material after elution.

[0046] Figure 7 Affinity purification packing material was used to purify S-RBD and S protein from cell expression supernatants. The RBD protein of the XBB.1.5 mutant was enriched and purified from the expression supernatant of SF9 cells (A), and the full-length S protein of XBB.1.5 was enriched and purified from the expression supernatant of 293F cells (B). Resin: Affinity purification packing material before binding to expression supernatant. Load: Cell expression supernatant. Bind: S-RBD nanobody affinity purification packing material after binding to supernatant and thorough washing. Elu: Elution solution containing the target protein obtained by treating the affinity purification packing material with pH 5.0 elution buffer. Resin post-Elu: Affinity purification packing material after elution.

[0047] Figure 8: Affinity purification packing material was used to repeatedly purify S-RBD from SF9 expression supernatant. WT-RBD was enriched and purified from SF9 cell expression supernatant using affinity purification packing material, repeated 10 times. Load: SF9 cell expression supernatant. Resin: Affinity purification packing material without supernatant binding. Bind: S-RBD nanobody affinity purification packing material after binding to supernatant and thorough washing. Elu: Elution solution containing the target protein obtained by treating the affinity purification packing material with pH 5.0 elution buffer. Resin postElu: Affinity purification packing material after elution. Detailed Implementation

[0048] Example 1: Design of Nanobody Histidine Mutation Introduction

[0049] This invention modifies Nb2-10 (PDB number: 8CYJ, C chain) to make it pH-dependent while maintaining a certain affinity, and finally designs combinatorial mutant nanobodies MNb-1 to MNb-14.

[0050] Based on the mutation sites designed in Table 1, site-directed mutations were performed on Nb2-10 (also known as Nb-WT in this invention), and a His tag and a translation stop codon were introduced at the C-terminus of the sequence. Subsequently, the insertion of the foreign fragment was verified to be completely correct by bacterial PCR identification, gene sequencing, etc.

[0051] Table 1: Design of Mutation Sites in Nanobody Mutants

[0052]

[0053] Nb2-10 and the mutant sequences MNb-1 to MNb-14 were cloned into the pET-30a vector, transformed into DH5α competent cells, plated on LB plates, and cultured overnight at 37°C. Positive clones were identified by PCR and confirmed by sequencing, yielding recombinant plasmids pET-30a-WT and pET-30a-MNb-1 to pET-30a-MNb-14.

[0054] The recombinant plasmids were transformed into Escherichia coli BL21(DE3), plated on LB plates and cultured overnight at 37°C. Single clones were picked and inoculated into LB liquid medium and cultured at 37°C for 3 hours. Then, β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM and expression was induced at 37°C for 12 hours. The expressed protein was expressed in the precipitate as inclusion bodies. The bacterial cells were lysed by sonication, and the precipitate was collected after centrifugation. The precipitate was washed with washing buffer (50 mM Tris, 300 mM NaCl, 0.5% Triton-100, 10 mM EDTA, 10 mM DTT, pH 8.0) and resuspension buffer (50 mM Tris, 100 mM NaCl, 10 mM EDTA, 10 mM DTT, pH 8.0), and then dissolved in dissolution buffer (6 M Mea-HCl, 50 mM Tris, 100 mM NaCl, 10% Glycerol, 10 mM EDTA, 10 mM DTT). The extracted inclusion bodies were then added dropwise to a stirred refolding buffer (600 mM L-Arginie HCl, 100 mM Tris, 10% Glycerol, 2 mM EDTA, 5 mM DTT). Incubate overnight at 4°C in GSH (0.5 mM GSSG). Subsequently, use a 5 kDa concentrating membrane to trap the refolded nanobodies, and further perform 1:10 buffer changes using neutral buffer (20 mM HEPES, pH 7.5, 150 mM NaCl). Utilize Superdex... TM A 75 10 / 300GL gel filtration chromatography column was used to further purify the protein, ultimately yielding high-purity, highly homogeneous nanobodies. Figure 1 ).

