An immunological composition containing a fatty acid-modified SpyCatcher protein and a method of preparing the same

Abstract

The application discloses an immunological composition containing a fatty acid modified SpyCatcher protein and a preparation method thereof, and belongs to the field of biological medicines; wherein the SpyCatcher protein (LipoSC) can self-polymerize into nanoparticles, and covalent connection with an antigen containing a SpyTag polypeptide through an isopeptide bond can improve the immunogenicity of the loaded antigen; taking the receptor binding domain (RBD) of the coronavirus spike protein (S protein) as an example, the immunological composition containing the fatty acid modified SpyCatcher protein and the coronavirus RBD antigen disclosed by the application can induce a strong RBD specific immune response, and in particular, as a mucosal subunit vaccine, can induce antigen specific mucosal immunity and humoral immunity. The application further provides a method for realizing the fatty acid modification of the SpyCatcher protein in Escherichia coli, and a method for preparing various immunological compositions by using the fatty acid modified protein.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an immune composition containing fatty acid-modified SpyCatcher protein and its preparation method. Background Technology

[0002] Currently, authorized vaccines mainly include live attenuated vaccines, inactivated vaccines, and subunit vaccines. However, whole-pathogen-based vaccines have many safety concerns and ineffective immune responses. With in-depth research into the life cycle, genetic characteristics, and pathogenic mechanisms of pathogens, the selection and design of vaccine antigens have transitioned from whole-pathogen antigens to one or several clearly defined protective components, thus ushering in the era of subunit vaccines. A successful vaccine must induce specific immune protection against the pathogen while ensuring safety. Subunit vaccines have excellent safety profiles, but their immunogenicity is relatively weak, and the antigens are easily cleared. Therefore, selecting appropriate antigens and antigen design schemes, immunostimulants, and antigen delivery systems are key factors for the success of subunit vaccines.

[0003] The SpyTag / SpyCatcher linker system is one of the most thoroughly studied protein linker systems. Due to its rapid reaction, specificity, broad tolerance to reaction conditions, and irreversibility, it 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 enables repeated display of antigens on the surface of self-polymerizing nanocarrier proteins, improving antigen presentation efficiency and immunogenicity. This linker system originates from the CnaB2 domain of the fibronectin-binding protein of *Streptococcus pyogenes*. The Lys31 and Asp117 regions of the CnaB2 domain can form intramolecular isopeptide bonds. Even after splitting the CnaB2 domain 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 linker 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 speed and efficiency of the linker reaction, the team artificially designed the SpyTag (13aa) / SpyCatcher (116aa) linker system, developing the SpyTag002 / SpyCatcher002 and SpyTag003 / SpyCatcher003 linker systems. These truncated and mutated SpyCatcher systems all have similar isopeptide bond linking functions, and the SpyCatcher described in this invention includes these SpyCatcher variants.

[0004] 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.

[0005] 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 its emergence, the World Health Organization has identified five major categories of variants of concern (VOCs) for SARS-CoV-2: Alpha, Beta, Gamma, Delta, and Omicron. More variants are expected to emerge in the future. Respiratory diastolic blood surface area (RBB) is the immunodominant region of the S protein, containing the vast majority of neutralizing epitopes, making it an ideal candidate antigen. Developing RBD-based vaccines facilitates immune focusing, particularly RBD-based mucosal subunit vaccines, which can induce specific mucosal immune responses and sIgA production, potentially blocking the infection and spread of mutant viruses at the respiratory mucosa. However, the small molecular weight and relatively low immunogenicity of RBDs hinder the development of subunit vaccines using RBDs as antigens, making mucosal vaccines using RBDs even more challenging.

[0006] Mycoplasma pneumoniae (MP) is the smallest naturally occurring microorganism capable of independent survival and is one of the main pathogens causing pneumonia in hospitalized children. Mycoplasma pneumoniae infection is transmitted through respiratory droplets. Tracheobronchitis is the most common type of lower respiratory tract infection, with approximately 10%-40% of cases developing into pneumonia. A small percentage of these cases can progress to severe or refractory pneumonia. Pneumonia caused by Mycoplasma pneumoniae infection is the most common cause of community-acquired pneumonia. Mycoplasma pneumoniae lacks a cell wall; its pathogenic mechanism primarily relies on cell adhesion. It colonizes the respiratory tract by attaching to specific organelles, thereby causing cell damage. The proteins responsible for adhesion are mainly P1 and P40 / 90 proteins. Therefore, P1 protein and / or P40 / 90 protein are candidate antigens for Mycoplasma pneumoniae subunit vaccines. Developing mucosal subunit vaccines based on P1 protein and / or P40 / 90 protein can induce specific mucosal immune responses and sIgA production, and is expected to block the adhesion and spread of mycoplasma at the respiratory mucosa in the first instance.

[0007] In existing technologies, displaying protective antigens on the surface of protein nanoparticles, and / or physically mixing or embedding adjuvants, and using attenuated viral or bacterial vectors to deliver antigens are effective means to improve antigen mucosal delivery efficiency and enhance immunogenicity. However, issues such as pre-existing immunity caused by the vector, immune focusing imbalance, and safety concerns arising from adjuvants and excipients hinder 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 minimize the side effects caused by adjuvants. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides an immune composition containing fatty acid-modified SpyCatcher protein and a method for its preparation.

[0009] According to a first aspect of the technical solution of the present invention, the present invention provides a fatty acid-modified SpyCatcher protein.

[0010] The fatty acid-modified SpyCatcher protein is characterized by containing an N-terminal cysteine ​​residue (Cys) modified with fatty acid.

[0011] In some embodiments, the fatty acid-modified N-terminal cysteine ​​(Cys) is specifically a Cys thiol group modified with diacylglycerol, or a Cys thiol group modified with diacylglycerol and an α-amino group modified with an acyl group.

[0012] The acyl carbon chain length is C16~C18.

[0013] The SpyCatcher protein is a polypeptide comprising any of the amino acid sequences shown in SEQ ID No. 1-3.

[0014] According to a second aspect of the present invention, the present invention provides an immune composition containing the above-mentioned fatty acid-modified SpyCatcher protein.

[0015] In some embodiments, the immune composition further comprises an antigen fused with the SpyTag polypeptide.

[0016] In some embodiments, the fatty acid-modified SpyCatcher protein is covalently linked to the antigen expressed fused with the SpyTag polypeptide via isopeptide bonds.

[0017] In some embodiments, the SpyTag polypeptide has an amino acid sequence as shown in any of SEQ ID No. 10-12.

[0018] When the fatty acid-modified SpyCatcher protein contains the SpyCatcher protein shown in SEQ ID No. 1, the SpyTag polypeptide shown in SEQ ID No. 10 shall be used; when the fatty acid-modified SpyCatcher protein contains the SpyCatcher protein shown in SEQ ID No. 2, the SpyTag polypeptide shown in SEQ ID No. 11 shall be used; when the fatty acid-modified SpyCatcher protein contains the SpyCatcher protein shown in SEQ ID No. 3, the SpyTag polypeptide shown in SEQ ID No. 12 shall be used.

[0019] In some embodiments, the antigen expressed in fusion with the SpyTag polypeptide is a respiratory virus antigen.

[0020] In some embodiments, the respiratory virus antigen includes coronavirus antigens.

[0021] In some embodiments, the coronavirus includes SARS-CoV-2, SARS-CoV, and MERS-CoV.

[0022] In some embodiments, the coronavirus antigen includes the spike protein (S protein) and / or functional fragments thereof.

[0023] In some embodiments, the functional fragment of the S protein is preferably a receptor-binding domain (RBD).

[0024] In some embodiments, the SARS-CoV-2 RBD includes domains from the SARS-CoV-2 S protein prototype and its various variants that can bind to the ACE2 receptor.

[0025] In some embodiments, the SARS-CoV S protein RBD includes the domains in the SARS-CoV S protein prototype and its various variants that can bind to the ACE2 receptor.

[0026] In some embodiments, the MERS-CoV S protein RBD includes a domain in the MERS-CoV S protein prototype and its various variants that can bind to the DPP4 receptor.

[0027] In some embodiments, the RBD amino acid sequence is as shown in any of SEQ ID No. 13-21.

[0028] In some embodiments, the antigen expressed in fusion with the SpyTag polypeptide is a mycoplasma protein antigen.

[0029] In some embodiments, the mycoplasma protein antigen is specifically Mycoplasma pneumoniae P1 protein or a fragment thereof, or P40 / P90 protein or a fragment thereof.

[0030] In some embodiments, the amino acid sequence of the Mycoplasma pneumoniae P1 protein is shown in SEQ ID No. 31.

[0031] In some embodiments, the amino acid sequence of the Mycoplasma pneumoniae P40 / P90 protein is shown in SEQ ID No. 32.

