Fatty acid-modified coronavirus antigen and use thereof

Abstract

Disclosed in the present invention are a fatty acid-modified coronavirus antigen and use thereof, pertaining to the field of biomedicine. The fatty acid-modified coronavirus antigen comprises a fatty acid-modified N-terminal cysteine (Cys) and a coronavirus antigen, and may comprise a linking domain. The coronavirus antigen is a coronavirus spike protein (S protein) receptor-binding domain (RBD). The fatty acid-modified coronavirus S protein RBD can self-assemble into nanoparticles. Compared with an RBD without fatty acid modification, the antigen can induce faster and higher humoral immunity by means of injection immunization, and can also induce mucosal immunity and humoral immunity by means of respiratory nebulization immunization, making it a candidate mucosal vaccine with potential medical value.

Description

A fatty acid-modified coronavirus antigen and its application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fatty acid-modified coronavirus antigen and its application. Background Technology

[0002] Coronaviruses are a class of enveloped, single-sense, positive-sense RNA viruses belonging to the Coronaviridae family. Seven species of coronaviruses are known to infect humans, of which SARS-CoV, SARS-CoV-2, and MERS-CoV currently cause severe illness and even death. The main structural proteins of coronaviruses include the spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The S protein, located on the surface of the coronavirus envelope, recognizes host cell receptors and mediates viral fusion with the host cell membrane through conformational changes. For example, the receptor for SARS-CoV and SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2), while the receptor for MERS-CoV is dipeptidyl peptidase-4 (DPP4). Within the S protein, the receptor-binding domain (RBD) acts as an independent structural domain capable of binding to the receptor. Due to the lack of corrective mechanisms during RNA virus replication, the encoded protein continuously mutates. Under selective pressure, the RBD of various mutant strains retains its receptor-specific binding ability. For example, since the emergence of the SARS-CoV-2 virus at the end of 2019, the World Health Organization has identified five major categories of variants of concern (VOCs): Alpha, Beta, Gamma, Delta, and Omicron. Various variants are expected to continue to emerge in the future. The RBD (Reactive Bronchodilator) is the immunodominant region of the S protein, containing the vast majority of neutralizing epitopes, making it an ideal candidate antigen. However, the small molecular weight and relatively low immunogenicity of RBDs hinder the development of subunit vaccines using RBDs as antigens, and mucosal vaccines using RBDs as antigens are even more challenging.

[0003] Bacteria possess a class of proteins whose N-terminal cysteine ​​(Cys) is modified with fatty acid and anchored to the cell surface via fatty acid chains; these are called bacterial lipoproteins. The in vivo synthesis of bacterial lipoproteins is guided by a signal peptide. Bioinformatics and statistical analysis of the signal peptide sequences of naturally occurring and non-fatty acid modified bacterial proteins revealed that the signal peptide of fatty acid modified proteins contains nonpolar amino acids near the signal peptidase cleavage site, exhibiting a degree of conservation. The conserved sequence of the last four amino acids in this region was summarized as [L / V / I][A / S / T / V / I][G / A / S][C], where the C-terminal cysteine ​​is the lipid modification site. Guided by the signal peptide, nascent peptide chains are localized to the extracellular space via the Sec or TAT secretion pathway. Lgt enzymes, using phosphatidylglycerol (PG) as a substrate, attach diacylglycerol to the sulfhydryl group of Cys. LspA enzymes cleave the portion of the signal peptide excluding the C-terminal Cys group. Lnt enzymes, using phosphatidylethanolamine (PE) as a substrate, transfer a fatty acid chain to the alpha-amino group of Cys. Bacterial fatty acid modification is heterogeneous, mainly manifested in the number and length of fatty acid chains. The acyl carbon chain length of bacterial lipoproteins is usually between C14 and C18, but the vast majority are C16.

[0004] Displaying antigens on the surface of protein nanocarriers is an effective way to improve antigen immunogenicity. A modular preparation scheme, which combines nanoparticles and loaded antigens through a universal, efficient, and controllable linking method, effectively solves problems such as low expression efficiency, particle assembly failure, structural instability, and loss of immunogenicity caused by carrier-antigen fusion expression. The SpyTag / SpyCatcher linking system is one of the most thoroughly studied protein linking systems. Due to its rapid reaction, specificity, broad tolerance to reaction conditions, and irreversibility, this system is widely used in vaccine development and research. By fusing it with pathogen antigens and suitable carrier proteins, precise covalent linking of antigens and carrier proteins can be achieved. In particular, it allows for repeated display of antigens on the surface of self-polymerizing nanocarrier proteins, improving antigen presentation efficiency and immunogenicity. This linkage system originates from the CnaB2 domain of the fibronectin-binding protein of Streptococcus pyogenes. Lys31 and Asp117 of the CnaB2 domain can form intramolecular isopeptide bonds. After the CnaB2 domain is split into an N-terminal domain containing Lys31 (SpyCatcher, 116aa) and a C-terminal polypeptide containing Asp117 (SpyTag, 13aa), the two fragments can still form specific isopeptide bonds. To further optimize the SpyTag / SpyCatcher ligation system, Mark Howarth's team analyzed the relationship between its structure and function, demonstrating the minimum SpyCatcher required to form isopeptide bonds. To improve the ligation reaction speed and efficiency, the team artificially designed the SpyTag(13aa) / SpyCatcher(116aa) ligation system, developing the SpyTag002 / SpyCatcher002 and SpyTag003 / SpyCatcher003 ligation systems.

[0005] In existing technologies, displaying protective antigens on the surface of protein nanoparticles, and / or physically mixing or embedding adjuvants, as well as using viral vectors to deliver antigens, are effective means to improve the immunogenicity of mucosal vaccines. However, issues such as pre-existing immunity caused by the vector, immune focusing imbalance, and safety concerns arising from excessive adjuvant use have hampered the development of mucosal vaccines. Therefore, it is necessary to develop a new strategy that can improve antigen immunogenicity while reducing the immunoimprinting effect and / or immune advantage of the carrier protein, and also reduce the side effects caused by adjuvants. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a fatty acid-modified coronavirus antigen and its applications.

[0007] According to a first aspect of the present invention, a fatty acid-modified coronavirus antigen is provided. The fatty acid-modified coronavirus antigen comprises a fatty acid-modified N-terminal cysteine ​​residue (Cys).

[0008] The fatty acid-modified N-terminal cysteine ​​(Cys) specifically refers to Cys whose thiol group is modified with diacylglycerol, or Cys whose thiol group is modified with diacylglycerol and whose α-amino group is modified with acyl group.

[0009] The coronaviruses include, but are not limited to, SARS-CoV-2, SARS-CoV, or MERS-CoV.

[0010] The preferred antigen of the coronavirus is the coronavirus S protein or a partial functional fragment thereof.

[0011] The functional fragment of the coronavirus S protein is either a receptor-binding domain (RBD) or an N-terminal domain (NTD), preferably an RBD.

[0012] In the SARS-CoV and SARS-CoV-2 coronavirus antigens, the receptor-binding domain (RBD) includes the region of the SARS-CoV-2 and SARS-CoV S protein that binds to ACE2 and its variants, and its amino acid sequence includes, but is not limited to, any of the sequences described in SEQ ID No. 1-9;

[0013] In the MERS-CoV coronavirus antigen, the receptor-binding domain (RBD) includes the region where the MERS-CoV S protein binds to DPP4 or a variant thereof; its amino acid sequence includes, but is not limited to, any of the sequences described in SEQ ID No. 10.

[0014] The fatty acid-modified coronavirus antigen provided by this invention may also contain a linker domain.

[0015] The linker domain is an isopeptide linker, preferably a SpyCatcher / SpyTag linker.

[0016] The coronavirus antigen is fused with the SpyTag polypeptide and covalently linked to the fatty acid-modified SpyCatcher protein via isopeptide bonds; or, the coronavirus antigen is fused with the SpyCatcher protein and covalently linked to the fatty acid-modified SpyTag polypeptide via isopeptide bonds.

[0017] The SpyCatcher protein is a polypeptide containing any of the amino acid sequences of SEQ ID No. 11-13, and the amino acid sequence of the SpyTag is any of SEQ ID No. 14-16.

[0018] When the SpyCatcher protein contains the amino acid sequence shown in SEQ ID NO.11, the SpyTag polypeptide contained in the corresponding fatty acid-modified recombinant RBD protein preferably uses the amino acid sequence shown in SEQ ID NO.14.

[0019] When the SpyCatcher protein contains the amino acid sequence shown in SEQ ID NO.12, the SpyTag polypeptide contained in the corresponding fatty acid-modified recombinant RBD protein preferably uses the amino acid sequence shown in SEQ ID NO.15.

[0020] When the SpyCatcher protein contains the amino acid sequence shown in SEQ ID NO.13, the SpyTag polypeptide contained in the corresponding fatty acid-modified recombinant RBD protein preferably uses the amino acid sequence shown in SEQ ID NO.16.

[0021] According to a second aspect of the present invention, a method for preparing the fatty acid-modified coronavirus antigen as described above is provided, the specific implementation steps of which are as follows:

[0022] 1) A DNA fragment containing a signal peptide coding sequence and a coronavirus antigen coding sequence was introduced into Escherichia coli, and recombinant E. coli was cultured to express recombinant coronavirus antigen with N-terminal fatty acid modification.

[0023] 2) Cultivate the recombinant Escherichia coli described in step 1) to express the recombinant coronavirus antigen modified with fatty acid;

[0024] 3) The recombinant Escherichia coli was lysed, a surfactant was added, and the fatty acid-modified recombinant coronavirus antigen was extracted and purified from the lysate;

[0025] With SARS-CoV-2 RBD JN.1 Taking the mutant strain as an example, the fatty acid-modified RBD... Delta The nucleotide sequence is shown in SEQ ID No. 23.

