Isolation and preparation method and application of shed spike protein s1 subunit

Abstract

This invention belongs to the field of biotechnology, specifically relating to a method and application for the isolation and preparation of detached spike protein S1 subunits. This invention successfully isolates detached S1 protein subunits using PEG precipitation, a rapid, convenient, and reliable method that yields structurally correct and stable S1 units. By simulating the binding of the spike protein to ACE2 during coronavirus infection, the S1 subunit is detached. The tag carried by the S1 subunit is then used to isolate and purify the naturally detached S1 subunit, resulting in structurally correct and stable S1 units. The S1 subunit protein is a core functional unit for viral invasion; drug design based on its naturally detached structure can efficiently inhibit viral fusion and is an important target for screening blocking agents (such as small molecules, antibodies, and nucleic acid aptamers). The detached S1 subunit retains an intact antigenic epitope and can be used as a core reagent in detection methods such as ELISA, immunochromatography, and biosensors for detecting viruses or neutralizing antibodies.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for isolating and preparing the S1 subunit of a detached spike protein and its application. Background Technology

[0002] The S1 subunit of the novel coronavirus spike protein is a key component for viral recognition and binding to the host cell ACE2 receptor, and is an important target for understanding viral receptor utilization, antibody antigen design, and vaccine development. Successfully purifying a structurally correct and stable S1 subunit allows for direct use as a vaccine antigen (such as in recombinant protein vaccines) or for high-throughput drug evaluation to identify neutralizing antibodies that can block viral binding to ACE2. It can also yield non-RBD blocking neutralizing antibodies. By "locking in" the S1 and S2 subunits, conformational changes in the spike protein can be prevented, thereby effectively neutralizing the virus.

[0003] Because the spike protein of the novel coronavirus carries a furin cleavage site, the binding between the naturally expressed mature S1 and S2 subunits is not very strong. During natural infection, after the spike protein binds to the ACE2 receptor, S1 detaches from the viral surface; this process is part of viral invasion. Currently, the mainstream and successful method for obtaining the S1 protein in scientific research and commercial fields bypasses the difficulty of isolating it from the whole virus by using recombinant DNA technology. Specifically, the gene encoding the S1 protein or a specific region of it (e.g., the full-length sequence of the S1 subunit, Val16-Arg685) is introduced into a highly efficient expression system (such as HEK293 cells or E. coli), allowing these systems to produce large quantities of the S1 subunit protein, which is then purified. These methods can obtain S1 protein subunits with purity (>95%), making them important tools for studying receptor-binding domain (RBD) function, developing diagnostic reagents, and vaccines.

[0004] The structural biological understanding of the S1 shedding mechanism is primarily based on receptor binding experiments under non-physiological conditions. For example, early cryo-electron microscopy captures of the S1 shedding structures of MERS-CoV and SARS-CoV-2 (Cai, Y. et al. Distinct conformational states of SARS-CoV-2 spike protein. Science 369, 1586-1592 (2020)., Yuan, Y. et al. Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains. Nature Communications 8, 15092 (2017).); and subsequent spike protein-ACE2 complex structures obtained based on 2P stable mutants (Benton, DJ et al. Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion. Nature (2020).), have not fully reflected the dynamics of wild-type viruses during actual infection. Currently, most studies on S1 subunit function focus on recombinantly expressed S1. However, expressed S1 may differ from naturally shed S1 protein in terms of origin, structural conformation, receptor affinity, and antibody binding effect, such as... Figure 1 As shown. More importantly, in the entire conformational change map of the spike protein from pre-fusion to post-fusion, the dynamic trajectory and dissociation path of the S1 subunit remain the most little-known core element.

[0005] Compared to recombinant expression of the S1 protein subunit, capturing the S1 subunit shed during coronavirus invasion presents significant technical challenges: 1. Difficult to capture: Natural shedding is a momentary event in the viral invasion process, making it difficult to accurately capture the S1 protein at this specific moment and state.

[0006] 2. Extremely low yield: The content of naturally detached S1 subunits in cell culture supernatant may be extremely low.

[0007] 3. Stability issues: The naturally detached S1 conformation may be unstable and prone to denaturation or degradation, which brings difficulties to its subsequent purification, storage and functional studies.