[0032] According to a third aspect of the present invention, the present invention provides a method for preparing the fatty acid-modified SpyCatcher protein as described above, the preparation method comprising the following steps: 1) A DNA fragment containing a signal peptide coding sequence and a SpyCatcher protein coding sequence was introduced into E. coli, and recombinant E. coli were cultured to express fatty acid-modified SpyCatcher protein. 2) Cultivate the recombinant Escherichia coli described in step 1), harvest the bacterial cells and lyse them, add an appropriate surfactant to extract and purify the fatty acid-modified SpyCatcher protein from the lysate.

[0033] In some embodiments, the signal peptide has an amino acid sequence as shown in any of SEQ ID No. 4-6.

[0034] The DNA fragment containing the signal peptide coding sequence and the SpyCatcher protein coding sequence can be introduced into E. coli via expression plasmids or integrated into the E. coli genome.

[0035] In some embodiments, the amino acid sequence of the fatty acid-modified SpyCatcher protein is shown in SEQ ID No. 36; to facilitate protein purification, a purification tag, such as a His tag, can be added, and the amino acid sequence of the fatty acid-modified SpyCatcher protein containing the His tag is shown in SEQ ID No. 35.

[0036] According to a fourth aspect of the present invention, an immune composition containing the above-mentioned fatty acid-modified SpyCatcher protein is provided. The preparation method includes the following steps: mixing the above-mentioned fatty acid-modified SpyCatcher protein and the above-mentioned antigen expressed by fusion with SpyTag polypeptide in a certain proportion, and reacting fully under suitable conditions to obtain an immune composition.

[0037] According to a fifth aspect of the present invention, the present invention provides the use of the above-described immune composition in the preparation of a product capable of inducing the production of antigen-specific antibodies in animals, wherein the immune composition can be administered by intramuscular injection, subcutaneous injection, nasal instillation, or nebulization.

[0038] In some embodiments, the product is a vaccine.

[0039] According to a sixth aspect of the present invention, the present invention provides a pharmaceutical composition comprising the above-described fatty acid-modified SpyCatcher protein.

[0040] According to a seventh aspect of the present invention, the present invention provides a pharmaceutical composition comprising a nucleic acid encoding the fatty acid-modified SpyCatcher protein described above.

[0041] According to an eighth aspect of the present invention, the present invention provides a pharmaceutical composition comprising a carrier encoding the fatty acid-modified SpyCatcher protein described above.

[0042] According to a ninth aspect of the present invention, the present invention provides a biomaterial, said biomaterial being any of the following: C1) The nucleic acid molecule encoding the fatty acid-modified SpyCatcher protein described above; 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).

[0043] Compared with the prior art, the present invention has at least the following beneficial effects: The immunocompositions disclosed in this invention, containing fatty acid-modified SpyCatcher protein and coronavirus RBD antigen, can induce strong RBD-specific immune responses. Particularly as mucosal subunit vaccines, they can induce antigen-specific mucosal and humoral immunity, laying the foundation for blocking the invasion and spread of pathogens at the infection site. This invention further provides a method for guiding the fatty acid modification of SpyCatcher protein in *E. coli* using a heterologous signal peptide, and a method for preparing various immunocompositions using this fatty acid-modified protein. Attached Figure Description

[0044] Figure 1 This is an SDS-PAGE image of SpyCatcher protein expression containing different signal peptides according to the present invention. Figure 2 This is an identification diagram of SDS-PAGE purified by MLSC Ni affinity chromatography according to the present invention; Figure 3 This is an SDS-PAGE identification image obtained by MLSC anion exchange chromatography according to the present invention; Figure 4 This is an SDS-PAGE identification image of SDS-PAGE purified by MLSC molecular size exclusion chromatography according to the present invention; Figure 5 The MLSC molecular size exclusion chromatography diagram according to the present invention; Figure 6 The rAgSC molecular size exclusion chromatography diagram according to the present invention; Figure 7 The P4SC molecular size exclusion chromatography diagram according to the present invention; Figure 8 This is a molecular exclusion chromatography diagram of human IgG according to the present invention; Figure 9 This is a deconvolution map for MLSC mass spectrometry detection according to the present invention; Figure 10 The SARS-CoV-2 Delta mutant strain RBD and SpyTag fusion protein (RBD) according to the present invention Delta ST) SDS-PAGE identification image of purified sample; Figure 11 LipoSC-RBD according to the present invention Delta ST molecular size exclusion chromatography; Figure 12 LipoSC-RBD according to the present invention Delta ST molecular size exclusion chromatography purification SDS-PAGE identification image; Figure 13 LipoSC-RBD according to the present invention Delta ST transmission electron microscopy (TEM) identification image; Figure 14 LipoSC-RBD according to the present invention Delta ST dynamic light scattering (DLS) identification diagram; Figure 15 Serum RBD levels in BALB / c mice before, 2 weeks after, 4 weeks after, and 6 weeks after lung delivery according to the present invention were measured. Delta Specific IgG titer; Figure 16 Serum RBD levels in BALB / c mice before, 2 weeks after, 4 weeks after, and 6 weeks after lung delivery according to the present invention were measured. Delta Specific IgA titer; Figure 17 For lung delivery of bronchoalveolar lavage fluid (BALF) RBD from BALB / c mice 2 weeks after triple immunization according to the present invention Delta Specific IgG titer; Figure 18 For lung delivery of bronchoalveolar lavage fluid (BALF) RBD from BALB / c mice 2 weeks after triple immunization according to the present invention Delta Specific IgA titer; Figure 19 The titers of SARS-CoV-2 Delta pseudovirus neutralizing antibodies in the serum of BALB / c mice before, 2 weeks after, 4 weeks after, and 6 weeks after lung delivery immunization according to the present invention; Figure 20 The titer of SARS-CoV-2 Delta pseudovirus neutralizing antibody in bronchoalveolar lavage fluid (BALF) of BALB / c mice 2 weeks after lung delivery of the present invention; Figure 21 Serum RBD after intramuscular injection of immunization according to the present invention Delta Combined with antibody titer; Figure 22 The titer of SARS-CoV-2 Delta pseudovirus neutralizing antibodies in serum after intramuscular injection immunization according to the present invention; Figure 23This is an SDS-PAGE image of recombinant Mycoplasma pneumoniae P1ST protein (MpP1ST) expression according to the present invention; Figure 24 This is a purified SDS-PAGE identification image of MpP1ST according to the present invention; Figure 25 This is a connection diagram of LipoSC and MpP1ST according to the present invention. Detailed Implementation

[0045] 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.

[0046] The specific content of the present invention will be further described below with reference to specific embodiments.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] pET30a(+) was purchased from Novagen.

[0049] Escherichia coli BL21(DE3) was purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number CB105.

[0050] Escherichia coli C43(DE3) was purchased from Sigma, catalog number CMC0019.

[0051] Pichia pastoris was deposited on March 18, 2020, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 19488.

[0052] The anti-His tag mouse monoclonal antibody was purchased from Sigma, catalog number A7058.

[0053] The chromatographic medium for Sepharose FF is from Cytiva, catalog number 17057502.

[0054] Sephadex G25 fine chromatography packing material was purchased from Cytiva, product catalog number 17003202.

[0055] SOURCE30Q chromatography packing material was purchased from Cytiva, product catalog number 17127503.

[0056] Capto MMC chromatography packing material was purchased from Cytiva, catalog number 17531710.

[0057] The Phenyl FF low sub chromatography packing material was purchased from Cytiva, catalog number 28926988.

[0058] The SOURCE 30S chromatography packing material was purchased from Cytiva, catalog number 17127302.

[0059] The Superdex™ 75 Increase10 / 300 GL pre-loaded column was purchased from Cytiva, catalog number 29148721.

[0060] The Superdex™ 200 Increase 10 / 300 GL pre-loaded column was purchased from Cytiva, catalog number 28990944.

[0061] BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0062] High-resolution mass spectrometer: TripleTOF 5600, AB Sciex.

[0063] Ultra-high performance liquid chromatograph: Waters Acquity UPLC.

[0064] Deconvolution software: IntactMass (Protein Metrics).