[0055] Example 2: Characterization of the interaction between nanobodies and S-RBD by gel filtration chromatography

[0056] To evaluate the binding affinity between the nanobodies MNb-1–MNb-14 obtained in Example 1 and wild-type S-RBD, mutants exhibiting strong pH dependence were screened. Nanobodies MNb-1–MNb-14 were incubated with S-RBD at a 1:1 molar ratio in neutral buffer (20 mM HEPES, pH 7.5, 150 mM NaCl) and weakly acidic buffer (100 mM NaAC, pH 5.0, 150 mM NaCl) at 4°C for 1 hour. After incubation, the samples were centrifuged and analyzed using Superdex. TMA 75 10 / 300GL gel filtration chromatography column was used. The supernatant after centrifugation was subjected to gel filtration chromatography in neutral buffer (20 mM HEPES, pH 7.5, 150 mM NaCl) and weakly acidic buffer (100 mM NaAC, pH 5.0, 150 mM NaCl). Finally, the binding ability of the nanobody to S-RBD under different pH conditions was determined by comprehensive analysis of the 280 nm absorption peak and SDS / PAGE results.

[0057] from Figure 2 It can be seen that the absorption peak at 280 nm is a single peak, and the SDS / PAGE results of the sample at the peak tip show two bands (14k and 25k), indicating a complex. Conversely, the absorption peak is a double peak, and the SDS / PAGE results of the sample at the peak tip show only a single band, indicating a monomer. Nanobodies MNb-11, MNb-13, and MNb-14 were screened and found to form a complex at pH 7.5 and a monomer at pH 5.0. Further determination of the complex formation of these three nanobodies at pH 6.0 and pH 5.5 was conducted. MNb-11 and MNb-14, with S-RBD, existed as monomers in buffer systems at pH 6.0 and pH 5.5, respectively; MNb-13, with S-RBD, existed as a complex in a buffer system at pH 6.0 and as a monomer in a buffer system at pH 5.5. The binding of these three nanobodies to S-RBD exhibits pH sensitivity and can be further validated as candidate nanobodies.

[0058] Example 3: Evaluation of the thermal stability of nanobodies using differential scanning fluoroscopy (DSF)

[0059] The thermal denaturation of proteins was detected using SYPRO orange dye (Sigma). Nb-WT, MNb-11, MNb-13, and MNb-14 were transferred to neutral buffer (20 mM HEPES, pH 7.5, 150 mM NaCl) and acidic buffer (100 mM NaAC, pH 4.5, 150 mM NaCl), respectively, and incubated at 4°C for 0, 6, 24, and 48 hours. DSF assays were performed using a CFX-connected real-time system (Bio-Rad) with samples heated at a temperature gradient of 25–95°C for 65 min. Fluorescence signal as a function of temperature was continuously monitored. Each sample was tested in triplicate, and the Boltzmann equation was fitted using GraphPad Prism 7.0 (GraphPad software). Finally, a comprehensive analysis of the thermal stability of the nanobodies under different conditions was conducted. Figure 3This demonstrates that the introduction of mutations did not reduce the thermal stability of the nanobody, and that treatment under acidic conditions (pH 4.5) for 48 hours had no effect on the thermal stability of the nanobody. This indicates that the modified nanobody is structurally stable and has good biological activity in general storage and regeneration scenarios.

[0060] Example 4: Surface plasmon resonance (SPR) determination of affinity between nanobodies and S-RBD under different conditions (pH, buffer composition, etc.)

[0061] To determine the affinity of wild-type S-RBD for Nb-WT, MNb-11, MNb-13, and MNb-14 under different pH conditions, wild-type S-RBD protein was immobilized on a chip surface (GE) via amino coupling. The binding and dissociation processes of S-RBD with nanobodies were measured in analytical buffers at pH 7.5 (20 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween 20), pH 5.5 (20 mM MES, 150 mM NaCl, 0.05% (v / v) Tween 20), pH 5.0 (100 mM NaAC, 150 mM NaCl, 0.05% (v / v) Tween 20), and pH 4.5 (100 mM NaAC, 150 mM NaCl, 0.05% (v / v) Tween 20), and the affinity was calculated.