[0008] However, the detached S1 protein subunits originate from the actual viral invasion process, undergoing dynamic conformational changes such as receptor binding. They more closely resemble their transient structure and modifications in the native state, potentially including specific cleaved native C-terminal structures. Therefore, obtaining the S1 subunits detached during coronavirus spike protein invasion directly and effectively reflects key steps in viral invasion (such as S1 detachment from the viral particle after receptor binding), making them ideal materials for studying viral invasion mechanisms and evaluating the neutralizing effect of antibodies on the natural invasion process. They are particularly suitable for assessing the dynamic mechanisms of coronavirus invasion and for screening or evaluating neutralizing antibodies or inhibitors that can effectively block the natural invasion process.

[0009] The natural shedding process is difficult to control precisely. The shed S1 protein itself is conformationally unstable, easily denatured or degraded, and its content in cell culture supernatant may be extremely low, requiring sophisticated separation and purification techniques. A method to simulate this process in vitro and capture the S1 protein in its native conformation, intact and detached from the virus particle, has not yet been achieved. Summary of the Invention

[0010] The purpose of this invention is to solve the above-mentioned problems existing in the prior art and to provide a method and application for the isolation and preparation of the detached spike protein S1 subunit.

[0011] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for isolating the detached spike protein S1 subunit, comprising the following steps: mixing a biological sample with a PEG reagent to perform a precipitation reaction, separating the solid and liquid after the reaction is complete, separating and removing the mixture containing PEG and coronavirus particle precipitate, and obtaining the detached spike protein S1 subunit from the remaining supernatant; wherein the biological sample is any one of the viral culture supernatant after coronavirus infection of cells, or bronchoalveolar lavage fluid derived from human or mouse lung tissue.

[0012] Preferably, the coronavirus includes at least one of SARS-CoV-2, MERS-CoV, hCoV-OC43 and hCoV-HKU1, and the amino acid sequence of the corresponding detached spike protein S1 subunit is shown in at least one of SEQ ID NO 1-SEQ ID NO 4.

[0013] Preferably, SARS-CoV-2 S1: (SEQ ID NO 1): 。

[0014] MERS-CoV S1: (SEQ ID NO 2): MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTR。

[0015] hCoV-OC43 S1: (SEQ ID NO 3): MFLILLISLPTAFAVIGDLKCTSDNINDKDTGPPPISTDTVDVTNGLGTYYVLDRVYLNTTLFLNGYYPTSGSTYRNMALKGSVLLSRLWFKPPFLSDFINGIFAKVKNTKVIKDRVMYSEFPAITIGSTFVNTSYSVVVQPRTINSTQDGDNKLQGLLEVSVCQYNMCEYPQTICHPNLGNHRKELWHLDTGVVSCLYKRNFTYDVNADYLYFHFYQEGGTFYAYFTDTGVVTKFLFNVYLGMALSHYYVMPLTCNSKLTLEYWVTPLTSRQYLLAFNQDGIIFNAEDCMSDFMSEIKCKTQSIAPPTGVYELNGYTVQPIADVYRRKPNLPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNKRFGFIEDSVFKPRPAGVLTNHDVVYAQHCFKAPKNFCPCKLNGSCVGSGPGKNNGIGTCPAGTNYLTCDNLCTPDPITFTGTYKCPQTKSLVGIGEHCSGLAVKSDYCGGNSCTCRPQAFLGWSADSCLQGDKCNIFANFILHDVNSGLTCSTDLQKANTDIILGVCVNYDLYGILGQGIFVEVNATYYNSWQNLLYDSNGNLYGFRDYIINRTFMIRSCYSGRVSAAFHANSSEPALLFRNIKCNYVFNNSLTRQLQPINYFDSYLGCVVNAYNSTAISVQTCDLTVGSGYCVDYSKNRRSR。

[0016] hCoV-HKU1 S1: (SEQ ID NO 4): .

[0017] The S1 protein subunits precipitated using PEG precipitation can be used in samples from various sources, such as bronchoalveolar lavage fluid from patients and mice, environmental or wastewater, and cell culture systems. The PEG precipitation method for separating the virus and the precipitated S1 protein supernatant does not require high-speed centrifugation gradient separation technology, making it faster, more convenient, and more reliable compared to the required initial viral supernatant concentration and P3 biosafety requirements.

[0018] Preferably, the PEG reagent is an aqueous solution of PEG, wherein the average molecular weight of PEG is 5000-7000 and the concentration of PEG is 7%-9% (w / v).

[0019] Preferably, the PEG is PEG6000 and the concentration of PEG is 8% (w / v).

[0020] Preferably, the precipitation reaction temperature is 0-4℃.