[0026] Alternatively, a method for preparing a fatty acid-modified coronavirus antigen containing a linker is provided, the method comprising:

[0027] a) A DNA fragment containing a signal peptide coding sequence and a SpyCatcher protein coding sequence was introduced into *E. coli*, and recombinant *E. coli* was cultured to express recombinant SpyCatcher protein with N-terminal fatty acid modification. The recombinant *E. coli* was lysed, a surfactant was added, and the fatty acid modified recombinant SpyCatcher protein was extracted and purified from the lysate. The nucleotide sequence of the fatty acid modified recombinant SpyCatcher protein is shown in SEQ ID No. 20.

[0028] b) A DNA fragment containing a signal peptide coding sequence, a SpyTag polypeptide coding sequence, and a coronavirus antigen coding sequence is introduced into engineered cells to culture a recombinant cell line that expresses the coronavirus antigen containing the SpyTag polypeptide and purifies the coronavirus antigen containing the SpyTag polypeptide; the cell line can be mammalian cells, insect cells, or yeast cells. (The text then abruptly shifts to a seemingly unrelated topic: SARS-CoV-2 RBD...) Delta For example, the amino acid sequence of the coronavirus antigen containing the SpyTag polypeptide is shown in SEQ ID No. 26.

[0029] 3) The purified fatty acid-modified recombinant SpyCatcher protein was mixed with coronavirus antigen containing SpyTag peptide and reacted fully under certain conditions to obtain fatty acid-modified coronavirus antigen.

[0030] In the preparation method described above, the amino acid sequence of the signal peptide is any of the sequences in SEQ ID No. 17-19.

[0031] According to a third aspect of the technical solution of the present invention, the application of the fatty acid-modified coronavirus antigen as described above is provided, wherein the application is for the preparation of vaccines or for screening drugs for the treatment of coronaviruses.

[0032] According to a fourth aspect of the present invention, a vaccine containing the aforementioned fatty acid-modified coronavirus antigen is provided. The vaccine is a respiratory mucosal immunization vaccine or an injectable vaccine.

[0033] According to a fifth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising the above-described fatty acid-modified coronavirus antigen.

[0034] According to a sixth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising a nucleic acid encoding the above-mentioned fatty acid-modified coronavirus antigen.

[0035] According to a seventh aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising a carrier encoding the above-mentioned fatty acid-modified coronavirus antigen.

[0036] According to an eighth aspect of the present invention, a biomaterial is provided, wherein the biomaterial is any one of the following:

[0037] C1) Encodes a nucleic acid molecule of the fatty acid-modified coronavirus antigen as described in any one of claims 1-14;

[0038] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0039] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0040] C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3);

[0041] C5) Recombinant cells containing the nucleic acid molecule described in C1), or recombinant cells containing the expression cassette described in C2), or recombinant cells containing the recombinant vector described in C3).

[0042] According to the ninth aspect of the technical solution of the present invention, a method for preparing a vaccine using the above-mentioned fatty acid modification of coronavirus antigen is provided.

[0043] According to the tenth aspect of the technical solution of the present invention, a method for preparing a vaccine using the above-mentioned biological materials is provided.

[0044] Compared with the prior art, the present invention has at least the following beneficial effects:

[0045] 1. The fatty acid-modified coronavirus RBD antigen disclosed in this invention can induce high titers of IgG binding antibodies and neutralizing antibodies in animals after a single intramuscular injection. In animals immunized via the respiratory tract, high titers of RBD-specific IgG, IgA binding antibodies, and neutralizing antibodies can be detected in bronchoalveolar lavage fluid (BALF) after three immunizations. Compared with existing coronavirus protein antigens, this antigen not only provides faster and stronger immune protection induced by conventional intramuscular injection, but also induces respiratory mucosal immunity, providing a basis for blocking coronavirus infection and spread at the infection site and forming comprehensive immune protection. Furthermore, respiratory mucosal immunization, as a non-invasive method, can improve patient compliance and is suitable for large-scale immunization during pandemics.

[0046] 2. The fatty acid-modified coronavirus antigen of the present invention is a fatty acid-modified coronavirus RBD antigen, including two structures: directly fatty acid-modified RBD and RBD displayed on a fatty acid-modified SpyCatcher protein via a SpyCatcher / SpyTag linker. The fatty acid-modified coronavirus RBD antigen provided by the present invention can be polymerized into nanoparticles, inducing faster and higher RBD-specific antibody responses. In particular, when used as a main component of inhaled vaccines, it can induce specific IgG, IgA, and sIgA antibodies, significantly improving mucosal immune efficacy and demonstrating extremely high practical application potential. Attached Figure Description

[0047] Figure 1 shows the plasmid map of the pET30-MLSARS2RBD expression vector;

[0048] Figure 2 shows the SDS-PAGE and Western blot expression verification of lipid-modified MLSARS2RBD protein; Samples 1-6: Single clones 1-6;

[0049] Figure 3 shows the purification of lipotropic MLSARS2RBD protein using Chelating affinity chromatography; A: Chromatogram of MLSARS2RBD protein purified by Chelating affinity chromatography; B: SDS-PAGE of MLSARS2RBD protein purification, lanes 1: 1% Triton dissolves sample, 2: Dissolved sample is loaded, 3: Passes through lane 1, 4: Passes through lane 2, 5: 2.5% B elutes sample, 6: 15% B elutes sample, 7: 50% B elutes sample, 8: 100% B elutes sample, 9: 0.5M NaOH elutes sample;

[0050] Figure 4 shows the Superdex 200 size exclusion chromatography purification of the lipid-modified MLSARS2RBD protein; lanes 1-8: eluted samples at different time points;

[0051] Figure 5 shows the MALDI-TOF detection spectrum of lipid-modified MLSARS2RBD protein;

[0052] Figure 6 shows the plasmid map of the pET30-MLSARSRBD expression vector;

[0053] Figure 7 shows the SDS-PAGE and Western blot expression verification of MLSARSRBD protein; A: 1-4 are SDS-PAGE of C43DE3 / pET30-MLSARSRBD monoclonal bacterial cultures 1-4, B: 1-4 are Western blot of C43DE3 / pET30-MLSARSRBD monoclonal bacterial cultures 1-4.

[0054] Figure 8 shows the purification of lipid-modified MLSARSRBD protein by Chelating affinity chromatography; 1: C43DE3 / pET30-MLSARSRBD bacterial culture, 2: ultrasonic sample of bacterial culture, 3: ultrasonic precipitation of bacterial culture, 4: ultrasonic supernatant of bacterial culture, 5: supernatant of PEG20000 precipitation, 6: sample dissolved in 1% Triton, 7: eluted sample 1, 8: eluted sample 2.

[0055] Figure 9 shows the plasmid map of the pET30-MLMERSRBD expression vector;

[0056] Figure 10 shows the SDS-PAGE and Western blot validation of MLMERSRBD protein expression; A: 1-4 are SDS-PAGE of C43DE3 / pET30-MLSARSRBD monoclonal bacterial culture 1-4, 5-7 are SDS-PAGE of C43DE3 / pET30-MLMERSRBD monoclonal bacterial culture 5-7; B: 1-4 are Western blot of C43DE3 / pET30-MLSARSRBD monoclonal bacterial culture 1-4, 5-7 are Western blot of C43DE3 / pET30-MLMERSRBD monoclonal bacterial culture 5-7.

[0057] Figure 11 shows the purification of lipidized MLMERSRBD protein by Chelating affinity chromatography column; 1: C43DE3 / pET30-MLMERSRBD bacterial culture, 2: ultrasonic sample of bacterial culture, 3: ultrasonic precipitation of bacterial culture, 4: ultrasonic supernatant of bacterial culture, 5: supernatant of PEG20000 precipitation, 6: sample dissolved in 1% Triton, 7: eluted sample 1, 8: eluted sample 2.

[0058] Figure 12 shows the SDS-PAGE identification of recombinant SpyCatcher protein expression containing different signal peptides;

[0059] Figure 13 shows the SDS-PAGE identification of the esterified SpyCatcher (SC) Ni affinity chromatography purification.

[0060] Figure 14 shows the SDS-PAGE identification diagram of the lipid-modified SpyCatcher (SC) anion exchange chromatography purification.

[0061] Figure 15 shows the SDS-PAGE identification of the size exclusion chromatography purified esterified SpyCatcher (SC) molecules.

[0062] Figure 16 is a size exclusion chromatography diagram of SpyCatcher (SC) with ML esterification signal peptide;

[0063] Figure 17 shows the size exclusion chromatography of SpyCatcher (SC) molecules with rAg esterification signal peptides.

[0064] Figure 18 is a size exclusion chromatography diagram of SpyCatcher (SC) with P4 esterified signal peptide.

