Immune composition containing fatty acid modified SpyCatcher protein and preparation method thereof

Abstract

The invention discloses an immune composition containing a fatty acid modified SpyCatcher protein and a preparation method of the immune composition, and belongs to the field of biological medicines. Wherein the SpyCatcher protein (LipoSC) can be self-polymerized into nanoparticles, the SpyCatcher protein (LipoSC) is covalently linked with an antigen containing SpyTag polypeptide through an isopeptide bond, so that the immunogenicity of the loaded antigen can be improved, and a coronavirus spike protein (S protein) receptor binding domain (RBD) is taken as an example. The immune composition containing the fatty acid modified SpyCatcher protein and the coronavirus RBD antigen disclosed by the invention can induce strong RBD specific immune response, and particularly can induce mucosal immunity and humoral immunity of antigen specificity as a mucosal subunit vaccine. The invention further provides a method for finishing fatty acid modification of the SpyCatcher protein in escherichia coli and a method for preparing various immune compositions by using the fatty acid modified protein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an immune composition containing a fatty acid-modified SpyCatcher protein and a preparation method thereof. Background Art

[0002] Currently, the vaccines authorized for marketing mainly include live attenuated vaccines, inactivated vaccines, subunit vaccines, etc. However, vaccines based on whole pathogens have many safety risks and ineffective immune response problems. With the in-depth study of the life cycle, genetic characteristics, pathogenic mechanism, etc. of pathogens, the selection and design of vaccine antigens have transitioned from whole pathogen antigens to one or several clear protective components, thus entering the era of subunit vaccines. A successful vaccine must be able to induce specific immune protection against pathogens while ensuring safety. Subunit vaccines have excellent safety, but weak immunogenicity and antigens are easily cleared. Therefore, the selection of appropriate antigens and antigen design schemes, immune stimulants and antigen delivery systems are key factors for the success of subunit vaccines.

[0003] The SpyTag / SpyCatcher connection system is one of the most intensively studied protein connection systems. This system is widely used in vaccine development and research due to its rapid reaction, specificity, wide range of reaction conditions, and irreversible reaction. It can be fused with pathogen antigens and suitable carrier proteins to achieve precise covalent connection between antigens and carrier proteins, especially to achieve repeated display of antigens on the surface of self-polymerized nanocarrier proteins, thereby improving the presentation efficiency and immunogenicity of antigens. This connection system is derived 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 the N-terminal domain containing Lys31 (SpyCatcher, 116aa) and the 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 and demonstrated the minimum SpyCatcher required to form an isopeptide bond. To improve the speed and efficiency of the ligation reaction, the team artificially designed the SpyTag (13aa) / SpyCatcher (116aa) ligation system and developed the SpyTag002 / SpyCatcher002 and SpyTag003 / SpyCatcher003 ligation systems. These truncated and mutated SpyCatcher systems all have similar isopeptide bond ligation functions, and the SpyCatcher described in the present invention all include these SpyCatcher variants.

[0004] There is a class of proteins in bacteria whose N-terminal cysteine ​​(Cys) is modified by fatty acid and fixed to the cell surface by fatty acid chains, called bacterial lipoproteins. The in vivo synthesis of bacterial lipoproteins is completed under the guidance of signal peptides. Bioinformatics and statistical analysis of the signal peptide sequences of bacterial natural fatty acid-modified and non-fatty acid-modified proteins revealed that the signal peptides of fatty acid-modified proteins are non-polar amino acids near the signal peptidase cleavage site and have a certain degree of conservation. In summary, it was found that the conservative sequence of the last four amino acids in this region is [L / V / I][A / S / T / V / I][G / A / S][C], among which the C-terminal cysteine ​​is the lipidation modification site. Under the guidance of the signal peptide, the nascent peptide chain is localized to the outside of the cell membrane through the Sec or TAT secretion pathway. The Lgt enzyme uses phosphatidylglycerol (PG) as a substrate to connect diacylglycerol to the sulfhydryl group of Cys. The LspA enzyme removes the signal peptide except the C-terminal Cys. The Lnt enzyme uses phosphatidylethanolamine (PE) as a substrate to transfer a fatty acid chain to the alpha-amino group of Cys. Bacterial fatty acid modification is heterogeneous, mainly in terms of the number and length of fatty acid chains. The length of the fatty acyl carbon chain of bacterial lipoproteins is usually between C14 and C18, but the vast majority of fatty acyl carbon chains are C16.

[0005] Coronavirus is a type of enveloped single-stranded positive-strand RNA virus belonging to the Coronaviridae family. There are seven known coronaviruses that can infect humans. Currently, SARS-CoV, SARS-CoV-2, and MERS-CoV can cause severe illness or even death. The main structural proteins of coronaviruses include spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The S protein is located on the surface of the coronavirus envelope and can recognize host cell receptors and mediate membrane fusion between the virus and the host cell through conformational changes. For example, the receptors of SARS-CoV and SARS-CoV-2 are angiotensin converting enzyme 2 (ACE2), and the receptor of MERS-CoV is dipeptidyl peptidase 4 (DPP4). In the S protein, RBD (receptor-binding domain) has the ability to bind to the receptor as an independent domain. Due to the lack of correction mechanism in the RNA virus replication process, the encoded protein continues to mutate. Under the selection pressure, the RBD of various mutant strains still maintains the ability to bind specifically to the receptor. For example, since the emergence of the SARS-CoV-2 virus, the World Health Organization has identified five major types of variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and Omicron, and various variants will continue to appear in the future. RBD is the immunodominant region of the S protein, containing the vast majority of neutralizing epitopes, and is an ideal candidate antigen. The development of RBD-based vaccines is conducive to immune focusing, especially the development of RBD-based mucosal subunit vaccines, which can induce specific mucosal immune responses and the production of sIgA, and is expected to block the infection and spread of mutant viruses in the respiratory mucosa in the first place. However, the small molecular weight and relatively low immunogenicity of RBD have hindered the development of subunit vaccines with RBD as the antigen, and mucosal vaccines with RBD as the antigen are even more difficult.

[0006] Mycoplasma pneumoniae (MP) is the smallest microorganism in nature that can survive independently. It is one of the main pathogens that cause pneumonia in hospitalized children. Mycoplasma pneumoniae infection is transmitted through droplets through the respiratory tract. Tracheobronchitis is the most common type of lower respiratory tract infection. About 10%-40% will develop into pneumonia. A small number of cases will also develop into severe pneumonia and refractory pneumonia. Pneumonia caused by Mycoplasma pneumoniae infection is the most common cause of community-acquired pneumonia. Mycoplasma pneumoniae has no cell wall, and its pathogenic mechanism mainly relies on cell adhesion. It colonizes in the respiratory tract by specifically attaching to organelles, thereby causing cell damage. The proteins that play an adhesion role are mainly P1 protein and P40 / 90 protein. Therefore, P1 protein and / or P40 / 90 protein are candidate antigens for Mycoplasma pneumoniae subunit vaccines. The development of mucosal subunit vaccines based on P1 protein and / or P40 / 90 protein can induce specific mucosal immune responses and the production of sIgA, which is expected to block the adhesion and spread of mycoplasma in the respiratory mucosa as soon as possible.