[0065] SEQ ID No. 1: Amino acid sequence of the SpyCatcher truncated form: DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG; SEQ ID No. 2: Amino acid sequence of SpyCatcher002 GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHT; SEQ ID No. 3: SpyCatcher003 amino acid sequence: GAMVTTLSGLSGEQGPSGDMTTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT; SEQ ID No.4:ML: MKATKLVLGAVILGSTLLAGC; SEQ ID No.5:rAg is a source of oxygen MKKLLIAAMMAAALAAC; SEQ ID No.6:P4 Responsible Device MKTTLKMTALAALSAFVLAGC; SEQ ID No.7:ML-SC(NdeⅠ,NotⅠ): catATGaaagctactaactggtactgggcgcggtaatcctgggttctactctgctggcaggttgGGATCCCAcCAtCAtCAtCAcCAcCAcCAtggcgccatggttg ataccttatcaggtttatcaagtgagcaaggtcagtccggtgatatgacaattgaagatagtgctacccattattaaattctcaaaacgtgatgatgacggcaaagagttagct ggtgcaactatggagttgcgtgattcatctggtaaaactattagtacatggattcagatggacaagtgaaagatttctacctgtatccaggaaaatacatttgtcgaaaccg cagcaccagacggttatgaggtagcaactgctattacctttacagttaatgagcaaggtcaggttactgtaaatggcaaagcaactaaaggtgacgctcatattTGAgcggccgc; SEQ ID No.8:rAg-SC(NdeⅠ,NotⅠ): catATGaaaaaattattgattgccgcaatgatggcggctgccttggcagcttgcGGATCCCAcCAtCAtCAtCAcCAcCAcCAtCACCAtggcgccatggttgataccttatcaggtttatcaagtgagcaaggtcagtccggtgatatgacaattgaagaagatagtgctacccatattaaattctcaaaacgtgatgaggacggcaaagagttagctggtgcaactatggagttgcgtgattcatctggtaaaactattagtacatggatttcagatggacaagtgaaagatttctacctgtatccaggaaaatatacatttgtcgaaaccgcagcaccagacggttatgaggtagcaactgctattacctttacagttaatgagcaaggtcaggttactgtaaatggcaaagcaactaaaggtgacgctcatattTGAgcggcgc; SEQ ID No.9: P4-SC nucleotide sequence (NdeⅠ, NotⅠ): ctaATGAAAACCACCCTGAAAATGACCGCTCTGGCTGCTCTGTCTGCTTTCGTTCTGGCTGGTtgcGGATCCCAcCAtCAtCAtCAcCAcCAcCAtCACCAtggcgccatggttgataccttatcaggtttatcaagtgagcaaggtcagtccggtgatatgacaattgaagaagatagtgctacccatattaaattctcaaaacgtgatgaggacggcaaagagttagctggtgcaactatggagttgcgtgattcatctggtaaaactattagtacatggatttcagatggacaagtgaaagatttctacctgtatccaggaaaatatacatttgtcgaaaccgcagcaccagacggttatgaggtagcaactgctattacctttacagttaatgagcaaggtcaggttactgtaaatggcaaagcaactaaaggtgacgctcatattTGAgcggccgc; SEQ ID No. 10: Amino acid sequence of SpyTag: AHIVMVDAYKPTK; The amino acid sequence of SEQ ID No. 11: SpyTag002: VPTIVMVDAYKRYK; The amino acid sequence of SEQ ID No. 12: SpyTag003: RGVPHIVMVDAYKRYK; SEQ ID No.13: SARS-CoV-2 S-RBD WT amino acid sequence: RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLP DDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.14: SARS-CoV-2 S-RBD Beta amino acid sequence: RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLP DDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.15: SARS-CoV-2 S-RBD Delta (R319-V534) amino acid sequence: RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.16: SARS-CoV-2 S-RBD JN.1 Amino acid sequence: RVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.17: SARS-CoV-2 S-RBD Kp.2.3 Amino acid sequence: RVQPTESIVRFPNVTNLCPFHEVFNATTFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSLRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.18: SARS-CoV-2 S-RBD Kp.3.2.3 Amino acid sequence: RVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSLRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLESYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.19: SARS-CoV-2 S-RBD XDV.1 Amino acid sequence: RVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSLRKSKLKPFERDISTEIYQAGNIPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.20: Amino acid sequence of SARS-CoV S-RBD RFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLI; SEQ ID No.21: Amino acid sequence of MERS-CoV S-RBD AKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL; SEQ ID No.22: SARS-CoV-2 S-RBD WT -ST nucleotide sequence: CTCGAGAAAAGAAGAGTACAACCAACTGAATCCATTGTTAGATTTCCTAATATCACTAACCTGTGCCCATTTGGTGAAGTTTTTAACGCTACTAGATTTGCTTCTGTTTACGCCTGGAACAGAAAGAGAATTTCTAACTGTGTTGCTGATTACTCTGTTCTTTACAACTCTGCCTCTTTTTCTACTTTTAAGTGTTATGGTGTCTCTCCAACCAAGTTGAACGATTTGTGTTTTACCAACGTTTACGCTGATTCTTTTGTTATTAGAGGTGATGAGGTTAGACAAATTGCTCCTGGTCAAACTGGTAAGATTGCTGATTATAACTACAAGTTGCCTGATGATTTTACTGGTTGCGTCATTGCTTGGAACTCTAATAATTTGGATTCTAAGGTTGGTGGAAATTACAACTACTTGTACAGATTGTTTAGAAAGAGTAACTTGAAGCCATTTGAAAGAGATATTTCTACTGAAATCTACCAAGCTGGATCTACTCCTTGTAACGGTGTCGAAGGTTTTAACTGCTACTTTCCTTTGCAGTCTTACGGTTTTCAACCCACTAACGGTGTTGGTTACCAGCCCTACAGAGTTGTTGTTTTGTCTTTTGAGTTGCTTCATGCTCCAGCTACTGTTTGTGGTCCTAAGAAGTCTACTAACTTGGTTGGTGGTGGTGGTTCTGctCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; SEQ ID No.23: SARS-CoV-2 S-RBD Beta -ST nucleotide sequence: CTCGAGAAAAGAAGAGTACAACCAACTGAATCCATTGTTAGATTTCCTAATATCACTAACCTGTGCCCATTTGGTGAAGTTTTTAACGCTACTAGATTTGCTTCTGTTTACGCCTGGAACAGAAAGAGAATTTCTAACTGTGTTGCTGATTACTCTGTTCTTTACAACTCTGCCTCTTTTTCTACTTTTAAGTGTTATGGTGTCTCTCCAACCAAGTTGAACGATTTGTGTTTTACCAACGTTTACGCTGATTCTTTTGTTATTAGAGGTGATGAGGTTAGACAAATTGCTCCTGGTCAAACTGGTAAcATTGCTGATTATAACTACAAGTTGCCTGATGATTTTACTGGTTGCGTCATTGCTTGGAACTCTAATAATTTGGATTCTAAGGTTGGTGGAAATTACAACTACTTGTACAGATTGTTTAGAAAGAGTAACTTGAAGCCATTTGAAAGAGATATTTCTACTGAAATCTACCAAGCTGGATCTACTCCTTGTAACGGTGTCAAgGGTTTTAACTGCTACTTTCCTTTGCAGTCTTACGGTTTTCAACCCACTTACGGTGTTGGTTACCAGCCCTACAGAGTTGTTGTTTTGTCTTTTGAGTTGCTTCATGCTCCAGCTACTGTTTGTGGTCCTAAGAAGTCTACTAACTTGGTTGGTGGTGGTGGTTCTGctCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; SEQ ID No.24: SARS-CoV-2 S-RBD Delta -ST nucleotide sequence: CTCGAGAAAAGAAGAGTACAACCAACTGAATCCATTGTTAGATTTCCTAATATCACTAACCTGTGCCCATTTGGTGAAGTTTTTAACGCTACTAGATTTGCTTCTGTTTACGCCTGGAACAGAAAGAGAATTTCTAACTGTGTTGCTGATTACTCTGTTCTTTACAACTCTGCCTCTTTTTCTACTTTTAAGTGTTATGGTGTCTCTCCAACCAAGTTGAACGATTTGTGTTTTACCAACGTTTACGCTGATTCTTTTGTTATTAGAGGTGATGAGGTTAGACAAATTGCTCCTGGTCAAACTGGTAAGATTGCTGATTATAACTACAAGTTGCCTGATGATTTTACTGGTTGCGTCATTGCTTGGAACTCTAATAATTTGGATTCTAAGGTTGGTGGAAATTACAACTACagaTACAGATTGTTTAGAAAGAGTAACTTGAAGCCATTTGAAAGAGATATTTCTACTGAAATCTACCAAGCTGGATCTAagCCTTGTAACGGTGTCGAAGGTTTTAACTGCTACTTTCCTTTGCAGTCTTACGGTTTTCAACCCACTAACGGTGTTGGTTACCAGCCCTACAGAGTTGTTGTTTTGTCTTTTGAGTTGCTTCATGCTCCAGCTACTGTTTGTGGTCCTAAGAAGTCTACTAACTTGGTTGGTGGTGGTGGTTCTGctCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; SEQ ID No.25: SARS-CoV-2 S-RBD JN.1 -ST nucleotide sequence: CTCGAGAAAAGAAGAGTTCAACCAACTGAATCTATTGTTAGATTTCCAAACGTTACTAACTTGTGTCCATTTCATGAAGTTTTTAACGCTACTAGATTTGCTTCTGTTTACGCTTGGAACAGAACTAGAATTTCTAACTGTGTTGCTGATTACTCTGTTTTGTACAACTTTGCTCCATTCTTTGCTTTTAAGTGTTACGGTGTTTCTCCAACTAAGTTGAATGATTTGTGTTTTACTAATGTTTACGCTGATTCTTTTGTTATCAAAGGTAATGAAGTTTCTCAAATTGCTCCAGGTCAAACTGGAAACATTGCTGATTATAACTACAAGTTGCCAGATGATTTTACTGGTTGTGTTATTGCTTGGAATTCAAATAAGTTGGATTCTAAGCATTCTGGTAACTACGATTATTGGTACAGAAGTTTTAGAAAGTCTAAGTTGAAGCCATTTGAAAGAGATATTTCTACTGAAATCTACCAAGCTGGTAATAAGCCTTGTAAGGGTAAAGGTCCAAACTGTTACTTTCCACTTCAATCTTACGGTTTTAGACCTACTTACGGTGTTGGTCATCAACCATACAGAGTTGTTGTTTTGTCTTTTGAATTGTTGCATGCTCCAGCTACTGTTTGTGGTCCAAAGAAGTCTACTAACTTGGTTGGTGGTGGTGGTTCTGctCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; SEQ ID No.26: SARS-CoV-2 S-RBD Kp.2.3 -ST nucleotide sequence: CTCGAGAAAAGAAGAGTTCAACCAACTGAATCTATTGTTAGATTTCCAAACGTTACTAACTTGTGTCCATTTCATGAAGTTTTTAACGCTACTACTTTTGCTTCTGTTTACGCTTGGAACAGAACTAGAATTTCTAACTGTGTTGCTGATTACTCTGTTTTGTACAACTTTGCTCCATTCTTTGCTTTTAAGTGTTACGGTGTTTCTCCAACTAAGTTGAATGATTTGTGTTTTACTAATGTTTACGCTGATTCTTTTGTTATCAAAGGTAATGAAGTTTCTCAAATTGCTCCAGGTCAAACTGGAAACATTGCTGATTATAACTACAAGTTGCCAGATGATTTTACTGGTTGTGTTATTGCTTGGAATTCAAATAAGTTGGATTCTAAGCATTCTGGTAACTACGATTATTGGTACAGAAGTTTGAGAAAGTCTAAGTTGAAGCCATTTGAAAGAGATATTTCTACTGAAATCTACCAAGCTGGTAATAAGCCTTGTAAGGGTAAAGGTCCAAACTGTTACTTTCCACTTCAATCTTACGGTTTTAGACCTACTTACGGTGTTGGTCATCAACCATACAGAGTTGTTGTTTTGTCTTTTGAATTGTTGCATGCTCCAGCTACTGTTTGTGGTCCAAAGAAGTCTACTAACTTGGTTGGTGGTGGTGGTTCTGctCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; SEQ ID No.27: SARS-CoV-2 S-RBD Kp.3.2.3 -ST nucleotide sequence: CTCGAGAAAAGAAGAGTTCAACCAACTGAATCTATTGTTAGATTTCCAAACGTTACTAACTTGTGTCCATTTCATGAAGTTTTTAACGCTACTAGATTTGCTTCTGTTTACGCTTGGAACAGAACTAGAATTTCTAACTGTGTTGCTGATTACTCTGTTTTGTACAACTTTGCTCCATTCTTTGCTTTTAAGTGTTACGGTGTTTCTCCAACTAAGTTGAATGATTTGTGTTTTACTAATGTTTACGCTGATTCTTTTGTTATCAAAGGTAATGAAGTTTCTCAAATTGCTCCAGGTCAAACTGGAAACATTGCTGATTATAACTACAAGTTGCCAGATGATTTTACTGGTTGTGTTATTGCTTGGAATTCAAATAAGTTGGATTCTAAGCATTCTGGTAACTACGATTATTGGTACAGAAGTTTGAGAAAGTCTAAGTTGAAGCCATTTGAAAGAGATATTTCTACTGAAATCTACCAAGCTGGTAATAAGCCTTGTAAGGGTAAAGGTCCAAACTGTTACTTTCCACTTGAATCTTACGGTTTTAGACCTACTTACGGTGTTGGTCATCAACCATACAGAGTTGTTGTTTTGTCTTTTGAATTGTTGCATGCTCCAGCTACTGTTTGTGGTCCAAAGAAGTCTACTAACTTGGTTGGTGGTGGTGGTTCTGctCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; SEQ ID No.28: SARS-CoV-2 S-RBD XDV.1 -ST nucleotide sequence: CTCGAGAAAAGAAGAGTTCAACCAACTGAATCTATTGTTAGATTTCCAAACGTTACTAACTTGTGTCCATTTCATGAAGTTTTTAACGCTACTAGATTTGCTTCTGTTTACGCTTGGAACAGAACTAGAATTTCTAACTGTGTTGCTGATTACTCTGTTTTGTACAACTTTGCTCCATTCTTTGCTTTTAAGTGTTACGGTGTTTCTCCAACTAAGTTGAATGATTTGTGTTTTACTAATGTTTACGCTGATTCTTTTGTTATCAAAGGTAATGAAGTTTCTCAAATTGCTCCAGGTCAAACTGGAAACATTGCTGATTATAACTACAAGTTGCCAGATGATTTTACTGGTTGTGTTATTGCTTGGAATTCAAATAAGTTGGATTCTAAGCATTCTGGTAACTACGATTATTGGTACAGAAGTTTGAGAAAGTCTAAGTTGAAGCCATTTGAAAGAGATATTTCTACTGAAATCTACCAAGCTGGTAATATCCCTTGTAAGGGTAAAGGTCCAAACTGTTACTTTCCACTTCAATCTTACGGTTTTAGACCTACTTACGGTGTTGGTCATCAACCATACAGAGTTGTTGTTTTGTCTTTTGAATTGTTGCATGCTCCAGCTACTGTTTGTGGTCCAAAGAAGTCTACTAACTTGGTTGGTGGTGGTGGTTCTGctCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; SEQ ID No.29: Nucleotide sequence of SARS-CoV S-RBD-ST: CTCGAGAAAAGAAGATTCCCTAATATCACCAACCTTTGCCCTTTCGGAGAAGTTTTCAACGCTACCAAGTTCCCAAGTGTTTACGCTTGGGAAAGAAAGAAAATTTCTAATTGTGTCGCCGATTACTCCGTTTTGTACAACTCAACATTTTTCAGTACCTTTAAGTGTTACGGTGTCTCTGCTACTAAATTGAACGATCTTTGCTTCTCAAACGTTTATGCTGACAGTTTCGTTGTCAAGGGAGATGACGTCAGACAAATTGCACCAGGTCAAACCGGAGTTATCGCTGATTACAACTACAAATTGCCTGATGACTTCATGGGTTGTGTTTTGGCCTGGAATACTAGAAACATTGACGCAACTTCCACAGGAAACTACAACTACAAGTACAGATATTTGAGACATGGAAAACTTAGACCATTTGAAAGAGATATCTCTAACGTTCCATTCTCCCCTGACGGTAAACCATGTACACCACCTGCTTTGAATTGCTATTGGCCTCTTAACGATTACGGATTTTATACTACAACCGGTATTGGATACCAACCTTATAGAGTTGTCGTTTTGTCATTCGAGTTGCTTAATGCTCCAGCCACTGTTTGTGGTCCTAAGTTGAGTACAGACCTTATCGGTGGTGGTGGTTCTGctCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; SEQ ID No.30: Nucleotide sequence of MERS-CoV S-RBD-ST: CTCGAGAAAAGAGCCAAGCCATCCGGATCAGTTGTCGAACAGGCAGAGGGTGTTGAATGTGATTTTAGTCCTTTGCTTTCTGGTACTCCACCTCAAGTTTACAACTTCAAGAGATTGGTCTTCACTAACTGTAACTACAACTTGACAAAGTTGCTTTCTTTGTTTTCTGTTAACGATTTCACTTGTTCACAAATTAGTCCAGCAGCTATCGCTTCAAATTGCTATTCAAGTTTGATCCTTGACTACTTCTCTTACCCATTGTCTATGAAGTCCGATCTTTCAGTTTCTTCCGCAGGTCCTATCTCTCAGTTCAATTACAAGCAAAGTTTTTCTAACCCTACTTGTTTGATCCTTGCTACAGTTCCACACAACTTGACCACTATCACCAAGCCTCTTAAGTACTCTTACATCAACAAGTGTTCCAGATTGCTTTCAGATGACAGAACTGAGGTTCCACAGTTGGTCAATGCTAACCAATACAGTCCTTGTGTTTCTATTGTCCCATCCACTGTTTGGGAGGATGGTGACTACTATAGAAAGCAATTGTCTCCTCTTGAAGGTGGAGGTTGGTTGGTTGCATCCGGATCAACCGTCGCTATGACTGAACAATTGCAGATGGGATTTGGTATTACAGTTCAATACGGTACAGACACCAATTCAGTCTGTCCAAAGTTGGGTGGTGGTGGTTCTGctCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; SEQ ID No.31: Amino acid sequence of Mycoplasma pneumoniae P1 protein: SEQ ID No. 32: Amino acid sequence of Mycoplasma pneumoniae P40 / P90 protein: SEQ ID No. 33: Nucleotide sequence of Mycoplasma pneumoniae P1-ST protein: SEQ ID No. 34: Nucleotide sequence of Mycoplasma pneumoniae P40 / 90-ST protein: SEQ ID No. 35: Amino acid sequence of fatty acid-modified SpyCatcher protein: CGSHHHHHHHHGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI; SEQ ID No. 36: Amino acid sequence of fatty acid-modified SpyCatcher protein: CGSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI; Example 1: Preparation and characterization of fatty acid-modified SpyCatcher protein (1.1) Construction of the fatty acid-modified SpyCatcher protein expression vector Based on the Escherichia coli Murein-lipoprotein signal peptide sequence (EU900370.1), Neisseria meningitidis Ag473 signal peptide sequence (AY566590.1), and Haemophilus influenzae P4 signal peptide sequence (M68502.1) published in GenBank, these sequences were fused to the N-terminus of a polypeptide containing the sequence shown in SEQ ID No. 1, and a His tag was added to facilitate purification. 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.