[0065] Figure 19 shows the size exclusion chromatography diagram of human IgG molecules;

[0066] Figure 20 shows the deconvolution plot of the lipolysis SpyCatcher (SC) mass spectrometry detection;

[0067] Figure 21 shows the fusion protein (RBD) of the SARS-CoV-2 Delta mutant strain RBD and SpyTag (RBD). Delta ST) SDS-PAGE identification image of purified sample;

[0068] Figure 22 shows LipoSC-RBD Delta ST molecular size exclusion chromatography;

[0069] Figure 23 shows LipoSC-RBD Delta ST molecular size exclusion chromatography purification SDS-PAGE identification image;

[0070] Figure 24 shows LipoSC-RBD Delta ST transmission electron microscopy (TEM) identification image;

[0071] Figure 25 shows LipoSC-RBD Delta ST dynamic light scattering (DLS) identification diagram;

[0072] Figure 26 shows the serum RBD levels of BALB / c mice before lung delivery immunization, 2 weeks after immunization, 4 weeks after immunization, and 6 weeks after immunization. Delta Specific IgG titer;

[0073] Figure 27 shows the serum RBD levels of BALB / c mice before lung delivery immunization, 2 weeks after immunization, 4 weeks after immunization, and 6 weeks after immunization. Delta Specific IgA titer;

[0074] Figure 28 shows the bronchoalveolar lavage fluid (BALF) RBD of BALB / c mice 2 weeks after lung delivery for triple immunization. Delta Specific IgG titer;

[0075] Figure 29 shows the bronchoalveolar lavage fluid (BALF) RBD of BALB / c mice 2 weeks after lung delivery for triple immunization. Delta Specific IgA titer;

[0076] Figure 30 shows the serum SARS-CoV-2 Delta pseudovirus neutralizing antibody titers of BALB / c mice before lung delivery immunization, 2 weeks after immunization, 4 weeks after immunization, and 6 weeks after immunization.

[0077] Figure 31 shows the titer of SARS-CoV-2 Delta pseudovirus neutralizing antibody in bronchoalveolar lavage fluid (BALF) of BALB / c mice 2 weeks after lung delivery for triple immunization;

[0078] Figure 32 shows serum RBD after intramuscular injection of immunization. Delta Combined with antibody titer;

[0079] Figure 33 shows the serum SARS-CoV-2 Delta pseudovirus neutralizing antibody titer after intramuscular injection immunization; Detailed Implementation

[0080] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0081] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0082] pET30a was purchased from Novagen.

[0083] Escherichia coli BL21DE3 was purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number CB105.

[0084] Escherichia coli C43DEDE3 was purchased from Sigma, catalog number CMC0019.

[0085] Pichia pastoris is deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 19488.

[0086] Anti-His tag mouse monoclonal antibody was purchased from Sigma, catalog number A7058.

[0087] The chromatographic medium for Sepharose FF is from Cytiva, catalog number 17057502.

[0088] Sephadex G25 fine chromatography packing material was purchased from Cytiva, product catalog number 17003202.

[0089] SOURCE30Q chromatography packing material was purchased from Cytiva, product catalog number 17127503.

[0090] Capto MMC chromatography packing material was purchased from Cytiva, catalog number 17531710.

[0091] The Phenyl FF low sub chromatography packing material was purchased from Cytiva, catalog number 28926988.

[0092] The SOURCE 30S chromatography packing material was purchased from Cytiva, catalog number 17127302.

[0093] Superdex TM The 75 Increase10 / 300 GL pre-loaded column was purchased from Cytiva, catalog number 29148721.

[0094] Superdex TM The 200 Increase 10 / 300 GL pre-loaded column was purchased from Cytiva, catalog number 28990944.

[0095] Balb / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0096] SEQ ID No.1: SARS-CoV-2 S-RBD WT amino acid sequence

[0097] SEQ ID No.2: SARS-CoV-2 S-RBD Alpha amino acid sequence

[0098] SEQ ID No.3: SARS-CoV-2 S-RBD Beta amino acid sequence

[0099] SEQ ID No.4: SARS-CoV-2 S-RBD Kappa amino acid sequence

[0100] SEQ ID No.5: SARS-CoV-2 S-RBD Delta (R319-V534) amino acid sequence

[0101] SEQ ID No.6: SARS-CoV-2 S-RBD Omicron amino acid sequence

[0102] SEQ ID No.7: SARS-CoV-2 S-RBD XBB1.5 amino acid sequence

[0103] SEQ ID No.8: SARS-CoV-2 S-RBD JN1 amino acid sequence

[0104] SEQ ID No. 9: SARS-CoV S-RBD amino acid sequence

[0105] SEQ ID No. 10: MERS-CoV S-RBD amino acid sequence

[0106] SEQ ID No. 11: Amino acid sequence of SpyCatcher

[0107] SEQ ID No. 12: Amino acid sequence of SpyCatcher002

[0108] SEQ ID No. 13: Amino acid sequence of SpyCatcher003

[0109] SEQ ID No. 14: Amino acid sequence of SpyTag

[0110] SEQ ID No. 15: Amino acid sequence of SpyTag002

[0111] Amino acid sequence of SEQ ID No. 16: SpyTag003

[0112] SEQ ID No. 17: ML fatty acid-modified signal peptide amino acid sequence

[0113] SEQ ID No. 18: amino acid sequence of rAg signal peptide

[0114] SEQ ID No. 19: P4 signal peptide amino acid sequence

[0115] SEQ ID No. 20: ML-SC nucleotide sequence (NdeⅠ, NotⅠ)

[0116] SEQ ID No. 21: rAg-SC nucleotide sequence (NdeⅠ, NotⅠ)

[0117] SEQ ID No. 22: P4-SC nucleotide sequence (NdeⅠ, NotⅠ)

[0118] SEQ ID No. 23: MLSARS2RBD nucleotide sequence

[0119] SEQ ID No. 24: MLSARSRBD nucleotide sequence

[0120] SEQ ID No. 25: MLMERSRBD nucleotide sequence

[0121] SEQ ID No.26: SARS-CoV-2 S-RBD Delta -ST nucleotide sequence

[0122] Example 1: Preparation of fatty acid-modified SARS-CoV-2 RBD

[0123] (1.1) Construction of fatty acid-modified SARS-CoV-2 RBD expression vector

[0124] Based on the E. coli Murein-lipoprotein signal peptide sequence (EU900370.1) and the sequence of the SARS-CoV-2 S protein JN1 mutant strain published in GenBank (supplementary GenBank number), the signal peptide was fused to the N-terminus of RBDJN1 to form the MLSARS2RBD sequence. This sequence, SEQ ID No. 23, was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequence is as follows: nucleotides 1-6 from the 5' end are NdeⅠ restriction site sequences; nucleotides 4-66 are the signal peptide coding sequence; nucleotides 67-84 are the adapter coding sequence; nucleotides 85-729 are the RBDJN.1 coding sequence; nucleotides 730-744 are the adapter coding sequence; and nucleotides 745-750 are the XhoⅠ restriction site sequences. SEQ ID No. 23 was cloned into the pET30a vector between the NdeⅠ and XhoⅠ restriction sites to construct the pET30-MLSARS2RBD expression vector, as shown in Figure 1.

[0125] (1.2) Construction of fatty acid-modified SARS-CoV-2 RBD expression strain

[0126] The constructed expression vector pET30-MLSARS2RBD was introduced into Escherichia coli C43DE3 (purchased from Sigma, catalog number CMC0019) host cells and plated on LB solid medium containing a final concentration of 50 μg / mL kanamycin. The positive clone was the expression strain C43DE3 / pET30-MLSARS2RBD.

[0127] (1.3) Expression and purification of fatty acid-modified SARS-CoV-2 RBD

[0128] A single clone of recombinant strain C43DE3 / pET30-MLSARS2RBD was picked and inoculated into 3 mL of LB medium containing a final concentration of 50 μg / mL kanamycin. The medium was cultured at 37°C until the OD600 was approximately 0.6. Then, IPTG was added to a final concentration of 0.5 mM, and the temperature was lowered to 25°C for induction for 6 h. Take 1 mL of the bacterial culture induced at 25℃ for 6 h, centrifuge to obtain bacterial cells, suspend the bacterial cells in distilled water at a ratio of 1:30 (W / V), sonicate to disrupt the bacterial cells, centrifuge at 12000 rpm for 5 min, aspirate the supernatant with a pipette, resuspend the precipitate in the original volume, prepare samples with 5X reducing buffer (250 mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500 mM DTT), boil in water for 10 min, perform 12% SDS-PAGE electrophoresis, transfer to PVDF membrane after electrophoresis, maintain constant voltage at 20 V for 1 h, and detect with mouse-derived Anti-His tag monoclonal antibody (Sigma, A7058) (as shown in Figure 2).

[0129] Figure 2 shows the SDS-PAGE electrophoresis results of the supernatant and precipitate after the six selected monoclonal clones were induced to express MLSARS2RBD. The right figure shows the corresponding Western blot results. The results show that all six monoclonal clones successfully expressed the MLSARS2RBD protein, and the content of the target protein in the supernatant was higher.

[0130] After SDS-PAGE and WB identification of expression, the culture volume was expanded and the recombinant fatty acid-modified SARS-CoV-2 RBD was purified.

[0131] The culture volume was increased to 1.6 L. The C43DE3 / pET30-MLSARS2RBD bacterial cell precipitate was collected by centrifugation at 8000 rpm for 20 min, resuspended in 100 mL of water, sonicated, and centrifuged at 8000 rpm for 10 min to collect the supernatant. 1% PEG 20000, 0.9% NaCl, and 50 mM PB 7.0 were added, and the mixture was stirred at room temperature for 2 h. The precipitate was collected by centrifugation at 8000 rpm for 10 min, resuspended in 100 mL of water, and resuspended in 1% Triton. The mixture was stirred at room temperature for 2 h, and then adjusted to pH 7.5 with 20 mM Tris-HCl, 0.25 M NaCl, and 5 mM imidazole. The mixture was centrifuged at 8000 rpm for 10 min to collect the supernatant. The sample was purified using a Chelating affinity chromatography column (Φ1.6 cm * 15 cm).