[0007] In the existing technical solutions, displaying protective antigens on the surface of protein nanoparticles, and / or physically mixing or embedding adjuvants, and delivering antigens using attenuated viral vectors or bacterial vectors are effective means to improve the efficiency of antigen mucosal delivery and immunogenicity. However, the pre-existing immunity problems caused by the vectors, the imbalance of immune focus, and the safety problems caused by adjuvants and auxiliary agents have plagued the development of mucosal vaccines. Therefore, it is necessary to develop a new strategy that can reduce the immunogenicity of the antigen while reducing the immunological imprinting effect and / or immune advantage of the carrier protein and the side effects caused by the adjuvant. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides an immune composition containing a fatty acid-modified SpyCatcher protein and a preparation method thereof.

[0009] According to the 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 a fatty acid-modified N-terminal cysteine ​​(Cys).

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

[0012] The carbon chain length of the fatty acyl group is C16~C18.

[0013] The SpyCatcher protein is a polypeptide comprising an amino acid sequence as shown in any one of SEQ ID No. 1-3.

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

[0015] In certain embodiments, the immune composition further comprises an antigen expressed as a fusion with the SpyTag polypeptide.

[0016] In certain embodiments, the fatty acid-modified SpyCatcher protein is covalently linked to the antigen expressed by fusion with the SpyTag polypeptide via an isopeptide bond.

[0017] In certain embodiments, the amino acid sequence of the SpyTag polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID No. 10-12.

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

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

[0020] In certain embodiments, the respiratory virus antigen comprises a coronavirus antigen.

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

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

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

[0024] In certain embodiments, the SARS-CoV-2 RBD includes a domain in the SARS-CoV-2 S protein prototype and its various variants that can bind to the ACE2 receptor.

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

[0026] In certain 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 certain embodiments, the RBD amino acid sequence is shown in any one of SEQ ID No. 13-21.

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

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

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

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

[0032] According to the third aspect of the technical solution of the present invention, the present invention provides a method for preparing the above-mentioned fatty acid-modified SpyCatcher protein, the preparation method comprising the following steps: 1) Introducing a DNA fragment containing a signal peptide coding sequence and a SpyCatcher protein coding sequence into Escherichia coli, and culturing the recombinant Escherichia coli to express the fatty acid-modified SpyCatcher protein; 2) Cultivating the recombinant E. coli described in step 1), harvesting and lysing the cells, and adding an appropriate surfactant to extract and purify the fatty acid-modified SpyCatcher protein from the lysate.

[0033] In certain embodiments, the amino acid sequence of the signal peptide is shown in any one 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 Escherichia coli through an expression plasmid, and can also be integrated into the Escherichia coli genome.

[0035] In certain 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. The amino acid sequence of the fatty acid-modified SpyCatcher protein containing a His tag is shown in SEQ ID No. 35.

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

[0037] According to the fifth aspect of the technical solution of the present invention, the present invention provides the use of the above-mentioned immune composition in the preparation of a product that can induce animals to produce antigen-specific antibodies. The immune composition can be injected intramuscularly, subcutaneously, or by nasal drops or aerosolization.

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

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

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

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

[0042] According to the ninth aspect of the technical solution of the present invention, the present invention provides a biomaterial, wherein the biomaterial is any one of the following: C1) a nucleic acid molecule encoding the above-mentioned fatty acid-modified SpyCatcher protein; 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) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) A recombinant cell containing the nucleic acid molecule described in C1), or a recombinant cell containing the expression cassette described in C2), or a recombinant cell containing the recombinant vector described in C3).

[0043] Compared with the prior art, the present invention has at least the following beneficial effects: The immune composition containing fatty acid-modified SpyCatcher protein and coronavirus RBD antigen disclosed in the present invention can induce a strong RBD-specific immune response, especially as a mucosal subunit vaccine, which can induce antigen-specific mucosal immunity and humoral immunity, laying the foundation for blocking the invasion and spread of pathogens at the infection site. The present invention further provides a method for guiding the fatty acid modification of SpyCatcher protein in Escherichia coli using a heterologous signal peptide, and a method for preparing various immune compositions using the fatty acid-modified protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is an SDS-PAGE identification diagram of the expression of SpyCatcher proteins containing different signal peptides according to the present invention; Figure 2 This is an SDS-PAGE identification diagram of MLSC Ni affinity chromatography purification according to the present invention; Figure 3 This is an SDS-PAGE identification diagram of MLSC anion exchange chromatography purification according to the present invention; Figure 4 This is an SDS-PAGE identification diagram of MLSC molecular exclusion chromatography purification according to the present invention; Figure 5 is a molecular exclusion chromatogram of MLSC according to the present invention; Figure 6 is a molecular exclusion chromatogram of rAgSC according to the present invention; Figure 7 is a molecular exclusion chromatogram of P4SC according to the present invention; Figure 8 is a size exclusion chromatogram of human IgG molecules according to the present invention; Fig. 9 It is a deconvolution diagram of MLSC mass spectrometry detection according to the present invention; Fig.10 According to the SARS-CoV-2 Delta mutant strain RBD and SpyTag fusion protein (RBD Delta ST) SDS-PAGE identification of purified samples; Fig.11 LipoSC-RBD according to the present invention Delta ST molecular size exclusion chromatography; Fig.12 LipoSC-RBD according to the present invention Delta ST molecular size exclusion chromatography purification SDS-PAGE identification diagram; Fig.13 LipoSC-RBD according to the present invention Delta Transmission electron microscopy (TEM) identification image of ST; Fig.14 LipoSC-RBD according to the present invention Delta ST dynamic light scattering (DLS) identification diagram; Fig.15 For lung delivery according to the present invention, serum RBD of BALB / c mice before immunization, 2 weeks after immunization, 4 weeks after immunization, and 6 weeks after immunization Delta Specific IgG titer; Fig.16 For lung delivery according to the present invention, serum RBD of BALB / c mice before immunization, 2 weeks after immunization, 4 weeks after immunization, and 6 weeks after immunization Delta Specific IgA titer; Fig.17 The RBD was delivered to the lungs of BALB / c mice bronchoalveolar lavage fluid (BALF) 2 weeks after triple immunization according to the present invention. Delta Specific IgG titer; Fig.18 The RBD was delivered to the lungs of BALB / c mice bronchoalveolar lavage fluid (BALF) 2 weeks after triple immunization according to the present invention. Delta Specific IgA titer; Fig.19 Serum SARS-CoV-2 Delta pseudovirus neutralizing antibody titers of BALB / c mice before, 2 weeks after, 4 weeks after, and 6 weeks after lung delivery according to the present invention; Fig. 20 Neutralizing antibody titer of SARS-CoV-2Delta pseudovirus in bronchoalveolar lavage fluid (BALF) of BALB / c mice 2 weeks after three immunizations of lung delivery according to the present invention; Fig.21 The serum RBD after intramuscular injection immunization according to the present invention Delta Binding antibody titer; Fig. 22 is the serum SARS-CoV-2 Delta pseudovirus neutralizing antibody titer after intramuscular injection immunization according to the present invention; Fig.23This is an SDS-PAGE identification diagram of the expression of the recombinant Mycoplasma pneumoniae P1ST protein (MpP1ST) according to the present invention; Fig.24 This is an SDS-PAGE identification diagram of the MpP1ST purification according to the present invention; Fig.25 FIG. 4 is a diagram showing the connection between LipoSC and MpP1ST according to the present invention. DETAILED DESCRIPTION