[0062] Figure 4 As can be seen, Nb-WT and S-RBD exhibit a slow binding and slow dissociation mode, and the affinity was calculated using a 1:1 binding model; MNb-11, MNb-13, and MNb-14 exhibit a fast binding and fast dissociation mode with S-RBD, and the affinity was calculated using a steady-state method. Finally, the differences in affinity between nanobodies and S-RBD under different pH conditions are summarized (Table 2).

[0063] Nb-WT shows minimal change in affinity as pH decreases, maintaining nanomolar affinity even at pH 4.5. Therefore, it is not pH-dependent and cannot be used for protein purification by adjusting solution pH. In contrast, the affinity of mutant nanobodies MNb-11, MNb-13, and MNb-14 for S-RBD decreases significantly with decreasing pH. Compared to Nb-WT, the binding of nanobodies MNb-11, MNb-13, and MNb-14 to S-RBD is pH-dependent.

[0064] In summary, the binding states of nanobodies and S-RBDs at different pH values ​​were characterized by gel filtration chromatography and SPR. It was observed that the binding affinity of MNb-11 and MNb-14 to S-RBD decreased most significantly with decreasing pH compared to MNb-13. Furthermore, at pH 6.0, the affinity of MNb-11 and MNb-14 to S-RBD was several times lower than that of MNb-13, and they failed to form a complex, exhibiting the strongest pH sensitivity. Therefore, MNb-11 and MNb-14 are preferred nanobodies for further development as affinity purification packing materials.

[0065] Table 2: Factor reduction in S-RBD and nanobody binding kinetics

[0066]

[0067] Example 5: Preparation of affinity purification packing material and efficiency evaluation of S-RBD purification.

[0068] Following the method provided in the NHS-Activated LA Beads 4FF product manual, 1 mg of MNb-11 and MNb-14 nanoantibody proteins were coupled to 1 mL of NHS-Activated LA Beads 4FF (blank resin) to prepare S-RBD protein affinity packing material. At pH 7.5 (20 mM HEPES, 150 mM NaCl) and 4°C, 200 μL of the prepared S-RBD protein affinity packing material was mixed with 0.1 mg of S-RBD protein solution by rotation for 1 h. The mixture was then passed through a gravity column to retain the packing material. The resin was first washed with an equal volume of pH 7.5 (20 mM HEPES, 1 M NaCl) high-salt buffer. The packing material was then divided into four equal portions and eluted four times each with elution buffers at pH 6.0, pH 5.5, pH 5.0, and pH 4.5 at room temperature.

[0069] like Figure 5 As shown, the S-RBD protein affinity packing material was mixed with the S-RBD solution and washed. The treated packing material was then prepared for electrophoresis. The gel image showed two protein bands: the S-RBD band at 25KD and the nanobody packing material band at 15KD. It can be seen that the MNb-11 and MNb-14 nanobodies can fully bind to the S-RBD in the solution under pH 7.5 conditions. The bound packing material was eluted four times using different elution buffers. The elution buffers were then used to prepare samples for electrophoresis. As the number of elutions increased, the amount of eluted protein decreased. Finally, the eluted packing material was prepared for electrophoresis. Figure 5As can be seen, the elution efficiency increases with decreasing pH. At pH 6.0, the elution efficiency of MNb-11 is above 80%, and that of MNb-14 is above 65%. At pH 5.5, the elution efficiency of MNb-11 is above 85%, and that of MNb-14 is above 75%. At pH 5.0, the elution efficiency of both packing materials is above 95%. At pH 4.5, the elution efficiency of both packing materials is close to 100%. Therefore, pH 5.0–6.0 can be used as elution conditions for protein purification, and pH 4.5 can be used as a regeneration condition for the packing materials.