[0132] Prepare buffer A (20 mM Tris HCl 7.5 + 0.5 M NaCl + 5 mM imidazole + 1% Triton) and buffer B (20 mM Tris HCl 7.5 + 0.5 M NaCl + 1 M imidazole + 1% Triton). First, wash the column bed with 0.5 M NaOH aqueous solution for 3 column volumes, then equilibrate to pH neutral with deionized water, then equilibrate with 0.2 M NiSO4 aqueous solution for 3 column volumes, then equilibrate with buffer B for 1 column volume, and finally equilibrate with buffer A for 3 column volumes. Load the solution containing recombinant MLSARS2RBD protein onto a Chelating affinity chromatography column, wash away unbound protein with buffer A, equilibrate for 2 column volumes, then elute with 2.5%, 15%, 50%, and 100% buffer B; collect the sample eluted with 15% buffer B to obtain the preliminarily purified sample, as shown in Figure 3.

[0133] Figure 3 (left) shows the chromatogram of MLSARS2RBD protein purified by Chelating affinity chromatography column; the results show that a UV absorption peak appeared when eluted with 15% B, indicating that the target protein was eluted with 15% B (150 mM imidazole).

[0134] Figure 3 (right) shows the SDS-PAGE of MLSARS2RBD protein purification. Lane 6 is for protein elution with 15% B. According to the previous Western blot results, the target protein band is located at 25 kDa.

[0135] A: Chromatogram of MLSARS2RBD protein purified by Chelating affinity chromatography column; B: SDS-PAGE of MLSARS2RBD protein purification, lanes 1: 1% Triton dissolves sample, lane 2: Dissolves sample and loads, lane 3: Passes through lane 1, lane 4: Passes through lane 2, lane 5: 2.5% B elutes sample, lane 6: 15% B elutes sample, lane 7: 50% B elutes sample, lane 8: 100% B elutes sample, lane 9: 0.5M NaOH elutes sample.

[0136] The sample was purified using Superdex 200. The Superdex 200 column (φ1×30cm, Cytiva, 28990944) was equilibrated with SEC buffer (5mM pH7.4 PB + 0.9% NaCl + 0.5% Tween). 1 mL of the 15% PB purified sample was loaded for purification. SDS-PAGE showed that high-purity MLSARS2RBD protein was obtained, as shown in Figure 4.

[0137] (1.4) Mass spectrometry characterization and detection

[0138] To further confirm whether the N-terminus of the purified recombinant MLSARS2RBD protein was modified with fatty acids, MLSARS2RBD was used as the research object. The relative molecular mass was determined by Shanghai Zhongke New Life Biotechnology Co., Ltd. using the MALDI-TOF method, and the measured relative molecular mass of the sample was 27089.86 Da. Given that the theoretical molecular weight of unmodified SARS2RBD is 26187.48 Da and the molecular weight of Pam3Cys is 910.46 Da, it was deduced that the recombinant MLSARS2RBD with a molecular weight of 27089.86 Da contains an additional molecule of Pam3Cys (26187.48 + 910.46 - 18) (error 9.92 Da). The recombinant MLSARS2RBD protein prepared by this method was named LipoSARS2RBD. Figure 5 shows the MALDI-TOF detection spectrum of the lipid-modified MLSARS2RBD protein.

[0139] Example 2: Preparation of fatty acid-modified SARS-CoV RBD

[0140] (2.1) Construction of fatty acid-modified SARS-CoV RBD expression vector

[0141] The signal peptide sequence (EU900370.1) was fused to the N-terminus of the SARS RBD to form the MLSARSRBD sequence. This sequence, SEQ ID No. 24, was synthesized by Sangon Biotech (Shanghai) Co., Ltd. In this sequence, nucleotides 1-6 from the 5' end are NdeI restriction site sequences, nucleotides 4-66 are signal peptide coding sequences, nucleotides 67-84 are adapter coding sequences, nucleotides 85-708 are SARSRBD coding sequences, nucleotides 709-723 are adapter coding sequences, and nucleotides 724-729 are XhoI restriction site sequences. SEQ ID No. 24 was cloned into the pET30a vector between the NdeI and XhoI restriction sites to construct the pET30-MLSARSRBD expression vector, as shown in Figure 6.

[0142] (2.2) Construction of fatty acid-modified SARS-CoV RBD expression strain

[0143] The constructed expression vector PET30-MLSARSRBD was introduced into Escherichia coli C43DE3 (purchased from Sigma, catalog number CMC0019) host cells and plated on LB solid medium containing a final concentration of 50 μg / mL kanamycin. The positive clone was the expression strain C43DE3 / pET30-MLSARSRBD.

[0144] (2.3) Expression and purification of fatty acid-modified SARS-CoV RBD

[0145] Recombinant bacterial strain C43DE3 / pET30-MLSARSRBD was picked and inoculated into 3 mL of LB medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C until the OD600 reached approximately 0.6. IPTG was then added to a final concentration of 0.5 mM, and the culture was cooled to 25°C for 6 h of induction. 1 mL of the incubated bacterial culture was centrifuged, and the cells were resuspended in distilled water at a ratio of 1:30 (w / v). The cells were prepared with 5X reducing buffer (250 mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500 mM DTT), boiled for 10 min, and subjected to 12% SDS-PAGE electrophoresis. After electrophoresis, the cells were transferred to a PVDF membrane and transferred at a constant voltage of 20 V for 1 h. The results were detected using a mouse-derived anti-His tag monoclonal antibody (Sigma, A7058), as shown in Figure 7.

[0146] In Figure 7, the left image (1-4) shows the SDS-PAGE of the C43DE3 / pET30-MLSARSRBD monoclonal bacterial suspension (1-4), and the right image (1-4) shows the Western blot of the C43DE3 / pET30-MLSARSRBD monoclonal bacterial suspension (1-4).

[0147] After SDS-PAGE and WB analysis to identify the expression, the culture volume was expanded to purify the recombinant fatty acid-modified SARS-CoV RBD.

[0148] The culture volume was increased to 1.6 L. The C43DE3 / pET30-MLSARSRBD bacterial pellet was collected by centrifugation at 8000 rpm for 20 min. The pellet was resuspended in 100 mL of water, sonicated, and centrifuged at 8000 rpm for 10 min to collect the supernatant. 1% Triton was added, and the mixture was stirred at room temperature for 2 h. Then, 20 mM Tris-HCl (pH 7.5), 0.25 M NaCl, and 5 mM imidazole were added, and the mixture was centrifuged at 8000 rpm for 10 min to collect the supernatant. The sample was purified using a Chelating affinity chromatography column (Φ1.6 cm * 15 cm).

[0149] Prepare buffer A (20 mM Tris HCl 7.5 + 0.5 M NaCl + 5 mM imidazole + 1% Triton) and buffer B (20 mM Tris HCl 7.5 + 0.5 M NaCl + 1 M imidazole + 1% Triton). First, wash the column bed with 0.5 M NaOH aqueous solution for 3 column volumes, then equilibrate to pH neutral with deionized water, then equilibrate with 0.2 M NiSO4 aqueous solution for 3 column volumes, then equilibrate with buffer B for 1 column volume, and finally equilibrate with buffer A for 3 column volumes. Load the sample containing recombinant MLSARSRBD onto a Chelating affinity chromatography column, wash away unbound protein with buffer A, equilibrate for 2 column volumes, and then elute with 100% buffer B to obtain the preliminarily purified sample. Figure 8 shows the purification of lipid-modified MLSARSRBD protein using a Chelating affinity chromatography column, where 1: C43DE3 / pET30-MLSARSRBD bacterial culture, 2: ultrasonic sample of bacterial culture, 3: ultrasonic precipitation of bacterial culture, 4: ultrasonic supernatant of bacterial culture, 5: supernatant of PEG20000 precipitation, 6: sample dissolved in 1% Triton, 7: eluted sample 1, and 8: eluted sample 2.

[0150] Example 3: Preparation of fatty acid-modified MERS-CoV RBD

[0151] (3.1) Construction of fatty acid-modified MERS-CoV RBD expression vector

[0152] The signal peptide sequence (EU900370.1) was fused to the N-terminus of the MERS RBD to form the MLSARSRBD sequence. This sequence, SEQ ID No. 25, was synthesized by Sangon Biotech (Shanghai) Co., Ltd. In this sequence, nucleotides 1-6 from the 5' end are NdeI restriction site sequences, nucleotides 4-66 are the signal peptide coding sequence, nucleotides 67-84 are the adapter coding sequence, nucleotides 85-747 are the SARSRBD coding sequence, nucleotides 748-762 are the adapter coding sequence, and nucleotides 763-768 are the XhoI restriction site sequences. SEQ ID No. 25 was cloned into the pET30a vector between the NdeI and XhoI restriction sites to construct the pET30-MLMERSRBD expression vector, as shown in Figure 9.

[0153] (3.2) Construction of fatty acid-modified MERS-CoV RBD expression strain

[0154] The constructed expression vector PET30-MLMERSRBD was introduced into Escherichia coli C43DE3 (purchased from Sigma, catalog number CMC0019) host cells and plated on LB solid medium containing a final concentration of 50 μg / mL kanamycin. The positive clone was the expression strain C43DE3 / pET30-MLMERSRBD.