[0021] Preferably, the precipitation reaction temperature is 4°C.

[0022] Preferably, the viral culture supernatant after the coronavirus infects cells, or the bronchoalveolar lavage fluid derived from human or mouse lung tissue, is first centrifuged and clarified or filtered to remove cell debris before being mixed with PEG reagent for precipitation reaction.

[0023] Preferably, the solid-liquid separation method is centrifugal separation or membrane filtration separation; the centrifugal force of the centrifugal separation is 8000-10000 xg.

[0024] Secondly, the present invention provides a nucleic acid molecule for preparing the detached spike protein S1 subunit, wherein the nucleic acid molecule encodes a full-length spike protein with a purified tag inserted after the N-terminal signal peptide and before the NTD domain sequence.

[0025] Preferably, the tag sequence is directly fused after the cleavage site of the N-terminal signal peptide and directly fused before the first amino acid of the NTD domain.

[0026] Preferably, the full-length spike protein is derived from SARS-CoV-2, MERS-CoV, HCoV-HKU1, or HCoV-OC43.

[0027] Preferably, the full-length spike protein is derived from SARS-CoV-2, with the Uniprot number P0DTC2 (SARS-CoV-2 Spike).

[0028] Preferably, the purification tag is a Flag tag, a His tag, or a Myc tag.

[0029] Preferably, the Flag-tag sequence is: DYKDDDDK (SEQ ID NO 5); the His-tag sequence is: HHHHHH (SEQ ID NO 6); and the Myc-tag sequence is: EQKLISEEDL (SEQ ID NO 7).

[0030] Thirdly, the present invention provides a plasmid for preparing the detached spike protein S1 subunit, comprising the aforementioned nucleic acid molecule.

[0031] Fourthly, the present invention provides a plasmid combination for preparing detached spike protein S1 subunits, comprising the plasmids described above and a plasmid expressing human ACE2.

[0032] Fifthly, the present invention provides a cell for preparing detached spike protein S1 subunits, said cell being capable of expressing full-length spike protein and human ACE2 with a purified tag inserted after the N-terminal signal peptide and before the NTD domain sequence.

[0033] Preferably, the cell contains the plasmid combination described above.

[0034] In a sixth aspect, the present invention provides the use of the described nucleic acid molecule, the described plasmid, the described plasmid combination, or the described cell in the preparation of the detached spike protein S1 subunit.

[0035] In a seventh aspect, the present invention provides a method for preparing detached spike protein S1 subunits, comprising the following steps: constructing the aforementioned nucleic acid molecule or plasmid, co-transfecting it with a plasmid expressing human ACE2 into a host cell to detach the spike protein S1 subunits, and separating and purifying the naturally detached spike protein S1 subunits using a carried tag, wherein the amino acid sequence of the detached spike protein S1 subunits is shown in at least one of SEQ ID NO 1-SEQ ID NO 4.

[0036] Preferably, the host cell is a HEK293F cell, a HEK293T cell, or a CHO cell.

[0037] Eighthly, the present invention provides an application of the detached spike protein S1 subunit obtained by the separation method or the preparation method described above as a target for screening blocking agents.

[0038] In a ninth aspect, the present invention provides an application of the detached spike protein S1 subunit obtained by the separation method or the preparation method described above for the preparation of products for detecting viruses or neutralizing antibodies.

[0039] In a tenth aspect, the present invention provides an application of the detached spike protein S1 subunit obtained by the separation method or the preparation method described above for the preparation of a vaccine.

[0040] The advantages and beneficial effects of this invention are as follows: 1. The separation method of this invention, which uses PEG precipitation to precipitate the detached S1 protein subunit, can be applied to samples from various sources, such as bronchoalveolar lavage fluid from patients and mice, environmental or wastewater, and cell culture systems. The PEG precipitation method for separating the virus and the detached S1 protein supernatant does not require high-speed centrifugation gradient separation technology. Compared to other methods, it is faster, more convenient, and more reliable, and can yield structurally correct and stable S1.

[0041] 2. The preparation method of the present invention simulates the binding of the spike protein to ACE2 during coronavirus infection, causing the S1 subunit to detach. The naturally detached S1 subunit is isolated and purified using the tag carried by the S1 subunit, resulting in a structurally correct and stable S1.

[0042] 3. Drug design and screening: The S1 subunit protein is the core functional unit for viral invasion. Drugs designed based on its naturally detached structure can effectively inhibit viral fusion and are an important target for screening blocking agents (such as small molecules, antibodies, and nucleic acid aptamers).