[0045] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0046] The specific contents of the present invention are further described below in conjunction with specific embodiments.

[0047] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

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

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

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

[0051] Pichia pastoris was deposited in the General Microbiology Center of China Culture Collection Administration on March 18, 2020, with the deposit number CGMCC No.19488.

[0052] Anti-His tag mouse monoclonal antibody was purchased from Sigma with the catalog number A7058.

[0053] Chelating Sepharose FF chromatography medium is from Cytiva, product catalog number 17057502.

[0054] Sephadex G25 fine chromatography medium was purchased from Cytiva with the product catalog number being 17003202.

[0055] SOURCE30Q chromatography medium was purchased from cytiva, product catalog number 17127503.

[0056] Capto MMC chromatography medium was purchased from Cytiva with catalog number 17531710.

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

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

[0059] SuperdexTM 75 Increase10 / 300 GL prepacked column was purchased from Cytiva with product catalog number 29148721.

[0060] SuperdexTM 200 Increase 10 / 300 GL prepacked column was purchased from Cytiva, product catalog number 28990944.

[0061] BALB / c mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

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

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

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

[0065] SEQ ID No.1: Amino acid sequence of SpyCatcher truncation: DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG; SEQ ID No.2: SpyCatcher002 amino acid sequence GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHT; SEQ ID No.3: SpyCatcher003 amino acid sequence: GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT; 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): catATGaaagctactaactggtactgggcgcggtaatcctgggttctactctgctggcaggttcGGATCCCAcCAtCAtCAtCAcCAcCAcCAtggcgccatggttg ataccttatcaggtttatcaagtgagcaaggtcagtccggtgatatgacaattgaagatagtgctacccatattaaattctcaaaacgtgatgatgagacggcaaagagttagct ggtgcaactatggagttgcgtgattcatctggtaaaactattagtacatggattcagatggacaagtgaaagatttctacctgtatccaggaaaatacatttgtcgaaaccg cagcaccagacggttatgaggtagcaacctgctattacctttacagttaatgagcaaggtcaggttactgtaaatggcaaagcaactaaaggtgacgctcatattTGAgcggccgc; 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; SEQ ID No.11: Amino acid sequence of SpyTag002: VPTIVMVDAYKRYK; SEQ ID No.12: Amino acid sequence of 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: Mycoplasma pneumoniae P1-ST protein nucleotide sequence: SEQ ID No.34: Mycoplasma pneumoniae P40 / 90-ST protein nucleotide sequence: SEQ ID No.35: Fatty acid-modified SpyCatcher protein amino acid sequence: CGSHHHHHHHHGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI; SEQ ID No.36: Fatty acid-modified SpyCatcher protein amino acid sequence: CGSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI; Example 1: Preparation and characterization of fatty acid-modified SpyCatcher protein (1.1) Construction of the expression vector for fatty acid-modified SpyCatcher protein According to 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, they were fused to the N-terminus of the polypeptide containing the sequence shown in SEQ ID No.1, and a His tag was added to facilitate purification. They were named MLSC, rAgSC, and P4SC, respectively, and the DNA sequences were optimized and synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. according to the preferred codons of Escherichia coli.

[0066] The optimized MLSC nucleotide sequence is shown in SEQ ID No.7, wherein the nucleotides 1 to 6 from the 5' end are NdeⅠ restriction site sequences, the nucleotides 4 to 66 are Murein-lipoprotein lipoprotein signal peptide coding sequences, the nucleotides 67 to 102 are linker and His tag coding sequences, the nucleotides 103 to 453 are SpyCatcher (SC) coding sequences, and the nucleotides 454 to 461 are NotⅠ restriction site sequences; The optimized rAgSC nucleotide sequence is shown in SEQ ID No. 8, wherein the nucleotides 1 to 6 from the 5' end are NdeⅠ restriction site sequences, the nucleotides 4 to 54 are Ag473 lipoprotein signal peptide coding sequences, the nucleotides 55 to 90 are linker and His tag coding sequences, the nucleotides 91 to 441 are SpyCatcher (SC) coding sequences, and the nucleotides 442 to 449 are NotⅠ restriction site sequences; The optimized P4SC nucleotide sequence is shown in SEQ ID No. 9, wherein the nucleotides 1 to 6 from the 5' end are NdeⅠ restriction site sequences, the nucleotides 4 to 66 are P4 lipoprotein signal peptide coding sequences, the nucleotides 67 to 102 are linker and His tag coding sequences, the nucleotides 103 to 453 are SpyCatcher (SC) coding sequences, and the nucleotides 454 to 461 are NotⅠ restriction site sequences; The optimized sequences were cloned into the NdeⅠ and NotⅠ restriction sites of the pET30a vector, respectively, to construct the pET30-MLSC, pET30-rAgSC, and pET30-P4SC expression vectors.

[0067] (1.2) Construction of a strain expressing fatty acid-modified SpyCatcher protein The above-constructed expression vectors pET30-MLSC, pET30-rAgSC, and pET30-P4SC were introduced into Escherichia coli C43 (DE3) (purchased from Sigma, Catalog No. CMC0019) or BL21 (DE3) (purchased from Tiangen Biochemical Technology Co., Ltd., Catalog No. CB105; Thermo Fisher Scientific Co., Ltd., Catalog No. EC0114) host cells, preferably C43 (DE3) host cells, and coated with LB solid medium (0.5% yeast extract, 1% tryptone, 1% NaCl) containing kanamycin at a final concentration of 50 μg / mL. The positive clones were expression strains C43 (DE3) / pET30-MLSC, C43 (DE3) / pET30-rAgSC, C43 (DE3) / pET30-P4SC.

[0068] (1.3) Expression and purification of recombinant SpyCatcher protein Recombinant bacteria C43(DE3) / pET30-MLSC, C43(DE3) / pET30-rAgSC, and C43(DE3) / pET30-P4SC monoclonal clones were picked and inoculated into LB medium containing kanamycin with a final concentration of 50 μg / mL, cultured at 37°C until OD600 was approximately 0.6, IPTG with a final concentration of 0.5 mM was added, and the temperature was lowered to 25°C for induction for 6h-20h.