[0155] (3.3) Expression and purification of fatty acid-modified MERS-CoV RBD

[0156] Recombinant bacterial strain C43DE3 / pET30-MLMERSRBD was picked and inoculated into 3 mL of LB medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C until the OD600 reached approximately 0.6. IPTG was then added to a final concentration of 0.5 mM, and the culture was cooled to 25°C for 6 h of induction. 1 mL of the incubated bacterial culture was centrifuged to obtain bacterial cells. The cells were resuspended in distilled water at a ratio of 1:30 (w / v). A 5X reducing buffer (250 mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500 mM DTT) was prepared, and the mixture was boiled for 10 min. Electrophoresis was performed using 12% SDS-PAGE. After electrophoresis, the sample was transferred to a PVDF membrane and transferred at a constant voltage of 20 V for 1 h. The sample was then detected using a mouse-derived anti-His tag monoclonal antibody (Sigma, A7058). Figure 10 shows the validation of MLMERSRBD protein expression. In the left image of Figure 10, 1-4 are SDS-PAGE of C43DE3 / pET30-MLSARSRBD monoclonal bacterial culture 1-4, and 5-7 are SDS-PAGE of C43DE3 / pET30-MLMERSRBD monoclonal bacterial culture 5-7. In the right image of Figure 10, 1-4 are Western blot of C43DE3 / pET30-MLSARSRBD monoclonal bacterial culture 1-4, and 5-7 are Western blot of C43DE3 / pET30-MLMERSRBD monoclonal bacterial culture 5-7.

[0157] After SDS-PAGE and WB analysis to identify the expression, the culture volume was expanded to purify the recombinant fatty acid-modified MERS-CoV RBD.

[0158] The culture volume was increased to 1.6 L. The C43DE3 / pET30-MLMERSRBD bacterial pellet was collected by centrifugation at 8000 rpm for 20 min. The pellet was resuspended in 100 mL of water, sonicated, and centrifuged at 8000 rpm for 10 min to collect the supernatant. 1% Triton was added, and the mixture was stirred at room temperature for 2 h. Then, 20 mM Tris-HCl (pH 7.5), 0.25 M NaCl, and 5 mM imidazole were added, and the mixture was centrifuged at 8000 rpm for 10 min to collect the supernatant. The sample was purified using a Chelating affinity chromatography column (Φ1.6 cm * 15 cm).

[0159] Prepare buffer A (20mM Tris HCl 7.5 + 0.5M NaCl + 5mM imidazole + 1% Triton) and buffer B (20mM Tris HCl 7.5 + 0.5M NaCl + 1M imidazole + 1% Triton). First, wash the column bed with 0.5M NaOH aqueous solution for 3 column volumes, then equilibrate to pH neutral with deionized water, then equilibrate with 0.2M NiSO4 aqueous solution for 3 column volumes, then equilibrate with buffer B for 1 column volume, and finally equilibrate with buffer A for 3 column volumes. The sample containing recombinant MLMERSRBD was loaded onto a Chelating affinity chromatography column. Unbound protein was washed away with buffer A, and the column volume was equilibrated to 2 column volumes. Then, the sample was eluted with 100% buffer B to obtain a preliminarily purified sample. As shown in Figure 11, the lipidized MLMERSRBD protein was purified by Chelating affinity chromatography. In the figure, 1: C43DE3 / pET30-MLMERSRBD bacterial culture, 2: bacterial culture sonicated sample, 3: bacterial culture sonicated precipitation, 4: bacterial culture sonicated supernatant, 5: PEG20000 precipitation supernatant, 6: 1% Triton dissolved sample, 7: eluted sample 1, and 8: eluted sample 2.

[0160] Example 4: Preparation of recombinant SpyCatcher protein

[0161] (4.1) Construction of the fatty acid-modified SpyCatcher protein expression vector

[0162] Based on the Escherichia coli Murein-lipoprotein signal peptide sequence (EU900370.1) (SEQ ID No. 17), the Neisseria meningitidis Ag473 signal peptide sequence (AY566590.1) (SEQ ID No. 18), and the Haemophilus influenzae P4 signal peptide sequence (M68502.1) (SEQ ID No. 19) published in GenBank, these sequences were fused to the N-terminus of the SpyCatcher protein (amino acid sequences are shown in SEQ ID No. 11). A His tag was added to facilitate purification, and these sequences were named MLSC, rAgSC, and P4SC, respectively. Sangon Biotech (Shanghai) Co., Ltd. was commissioned to optimize and synthesize the DNA sequences based on the codons preferred by Escherichia coli.

[0163] The optimized MLSC nucleotide sequence is shown in SEQ ID No. 20, wherein nucleotides 1-6 from the 5' end are NdeI restriction site sequences, nucleotides 4-66 are Murein-lipoprotein signal peptide coding sequences, nucleotides 67-102 are adapter and His tag coding sequences, nucleotides 103-453 are SpyCatcher (SC) coding sequences, and nucleotides 454-461 are NotI restriction site sequences;

[0164] The optimized rAgSC nucleotide sequence is shown in SEQ ID No. 21, wherein nucleotides 1-6 from the 5' end are the NdeⅠ restriction site sequence, nucleotides 4-54 are the Ag473 signal peptide coding sequence, nucleotides 55-90 are the linker and His tag coding sequence, nucleotides 91-441 are the SpyCatcher (SC) coding sequence, and nucleotides 442-449 are the NotⅠ restriction site sequence;

[0165] The optimized P4SC nucleotide sequence is shown in SEQ ID No. 22, wherein nucleotides 1-6 from the 5' end are the NdeⅠ restriction site sequence, nucleotides 4-66 are the P4 signal peptide coding sequence, nucleotides 67-102 are the linker and His tag coding sequence, nucleotides 103-453 are the SpyCatcher (SC) coding sequence, and nucleotides 454-461 are the NotⅠ restriction site sequence;

[0166] The optimized sequences were cloned into the NdeⅠ and NotⅠ restriction sites of the pET30a vector to construct the pET30-MLSC, pET30-rAgSC, and pET30-P4SC expression vectors.

[0167] (4.2) Construction of a strain expressing SpyCatcher protein modified with fatty acid esterification

[0168] The constructed expression vectors pET30-MLSC, pET30-rAgSC, and pET30-P4SC were introduced into Escherichia coli C43(DE3) (purchased from Sigma, catalog number CMC0019) or BL21(DE3) (purchased from Tiangen Biotech, catalog number CB105; Thermo Fisher Scientific, catalog number EC0114) host cells, preferably C43(DE3) host cells. The cells were plated on LB solid medium containing a final concentration of 50 μg / mL kanamycin. Positive clones, namely the expression strains C43(DE3) / pET30-MLSC, C43(DE3) / pET30-rAgSC, and C43(DE3) / pET30-P4SC, were obtained.

[0169] (4.3) Expression and purification of recombinant SpyCatcher protein

[0170] Single clones of recombinant bacteria C43(DE3) / pET30-MLSC, C43(DE3) / pET30-rAgSC, and C43(DE3) / pET30-P4SC were inoculated into LB medium containing kanamycin at a final concentration of 50 μg / mL and cultured at 37°C until the OD600 was approximately 0.6. Then, IPTG at a final concentration of 0.5 mM was added, and the temperature was lowered to 25°C for induction for 20 h.

[0171] The following day, 1 mL of each bacterial culture induced at 25℃ for 20 h was taken, centrifuged to obtain bacterial cells, and the cells were resuspended in distilled water at a ratio of 1:30 (W / V). The cells were sonicated and centrifuged to separate the precipitate and supernatant. The precipitate was resuspended in an equal volume of water and prepared with 5X reducing buffer (250 mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500 mM DTT). The sample was boiled in a water bath for 10 min and electrophoresed with 15% SDS-PAGE. After electrophoresis, the sample was transferred to a PVDF membrane and transferred at a constant voltage of 20 V for 1 h. The results were detected with a mouse-derived anti-His tag monoclonal antibody (Sigma, A7058). The results are shown in Figure 12.

[0172] After SDS-PAGE and WB analysis to identify the expression, the culture volume was expanded to purify the fatty acid-modified SpyCatcher protein, using C43(DE3) / pET30-MLSC as an example.

[0173] The sample was purified using a Chelating affinity chromatography column (Φ1.6cm*15cm). After harvesting the bacterial cells, the cells were suspended in Ni-A1 buffer (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole + 1% Triton X100) at a ratio of 1:20 (w / v), sonicated, and the supernatant was collected by centrifugation and purified by Chelating affinity chromatography.

[0174] First, wash the column bed with at least 3 column volumes of 0.5M NaOH aqueous solution. Then, equilibrate to pH neutral with deionized water. Next, equilibrate with at least 3 column volumes of 0.2M NiSO4 aqueous solution. Then, equilibrate with one column volume of Ni-B buffer (20mM pH 7.5 Tris-HCl, 0.3M NaCl, 500mM imidazole + 1% Triton X100). Finally, equilibrate with at least 3 column volumes of Ni-A1 buffer (20mM pH 7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole + 1% Triton X100). Samples containing recombinant SpyCatcher were loaded onto a Chelating affinity chromatography column and washed with Ni-A1 (20 mM pH 7.5 Tris-HCl + 0.3 M NaCl + 5 mM imidazole + 1% Triton X100) buffer to remove unbound proteins, equilibrating for at least 5 column volumes. Then, the sample was eluted with 10%, 30%, and 100% Ni-B buffer. The sample eluted with 30% Ni-B buffer was collected to obtain a preliminarily purified sample, as shown in Figure 13, which is an SDS-PAGE identification image of the esterified SpyCatcher (SC) purified by Ni affinity chromatography.

[0175] Desalting was performed using a Sephadex G-25 Fine chromatography column. First, the column bed was flushed with one column volume of 0.5M NaOH aqueous solution, and then equilibrated to pH neutral with deionized water. Next, one column volume was equilibrated with 30Q-A1 buffer (20mM pH7.5 Tris-HCl, +1% Triton X100). Finally, the sample was eluted with 30% B from the Chelating affinity chromatography column to remove salt, with the sample loading volume not exceeding 1 / 3 of the column volume.