[0043] 4. Diagnostic detection technology: The detached S1 subunit maintains the complete antigen epitope and can be used as a core reagent in detection methods such as ELISA, immunochromatography, and biosensors to detect viruses or neutralizing antibodies.

[0044] 5. As a safe vaccine antigen: Compared with the full-length spike protein of coronavirus, the S1 subunit that is detached after fusion does not contain the S2 subunit that mediates membrane fusion, which is theoretically safer and can more effectively stimulate neutralizing antibodies against the viral receptor binding domain. Attached Figure Description

[0045] Figure 1 The difference between expressed S1 and naturally shed S1 protein; Figure 2 The following are the isolation routes of the soluble spike protein S1 subunit protein shed during coronavirus infection in Example 1 of this invention (A), the immunoblot map of the shed S1 isolated after infection with wild-type SARS-CoV-2 (B), the immunoblot map of the shed S1 isolated after infection with Omicron XBB.1.5 strain (C), and the immunoblot map of the product isolated after infection with human coronavirus OC43 (D). Figure 3 The diagram shows the structure of the N-terminus of the full-length spike protein expressed in Example 2 of the present invention (A), the electrophoresis diagram of the product separated and purified by anti-flag protein purification tag agarose column in Example 3 (B), and the electrophoresis diagram of the Flag-S1 product further purified by concanavalin A agarose strain in Example 3 (C). Figure 4 The following are the chromatographic patterns (A) of the detached S1 and full-length spike protein obtained by Superose 6-column molecular sieve in Example 4 of this invention, the molecular sieve component identification diagram (B) of the detached S1 and full-length spike protein, the electrophoresis diagram of the detached S1 and full-length spike protein obtained by DSS chemical coupling (C) the affinity diagram of ACE2 for protease-linked immunosorbent assay (PISA) of the detached S1 and full-length spike protein (D) the immunoblot diagram of ACE2 co-precipitation (E) and the flow cytometry analysis diagram of HEK293T-ACE2 cells (F). Figure 5 The diagram shows the discovery of S1 by hydrogen-deuterium exchange mass spectrometry in Example 5 of the present invention (A), the dynamic molecular dynamics simulation diagram of the SD1 peptide that has detached from S1 (B), and the dynamic bimodal spectrum of the SD1 peptide that has detached from S1 (C). Figure 6 The diagram shows the neutralization epitope map of the SD1 peptide segment that sheds S1 in Example 5 of the present invention (A), the result of SD1-1 inhibiting the cell fusion of the novel coronavirus (B), and the result of SD1-1 inhibiting the shedding of S1 from the full-length spike protein (C). Figure 7 The image shows the immunogenicity results of mice induced by the detached spike protein S1 subunit in Example 6. In the image, A shows the results of neutralizing antibodies against wild-type SARS-CoV-2 pseudovirus; and B shows the results of the half-neutralizing concentration (IC50 / mL) of the SARS-CoV-2 pseudovirus. Detailed Implementation

[0046] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that these embodiments are only some preferred technical solutions, and the scope of protection claimed by the present invention is not limited to the following embodiments.

[0047] Example 1: Purification Technique for Soluble Spike Protein S1 Subunit Dropped from Coronavirus Infection This embodiment uses the novel coronavirus (SARS-CoV-2) as an example for illustration. Figure 2 As shown in Figure A, this embodiment successfully isolated and confirmed that SARS-CoV-2 and human OC43 coronavirus infection leads to the detachment of the S1 subunit of the spike protein, which is present in soluble proteins in cell culture supernatant using PEG6000 viral precipitation and high molecular weight soluble protein separation technology. Specifically, the method involves using viral culture supernatant from infected cells and bronchoalveolar lavage fluid derived from human or mouse lung tissue. After removing cells by centrifugation at 2000 rpm, the mixture is incubated overnight at 4°C with 8% (w / v) PEG6000 reagent dissolved in water. Following this, the mixture is centrifuged at 9800 x g for 45 minutes to obtain a precipitate containing PEG6000 and coronavirus particles. Soluble proteins with a molecular weight greater than ~80 kDa and the detached S1 protein subunit are completely retained in the soluble culture medium or bronchoalveolar lavage supernatant. The S1 protein can be identified by immunoblotting of cell lysate, PEG precipitate, and PEG soluble protein. Mouse-derived primary antibodies include Beijing Sinocare Biotech Co., Ltd., 40592-MM117-100, which cross-detects S1 of multiple Omicron mutant strains; or 40591-MM42-100, which detects early SARS-CoV-2 strains from WT to Delta.