[0070] Example 6: Affinity purification packing material's effect on the purification of S-RBD mutant protein

[0071] Following the method provided in the NHS-Activated LA Beads 4FF product manual, 1 mg of MNb-11 and MNb-14 nanoantibody proteins were coupled to 1 mL of NHS-Activated LA Beads 4FF (blank resin) to prepare S-RBD protein affinity filler.

[0072] The S-RBD affinity packing material was bound to and eluted from the S-RBDs of mutant strains Beta, Delta, BA.4 / 5, XBB.1.5, CH.1.1, and BA.2.86, respectively, to verify its broad-spectrum purification of SARS-CoV-2 S-RBD. The binding conditions were pH 7.5 (20 mM HEPES, 150 mM NaCl), 4 °C, and vortexing for 1 h. The elution conditions were pH 5.0 (100 mM NaAC, 150 mM NaCl), and room temperature.

[0073] The SDS-PAGE results are shown below. Figure 6 The S-RBD affinity packing material was mixed with the S-RBD solution and washed. The treated packing material was then used for sample preparation and electrophoresis. The results showed two protein bands: the S-RBD band at 25 kDa and the nanobody packing material band at 15 kDa. Figure 6 (Left, pH 7.5 binding), it can be seen that the affinity purification packing can bind the mutant S-RBD protein; the bound packing was eluted four times, and the eluents were prepared for electrophoresis. With the increase of elution times, the amount of eluted protein decreased. Finally, the eluted packing was prepared for electrophoresis, and almost no S-RBD residue was found. Figure 6 (Right, eluted at pH 5.0), demonstrating that the affinity purification packing material can purify not only wild-type S-RBD but also mutant S-RBD protein, with a purification efficiency of over 95%.

[0074] Example 7: Evaluation of the purification capacity of affinity purification packing material for S-RBD and S protein in cell expression supernatant.

[0075] Following the method provided in the NHS-Activated LA Beads 4FF product manual, 5 mg of MNb-11 and MNb-14 nanoantibody proteins were coupled to 5 mL of NHS-Activated LA Beads 4FF (blank resin) to prepare S-RBD protein affinity filler.

[0076] To comprehensively evaluate the ability of the S-RBD affinity purification packing material to purify S-RBD and S protein from cell expression supernatant, the S-RBD protein of the XBB.1.5 mutant was used. Figure 7 A) and XBB.1.5 full-length S protein ( Figure 7 Taking B) as an example, the supernatant from the expression of the two proteins was collected after 72 hours and mixed with affinity purification packing material at 4°C for 1 hour. An empty gravity column was fixed on a ring support, allowing the mixture of cell expression supernatant and packing material to flow through the column. The expression supernatant flowed out by gravity, while the packing material was retained by the gravity column membrane. After all the cell expression supernatant had flowed through, an equal volume of pH 7.5 (20 mM HEPES, 1 M NaCl) buffer was added, and this process was repeated three times to wash away non-specific proteins adsorbed onto the packing material. Then, pH 5.0 (100 mM NaAC, 150 mM NaCl) elution buffer was added, and the eluent was collected, repeating this process four times. Samples related to the above purification process were prepared and subjected to SDS-PAGE experiments. The results are shown below. Figure 7 As shown, both affinity purification packing materials have strong enrichment capabilities for S-RBD and S proteins, and no impurity proteins remain on the enriched packing materials. The purity of the target protein in the elution buffer is above 99%, demonstrating strong binding specificity and ensuring the efficiency and purity of protein purification.

[0077] Example 8: Validation of the durability and reusability of affinity purification packing material

[0078] Following the method provided in the NHS-Activated LA Beads 4FF product manual, 5 mg of MNb-11 and MNb-14 nanoantibody proteins were coupled to 5 mL of NHS-Activated LA Beads 4FF (blank resin) to prepare S-RBD protein affinity filler.