[0176] The sample was purified using a SOURCE30Q anion exchange chromatography column (Φ1.6cm*15cm). First, wash the column bed with at least 3 column volumes of 0.5M NaOH aqueous solution, and then equilibrate to pH neutral with deionized water. Next, equilibrate with at least 3 column volumes of 30Q-A1 buffer (20mM pH 7.5 Tris-HCl, +1% Triton X100). Then, load the desalted sample containing recombinant SpyCatcher onto a SOURCE30Q anion exchange chromatography column, equilibrate with 10 column volumes of 30Q-A1 buffer, and then equilibrate with 10 column volumes of 30Q-A2 buffer (20mM pH 7.5 Tris-HCl, +0.1% Tween 80). Finally, elute linearly with 0-50% 30Q-B buffer (20mM pH 7.5 Tris-HCl + 1M NaCl + 0.1% Tween 80). Collect the eluted sample, as shown in Figure 14, which is an SDS-PAGE identification image of the purified esterified SpyCatcher (SC) via anion exchange chromatography.

[0177] The sample was purified using Superdex 200 increase. The Superdex 200 increase column (φ1×30cm, Cytiva, 28990944) was equilibrated with SEC (5mM pH7.4 PB + 0.9% NaCl) buffer. 1 mL of the purified sample from SOURCE30Q was loaded for further purification, with human IgG used as a molecular weight control. The gel chromatography showed that the retention volumes of MLSC, rAgSC, and P4SC were between 10.1 mL and 10.3 mL, while the retention volume of IgG was 13.1 mL. This indicates that the molecular weights of the three recombinant proteins MLSC, rAgSC, and P4SC are greater than 150 kDa, significantly larger than the 16 kDa of the SC monomer. Therefore, it was determined that the three recombinant proteins MLSC, rAgSC, and P4SC exist in multimeric form (see Figures 15, 16, 17, and 18). Figure 15 shows the SDS-PAGE identification of the purified lipotropic SpyCatcher (SC) molecules by size exclusion chromatography. Figure 16 shows the size exclusion chromatography of SpyCatcher (SC) molecules with the ML lipotropic signal peptide. Figure 17 shows the size exclusion chromatography of SpyCatcher (SC) molecules with the rAg lipotropic signal peptide. Figure 18 shows the size exclusion chromatography of SpyCatcher (SC) molecules with the P4 lipotropic signal peptide. Figure 19 shows the size exclusion chromatography of human IgG molecules, with a retention volume of 13.1 mL, which is larger than the retention volumes of MLSC, rAgSC, and P4SC. This indicates that the molecular weights of the three recombinant proteins MLSC, rAgSC, and P4SC are greater than 150 kDa, and that the three proteins exist in a multimeric form.

[0178] (4.4) Characterization of MLSC

[0179] To further confirm whether the N-terminus of the purified recombinant SpyCatcher protein was modified with fatty acids, MLSC was used as the research object. Shanghai Zhongke New Life Biotechnology Co., Ltd. was commissioned to perform N-terminal sequencing using the Edman degradation method. The N-terminal sequence could not be detected, indicating that the N-terminus of the recombinant protein was modified post-translationally.

[0180] The molecular weight of recombinant SpyCatcher was determined by Beijing Mingde Zhengkang Technology Co., Ltd. using mass spectrometry (LC / Q-TOF-MS) (as shown in Figure 20, deconvolution plot of esterified SpyCatcher (SC) mass spectrometry detection). The results showed that the molecular weight of recombinant SpyCatcher was concentrated in two clusters: the first cluster consisted of 14810.1 Da, 14824.2 Da, and 14838.0 Da, and the second cluster consisted of 14585.8 Da and 14600.0 Da. Given that the theoretical molecular weight of unmodified SpyCatcher is 13919.11 Da, and the molecular weight of Pam3Cys is 910.46 Da, it can be inferred that the recombinant SpyCatcher with a molecular weight of 14810.1 Da contains an additional molecule of Pam3Cys (13919.11+).

[0181] 910.46-18); According to the technical solution of the present invention, the recombinant SpyCatcher with a molecular weight of 14810.1 Da is presumably Pam3C-SpyCatcher, that is, SpyCatcher with the N-terminus modified by fatty acid modification, wherein the thiol group of the N-terminal Cys is modified by diacylglycerol (C16:0,C16:0), and the α-amino group is modified by palmitic acid (C16:0); the molecular weight of the recombinant SpyCatcher with a molecular weight of 14824.2 Da increases by about 14 Da, which is consistent with the molecular weight of CH2, and it is presumed that one fatty acid chain is extended by one CH2; the molecular weight of the recombinant SpyCatcher with a molecular weight of 14838 Da increases by about 28 Da, which is consistent with the molecular weight of (CH2)2, and it is presumed that the fatty acid chain is extended by two CH2. The molecular weights of the second cluster peaks, 14585.8 Da and 14600.0 Da, correspond to the molecular weights of the first cluster peaks, 14824.2 Da and 14838.0 Da, respectively. The molecular weights are reduced by 238.4 Da and 238 Da, which is consistent with the molecular weight of palmitoylated (C16:0).

[0182] Based on the above calculations and the technical solution of this invention, the N-terminus of the recombinant SpyCatcher prepared by this invention is modified with fatty acid esterification, specifically, the thiol group of Cys is modified with diacylglycerol, and / or the α-amino group of Cys is modified with palmitoylation (C16:0), wherein the fatty acid chain structure of the diacylglycerol is C16:0+C16:0, or C16:0+C17:0, or C16:0+C18:0, or C17:0+C17:0. The recombinant SpyCatcher protein prepared by this method is named LipoSC.

[0183] Example 5: Preparation of recombinant SARS-CoV-2 viral S protein RBD

[0184] This invention uses the SARS-CoV-2S protein delta mutant strain RBD as an example to elucidate the antigen containing the SpyTag polypeptide.

[0185] (5.1) Construction of the SARS-CoV-2 S protein RBD-SpyTag yeast expression vector

[0186] Based on the amino acid sequence from position 319 to 534 of the S protein of the SARS-CoV-2 Delta mutant strain (GenBank accession number OK091006.1), a SpyTag sequence (amino acid sequence 12) was added to its C-terminus. The DNA sequence was then optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. based on the Pichia pastoris' preferred codons. The nucleotide sequence is shown in SEQ ID No. 26 and named SARS-CoV-2RBD. Delta ST was inserted between the XhoI and NotI restriction sites of the pPICZαA vector to obtain the recombinant expression vector pPICZαA-RBD. Delta ST.

[0187] (5.2) Recombinant expression vector pPICZαA-RBD Delta ST-transformed Pichia pastoris strain CGMCC No. 19488;

[0188] Pichia pastoris strain CGMCC No. 19488 was streaked onto YPD plates for resuscitation, and single colonies were isolated. Resuscitated single colonies were picked and inoculated into YPD (1% yeast extract, 2% tryptone, 2% glucose) liquid medium. After incubation in test tubes until the logarithmic growth phase, 1 mL was transferred to a 100 mL YPD shake flask and cultured at 25°C and 200 rpm until the OD600 reached 1.3-1.5. The culture was then rapidly cooled on ice and centrifuged at 4°C, 1500 g × 5 min. The cells were resuspended in an equal volume of pre-chilled distilled water and centrifuged at 4°C, 1500 g × 5 min, and the supernatant was discarded. This step was repeated 3 times. The cells were then resuspended in an equal volume of pre-chilled 1M sorbitol and centrifuged at 4°C, 1500 g × 5 min, and the supernatant was discarded. This step was repeated 3 times. The bacterial precipitate, after being washed three times with distilled water and three times with sorbitol, was resuspended by adding 1 mL of 1M sorbitol. 100 μL of each precipitate was dispensed into sterile centrifuge tubes and stored at -80℃ to obtain Pichia pastoris strain CGMCC No.19488 electroporation-transformed competent cells.

[0189] The constructed expression plasmid pPICZαA-RBD Delta Approximately 10 μg of ST was linearized by restriction endonuclease BglII. The digestion system (50 μL) is as follows: expression plasmid pPICZαA-RBD Delta 43 μL of ST, 2 μL of BglII, and 5 μL of 10×NEB3.1 buffer were added. After digestion at 37℃ for 1 h, samples were collected and separated by 1% agarose gel electrophoresis to analyze whether the plasmid was completely linearized. The results showed that the completely linearized digestion products were recovered using a centrifugal column-type DNA fragment recovery kit. Finally, the linearized plasmid was eluted with 25 μL of pure water.

[0190] Take the linearized expression plasmid pPICZαA-RBD Delta Add 15 μL of ST solution to 100 μL of Pichia pastoris strain CGMCC No. 19488 for electroporation transformation of competent cells. After mixing, transfer to a pre-chilled 0.2 cm electroporation cuvette and electroporate at 2 kV. Immediately add 900 μL of pre-chilled 1 M sorbitol and transfer to a clean test tube. Incubate at 25°C for 2 hours. Then add 1 mL of antibiotic-free YPD liquid medium and incubate at 25°C and 200 rpm for 3-4 hours. Spread 300 μL of the bacterial culture obtained from the above shaking culture onto YPD plates selected for Zeocin resistance and incubate upside down at 25°C for 60-72 hours.