[0066] The optimized MLSC nucleotide sequence is shown in SEQ ID No. 7, 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; The optimized rAgSC nucleotide sequence is shown in SEQ ID No. 8, wherein nucleotides 1-6 from the 5' end are NdeⅠ restriction site sequences, nucleotides 4-54 are Ag473 lipoprotein signal peptide coding sequences, nucleotides 55-90 are adapter and His tag coding sequences, nucleotides 91-441 are SpyCatcher (SC) coding sequences, and nucleotides 442-449 are NotⅠ restriction site sequences; The optimized P4SC nucleotide sequence is shown in SEQ ID No. 9, wherein nucleotides 1-6 from the 5' end are NdeⅠ restriction site sequences, nucleotides 4-66 are P4 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; 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.

[0067] (1.2) Construction of a strain expressing SpyCatcher protein modified with fatty acid esterification 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 (0.5% yeast extract, 1% tryptone, 1% NaCl) containing a final concentration of 50 μg / mL kanamycin. Positive clones, i.e., expression strains C43(DE3) / pET30-MLSC, C43(DE3) / pET30-rAgSC, and C43(DE3) / pET30-P4SC, were obtained.

[0068] (1.3) Expression and purification of recombinant SpyCatcher protein 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 6-20 h.

[0069] Take 1 mL of each bacterial culture induced at 25℃ for 20 h, centrifuge to obtain bacterial cells, suspend the cells in distilled water at a ratio of 1:30 (w / v), sonicate to disrupt, centrifuge to separate the precipitate and supernatant, suspend the precipitate in an equal volume of water, prepare the sample with 5× 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 15% SDS-PAGE electrophoresis, transfer to a PVDF membrane, maintain constant voltage at 20 V for 1 h, and detect with mouse-derived anti-His tag monoclonal antibody (Sigma, A7058). The results are as follows. Figure 1 As shown.

[0070] 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.

[0071] 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.

[0072] 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 pH7.5 Tris-HCl, 0.3M NaCl, 500mM imidazole + 1% Triton X100). Finally, equilibrate with at least 3 column volumes of Ni-A1 buffer (20mM pH7.5 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 buffer (20 mM pH 7.5 Tris-HCl + 0.3 M NaCl + 5 mM imidazole + 1% Triton X100) 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 the preliminarily purified sample. Figure 2 ).

[0073] 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 a sample loading volume not exceeding 1 / 3 of the column volume.

[0074] 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, then equilibrate to pH neutral with deionized water; then, equilibrate with at least 3 column volumes of 30Q-A1 buffer (20mM pH 7.5 Tris-HCl, + 1% Triton X100); next, load the desalted sample containing recombinant SpyCatcher onto a SOURCE30Q anion exchange chromatography column, equilibrate with 10 column volumes of 30Q-A1 buffer, 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 + 1 M NaCl + 0.1% Tween 80); collect the eluted sample ( Figure 3 ).

[0075] The sample was purified using Superdex 200 increase. The Superdex 200 increase column (φ1×30 cm, Cytiva, 28990944) was equilibrated with SEC (5 mM pH 7.4 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 chromatogram 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. Figure 8 This indicates that the molecular weights of the three recombinant proteins MLSC, rAgSC, and P4SC are greater than 150 kDa, which is much larger than the 16 kDa of the SC monomer. Therefore, it is determined that the three recombinant proteins MLSC, rAgSC, and P4SC exist in multimeric form. Figure 4 , 5 6, 7).

[0076] (1.4) Characterization of MLSC To further confirm whether the N-terminus of the purified recombinant SpyCatcher protein was modified with fatty acid esterification, 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.

[0077] Beijing Mingde Zhengkang Technology Co., Ltd. was commissioned to use mass spectrometry (LC / Q-TOF-MS) Figure 9 The molecular weight of recombinant SpyCatcher was determined, and the results showed that the molecular weight of recombinant SpyCatcher was concentrated in two clusters: the first cluster had molecular weights of 14810.1 Da, 14824.2 Da, and 14838.0 Da, and the second cluster had molecular weights 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 is inferred that the recombinant SpyCatcher with a molecular weight of 14810.1 Da has an additional molecule of Pam3Cys (13919.11 + 910.46 - 18). Based on the technical solution of this invention, it is speculated that the recombinant SpyCatcher with a molecular weight of 14810.1 Da is Pam3C-SpyCatcher, that is, a SpyCatcher with an N-terminal fatty acid modification, wherein the thiol group of the N-terminal Cys group is modified with diacylglycerol (C16:0, The recombinant SpyCatcher with a molecular weight of 14824.2 Da increased in molecular weight by approximately 14 Da, consistent with the molecular weight of CH2, suggesting that one fatty acid chain is extended by one CH2. The recombinant SpyCatcher with a molecular weight of 14838 Da increased in molecular weight by approximately 28 Da, consistent with the molecular weight of (CH2)2, suggesting 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, with a decrease in molecular weight of 238.4 Da and 238 Da, consistent with the molecular weight of palmitoylated (C16:0), indicating that the N-terminus of the SpyCatcher is modified with fatty acid, wherein the thiol group of the N-terminal Cys is modified with diacylglycerol.

[0078] 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.

[0079] Example 2: Preparation of recombinant SARS-CoV-2 viral S protein RBD containing SpyTag peptide This invention uses the SARS-CoV-2 Delta mutant strain S protein RBD as an example to elucidate the antigen containing the SpyTag polypeptide.

[0080] (2.1) SARS-CoV-2 S protein RBD Delta Construction of the SpyTag yeast expression vector 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 was added to its C-terminus. The DNA sequence was then optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. based on Pichia pastoris preferred codons. The nucleotide sequence is shown in SEQ ID No. 24, and the DNA is named SARS-CoV-2 RBD. 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.

[0081] (2.2) Recombinant expression vector pPICZαA-RBD Delta ST-transformed Pichia pastoris strain CGMCC No. 19488 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 liquid medium (1% yeast extract, 2% tryptone, 2% glucose). 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 at 1500 g × 5 min. The cells were resuspended in an equal volume of pre-chilled distilled water and centrifuged at 4°C at 1500 g × 5 min, discarding the supernatant. 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 at 1500 g × 5 min, discarding the supernatant. This step was repeated 3 times. The bacterial precipitate, after being washed three times with distilled water and three times with sorbitol, was resuspended with 1 mL of 1M sorbitol, and 100 μL was dispensed into each sterile centrifuge tube and stored at -80℃ to obtain Pichia pastoris strain CGMCCNo.19488 electroporation-transformed competent cells.

[0082] 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 digested at 37℃ for 1 h. Samples were then 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.

[0083] 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. Mix well and transfer to a pre-chilled 0.2 cm electroporation cuvette. Electroporate at 2 kV and immediately add 900 μL of pre-chilled 1 M sorbitol. Transfer to a clean test tube and 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 (100 μg / mL) and incubate upside down at 25°C for 60-72 hours.

[0084] (2.3) Screening of recombinant expression strains 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 the culture 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.

[0085] 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).

[0086] (2.4) Recombinant strain CGMCC No.19488 / pPICZαA-RBD Delta ST cultivation 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 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.