[0069] Take 1 mL of each bacterial solution induced at 25°C for 20 h, centrifuge to obtain bacteria, suspend the bacteria in distilled water at a ratio of 1:30 (W / V), ultrasonically disrupt, centrifuge to separate the precipitate and supernatant, resuspend 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 a boiling water bath for 10 min, perform 15% SDS-PAGE electrophoresis, transfer to PVDF membrane after electrophoresis, transfer at 20 V constant voltage for 1 h, and detect with mouse Anti-His tag monoclonal antibody (Sigma, A7058). The results are as follows: Figure 1 shown.

[0070] After the expression was identified by SDS-PAGE and WB, the culture volume was expanded and the fatty acid-modified SpyCatcher protein was purified 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 bacteria, the bacteria 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), ultrasonically disrupted, centrifuged to obtain the supernatant, and purified using Chelating affinity chromatography.

[0072] First, rinse the column bed with a 0.5 M NaOH aqueous solution for at least 3 column volumes, then equilibrate with deionized water to neutral pH, then equilibrate with a 0.2 M NiSO4 aqueous solution for at least 3 column volumes, then equilibrate with Ni-B buffer (20 mM pH 7.5 Tris-HCl, 0.3 M NaCl, 500 mM imidazole + 1% Triton X100) for one column volume, and finally equilibrate with Ni-A1 buffer (20 mM pH 7.5 Tris-HCl + 0.3 M NaCl + 5 mM imidazole + 1% Triton X100) for at least 3 column volumes. The sample containing recombinant SpyCatcher was loaded onto a Chelating affinity chromatography column, and unbound proteins were washed away with Ni-A1 (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole + 1% Triton X100) buffer, and equilibrated for at least 5 column volumes; then eluted with 10%, 30%, and 100% Ni-B buffer; the sample eluted with 30% Ni-B buffer was collected to obtain a preliminary purified sample ( Figure 2 ).

[0073] Desalting with Sephadex G-25 Fine chromatography column. First, wash the column bed with 0.5M NaOH aqueous solution for 1 column volume, and then balance it with deionized water to neutral pH; then, balance it with 30Q-A1 buffer (20mM pH7.5 Tris-HCl, +1% Triton X100) for one column volume; finally, desalt the sample eluted with 30%B of Chelating affinity chromatography column, and the loading volume should not exceed 1 / 3 column volume.

[0074] The sample was purified using a SOURCE30Q anion exchange chromatography column (Φ1.6cm*15cm). First, the column bed was rinsed with a 0.5 M NaOH aqueous solution for at least 3 column volumes, and then equilibrated with deionized water to a neutral pH; then, equilibrated with 30Q-A1 buffer (20 mM pH7.5 Tris-HCl, + 1% Triton X100) for at least 3 column volumes; then, the desalted sample containing the recombinant SpyCatcher was loaded onto the SOURCE30Q anion exchange chromatography column, equilibrated with 30Q-A1 buffer for 10 column volumes, and then equilibrated with 30Q-A2 buffer (20 mM pH7.5 Tris-HCl, + 0.1% Tween 80) for 10 column volumes; finally, linearly eluted with 0-50% 30Q-B buffer (20 mM pH7.5 Tris-HCl + 1 M NaCl + 0.1% Tween 80); 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 (5mM pH7.4 PB + 0.9% NaCl) buffer, and 1 mL of the above SOURCE30Q purified sample was loaded for purification. Human IgG was used as a molecular weight control. From the gel chromatogram, it can be seen that the retention volumes of MLSC, rAgSC, and P4SC are between 10.1 mL and 10.3 mL, while the retention volume of IgG is 13.1 mL ( Figure 8 ), indicating that the molecular weight of the three recombinant proteins MLSC, rAgSC, and P4SC is greater than 150kDa, which is much larger than the 16kDa of the SC monomer. Therefore, it is judged that the three recombinant proteins MLSC, rAgSC, and P4SC exist in the form of polymers ( Figure 4 , 5 , 6, 7).

[0076] (1.4) Characterization of MLSC In order to further confirm whether the N-terminus of the purified recombinant SpyCatcher protein was modified by fatty acid, MLSC were used as the research object, and 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 after translation.

[0077] Beijing Mingde Zhengkang Technology Co., Ltd. was commissioned to use mass spectrometry (LC / Q-TOF-MS) ( Fig. 9 ) was used to detect the molecular weight of the recombinant SpyCatcher. The results showed that the molecular weight of the recombinant SpyCatcher was concentrated into two clusters. The first cluster was 14810.1 Da, 14824.2 Da and 14838.0 Da, and the second cluster was 14585.8 Da and 14600.0 Da. It is known 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). According to the technical solution of the present invention, it is inferred that the recombinant SpyCatcher with a molecular weight of 14810.1 Da is Pam3C-SpyCatcher, that is, SpyCatcher with fatty acid modification at the N-terminus, in which the thiol group of the Cys at the N-terminus is modified with diacylglycerol (C16:0, C16:0), and the α-amino group was modified with palmitic acid (C16:0); the molecular weight of the recombinant SpyCatcher with a molecular weight of 14824.2 Da increased by about 14 Da, which was consistent with the molecular weight of CH2, and it was speculated that one fatty acid chain was extended by one CH2; the molecular weight of the recombinant SpyCatcher with a molecular weight of 14838 Da increased by about 28 Da, which was consistent with the molecular weight of (CH2)2, and it was speculated that the fatty acid chain was extended by two CH2. The molecular weights of the second cluster peaks of 14585.8 Da and 14600.0 Da corresponded to the molecular weights of the first cluster peaks of 14824.2 Da and 14838.0 Da, and the molecular weights decreased by 238.4 Da and 238 Da, which were consistent with the molecular weight of palmitoylation (C16:0), that is, the SpyCatcher with fatty acid modification at the N-terminus, in which the thiol group of the N-terminal Cys was modified by diacylglycerol.

[0078] Based on the above calculations and the technical solution of the present invention, the N-terminus of the recombinant SpyCatcher prepared by the present invention is modified by lipidation, specifically, the thiol group of Cys is modified by diacylglycerol, and / or the α-amino group of Cys is modified by palmitoylation (C16:0), wherein the fatty acid chain structure of 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 virus S protein RBD containing SpyTag polypeptide The present invention takes the S protein RBD of the SARS-CoV-2 Delta mutant strain as an example to illustrate the antigen containing the SpyTag polypeptide.