[0191] (5.3) Screening of recombinant expression strains

[0192] After single colonies have grown on the plate, pick one colony and streak it onto a new YPD plate containing 100 μg / mL Zeocin. Incubate at 25°C in an inverted incubator. Once colonies have grown, inoculate into 3 mL of YPD liquid medium containing 100 μg / mL Zeocin and incubate at 25°C with a shaker at 200 rpm for 72 h. Then, transfer to 3 mL of BMGY medium (1% yeast extract, 2% tryptone, 100 mM PB6.5, 100 mM YNB, 1% glycerol) at a 5% (v / v) inoculation rate and incubate at 25°C with a shaker at 200 rpm for 48 hours. After 48 hours of induction, add 0.5% (v / v) methanol every 12 hours for induction. After 48 h of induction, collect the culture supernatant at 12000 rpm for 3 min.

[0193] The culture supernatant collected after 48 hours of methanol induction was subjected to SDS-PAGE and Western Blot (WB) screening. The Western Blot steps were as follows: (1) Separate the sample with 12% SDS-PAGE gel; (2) Transfer the sample on the SDS-PAGE gel to a PVDF membrane; (3) Block the PVDF membrane with the target protein transferred with 5% milk blocking solution and block at room temperature for 1 hour; (4) Transfer to anti-His tag antibody (Sigma A7058) diluted with 5% milk at a dilution of 1:2500 and incubate for 1 hour; (5) Wash with PBST for 5 min and wash 5 times; (6) Wash with PBST for 5 min and wash 5 times; (7) Develop with Pro-light HRP Chemiluminescent chromogenic solution (Tiangen Biotech, PA112-02).

[0194] (5.4) Recombinant strain CGMCC No.19488 / pPICZαA-RBD Delta ST cultivation

[0195] Select the positive clones obtained from the identification (i.e., recombinant strain CGMCC No.19488 / pPICZαA-RBD) Delta ST) was inoculated into YPD liquid medium (containing 100 μg / mL Zeocin) and cultured at 25°C and 200 rpm until the OD600 reached 15-20. Then, it was transferred to BMGY medium at a 5% (v / v) inoculation rate and fermented at 25°C and 200 rpm for 24 hours. RBD was then induced with 0.5% (v / v) methanol. Delta ST expression was induced every 12 hours, and the culture supernatant was collected by centrifugation after 72 hours of induction.

[0196] (5.5)RBD Delta purification of ST

[0197] 1. Capto MMC chromatography purification

[0198] The culture supernatant, after 72 hours of induced expression, was adjusted to pH 5.5 and purified using Capto MMC (Cytiva, 17531710) chromatography medium. The mobile phase composition was as follows:

[0199] MMC-A: 20mM pH 5.5 PB (phosphate buffer);

[0200] MMC-B: 100mM pH8.5 Tris-HCl+1M NaCl.

[0201] After loading the sample, equilibrate with MMC-A and then elute with MMC-B.

[0202] 2. Hydrophobic chromatography purification of Phenyl FF low sub

[0203] The Capto MMC purified sample was purified using Phenyl FF low sub (Cytiva, 28926988). The mobile phase composition was as follows:

[0204] Phenyl FF-A: 20mM pH 7.5 Tris-HCl + 1M AS (ammonium sulfate);

[0205] Phenyl FF-B: 20mM p H7.5 Tris-HCl.

[0206] First, elute the target protein with 40% Phenyl FF-B, then elute other proteins with 40-100% Phenyl FF-B.

[0207] 3. Sephadex G-25 Fine desalination

[0208] The Phenyl FF low sub purified sample was desalted using Sephadex G-25 Fine (Cytiva, 17003201) chromatography medium, and the protein sample was collected. The mobile phase composition was 20 mM pH 8.5 Tris-HCl.

[0209] 4. SOURCE 30Q anion exchange chromatography purification

[0210] The desalted sample was purified using SOURCE30Q (Cytiva, 17127502) chromatography medium. The mobile phase composition was as follows:

[0211] 30Q-A: 20mM p H8.5 Tris-HCl;

[0212] 30Q-B: 20mM pH8.5 Tris-HCl+1M NaCl.

[0213] After loading, equilibrate with 30Q-A, then elute with 30Q-B, with the target protein in the flow-through buffer.

[0214] 5. SOURCE 30S cation exchange chromatography

[0215] The sample was eluted with SOURCE 30S, the pH was adjusted to 6.5 with HCl, and then purified using a SOURCE 30S (Cytiva, 17127302) column. The mobile phase composition was as follows:

[0216] 30S-A: 20mM pH 6.5 PB;

[0217] 30S-B: 20mM pH7.0 PB+1M NaCl.

[0218] After loading the sample, equilibrate with A, and then elute the target protein with 50% B.

[0219] 6. Superdex™ 75 Increase 10 / 300 GL Gel Filtration Chromatography

[0220] The sample purified by SOURCE 30S was processed using Superdex™ 75 Increase 10 / 300 GL (Cytiva, 29148721) with a mobile phase composition of SEC: 5mM PB7.4 + 0.9% NaCl. After loading the sample, the mobile phase was equilibrated, and the sample was collected in fractions.

[0221] After the above purification steps, the RBDDeltaST sample was obtained. The SDS-PAGE identification of the purification process is shown in Figure 21. Figure 21 is the SDS-PAGE identification of the purified sample of SARS-CoV-2 Delta mutant strain RBD and SpyTag fusion protein (RBDDeltaST).

[0222] Example 6: Preparation of fatty acid-modified RBD nanoparticles

[0223] The LipoSC nanoparticles prepared above and SARS-CoV-2 RBD were mixed using the BCA method (Thermo Scientific, Cat. No. A55864, Related BCA Kits: 23225 (1000 mL)). DeltaProtein quantification was performed using ST, with the mixture at a molar ratio of 2:1, and ligation was carried out at 4°C for 12 hours. The Superdex 200 Increase column (φ1×30cm, Cytiva, 28990944) was equilibrated with SEC200 (5mM pH7.0 PB + 0.9% NaCl) buffer. 1 mL of the ligation product was then used to remove unligated RBD using Superdex 200 Increase. Delta ST, LipoSC-RBD Delta The retention volume of ST was 9.42 mL, slightly smaller than that of LipoSC (see Figure 22); SDS-PAGE reduction electrophoresis showed (see Figure 23) that RBDST was linked to LipoSC, with a molecular weight of approximately 43 kDa; TEM analysis showed that MLSC-RBDST was composed of nanoparticles (see Figure 24); DLS analysis showed (see Figure 25) that LipoSC-RBDST... Delta The ST diameter is approximately 13–20 nm (Number (Percent)). The purified LipoSC-RBD was obtained using the BCA method. Delta ST was used for protein quantification.

[0224] Example 7: Immunogenicity Study of LipoSC-RBDST

[0225] (7.1) Lung delivery immunization regimen

[0226] The LipoSC-RBD obtained in Example 6 Delta ST prepared the vaccine using physiological saline according to the concentration, so that 50 μL volume contained 10 μg of LipoSC-RBD. Delta ST, using SC-RBD Delta ST and saline were used as controls. Female BALB / c mice aged 6–8 weeks were immunized via lung delivery of 50 μL of vaccine on days 0, 14, and 28 (n=5). Blood was collected from the orbital sinus venosus of immunized mice before immunization and two weeks after the first, second, and third immunizations. See Figure 26.

[0227] (7.2) Detection of RBD-specific binding antibodies after lung delivery immunization

[0228] The serum RBD level in each group of mice was measured using an indirect ELISA method. Delta Specific IgG antibody titers. For operational procedures, please refer to the Concise Guide to Molecular Biology Experiments [M]. Science Press, 2008.

[0229] The results are shown in Figure 26: The titer of anti-RBDDelta-specific IgG antibody in the serum of mice immunized for two weeks after the first immunization was 1:371; the titer of RBD-specific IgG antibody in the serum of mice immunized for two weeks after the second immunization was 1:371. DeltaThe specific IgG antibody titer was 1:407380; RBD levels in the serum of mice immunized for two weeks were... Delta The specific IgG antibody titer was 1:645654.

[0230] RBD in serum Delta Specific IgA antibody titers were detected. The results are shown in Figure 27. The RBD levels in the serum of mice two weeks after secondary immunization were... Delta The titer of specific IgA antibody was 1:417, and the titer of RBDDelta-specific IgA antibody in the serum of mice two weeks after triple immunization was 1:4677.

[0231] Secretory IgA (sIgA) is an important indicator of mucosal immune response. Two weeks after the third immunization, bronchoalveolar lavage fluid (BALF) was collected from mice to detect renal endothelial growth factor (RBD) in the BALF. Delta Specific IgG titers (see Figure 28), IgA titers (see Figure 29). RBD Delta The specific IgG antibody titers were 1:6026 and the specific IgA titer was 1:3162.

[0232] (7.3) Detection of neutralizing antibody titers in serum of BALB / c mice after lung delivery immunization using a pseudovirus neutralization assay

[0233] 1) Cell plating: Digest 293T-ACE2 cells with 1 mL trypsin, add 3 mL of culture medium to stop digestion, centrifuge at 1000 rpm for 5 min, resuspend in 1 mL of culture medium, count the cells and dilute to 2×105 cells / mL, add 100 μL of cells to each well so that each well contains 2×104 cells / well, and incubate overnight.

[0234] 2) Sample preparation: 10 μL of serum from each group of mice was inactivated in a 56℃ water bath for 30 min.

[0235] 3) In addition to the cell control CC with 150 μL of culture medium, add 100 μL of culture medium to each well, and add 144 μL of culture medium to the first well. Take 6 μL of serum / bronchoalveolar lavage fluid from each group and add it (i.e., dilute 1:25). Take 50 μL from the first well and dilute it three times downward.