[0048] In this embodiment, it was found that after Vero-E6 cells were infected with the wild-type SARS-CoV-2 strain (WT) at 0.01 MOI for 48 hours, significantly detached S1 protein subunits were observed in the cell supernatant. Figure 2 (Detection method B, 40591-MM42-100). Unlike the surface of cells or virus particles, the detached S1 subunit does not contain the full-length S or viral riboprotein N bands (Sinochem, 40143-R001-H-20), indicating that this technique can effectively separate the S1 protein subunit from the cell surface or virus particles and from the soluble supernatant. This technique also revealed that different SARS-CoV-2 mutant strains, such as the 0.01 MOI OmicronXBB.1.5 strain, also shed the S1 protein subunit after infection with Vero-E6. Figure 2 The virus was detected by 40591-MM42-100 and was similar to the wild-type strain. To confirm that the shedding of the S1 subunit of the coronavirus spike protein is a conserved molecular mechanism, human colon cancer Caco-2 cells were further infected with human coronavirus OC43. The results showed that the cell infection supernatant obtained at different time points after infection, in addition to carrying human OC43 virus, also contained an equal amount of shed OC43 spike protein S1 subunit (C, detected by 40591-MM42-100), and was similar to the wild-type strain. Figure 2 (Tested by Sinocare, 40607-T62-20). These data confirm that under real coronavirus infection conditions, the S1 subunit of the spike protein can detach from the virus or cell surface and remain in the viral culture supernatant.

[0049] The inventors have used this method to test SARS-CoV-2, MERS-CoV, hCoV-OC43 and hCoV-HKU1, and all of them can effectively identify the detached S1; in other embodiments, the desired coronavirus can be selected for the experiment.

[0050] Example 2: Construction of an expression system capable of capturing the S1 subunit of the SARS-CoV-2 spike protein. Example 1 demonstrated that the shed S1 protein subunit of the coronavirus is closely related to viral infection and fusion. To capture the shed S1 protein subunit, the following two points are required: 1. By constructing a spike protein purification tag, the detached S1 protein subunit is efficiently captured and separated and purified from the complex environment.

[0051] A full-length spike protein expression plasmid was used, with a protein purification tag (Flag) inserted after the N-terminal signal peptide and before the NTD domain sequence of the spike protein. If the signal peptide sequence of the coronavirus spike protein is unknown and has not yet been studied, the N-terminal sequence of the spike protein can be predicted using the online SignalP-6.0 website (https: / / services.healthtech.dtu.dk / services / SignalP-6.0). The final result was a plasmid expressing the full-length spike protein at the N-terminus (pcDNA3.1-Flag-S). Figure 3 (A)

[0052] 2. It efficiently simulates the membrane fusion effect of coronaviruses during infection and actively mediates the shedding of S1.

[0053] In 2 x 10 6 On 293F cells at a cell density of 1 / mL, pcDNA3.1-Flag-S and human pcDNA3.1-ACE2 expression plasmids were transfected using low molecular weight PEI reagent (MW 40000, Polysciences, 24765-100), with the spike protein to ACE2 transfection ratio strictly controlled at 2:1. The cells were stored at 5% CO2 and 37°C. o After culturing and expressing the protein for 72 hours on a C shaker at 120 rpm, the cell pellet was removed by centrifugation at 2000g to obtain a supernatant containing the detached Flag-S1 protein subunit.

[0054] Example 3: Purification technology of S1 subunit shed from SARS-CoV-2 spike protein The viral supernatant containing detached S1 obtained in Example 2 was centrifuged at 3,000 × g for 15 minutes at 4°C to remove cell debris and precipitate, and the supernatant was used for subsequent purification. First, the supernatant containing Flag-S1 protein was affinity purified using an anti-flag protein purification tag agarose column.

[0055] The Flag-S1 protein in the supernatant obtained in step 2.1 was purified using an anti-Flag affinity gel (e.g., Sigma-Aldrich, catalog number A2220). The specific procedure is as follows: (1) Column equilibration: Load the anti-Flag affinity gel into the chromatography column (column volume 1 mL), and equilibrate the chromatography column with 10 column volumes of PBS buffer (pH 7.4) at a flow rate of 1 mL / min.