[0080] (2.1) SARS-CoV-2 S protein RBD Delta -Construction of SpyTag yeast expression vector According to the amino acid sequence of SARS-CoV-2 Delta mutant strain S protein from position 319 to 534 with GenBank number OK091006.1, SpyTag sequence was added to its C-terminus, and Sangon Biotech (Shanghai) Co., Ltd. was commissioned to optimize and synthesize the DNA sequence according to the preferred codon of Pichia pastoris. The nucleotide sequence is shown in SEQ ID No. 24 and 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 transformation of Pichia pastoris strain CGMCC No.19488 The Pichia pastoris strain CGMCC No.19488 was streaked on a YPD plate to revive and isolate a single clone. The revived single clone was picked and inoculated into a YPD (1% yeast extract, 2% tryptone, 2% glucose) liquid medium. After the test tube culture reached its logarithmic phase, 1 mL was transferred to a 100 mL YPD shake flask and cultured at 25°C 200rpm shaking incubator until OD600 reached 1.3-1.5, and then quickly cooled on ice and centrifuged at 4°C 1500g × 5min. The bacteria were resuspended with an equal volume of pre-cooled distilled water and centrifuged at 4°C 1500g × 5min, and the supernatant was discarded. This step was repeated 3 times; then the bacteria were resuspended with an equal volume of pre-cooled 1M sorbitol and centrifuged at 4°C 1500g × 5min, and the supernatant was discarded. This step was repeated 3 times. The bacterial precipitate washed three times with distilled water and three times with sorbitol was suspended by adding 1 mL of 1 M sorbitol, and 100 μL of each was dispensed into sterile centrifuge tubes and stored at -80°C to obtain the electroporation transformation competent cells of Pichia pastoris strain CGMCC No. 19488.

[0082] The constructed expression plasmid pPICZαA-RBD Delta About 10 μg of ST was linearized by restriction endonuclease BglII. The restriction enzyme system (50 μL) was as follows: expression plasmid pPICZαA-RBD Delta ST 43μL, BglII 2μL, 10×NEB3.1buffer 5μL, digest at 37℃ for 1h, take samples, separate by 1% agarose gel electrophoresis, and analyze whether the plasmid is completely linearized. The separation results show that the linearized digestion product is completely linearized. Use a centrifugal column type DNA fragment recovery kit to recover the fragments, and finally elute the linearized plasmid with 25μL pure water.

[0083] Take the linearized expression plasmid pPICZαA-RBD Delta 15μL of ST was added to 100μL of Pichia pastoris strain CGMCCNo.19488 electroporation competent cells, mixed and transferred to a pre-cooled 0.2cm electroporation cup, and quickly added 900μL of pre-cooled 1M sorbitol after 2kV voltage electroporation, transferred to a clean test tube, and placed in a 25℃ incubator for 2 hours. Then, 1mL of YPD liquid culture medium without antibiotics was added, and the culture was placed at 25℃ and 200rpm for 3-4 hours. 300μL of the bacterial solution obtained by the above shaking culture was spread on a YPD plate with Zeocin resistance (100μg / mL) and incubated in a 25℃ incubator for 60-72h.

[0084] (2.3) Screening of recombinant expression strains After a single colony grows on the plate, pick one single colony and streak it onto a new YPD plate containing 100μg / mL Zeocin, and invert it in a 25℃ incubator. After the colony grows, inoculate it into 3mL of YPD liquid medium containing 100μg / mL Zeocin, and culture it at 25℃, 200rpm shaking for 72h, and transfer it to 3mL BMGY medium (1% yeast extract, 2% tryptone, 100mM PB6.5, 100mM YNB, 1% glycerol) according to the inoculum volume of 5% (V / V), and culture it at 25℃ 200rpm shaking. After 48 hours, add 0.5% (V / V) methanol every 12 hours for induction. After 48 hours of induction, collect the culture supernatant at 12000rpm for 3min.

[0085] The culture supernatant collected after 48 hours of methanol induction was screened by SDS-PAGE and Western Blot (WB). The Western Blot steps are as follows: (1) Separate the samples on 12% SDS-PAGE gel; (2) Transfer the samples on the SDS-PAGE gel to a PVDF membrane; (3) Block the PVDF membrane with the target protein transferred with 5% milk blocking solution for 1 hour at room temperature; (4) Transfer to 5% milk with a dilution of 1:2500 and incubate for 1 hour with an anti-His tag antibody (Sigma A7058); (5) Wash with PBST for 5 minutes and wash 5 times; (6) Wash with PBST for 5 minutes and wash 5 times; (7) Use Pro-light HRP Chemiluminescent colorimetric solution (Tiangen Biochemical, PA112-02) for color development.

[0086] (2.4) Recombinant strain CGMCC No.19488 / pPICZαA-RBD Delta ST Cultivation Pick the positive clones identified (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 was 15-20. Then, the inoculum was transferred to BMGY medium at a 5% (V / V) inoculum and fermented at 25°C and 200 rpm for 24 hours. RBD was induced by 0.5% (V / V) methanol. Delta The expression of ST was induced every 12 hours, and the culture supernatant was collected by centrifugation 72 hours after induction.

[0087] (2.5) RBD Delta Purification of ST 1. Capto MMC chromatography purification The pH of the culture supernatant after induced expression for 72 hours was adjusted to 5.5 and purified using Capto MMC (Cytiva, 17531710) chromatography medium. The mobile phase composition was: MMC-A: 20 mM pH 5.5 PB (phosphate buffer); MMC-B: 100mM pH8.5 Tris-HCl+1M NaCl.

[0088] After loading, balance with MMC-A and then elute with MMC-B.

[0089] 2. Phenyl FF low sub hydrophobic chromatography purification The Capto MMC purified samples were purified using Phenyl FF low sub (Cytiva, 28926988) with the following mobile phase components: Phenyl FF-A: 20 mM pH 7.5 Tris-HCl + 1 M AS (ammonium sulfate); Phenyl FF-B: 20mM p H7.5 Tris-HCl.

[0090] First, the target protein was eluted with 40% Phenyl FF-B, and then the impurity proteins were eluted with 40-100% Phenyl FF-B.

[0091] 3. Sephadex G-25 Fine desalting 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 samples were purified using SOURCE30Q (Cytiva, 17127502) chromatography medium with the mobile phase composition: 30Q-A: 20mM p H8.5 Tris-HCl; 30Q-B: 20mM pH8.5 Tris-HCl+1M NaCl.

[0093] After loading, balance with 30Q-A and then elute with 30Q-B. The target protein is in the flow-through.

[0094] 5. SOURCE 30S cation exchange chromatography The sample was eluted with SOURCE 30S and its pH was adjusted to 6.5 with HCl. It was then purified using a SOURCE 30S (Cytiva, 17127302) column with the following mobile phase components: 30S-A: 20 mM pH 6.5 PB; 30S-B: 20mM pH7.0 PB+1M NaCl.

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

[0096] 6. SuperdexTM 75 Increase10 / 300 GL gel filtration chromatography The SOURCE 30S sample was purified using SuperdexTM 75 Increase10 / 300 GL (Cytiva, 29148721) with the mobile phase composition of SEC: 5mM PB7.4 + 0.9%NaC1. After the sample loading was completed, the mobile phase was used for balance and the samples were collected in segments.