[0236] 4) Dilute the pseudovirus to 1.34×10⁴ TCID₅₀ / mL, add 50 μL / well except for the cell control, shake the mixture for 30 s, and then place the 96-well plate in a cell culture incubator (37℃, 5% CO₂) for 1 hour.

[0237] 5) Discard the 96-well plate culture medium containing cells that has been incubated overnight, transfer 120 μL of serum-virus mixture into it, and incubate at 37°C with 5% CO2.

[0238] 6) After culturing for 8 hours, discard the culture medium in the 96-well plate, add 150 μL of DMEM complete culture medium per well, and incubate at 37°C with 5% CO2 for 48 hours.

[0239] 7) After the culture is complete, aspirate 100 μL of supernatant, add 100 μL of Bright-Glo luciferase assay reagent (Vazyme, DD1204-02), shake for 2 min, react at room temperature in the dark for 5 min, repeatedly pipet and transfer 100 μL of liquid to an opaque white plate (Perkins Elmer, 6005290).

[0240] 8) Use the PerkinElmer EnSight multi-function imaging microplate reader to read the luminescence value (RLU).

[0241] Neutralizing antibody titers are expressed as the reciprocal of the serum dilution corresponding to an inhibition rate of 50% or the antibody concentration corresponding to an inhibition rate of 50%.

[0242] The results are shown in Figures 30 and 31. Two weeks after each immunization, the neutralizing antibody titers in mouse serum were 1:29, 1:1995, and 1:41687, respectively; two weeks after the third immunization, the neutralizing antibody titer in the bronchoalveolar lavage fluid of mice was 1:270.

[0243] (7.4) Immunogenicity and protective effect of lipid-modified RBD evaluated by intramuscular injection

[0244] The RBD obtained in Examples 5 and 6 Delta ST and LipoSC-RBD Delta ST prepares the vaccine using physiological saline according to the concentration, so that 100 μL volume contains an equal mass of RBD. Delta ST (5 μg), or in combination with Al(OH)3 adjuvant (100 μg) and / or CpG (50 μg) adjuvant. Female BALB / c mice aged 6–8 weeks were immunized intramuscularly on days 0 and 14 (n=5). Blood was collected from the orbital sinus venosus of the immunized mice two weeks after the first immunization and two weeks after the second immunization. See Figures 30 and 31.

[0245] Detection of RBD in serum using the above method Delta ST-specific IgG antibody titers and neutralizing antibody titers. The results showed that when using non-lipidated RBD... Delta When mice were immunized with ST in combination with Al(OH)3 (100 μg) and CpG (50 μg) as adjuvants, RBD was observed after a single immunization. Delta The titer of ST-specific IgG binding antibody was 1:3715, and the titer of neutralizing antibody was 1:44; when using lipotropic RBD... Delta ST(LipoSC-RBD DeltaWhen immunizing mice with ST, RBD occurs after a single immunization. Delta The titer of ST-specific IgG binding antibody was 1:2754, and the titer of neutralizing antibody was 1:47; when using lipotropic RBD... Delta When mice are immunized with a combination of ST (LipoSC-RBDDeltaST) and Al(OH)3 (100 μg), RBD... Delta The titer of ST-specific IgG binding antibody was 1:8511, and the titer of neutralizing antibody was 1:182; the specific results are shown in Figures 32 and 33.

[0246] In summary, the fatty acid-modified coronavirus S protein RBD antigen provided by this invention has good immunogenicity, especially after lung delivery immunization, it can induce RBD-specific sIgA in mice, laying the foundation for inhalation vaccination of subunit vaccines.

[0247] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fatty acid-modified coronavirus antigen.

2. The fatty acid-modified coronavirus antigen according to claim 1, characterized in that, It contains an N-terminal cysteine ​​(Cys) that has been fatty acid modified.

3. The fatty acid-modified coronavirus antigen according to claim 2, characterized in that, The fatty acid-modified N-terminal cysteine ​​(Cys) specifically refers to Cys whose thiol group is modified with diacylglycerol, or Cys whose thiol group is modified with diacylglycerol and whose α-amino group is modified with acyl group.

4. The fatty acid-modified coronavirus antigen according to any one of claims 1-3, characterized in that, The coronaviruses include, but are limited to, SARS-CoV-2, SARS-CoV, and MERS-CoV.

5. The fatty acid-modified coronavirus antigen according to any one of claims 1-4, characterized in that, The SARS-CoV-2, SARS-CoV, or MERS-CoV viral antigens are spike proteins or partial functional fragments thereof.

6. The fatty acid-modified coronavirus antigen according to claim 5, characterized in that, The functional fragment is a receptor-binding domain (RBD) and / or an N-terminal domain (NTD), preferably an RBD.

7. The fatty acid-modified coronavirus antigen according to claim 4, characterized in that, In the SARS-CoV and SARS-CoV-2 coronavirus antigens, the receptor-binding domain (RBD) includes regions and variants that have the ability to bind to ACE2.

8. The fatty acid-modified coronavirus antigen according to claim 4, characterized in that, In the MERS-CoV coronavirus antigen, the receptor-binding domain (RBD) includes a region capable of binding to DPP4 and its variants.

9. The fatty acid-modified coronavirus antigen according to any one of claims 6-8, characterized in that, The receptor-binding domain (RBD) includes any of the sequences described in SEQ ID No. 1-10.

10. The fatty acid-modified coronavirus antigen according to any one of claims 1-2, characterized in that, The fatty acid-modified coronavirus antigen also contains a linker domain.

11. The fatty acid-modified coronavirus antigen according to claim 10, characterized in that, The linker domain is an isopeptide linker, specifically a SpyCatcher / SpyTag linker. The coronavirus antigen is fused with the SpyTag polypeptide and covalently linked to the fatty acid-modified SpyCatcher protein via isopeptide bonds; or, the coronavirus antigen is fused with the SpyCatcher protein and covalently linked to the fatty acid-modified SpyTag polypeptide via isopeptide bonds.

12. The fatty acid-modified coronavirus antigen according to claim 11, characterized in that, The SpyCatcher protein is a polypeptide containing any of the amino acid sequences of SEQ ID No. 11-13, preferably a polypeptide containing the amino acid sequence shown in SEQ ID No.

11.

13. The fatty acid-modified coronavirus antigen according to claim 11, characterized in that, The amino acid sequence of the SpyTag polypeptide is shown in any one of SEQ ID No. 14-16, with the amino acid sequence shown in SEQ ID No. 14 being preferred.

14. A method for preparing a fatty acid-modified coronavirus antigen as described in any one of claims 1-9, the method comprising: 1) A DNA fragment containing a signal peptide coding sequence and a coronavirus antigen coding sequence was introduced into Escherichia coli, and recombinant E. coli was cultured to express recombinant coronavirus antigen with N-terminal fatty acid modification. 2) Cultivate the recombinant Escherichia coli described in step 1) to express the recombinant coronavirus antigen modified with fatty acid; 3) The recombinant Escherichia coli was lysed, a surfactant was added, and the fatty acid-modified recombinant coronavirus antigen was extracted and purified from the lysate.

15. A method for preparing a fatty acid-modified coronavirus antigen as described in any one of claims 10-13, the method comprising: 1) A DNA fragment containing a signal peptide coding sequence and a SpyCatcher protein coding sequence was introduced into Escherichia coli, and recombinant E. coli was cultured to express recombinant SpyCatcher protein with N-terminal fatty acid modification; the recombinant E. coli was lysed, a surfactant was added, and the fatty acid modified recombinant SpyCatcher protein was extracted and purified from the lysate. 2) A DNA fragment containing a signal peptide coding sequence, a SpyTag polypeptide coding sequence, and a coronavirus antigen coding sequence was introduced into engineered cells, and recombinant cell lines were cultured to express coronavirus antigen containing the SpyTag polypeptide and the coronavirus antigen containing the SpyTag polypeptide was purified. 3) The purified fatty acid-modified recombinant SpyCatcher protein was mixed with coronavirus antigen containing SpyTag peptide and reacted fully under certain conditions to obtain fatty acid-modified coronavirus antigen.

16. The method according to any one of claims 14-15, characterized in that, The conserved sequence of the signal peptide includes the amino acid sequence of the last four C-terminal positions as [L / V / I][A / S / T / V / I][G / A / S][C].

17. The method according to claim 15, characterized in that, The signal peptide is any sequence of SEQ ID No. 17-19.

18. The application of the fatty acid-modified coronavirus antigen as described in any one of claims 1-13, characterized in that, The application is for the preparation of vaccines or the screening of drugs to treat coronaviruses.

19. A vaccine comprising the fatty acid-modified coronavirus antigen as described in any one of claims 1-13.

20. The vaccine according to claim 19, characterized in that, The vaccine is a respiratory mucosal immunotherapy vaccine or an injectable vaccine.

21. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fatty acid-modified coronavirus antigen as described in any one of claims 1-13.

22. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a nucleic acid encoding a fatty acid-modified coronavirus antigen as described in any one of claims 1-13.

23. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a vector encoding a fatty acid-modified coronavirus antigen as described in any one of claims 1-13.

24. A biomaterial, characterized in that, The biomaterial is any one of the following: C1) Encoding a nucleic acid molecule of a fatty acid-modified coronavirus antigen as described in any one of claims 1-13; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) Recombinant cells containing the nucleic acid molecule described in C1), or recombinant cells containing the expression cassette described in C2), or recombinant cells containing the recombinant vector described in C3).

25. A method for preparing a vaccine using fatty acid-modified coronavirus antigens according to any one of claims 1-14.

26. A method for preparing a vaccine using the biomaterials of claim 25.

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