[0087] (2.5) RBD Delta purification of ST 1. Capto MMC chromatography purification 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: MMC-A: 20mM pH 5.5 PB (phosphate buffer); MMC-B: 100mM pH8.5 Tris-HCl+1M NaCl.

[0088] After loading the sample, equilibrate with MMC-A and then elute with MMC-B.

[0089] 2. Hydrophobic chromatography purification of Phenyl FF low sub-phosphorus The Capto MMC purified sample was purified using Phenyl FF low sub (Cytiva, 28926988). The mobile phase composition was as follows: Phenyl FF-A: 20mM pH 7.5 Tris-HCl + 1M AS (ammonium sulfate); Phenyl FF-B: 20mM p H7.5 Tris-HCl.

[0090] First, elute the target protein with 40% Phenyl FF-B, then elute other proteins with 40-100% Phenyl FF-B.

[0091] 3. Sephadex G-25 Fine desalination 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.

[0092] 4. SOURCE 30Q anion exchange chromatography purification The desalted sample was purified using SOURCE30Q (Cytiva, 17127502) chromatography medium. The mobile phase composition was as follows: 30Q-A: 20mM p H8.5 Tris-HCl; 30Q-B: 20mM pH8.5 Tris-HCl+1M NaCl.

[0093] After loading, equilibrate with 30Q-A, then elute with 30Q-B, with the target protein in the flow-through buffer.

[0094] 5. SOURCE 30S cation exchange chromatography 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: 30S-A: 20mM pH 6.5 PB; 30S-B: 20mM pH7.0 PB+1M NaCl.

[0095] After loading the sample, equilibrate with A, and then elute the target protein with 50% B.

[0096] 6. Superdex™ 75 Increase 10 / 300 GL Gel Filtration Chromatography The sample purified by SOURCE 30S was loaded with 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 was equilibrated with the mobile phase and collected in fractions.

[0097] RBD was obtained after the above purification steps. Delta ST samples, purification process, SDS-PAGE identification as follows: Figure 10 .

[0098] Example 3: Preparation of fatty acid-modified RBD nanoparticles The LipoSC nanoparticles prepared above and SARS-CoV-2 RBD were combined using the BCA method (Thermo Scientific, Cat. No. A55864, Related BCA Kits: 23225 (1000 mL)). Delta Protein quantification was performed using ST chromatography, with the mixture at a molar ratio of 2:1 and ligation at 4°C for 12 hours. The Superdex 200 Increase column (φ1×30 cm, cytiva, 28990944) was equilibrated with SEC200 buffer (5 mM pH 7.0 PB + 0.9% NaCl). 1 mL of the ligation product was then used to remove unligated RBDs using Superdex 200 Increase chromatography. Delta ST, LipoSC-RBD Delta The retention volume of ST was 9.42 mL, which was slightly smaller than that of LipoSC. Figure 11 SDS-PAGE reduction electrophoresis showed that ( Figure 12 ), RBD Delta ST is attached to LipoSC, with a molecular weight of approximately 43 kDa; TEM analysis shows that LipoSC-RBDDelta ST represents nanoparticles ( Figure 13 ); DLS detection indicates ( Figure 14 LipoSC-RBD 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.

[0099] Example 4: Immunogenicity study of fatty acid-modified RBD (4.1) Lung delivery immunization regimen The LipoSC-RBD obtained in Example 3 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 with 50 μL of vaccine via lung delivery on days 0, 14, and 28 (n=5). Blood was collected from the orbital sinus of the immunized mice before immunization and two weeks after the first, second, and third immunizations.

[0100] (4.2) Detection of RBD-specific binding antibodies after lung delivery immunization The serum RBD levels in mice of each group were 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.

[0101] The results are as follows Figure 5 As shown: Anti-RBD levels in the serum of mice two weeks after immunization Delta The specific IgG antibody titer was 1:371; RBD levels in the serum of mice two weeks after the second immunization were... Delta The 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.

[0102] RBD in serum Delta The specific IgA antibody titer was measured. The results are as follows: Figure 16 As shown, RBD levels in the serum of mice two weeks after immunization Delta The specific IgA antibody titer was 1:417, and the RBD level in the serum of mice two weeks after triple immunization was [missing information]. Delta The specific IgA antibody titer was 1:4677.

[0103] 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 the level of renal dialysis (RBD) in the BALF. Delta Specific IgG titer ( Figure 17 ), IgA titer ( Figure 18 RBD Delta The specific IgG antibody titer was 1:6026, and the specific IgA titer was 1:3162.

[0104] (4.3) Detection of neutralizing antibody titers in serum of BALB / c mice after lung delivery immunization using a pseudovirus neutralization test 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 × 10⁻⁶ cells / mL. 5 Add 100 μL of cells to each well to make a cell density of 2 × 10⁶ cells / mL. 4 Cell / well, incubate overnight.

[0105] 2) Sample preparation: 10 μL of serum from each group of mice was inactivated in a 56℃ water bath for 30 min.

[0106] 3) In addition to the cell control CC with 150 μL of culture medium, add 100 μL of culture medium to each well, add 144 μL of culture medium to the first well, add 6 μL of serum / bronchoalveolar lavage fluid from each group (i.e., 1:25 dilution), and take 50 μL from the first well for a three-fold dilution downwards.

[0107] 4) Dilute the fake virus to 1.34 × 10⁻⁶ 4 Add 50 μL of TCID50 / mL to each well except for the cell control. Shake the mixture for 30 seconds and then incubate the 96-well plate in a cell culture incubator (37°C, 5% CO2) for 1 hour.

[0108] 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.

[0109] 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.

[0110] 7) After the culture is complete, discard 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).

[0111] 8) Use the PerkinElmer EnSight multi-function imaging microplate reader to read the luminescence value (RLU).

[0112] 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%.

[0113] The results are as follows Figure 23 , 24 As shown, the serum neutralizing antibody titers in mice were 1:29, 1:1995, and 1:41687 two weeks after each immunization; the neutralizing antibody titer in bronchoalveolar lavage fluid of mice was 1:270 two weeks after the third immunization.

[0114] (4.4) Immunogenicity and protective effect of lipid-modified RBD evaluated by intramuscular injection The RBD obtained in Examples 2 and 3 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 venous sinus of the immunized mice two weeks after the first immunization and two weeks after the second immunization.

[0115] 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 lipo-derived RBD-DeltaST (LipoSC-RBD) Delta When 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 ST (LipoSC-RBD) Delta When mice were immunized with a combination of ST and Al(OH)3 (100 μg), RBD was observed after a single immunization. Delta The titer of ST-specific IgG binding antibody was 1:8511, and the titer of neutralizing antibody was 1:182; specific results are as follows. Figure 21 , 22 As shown.

[0116] 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.

[0117] Example 5: Preparation of recombinant Mycoplasma pneumoniae P1 protein containing SpyTag polypeptide (5.1) Construction of recombinant Mycoplasma pneumoniae P1ST protein expression vector Based on the P1 protein sequence of Mycoplasma pneumoniae strain M29 published in GenBank (GenBank: U00089.2, amino acid sequence as shown in SEQ ID No. 31), it was fused to the C-terminus of the pelB signal peptide, and a His tag was added to facilitate purification. The DNA sequence was optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. based on the codons preferred by Escherichia coli.

[0118] The optimized pelB-P1ST nucleotide sequence is shown in SEQ ID No. 33, wherein nucleotides 1-6 from the 5' end are NdeⅠ restriction site sequences, nucleotides 4-69 are pelB signal peptide coding sequences, nucleotides 70-75 are linker coding sequences, nucleotides 76-4554 are P1 protein coding sequences, nucleotides 4555-4578 are linker coding sequences, nucleotides 4579-4617 are SpyTag polypeptide coding sequences, nucleotides 4618-4629 are linker coding sequences, nucleotides 4630-4647 are His tag coding sequences, and nucleotides 4651 to 4658 are NotⅠ sequences; The optimized sequence was cloned between the NdeⅠ and NotⅠ restriction sites of the pET30a vector to construct the pET30-pelBMpP1ST expression vector.

[0119] (5.2) Construction of P1ST protein expression strain The constructed expression vector pET30-pelBMpP1ST was introduced into Escherichia coli BL21(DE3) host cells (purchased from Tiangen Biotech, catalog number CB105; Thermo Fisher Scientific, catalog number EC0114), and plated on LB solid medium containing kanamycin at a final concentration of 50 μg / mL. The positive clones were the expression strains BL21(DE3) / pET30-pelBMpP1ST.

[0120] (5.3) Expression and purification of Mycoplasma pneumoniae P1ST protein Recombinant BL21(DE3) / pET30-pelBMpP1ST single clones 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 20°C to induce the expression of the target protein.