[0097] After the above purification steps, RBD was obtained Delta ST samples, purification process SDS-PAGE identification as Fig.10 .

[0098] Example 3: Preparation of fatty acid modified RBD nanoparticles 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)). Delta ST was used for protein quantification, and the mixture was mixed at a molar ratio of 2:1 and connected at 4°C for 12 h. A Superdex 200 Increase column (φ1×30 cm, cytiva, 28990944) was equilibrated with SEC200 (5mM pH7.0 PB + 0.9% NaCl) buffer, and 1 mL of the above connection product was taken and used to remove the unconnected RBD using Superdex 200 Increase. Delta ST, LipoSC-RBD Delta The retention volume of ST is 9.42 mL, which is slightly smaller than that of LipoSC ( Fig.11 ); SDS-PAGE reduction electrophoresis detection showed that ( Fig.12 ), RBD Delta ST is connected to LipoSC, with a molecular weight of about 43 kDa; TEM analysis shows that LipoSC-RBDDelta ST is nanoparticles ( Fig.13 ); DLS test showed that ( Fig.14 ), LipoSC-RBD Delta The ST diameter is about 13~20 nm (Number (Percent)). The purified LipoSC-RBD was purified by BCA method. Delta Protein quantification was performed using ST.

[0099] Example 4: Immunogenicity study of fatty acid modified RBD (4.1) Lung delivery immunization protocol The LipoSC-RBD obtained in Example 3 Delta ST prepared the vaccine with 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 to 8 weeks were immunized with 50 μL of vaccine delivered via the lungs on days 0, 14, and 28 (n=5). Blood was collected from the orbital venous 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 indirect ELISA method was used to measure the RBD in the serum of each group of mice Delta Specific IgG antibody titer. For the operation steps, please refer to the Concise Molecular Biology Experiment Guide [M]. Science Press, 2008.

[0101] The results are as follows Figure 5 Shown: Anti-RBD in the serum of mice immunized for two weeks Delta The specific IgG antibody titer was 1:371; RBD in the serum of mice immunized for two weeks Delta The specific IgG antibody titer was 1:407380; RBD in the serum of mice immunized for two weeks Delta The specific IgG antibody titer was 1:645654.

[0102] RBD in serum Delta The specific IgA antibody titer was tested. Fig.16 As shown, RBD in the serum of mice immunized for two weeks Delta The specific IgA antibody titer was 1:417, and the RBD in the serum of mice immunized for two weeks was 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 was collected from mice to detect RBD in bronchoalveolar lavage fluid (BALF). Delta Specific IgG titer ( Fig.17 )、IgA titer( Fig.18 ). RBD Delta The specific IgG antibody titer was 1:6026, and the specific IgA titer was 1:3162.

[0104] (4.3) Pseudovirus neutralization assay to detect serum neutralizing antibody titers in BALB / c mice after lung delivery immunization 1) Cell plating: Digest 293T-ACE2 cells with 1 mL of trypsin, add 3 mL of culture medium, centrifuge at 1000 rpm for 5 min, resuspend with 1 mL of culture medium, and dilute the cells to 2×10 after cell counting. 5 Add 100 μL of cells to each well to make each well contain 2×10 cells / mL. 4 cell / well and culture overnight.

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

[0106] 3) In addition to the cell control CC which was added with 150 μL of culture medium, 100 μL of culture medium was added to each well, 144 μL of culture medium was added to the first well, 6 μL of serum / alveolar lavage fluid from each group was added (i.e., 1:25 dilution), and 50 μL was taken from the first well and diluted downward threefold.

[0107] 4) Dilute the pseudovirus to 1.34×10 4 TCID50 / mL, 50 μL / well was added except for the cell control, and the mixture was shaken for 30 s before placing the 96-well plate in a cell culture incubator (37 °C, 5% CO2) and incubating for 1 h.

[0108] 5) The culture medium of the 96-well plate containing cells incubated overnight was discarded, 120 μL of the serum-virus mixture was transferred thereto, and incubated at 37° C., 5% CO 2 .

[0109] 6) After 8 h of culture, discard the culture medium in the 96-well plate, add 150 μL / well of DMEM complete culture medium, and incubate at 37°C, 5% CO2 for 48 h.

[0110] 7) After the incubation, discard 100 μL of supernatant, add 100 μL of Bright-Glo luciferase detection reagent (Vazyme, DD1204-02), shake for 2 minutes, react at room temperature in the dark for 5 minutes, repeatedly pipette and transfer 100 μL of liquid to an opaque white plate (Perkins Elmer, 6005290).

[0111] 8) The luminescence value (RLU) was read using a PerkinElmer EnSight multi-function imaging microplate reader.

[0112] Neutralizing antibody titers were expressed as the reciprocal of the serum dilution that gave a 50% inhibition rate or as the antibody concentration that gave a 50% inhibition rate.

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

[0114] (4.4) Evaluation of the immunogenicity and protective effect of lipidated RBD by intramuscular immunization The RBD obtained in Examples 2 and 3 Delta ST and LipoSC-RBD Delta ST prepares the vaccine with saline according to the concentration so that 100 μL volume contains an equal mass of RBD Delta ST (5 μg), or combined with Al(OH)3 adjuvant (100 μg) and / or CpG (50 μg) adjuvant. Female BALB / c mice aged 6 to 8 weeks were immunized by intramuscular injection on days 0 and 14 (n=5). Blood was collected from the orbital venous sinus of the immunized mice two weeks after the first and second immunizations.

[0115] According to the above method, RBD in serum was detected Delta ST-specific IgG antibody titers and neutralizing antibody titers. The results showed that when non-lipidated RBD Delta When ST was combined with Al(OH)3 (100 μg) and CpG (50 μg) as double adjuvants to immunize mice, RBD Delta The ST-specific IgG binding antibody titer was 1:3715, and the neutralizing antibody titer was 1:44; when lipidated RBD DeltaST (LipoSC-RBD Delta When mice were immunized with ST, RBD Delta The ST-specific IgG binding antibody titer was 1:2754, and the neutralizing antibody titer was 1:47; when lipidated RBD was used Delta ST(LipoSC-RBD Delta When ST) and Al(OH)3 (100 μg) were combined to immunize mice, RBD Delta The ST-specific IgG binding antibody titer was 1:8511, and the neutralizing antibody titer was 1:182; the specific results are as follows Fig.21 , 22 shown.

[0116] In summary, the fatty acid-modified coronavirus S protein RBD antigen provided by the present invention has good immunogenicity, especially after lung delivery immunization, it can induce mice to produce RBD-specific sIgA, 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 According to the P1 protein sequence of human Mycoplasma pneumoniae M29 strain published in GenBank (GenBank: U00089.2, the amino acid sequence is 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 according to the preferred codons of Escherichia coli by Shanghai Biotechnology Co., Ltd.