[0056] (2) Sample loading: The cell culture supernatant collected in step (1) is slowly passed through the affinity chromatography column at a flow rate of 0.5 mL / min, so that the Flag-S1 protein in the supernatant specifically binds to the anti-Flag antibody on the column material. The flow-through is collected for SDS-PAGE detection.

[0057] (3) Washing: After sample loading, wash the chromatography column with 10 column volumes of PBS buffer (containing 0.05% Tween-20) at a flow rate of 1 mL / min to remove unbound or non-specifically bound contaminating proteins. Collect the washing buffer for SDS-PAGE detection.

[0058] (4) Competitive elution: To maintain the native conformation of the detached Flag-S1 protein and avoid using acidic elution solutions that could damage the protein conformation, this embodiment employs a competitive elution method. Specifically, a 3×Flag polypeptide (sequence of which is...) is used. DYKDDDDKDYKDDDDKDYKDDDDK PBS buffer (final concentration 25 mg / mL) was used as the elution buffer, and the column was passed through at a flow rate of 0.5 mL / min. The 3×Flag peptide competitively binds to the anti-Flag antibody immobilized on the column material, thereby displacing and eluting the bound Flag-S1 protein. The eluent was collected in fractions, 1 mL per tube, labeled E1, E2, E3, E4, E5, and E6.

[0059] (5) Detection of eluted fractions: Each eluted fraction was subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining or silver staining. The results showed that Flag-S1 protein was mainly found in fractions E2 to E6. Figure 3 B, E2-E6), with a molecular weight of approximately 110 kDa, consistent with the theoretical molecular weight of the S1 subunit.

[0060] Because the purification of Flag-S1 in cell supernatants, such as E2-E6, may leave residual HSP70 or HSP90 proteins secreted by cells, a method for further purification of Flag-S1 using Conagarose protein strain was further invented.

[0061] 1. Pretreatment of packing material: Take 1 mL of packing material (ConA Beads 4FF, Smart-lifesciences, SA028005), centrifuge to remove the packing material storage solution, add 1 mL of equilibration solution, wash once, short centrifuge to remove the supernatant, repeat twice, and then add 1 mL of equilibration solution to resuspend.

[0062] 2. Incubation: Add the pretreated packing material to the purified sample (E2-E5) from the previous step and incubate overnight in a rotary incubator at 4 ℃.

[0063] 3. Installation and pretreatment of the purification column: Place the sieve plate at the bottom of the purification column, open the bottom cap of the purification column, and connect it to the silicone tubing installed on the peristaltic pump. After installation, pre-pass the column with 3 mL of equilibration buffer (20 mM Tris-HCl, 0.5 M NaCl, 1 mM CaCl2, 1 mM MnCl2, adjusted to pH 7.4 with 2 M HCl, and then filtered through a 0.45 μm filter membrane).

[0064] 4. Sample loading: Use a pipette to gradually add the sample into the purification column (during which the sample is added while shaking to ensure that all the packing material is added into the transfer column; at the same time, prevent the column from drying out).

[0065] 5. Washing: Wash with 10-20 mL of equilibration buffer (5-10 column volumes).

[0066] 6. Elution: Stopper the bottom of the purification column, resuspend in 2 mL of eluent (20 mM Tris-HCl, 0.5 M NaCl, 1 mM CaCl2, 1 mM MnCl2, 0.2 M mannose), and incubate for 10 min. Open the bottom of the purification column and collect the eluent by gravity. Repeat the elution 10 times and label them E1-E10.

[0067] 8. Identification of purified protein: Take 20 μL of each of the eluents E1-E10 and add 6 μL of 5 × loading buffer to identify the purified S1 by codon staining.

[0068] The mechanism of this method is through the oligomannose binding properties of ConA, which affinity for Flag-S1 containing oligomannose glycosylation modification. Figure 3 (The injection solution does not contain S1). Finally, non-denaturing elution was performed using a buffer containing mannose to obtain the detached spike protein Flag-S1 subunit. Figure 3 (C, E1-E9).