[0121] The following day, 1 mL of each bacterial culture induced at 20℃ 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 then sonicated, centrifuged to separate the precipitate and supernatant, and the precipitate was resuspended in an equal volume of water. The sample was prepared with 5X reducing buffer (250 mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500 mM DTT), incubated in a boiling water bath for 10 min, and subjected to 15% SDS-PAGE electrophoresis. After electrophoresis, the sample was transferred to a PVDF membrane and transferred at a constant voltage of 20V for 1 h. The results were detected using a mouse-derived anti-His tag monoclonal antibody (Sigma, A7058). Figure 23 As shown.

[0122] After expression was identified by SDS-PAGE and WB, the culture volume was expanded and the MpP1ST protein was purified.

[0123] The sample was purified using a Chelating affinity chromatography column (Φ1.6cm*15cm). After harvesting the bacterial cells, the cells were suspended in Ni-A2 buffer (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole) at a ratio of 1:20 (w / v), sonicated, and the supernatant was collected by centrifugation and purified by Chelating affinity chromatography.

[0124] 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-B2 buffer (20mM pH 7.5 Tris-HCl, 0.3M NaCl, 500mM imidazole). Finally, equilibrate with at least 3 column volumes of Ni-A2 buffer (20mM pH 7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole). The lysed supernatant containing P1 protein was loaded onto a Chelating affinity chromatography column. Unbound protein was washed away with Ni-A2 buffer, followed by elution with 10%, 30%, and 100% Ni-B2 buffer. The sample eluted with 30% Ni-B buffer was collected to obtain a preliminarily purified sample. This sample was further purified using a Superdex 200 increase column (φ1×30 cm, cytiva, 28990944). Figure 24 ).

[0125] Example 6: Preparation of fatty acid-modified MpP1 nanoparticles Protein quantification of the MpP1ST prepared in Example 5 was performed using the BCA method (Thermo Scientific, Cat. No. A55864, Related BCA Kits: 23225 (1000 mL)). The mixture was inoculated at a molar ratio of 2:1 for 12 hours at 4°C. Figure 25 Equilibrate the Superdex 200 Increase column (φ1×30 cm, cytiva, 28990944) with SEC200 (5mM pH7.0 PB + 0.9% NaCl) buffer, and remove unlinked MpP1ST by taking 1 mL of the above ligation product with Superdex 200 Increase.

[0126] Additionally, the fatty acid-modified SpyCatcher protein and its preparation method are described below.

[0127] The fatty acid-modified SpyCatcher protein comprises a fatty acid-modified N-terminal cysteine ​​residue (Cys) and the SpyCatcher protein; the fatty acid-modified N-terminal cysteine ​​residue (Cys) is characterized in that the thiol group of Cys is modified with diacylglycerol, or the thiol group of Cys is modified with diacylglycerol and the α-amino group is modified with an acyl group; wherein the carbon chain length of the acyl group is C16-C18; the SpyCatcher protein comprises the amino acid sequence shown in SEQ ID No. 1. The fatty acid-modified SpyCatcher protein may also contain pathogen-specific T cell epitopes and / or B cell epitopes, and may also contain purification tag sequences, such as His-Tag tags, for ease of purification.

[0128] The method for preparing the fatty acid-modified SpyCatcher protein provided by the present invention includes the following steps: Step S1: A DNA fragment containing a signal peptide coding sequence and a SpyCatcher protein coding sequence is introduced into E. coli, and recombinant E. coli is cultured to express fatty acid-modified SpyCatcher protein. In step S1 above, the signal peptide is any amino acid sequence shown in SEQ ID No. 4, SEQ ID No. 5, or SEQ ID No. 6: SEQ ID No.4: MKATKLVLGAVILGSTLLAGC SEQ ID No.5: MKKLLIAAMMALAAC SEQ ID No.6: MKTTLKMTALAALSAFVLAGC The DNA fragment containing the signal peptide coding sequence and the SpyCatcher protein coding sequence is introduced into E. coli via a recombinant expression vector. The recombinant expression vector is obtained by inserting the DNA fragment containing the signal peptide coding sequence and the SpyCatcher protein coding sequence into the multiple cloning site of the expression vector. The expression vector is preferably a pET series vector, specifically, the recombinant expression vector is obtained by inserting the DNA fragment containing the signal peptide coding sequence and the SpyCatcher protein coding sequence into the NdeⅠ and NotⅠ sites of pET30a. The DNA fragment sequence containing the signal peptide coding sequence and the SpyCatcher protein coding sequence is any one of the DNA sequences shown in SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9: In SEQ ID No. 7, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 66 are the signal peptide coding sequence shown in SEQ ID No. 4, positions 67 to 72 are the linker coding sequence, positions 73 to 102 are the His tag coding sequence, positions 103 to 450 are the SpyCatcher tag coding sequence, and positions 454 to 461 are NotⅠ restriction sites; In SEQ ID No. 8, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 54 are the signal peptide coding sequence shown in SEQ ID No. 5, positions 55 to 60 are the linker coding sequence, positions 61 to 90 are the His tag coding sequence, positions 91 to 438 are the SpyCatcher tag coding sequence, and positions 442 to 448 are NotⅠ restriction sites; In SEQ ID No. 9, positions 1 to 6 are NdeⅠ restriction sites, positions 4 to 66 are the signal peptide coding sequence shown in SEQ ID No. 6, positions 67 to 72 are the linker coding sequence, positions 73 to 102 are the His tag coding sequence, positions 103 to 450 are the SpyCatcher tag coding sequence, and positions 454 to 461 are NotⅠ restriction sites.

[0129] The obtained recombinant expression vector is electroporated or chemically converted into Escherichia coli to obtain recombinant engineered Escherichia coli; The DNA fragments containing signal peptide coding sequences and SpyCatcher protein coding sequences can also be integrated into the E. coli genome; Step S2: Cultivate the recombinant *E. coli* described in step S1), harvest and lyse the bacterial cells, add an appropriate surfactant, and extract and purify the fatty acid-modified SpyCatcher protein from the lysate. The lysis of the recombinant *E. coli* can be performed using methods such as ultrasonication or a high-pressure homogenizer. The method for extracting and purifying the fatty acid-modified SpyCatcher protein from the lysate can be achieved through precipitation, centrifugation, or further purification using various chromatographic methods to obtain the fatty acid-modified SpyCatcher protein. The chromatographic purification method in step S2 above may include ion exchange chromatography, size exclusion chromatography, etc. The amino acid sequence of the fatty acid-modified SpyCatcher protein is shown in SEQ ID No. 36.

[0130] A recombinant fusion protein containing fatty acid-modified SpyCatcher protein was prepared using the above method. The recombinant fusion protein containing fatty acid-modified SpyCatcher protein may also contain pathogen T-cell epitopes and / or B-cell epitopes. The antigen containing the SpyTag polypeptide is an antigen containing the SpyTag polypeptide or an antigen fused with and expressed with the SpyTag polypeptide. The amino acid sequence of the SpyTag polypeptide is shown in SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12. SEQ ID No. 10: AHIVMVDAYKPTK SEQ ID No. 11: VPTIVMVDAYKRYK SEQ ID No.12: RGVPHIVMVDAYKRYK When the fatty acid-modified SpyCatcher protein contains the SpyCatcher protein shown in SEQ ID No. 1, the SpyTag polypeptide shown in SEQ ID No. 10 should be used; when the fatty acid-modified SpyCatcher protein contains the SpyCatcher protein shown in SEQ ID No. 2, the SpyTag polypeptide shown in SEQ ID No. 11 should be used; when the fatty acid-modified SpyCatcher protein contains the SpyCatcher protein shown in SEQ ID No. 3, the SpyTag polypeptide shown in SEQ ID No. 12 should be used.

[0131] The antigen expressed in fusion with the SpyTag polypeptide can be a viral antigen; The viral antigen is a respiratory viral antigen, specifically a coronavirus antigen; the coronavirus antigen is a spike protein (S protein) or a functional fragment thereof, wherein the functional fragment of the S protein is preferably a receptor-binding domain (RBD) or an RBD tandem; the coronavirus includes, but is not limited to, SARS-CoV-2, SARS-CoV, and MERS-CoV; The amino acid sequence of the S protein RBD is the SARS-CoV-2 S protein RBD. The SARS-CoV-2 RBD includes the ACE2 receptor-binding domains of the SARS-CoV-2 S protein prototype and its various variants, including but not limited to the prototype (RBD amino acid sequence as shown in SEQ ID No. 13), beta type (RBD amino acid sequence as shown in SEQ ID No. 14), delta type (RBD amino acid sequence as shown in SEQ ID No. 15), JN1 type (RBD amino acid sequence as shown in SEQ ID No. 16), Kp.2.3 type (RBD amino acid sequence as shown in SEQ ID No. 17), Kp.3.2.3 type (RBD amino acid sequence as shown in SEQ ID No. 18), XDV.1 type (RBD amino acid sequence as shown in SEQ ID No. 19), etc.