[0118] The optimized pelB-P1ST nucleotide sequence is shown in SEQ ID No. 33, wherein nucleotides 1 to 6 from the 5' end are NdeⅠ restriction site sequences, nucleotides 4 to 69 are pelB signal peptide coding sequences, nucleotides 70 to 75 are linker coding sequences, nucleotides 76 to 4554 are P1 protein coding sequences, nucleotides 4555 to 4578 are linker coding sequences, nucleotides 4579 to 4617 are SpyTag polypeptide coding sequences, nucleotides 4618 to 4629 are linker coding sequences, nucleotides 4630 to 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 expression vector pET30-pelBMpP1ST constructed above was introduced into the host cells of Escherichia coli BL21 (DE3) (purchased from Tiangen Biochemical Technology Co., Ltd., Catalog No. CB105; Thermo Fisher Scientific Inc., Catalog No. EC0114), and LB solid culture medium containing kanamycin at a final concentration of 50 μg / mL was applied. The positive clone was the expression strain BL21 (DE3) / pET30-pelBMpP1ST.

[0120] (5.3) Expression and purification of Mycoplasma pneumoniae P1ST protein The recombinant bacteria BL21 (DE3) / pET30-pelBMpP1ST single clone was picked and inoculated into LB medium containing kanamycin with a final concentration of 50 μg / mL. It was cultured at 37 ° C until OD600 was about 0.6, and IPTG with 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 next day, 1 mL of each bacterial solution induced at 20°C was taken, and the cells were centrifuged and suspended in distilled water at a ratio of 1:30 (W / V). The cells were ultrasonically disrupted, and the precipitate and supernatant were separated by centrifugation. The precipitate was resuspended in an equal volume of water, and 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). The sample was prepared by boiling water bath for 10 min, and 15% SDS-PAGE electrophoresis was performed. After the electrophoresis was completed, the cells were transferred to a PVDF membrane and transferred at a constant voltage of 20 V for 1 h. The cells were detected with mouse Anti-His tag monoclonal antibody (Sigma, A7058). The results are shown as follows: Fig.23 shown.

[0122] After the 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 the bacterial cells were harvested, they were suspended in Ni-A2 buffer (20mM pH7.5 Tris-HCl + 0.3M NaCl + 5mM imidazole) at a ratio of 1:20 (W / V), ultrasonically disrupted, centrifuged to obtain the supernatant, and purified using Chelating affinity chromatography.

[0124] First, the column bed was rinsed with a 0.5 M NaOH aqueous solution for at least 3 column volumes, then equilibrated with deionized water to neutral pH, then equilibrated with a 0.2 M NiSO4 aqueous solution for at least 3 column volumes, then equilibrated with Ni-B2 buffer (20 mM pH 7.5 Tris-HCl, 0.3 M NaCl, 500 mM imidazole) for one column volume, and finally equilibrated with Ni-A2 buffer (20 mM pH 7.5 Tris-HCl + 0.3 M NaCl + 5 mM imidazole) for at least 3 column volumes. The supernatant containing P1 protein was loaded onto a Chelating affinity chromatography column, unbound proteins were washed away with Ni-A2 buffer, and then eluted with 10%, 30%, and 100% Ni-B2 buffer; the sample eluted with 30% Ni-B buffer was collected to obtain a preliminary purified sample, which was further purified using a Superdex 200 increase chromatography column (φ1×30 cm, cytiva, 28990944) ( Fig.24 ).

[0125] Example 6: Preparation of fatty acid-modified MpP1 nanoparticles The MpP1ST prepared in Example 5 was quantified by BCA method (Thermo scientific, Cat. No. A55864, Related BCA Kits: 23225 (1000 mL)), mixed at a molar ratio of 2:1, and connected at 4°C for 12 h ( Fig.25 ). A Superdex 200Increase column (φ1×30 cm, cytiva, 28990944) was equilibrated with SEC200 (5 mM pH7.0 PB + 0.9% NaCl) buffer, and 1 mL of the above ligation product was taken and the unligated MpP1ST was removed using Superdex 200Increase.

[0126] Additionally, a description is given below of a fatty acid-modified SpyCatcher protein and a method for preparing the fatty acid-modified SpyCatcher protein.

[0127] The fatty acid-modified SpyCatcher protein comprises a fatty acid-modified N-terminal cysteine ​​(Cys) and a SpyCatcher protein; the fatty acid-modified N-terminal cysteine ​​(Cys) is characterized in that the thiol group of Cys is modified by diacylglycerol, or the thiol group of Cys is modified by diacylglycerol and the α-amino group is modified by acyl group; wherein the carbon chain length of the acyl group is C16 to 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 a purification tag sequence, such as a His-Tag tag, for ease of purification.

[0128] The method for preparing the fatty acid-modified SpyCatcher protein provided by the present invention comprises the following steps: Step S1, introducing a DNA fragment containing a signal peptide coding sequence and a SpyCatcher protein coding sequence into Escherichia coli, and culturing the recombinant Escherichia coli to express the fatty acid-modified SpyCatcher protein; In the above step S1, the signal peptide is any one of the amino acid sequences shown in SEQ ID No.4, SEQ ID No.5, and 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 Escherichia coli through a recombinant expression vector, and 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 linker coding sequences, positions 73 to 102 are His tag coding sequences, positions 103 to 450 are SpyCatcher tag coding sequences, 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 linker coding sequences, positions 61 to 90 are His tag coding sequences, positions 91 to 438 are SpyCatcher tag coding sequences, and positions 442 to 448 are NotⅠ restriction sites; In SEQ ID No.9, positions 1 to 6 are NdeⅠ restriction site, positions 4 to 66 are the signal peptide coding sequence shown in SEQ ID No.6, positions 67 to 72 are linker coding sequence, positions 73 to 102 are His tag coding sequence, positions 103 to 450 are SpyCatcher tag coding sequence, and positions 454 to 461 are NotⅠ restriction site.