[0069] Example 4: Synonyms of S1 removal and S1 expression Currently, functional studies in this field mainly rely on in vitro recombinant expression of the S1 protein, but its conformation, modification, and function may differ fundamentally from the S1 protein naturally shed by the virus during infection. Figure 1 The detached S1, purified via concanavalin Agarose, exhibited significant differences in basic functions (such as receptor binding) compared to the pre-fusion spike protein or recombinantly expressed S1. The detached S1 was in soluble protein monomer form, such as... Figure 4As shown in Figure A, the elution position of the detached S1 obtained by Superose 6-column molecular sieve is significantly different from that of the extracellular domain of the full-length spike protein before fusion. Compared to the 190 kDa position of the full-length spike protein, the molecular weight of the detached S1 is approximately 110 kDa. Figure 4 (B) By chemically coupling the detached S1 with 2 mM DSS (Sigma, A39267), we found that it could not form a trimer structure like the full-length spike protein, indicating that the protein had been converted into a free monomeric form. Figure 4 (C)

[0070] This embodiment not only verified the monomeric characteristics of detached S1 at the biochemical level, but also verified the receptor binding differences of detached S1. First, using enzyme-linked immunosorbent assay (ELISA), recombinant protein of the full-length HexaPro spike protein extracellular domain before fusion and detached S1 purified from concanavalin A agarose strain were coated on ELISA plates (1 μg / well). Then, the receptor binding ability of ACE2 was detected by His-HRP-tagged antibody using the free ACE2-6his extracellular domain. The results showed that only the full-length spike protein could bind to soluble ACE2, but detached S1 lost its ACE2 binding ability. Figure 4 (D). On the other hand, using rabbit-derived anti-ACE2 antibody to immunoprecipitate ACE2, it was found that only 2 μg / mL of the full-length HexaPro spike protein extracellular domain or commercially purchased (Wuhan Aibote Biotechnology Co., Ltd., RP01262) recombinantly expressed S1 bound to ACE2, while the binding effect of detached S1 to ACE2 was significantly reduced. Figure 4 (E). Further, at the cellular level, flow cytometry analysis of HEK293T-ACE2 cells also confirmed that the extracellular domain of the full-length HexaPro spike protein or commercially purchased recombinant S1 expressed in ACE2 cells had a significant affinity for ACE2-expressing cells, while the binding effect of detached S1 to ACE2 was significantly reduced under the same concentration of 10 μg / mL. Figure 4 The above experimental results highlight the necessity and urgency of using the "shed S1 subunit" as the gold standard for research.

[0071] Example 5: Explanation of the structural dynamic differences of detached S1 and the targetability of its sites. Because the detached S1 may lose structural rigidity, creating flexible structural domain connections and carrying important structural deformation features, a hydrogen-deuterium exchange mass spectrometer (C18trap column, Waters, Milford, MA) was used, employing 90% deuterium water (D2O, Sigma, 151882) at a final concentration of 37. oC was processed at four time-progressive deuterium-assisted sites: 10 s, 1 min, 10 min, and 30 min. The flexible peptide regions of the detached S1 subunit and the full-length S-trimer S1 subunit before fusion were compared. Hydrogen-deuterium exchange mass spectrometry captured several differences in the extracellular domain of the detached S1 compared to the full-length HexaPro spike protein before fusion. The SD1 peptide of the detached S1 (two peptide regions showed significantly more deuterium-assisted regions than the full-length spike protein S1 subunit) Figure 5 (B and C in the middle).

[0072] By searching existing antibody libraries, SD1-1 is a monoclonal neutralizing antibody from recovered patients that specifically recognizes the aforementioned flexible SD1 peptide (Zhou, D. et al. The SARS-CoV-2 neutralizing antibody response to SD1 and its evasion by BA.2.86. Nature Communications 15, 2734 (2024).). It does not neutralize by blocking the RBD region, but rather through an unknown mechanism to neutralize multiple SARS-CoV-2 mutant strains. Cell fusion experiments showed that SD1-1 can dose-dependently (IC50: 1.824 μg / mL) block SARS-CoV-2 spike protein-induced cell fusion. Figure 6 (B). Further analysis of cell supernatant and post-fusion lysate revealed that SD1-1 also dose-dependently blocked ACE2 cell-induced supernatant S1 shedding events. Figure 6 The detached S1 subunit (C) prevents downstream S2' protease cleavage. These data suggest that the detached S1 subunit contains potential, specific epitope information that can be used for the discovery of neutralizing antibody mechanisms and the screening of novel antiviral drugs.