[0079] Taking the purification of wild-type S-RBD protein from cell expression supernatant as an example, the supernatant of SF9 cells expressing S-RBD protein for 72 hours was collected and mixed with S-RBD protein affinity packing material at 4°C for 1 hour. An empty gravity column was fixed on a ring support, allowing the mixture of cell expression supernatant and S-RBD protein affinity packing material to flow through the gravity column. The cell expression supernatant flowed out by gravity, while the gravity column membrane retained the packing material. After all the cell expression supernatant had flowed through, an equal volume of pH 7.5 (20 mM HEPES, 1 M NaCl) buffer was added, and this process was repeated three times to wash away non-specific proteins adsorbed onto the packing material. Then, an equal volume of pH 5.0 (100 mM NaAC, 150 mM NaCl) elution buffer was added, and the eluent was collected. This process was repeated four times. After elution, the packing material was regenerated with pH 4.5 (100 mM NaAC, 150 mM NaCl) buffer, and then rinsed with pH 7.5 buffer before re-binding the cell expression supernatant. This process was repeated 10 times, and samples from the 1st, 5th, and 10th purification processes were prepared for SDS-PAGE experiments. The results are shown below. Figure 8 As shown, in the 1st, 5th, and 10th replicates, the electrophoretic bands of the same packing material were similar in terms of position, length, and thickness. This indicates that the enrichment capacity, binding specificity, and purification efficiency of S-RBD in the expression supernatant were not affected by repeated use and regeneration of the packing material.

[0080] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A nanobody, characterized in that, The amino acid sequence of the nanobody is shown in any one of SEQ ID NO. 1-3, the nanobody can bind to the spike protein of SARS-CoV-2 and / or the receptor binding domain of the spike protein of SARS-CoV-2; the novel coronavirus includes wild strains of SARS-CoV-2 and mutant strains of SARS-CoV-2; the mutant strain of SARS-CoV-2 is Beta, Delta, BA.4 / 5, XBB.1.5, CH.1.1 or BA.2.

86.

2. A gene encoding the nanobody of claim 1.

3. A vector or host cell carrying the gene of claim 2.

4. A purified filler characterized in that, The purification filler is used for purifying the spike protein of SARS-CoV-2 and / or the receptor binding domain of the spike protein of SARS-CoV-2, and the purification filler comprises the nanobody of claim 1.

5. The purification packing of claim 4, wherein, The nanobody is coupled to a solid-phase carrier.

6. Use of the purification filler of claim 4 or 5 in the purification of the spike protein of SARS-CoV-2 and / or the receptor binding domain of the spike protein of SARS-CoV-2, for purposes other than disease diagnosis and treatment.

7. Use according to claim 6, characterized in that, The use includes the preparation of a purification reagent or a purification kit.

8. A purification kit characterized in that, The kit contains the purification filler of claim 4 or 5.

9. A method for purifying a novel coronavirus SARS-CoV-2 spike protein purification and / or a novel coronavirus SARS-CoV-2 spike protein receptor binding domain, characterized in that, The method is a method for purifying the spike protein of SARS-CoV-2 and / or the receptor binding domain of the spike protein of SARS-CoV-2 using the purification filler of claim 4 or 5 or the purification kit of claim 8; the method is for purposes other than disease diagnosis and treatment.

10. The method of claim 9, wherein, The specific steps of the method are as follows: S1, binding: mixing and incubating the sample to be purified with the purification filler; S2, column loading: adding the mixed solution after incubation in S1 to a gravity column, and retaining the purification filler; S3, washing: adding a high-salt buffer with neutral pH to the gravity column containing the purification filler, and flowing through, repeating 3 times; S4, elution: adding an elution buffer with weakly acidic pH to the gravity column after washing, and collecting the flow-through, repeating 4 times.

11. The method of claim 10, wherein, After step S4 elution, the purification filler is regenerated using a regeneration buffer with pH 4.5, and the regeneration buffer comprises 80-120 mM NaAC and 120-180 mM NaCl.

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