[0132] The S protein RBD is the SARS-CoV S protein RBD, which includes the SARS-CoV S protein prototype and its various variants, including the domains that can bind to the ACE2 receptor, one of which is the RBD with an amino acid sequence as shown in SEQ ID No. 20.

[0133] In another embodiment, the S protein RBD is the MERS-CoV S protein RBD, including the domains of the MERS-CoV S protein prototype and its various variants that can bind to the DPP4 receptor, one of which has the amino acid sequence shown in SEQ ID No. 21.

[0134] The DNA fragment sequence containing SpyTag, SARS-CoV-2 S protein RBDWT, and / or His-tag is shown in SEQ ID No. 22. Positions 1 to 6 are XhoI restriction sites, positions 13 to 660 are the S protein RBDWT coding sequence, positions 661 to 675 are the linker coding sequence, positions 676 to 714 are the SpyTag coding sequence, positions 715 to 717 are the stop codon sequence, and positions 718 to 725 are NotI restriction sites.

[0135] The DNA fragment sequence containing SpyTag, SARS-CoV-2 S protein RBDbeta, and / or His-tag is shown in SEQ ID No. 23. Positions 1 to 6 are XhoI restriction sites, positions 13 to 660 are the S protein RBDbeta coding sequence, positions 661 to 675 are the linker coding sequence, positions 676 to 714 are the SpyTag coding sequence, positions 715 to 717 are the stop codon sequence, and positions 718 to 725 are NotI restriction sites.

[0136] The DNA fragment sequence containing SpyTag, SARS-CoV-2 S protein RBDdelta, and / or His-tag is shown in SEQ ID No. 24. Positions 1 to 6 are XhoI restriction sites, positions 13 to 660 are the S protein RBDdelta coding sequence, positions 661 to 675 are the linker coding sequence, positions 676 to 714 are the SpyTag coding sequence, positions 715 to 717 are the stop codon sequence, and positions 718 to 725 are NotI restriction sites.

[0137] The DNA fragment sequence containing SpyTag, SARS-CoV-2 S protein RBDJN1, and / or His-tag is shown in SEQ ID No. 25. Positions 1 to 6 are XhoI restriction sites, positions 13 to 657 are the coding sequence for S protein RBDJN1, positions 658 to 672 are the linker coding sequence, positions 673 to 711 are the SpyTag coding sequence, positions 712 to 714 are the stop codon sequence, and positions 715 to 722 are NotI restriction sites.

[0138] The DNA fragment sequence containing SpyTag, SARS-CoV-2 S protein RBDKp.2.3, and / or His-tag is shown in SEQ ID No. 26. Positions 1 to 6 are XhoI restriction sites, positions 13 to 657 are the S protein RBDJN1 coding sequence, positions 658 to 672 are the linker coding sequence, positions 673 to 711 are the SpyTag coding sequence, positions 712 to 714 are the stop codon sequence, and positions 715 to 722 are NotI restriction sites.

[0139] The DNA fragment sequence containing SpyTag, SARS-CoV-2 S protein RBDKp.3.2.3, and / or His-tag is shown in SEQ ID No. 27. Positions 1 to 6 are XhoI restriction sites, positions 13 to 657 are the S protein RBDJN1 coding sequence, positions 658 to 672 are the linker coding sequence, positions 673 to 711 are the SpyTag coding sequence, positions 712 to 714 are the stop codon sequence, and positions 715 to 722 are NotI restriction sites.

[0140] The DNA fragment sequence containing SpyTag, SARS-CoV-2 S protein RBDXDV.1, and / or His-tag is shown in SEQ ID No. 28. Positions 1 to 6 are XhoI restriction sites, positions 13 to 657 are the coding sequence for S protein RBDJN1, positions 658 to 672 are the linker coding sequence, positions 673 to 711 are the SpyTag coding sequence, positions 712 to 714 are the stop codon sequence, and positions 715 to 722 are NotI restriction sites.

[0141] The DNA fragment sequence containing SpyTag, SARS-CoV S protein RBD, and / or His-tag is shown in SEQ ID No. 29. Positions 1 to 6 are XhoI restriction sites, positions 13 to 630 are the SARS-CoV S protein RBD coding sequence, positions 634 to 645 are the linker coding sequence, positions 646 to 684 are the SpyTag coding sequence, positions 685 to 687 are the stop codon sequence, and positions 688 to 695 are NotI restriction sites. The DNA fragment sequence containing SpyTag, MERS-CoV S protein RBD, and / or His-tag is shown in SEQ ID No. 30. Positions 1 to 6 are XhoI restriction sites, positions 13 to 675 are the MERS-CoV S protein RBD coding sequence, positions 676 to 690 are the linker coding sequence, positions 691 to 729 are the SpyTag coding sequence, positions 730 to 732 are the stop codon sequence, and positions 733 to 740 are NotI restriction sites.

[0142] The viral antigen can be expressed using Escherichia coli, mammalian cells, yeast cells, or insect cells; preferably, it is expressed using yeast cells, and the specific technical solution can be prepared according to the method described in Chinese patent application CN105671109A; specifically, using RBD DeltaFor example, step S1) involves constructing the RBD. Delta 1) Transform the recombinant expression vector into Pichia pastoris strain CGMCC No. 19488 by electroporation; 2) Screen for recombinant expression strains; 3) Expand the culture of positive high-expression recombinant strains; 4) Purify RBD by cation exchange chromatography, hydrophobic chromatography, and anion exchange chromatography. Delta -SpyTag.

[0143] The antigen expressed by the SpyTag polypeptide can be a Mycoplasma pneumoniae protein antigen; The Mycoplasma pneumoniae protein antigen may be the Mycoplasma pneumoniae P1 protein and a partial fragment thereof, or the P40 / P90 protein and a partial fragment thereof. The P1 and P40 / P90 proteins of Mycoplasma pneumoniae exhibit significant sequence variations depending on the strain, but their structures and functions are similar. The P1 or P40 / P90 proteins described in this invention include these variants. Sequences 31 and 32 are the amino acid sequences of a typical P1 protein and P40 / P90 protein, respectively.

[0144] This invention also provides a method for preparing an immune composition containing fatty acid-modified SpyCatcher protein and SpyTag polypeptide antigen. The method involves mixing the fatty acid-modified SpyCatcher protein and the SpyTag polypeptide antigen in a specific molar ratio; preparing an immune composition in which the fatty acid-modified SpyCatcher protein and the SpyTag polypeptide antigen are covalently linked by isopeptide bonds. The method for preparing the immune composition in which the fatty acid-modified SpyCatcher protein and the SpyTag polypeptide antigen are covalently linked by isopeptide bonds may include purifying the final product using size exclusion chromatography after a sufficient reaction to obtain the immune composition. The immune composition may also be used in combination with aluminum adjuvants, oil-in-water emulsions, CpG adjuvants, or TLR4 agonists.

[0145] Furthermore, an injectable vaccine containing a fatty acid-modified SpyCatcher protein and an immune composition containing the SpyTag polypeptide antigen also falls within the scope of protection of this invention; the immune composition, when administered intramuscularly or subcutaneously to immunize animals, can induce antigen-specific humoral immunity in the animals.

[0146] Furthermore, a mucosal vaccine containing a fatty acid-modified SpyCatcher protein and an immune composition containing the SpyTag polypeptide antigen also falls within the scope of protection of this invention; the immune composition, administered via nasal drops or nebulized inhalation, can induce antigen-specific humoral immunity and mucosal immunity in animals.

[0147] The use of any of the above-described immune compositions in the preparation of products that can induce the production of antigen-specific antibodies in animals is also within the scope of protection of this invention; The use of any of the above-described immune compositions in the preparation of products for the prevention and / or treatment of diseases caused by corresponding pathogens is also within the scope of protection of this invention.

[0148] 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 method for preparing fatty acid-modified RBD nanoparticles, characterized in that, The RBD nanoparticles are composed of LipoSC nanoparticles and SARS-CoV-2 RBD. Delta ST was mixed at a molar ratio of 2:1, and then covalently linked at 4°C for 12 hours to form RBD nanoparticles through the SpyTag / SpyCatcher linkage system. The LipoSC was prepared by the following method: the nucleic acid molecule shown in SEQ ID No. 7 was used to construct an expression vector, which was introduced into Escherichia coli C43 (DE3) host cells. Positive clones were screened, cultured in LB solid medium, and induced to express and purify to obtain LipoSC. The SARS-CoV-2 RBD Delta ST is prepared by the following method: The nucleic acid molecule shown in SEQ ID No. 24 was used to construct an expression vector, which was transformed into Pichia pastoris. Positive clones were screened, inoculated into YPD liquid medium, and induced to express and purify to obtain SARS-CoV-2 RBD. Delta ST.

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