[0073] Example 6: Induction of immunogenicity in mice using shed spike protein S1 subunit To demonstrate the antigenic immunogenicity of the purified detached S1, a mouse subcutaneous injection model was used to evaluate neutralizing antibodies in C57BL6 mice inoculated with the detached S1 subunit. Aluminum hydroxide (aluminum adjuvant) and a dose of 20 μg of detached S1 per mouse (or the PBS control group) were mixed at a 1:1 ratio to obtain 40 μL of the protein subunit vaccine per mouse. Fourteen days after inoculation, mice were re-inoculated with the same dose of detached S1 (booster injection). Neutralizing serum containing detached S1 was obtained after mouse sacrifice. Results showed that, compared to the control group, serum from each mouse inoculated with the detached S1 protein subunit vaccine produced varying degrees of neutralizing antibodies against wild-type SARS-CoV-2 pseudovirus. Figure 7(A). Mice vaccinated with the exfoliated S1 protein subunit vaccine had an average IC50 / mL half-neutralization concentration (IC50 / mL) of ~87.096 for SARS-CoV-2 pseudovirus. Figure 7 (B). These data indicate that the shed S1 is immunogenic and can induce mice to produce effective neutralizing antibodies against the novel coronavirus.

[0074] The above description is only the best specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A method for isolating the detached S1 subunit of the spike protein, characterized in that, The method includes the following steps: mixing a biological sample with a PEG reagent to perform a precipitation reaction, separating the solid and liquid after the reaction is complete, separating and removing the mixture containing PEG and coronavirus particle precipitate, and obtaining the detached spike protein S1 subunit from the remaining supernatant; wherein the biological sample is any one of the viral culture supernatant after coronavirus infection of cells, or bronchoalveolar lavage fluid derived from human or mouse lung tissue.

2. The method for isolating the detached spike protein S1 subunit according to claim 1, characterized in that, The coronaviruses include at least one of SARS-CoV-2, MERS-CoV, hCoV-OC43 and hCoV-HKU1, and the amino acid sequence of the corresponding detached spike protein S1 subunit is shown in at least one of SEQ ID NO 1-SEQ ID NO 4.

3. The method for isolating the detached spike protein S1 subunit according to claim 1, characterized in that: The PEG reagent is an aqueous solution of PEG, wherein the average molecular weight of PEG is 5000-7000 and the concentration of PEG is 7%-9% (w / v). Preferably, the precipitation reaction temperature is 0-4℃.

4. A nucleic acid molecule for preparing the detached S1 subunit of the spike protein, characterized in that, The nucleic acid molecule encodes a full-length spike protein with a purified tag inserted after the N-terminal signal peptide and before the NTD domain sequence.

5. The nucleic acid molecule according to claim 4, characterized in that, The full-length spike protein is derived from at least one of SARS-CoV-2, MERS-CoV, hCoV-OC43, and hCoV-HKU1.

6. A plasmid for preparing detached spike protein S1 subunits, characterized in that, It includes the nucleic acid molecule as described in claim 4 or 5.

7. A plasmid assembly for preparing detached spike protein S1 subunits, characterized in that, The plasmid comprising the plasmid of claim 6 and the plasmid expressing human ACE2.

8. A cell for preparing detached spike protein S1 subunits, characterized in that, The cells are able to express full-length spike protein and human ACE2 with a purified tag inserted after the N-terminal signal peptide and before the NTD domain sequence. Preferably, the cell comprises the plasmid combination of claim 7.

9. The use of the nucleic acid molecule of claim 4 or 5, the plasmid of claim 6, the plasmid combination of claim 7, or the cell of claim 8 in the preparation of the exfoliated spike protein S1 subunit.

10. A method for preparing an exfoliated spike protein S1 subunit, characterized in that, The method includes the following steps: constructing the nucleic acid molecule of claim 4 or 5 or the plasmid of claim 6, co-transfecting it with a plasmid expressing human ACE2 into a host cell to cause the spike protein S1 subunit to detach, and using the carried tag to isolate and purify the naturally detached spike protein S1 subunit, wherein the amino acid sequence of the detached spike protein S1 subunit is shown as at least one of SEQ ID NO 1-SEQ ID NO 4.

11. The detached spike protein S1 subunit obtained by the separation method of any one of claims 1-3 or the preparation method of claim 10 is used as a target for screening blocking agents.

12. The use of the detached spike protein S1 subunit obtained by the separation method of any one of claims 1-3 or the preparation method of claim 10 for the preparation of products for detecting viruses or neutralizing antibodies.

13. The detached spike protein S1 subunit obtained by the separation method according to any one of claims 1-3 or the preparation method according to claim 10 is used for the preparation of vaccines.

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