[0129] The obtained recombinant expression vector is transformed into Escherichia coli by electroporation or chemical transformation to obtain recombinant engineered Escherichia coli; The DNA fragment containing the signal peptide coding sequence and the SpyCatcher protein coding sequence can also be integrated into the Escherichia coli genome; Step S2, culturing the recombinant E. coli described in step S1), harvesting and lysing the cells, adding an appropriate surfactant, and extracting and purifying the fatty acid modified SpyCatcher protein from the lysate. The recombinant E. coli lysis can be performed by ultrasound or high pressure homogenizer to break up the bacteria, and the method for extracting and purifying the fatty acid modified SpyCatcher protein from the lysate can be performed by precipitation, centrifugation, and further purification by multiple chromatography to obtain the fatty acid modified SpyCatcher protein; The chromatography purification method in step S2 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] The above preparation method is used to prepare a recombinant fusion protein containing a fatty acid modified SpyCatcher protein, and the recombinant fusion protein containing a fatty acid modified SpyCatcher protein can also contain pathogen T cell epitopes and / or B cell epitopes, etc. The antigen containing the SpyTag polypeptide is an antigen containing a SpyTag polypeptide and an antigen fused 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 includes the SpyCatcher protein shown in SEQ ID No.1, the SpyTag polypeptide shown in SEQ ID No.10 should be selected; when the fatty acid-modified SpyCatcher protein includes the SpyCatcher protein shown in SEQ ID No.2, the SpyTag polypeptide shown in SEQ ID No.11 should be selected; when the fatty acid-modified SpyCatcher protein includes the SpyCatcher protein shown in SEQ ID No.3, the SpyTag polypeptide shown in SEQ ID No.12 should be selected.

[0131] The antigen fused with the SpyTag polypeptide may be a viral antigen; The viral antigen is a respiratory virus antigen, specifically a coronavirus antigen; the coronavirus antigen is a spike protein (S protein) or a functional fragment thereof, and the S protein functional fragment is preferably a receptor binding domain (RBD), or an RBD concatemer; 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, and the SARS-CoV-2 RBD includes the domains in the SARS-CoV-2 S protein prototype and its various variants that can bind to the ACE2 receptor, 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 SEQID 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, including domains in the SARS-CoV S protein prototype and its various variants 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 domains in the MERS-CoV S protein prototype and its various variants that can bind to the DPP4 receptor, one of which has an amino acid sequence as shown in SEQ ID No.21.

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

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

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

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

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

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

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

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

[0142] The viral antigen can be expressed in Escherichia coli, mammalian cells, yeast cells or insect cells; preferably, yeast cells are used for expression. The specific technical scheme can be prepared by referring to the method described in Chinese patent application CN105671109A; specifically, RBD DeltaFor example, the step S1) constructs RBD Delta -SpyTag yeast recombinant expression vector; 2) electroporate the recombinant expression vector into Pichia pastoris strain CGMCC No.19488; 3) screen the recombinant expression strain; 4) expand the positive high-expression recombinant strain; 5) purify RBD by cation chromatography, hydrophobic chromatography, anion chromatography, etc. Delta -SpyTag.

[0143] The antigen fused with the SpyTag polypeptide for expression may be a Mycoplasma pneumoniae protein antigen; The Mycoplasma pneumoniae protein antigen can be Mycoplasma pneumoniae P1 protein and its partial fragments, or P40 / P90 protein and its partial fragments. The P1 protein and P40 / P90 protein of Mycoplasma pneumoniae vary greatly in sequence with different strains, but their structures and functions are similar. The P1 protein or P40 / P90 protein of the present invention includes these variants. Sequence 31 and sequence 32 are respectively a typical amino acid sequence of P1 protein and P40 / P90 protein.

[0144] The present invention also provides a method for preparing an immune composition containing a fatty acid-modified SpyCatcher protein and the SpyTag polypeptide antigen, wherein the fatty acid-modified SpyCatcher protein and the SpyTag polypeptide antigen are mixed in a certain molar ratio; and an immune composition in which the fatty acid-modified SpyCatcher protein and the SpyTag polypeptide antigen are covalently linked via an isopeptide bond is prepared. The method for preparing an immune composition in which the fatty acid-modified SpyCatcher protein and the SpyTag polypeptide antigen are covalently linked via an isopeptide bond may include purifying the final product by molecular exclusion chromatography after sufficient reaction to obtain an immune composition; the immune composition may also be used in combination with an aluminum adjuvant, an oil-in-water emulsion, a CpG adjuvant, a TLR4 agonist, or the like.

[0145] Furthermore, an injectable vaccine containing a fatty acid-modified SpyCatcher protein and the immune composition containing the SpyTag polypeptide antigen also falls within the scope of protection of the present invention; the immune composition can induce antigen-specific humoral immunity in animals by intramuscular or subcutaneous injection.

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

[0147] The use of any of the above-mentioned immune compositions in the preparation of products capable of inducing the production of antigen-specific antibodies in animals also falls within the scope of protection of the present invention; The use of any of the above-mentioned immune compositions in the preparation of products for preventing and / or treating diseases caused by corresponding pathogens also falls within the scope of protection of the present invention.

[0148] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fatty acid-modified SpyCatcher protein.

2. The fatty acid-modified SpyCatcher protein according to claim 1, characterized in that: It contains a fatty acid-modified N-terminal cysteine ​​(Cys).

3. The fatty acid-modified SpyCatcher protein according to claim 2, characterized in that The fatty acid-modified N-terminal cysteine ​​(Cys) specifically refers to a Cys thiol group modified by diacylglycerol, or a Cys thiol group modified by diacylglycerol and an α-amino group modified by an acyl group.

4. The fatty acid-modified SpyCatcher protein according to claim 3, characterized in that The carbon chain length of the fatty acyl group is C16~C18.

5. The fatty acid-modified SpyCatcher protein according to any one of claims 1 to 4, characterized in that: The SpyCatcher protein is a polypeptide comprising an amino acid sequence as shown in any one of SEQ ID No. 1-3.

6. An immune composition comprising the fatty acid-modified SpyCatcher protein according to any one of claims 1 to 4.

7. The immune composition according to claim 6, characterized in that The immune composition also comprises an antigen expressed by fusion with the SpyTag polypeptide.

8. The immune composition according to claim 6, characterized in that The fatty acid-modified SpyCatcher protein is covalently linked to the antigen expressed by fusion with the SpyTag polypeptide via an isopeptide bond.

9. The immune composition according to claim 8, characterized in that The amino acid sequence of the SpyTag polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID No. 10-12.

10. The immune composition according to any one of claims 6 to 9, characterized in that: The antigen fused with the SpyTag polypeptide for expression is a respiratory virus antigen.

11. The immune composition according to any one of claims 6 to 10, characterized in that: The respiratory virus antigens include coronavirus antigens.

12. The immune composition according to claim 11, characterized in that The coronaviruses include SARS-CoV-2, SARS-CoV, and MERS-CoV.

13. The immune composition according to claim 11, characterized in that The coronavirus antigen is a spike protein (S protein) and / or a functional fragment thereof.

14. The immune composition according to claim 13, characterized in that The functional fragment of the S protein is preferably a receptor binding domain (RBD).

15. The immune composition according to claim 14, characterized in that The SARS-CoV-2 RBD includes a domain in the SARS-CoV-2 S protein prototype and its various variants that can bind to the ACE2 receptor.

16. The immune composition according to claim 14, characterized in that The SARS-CoV S protein RBD includes a domain in the SARS-CoV S protein prototype and its various variants that can bind to the ACE2 receptor.

17. The immune composition according to claim 14, characterized in that The MERS-CoV S protein RBD includes a structural domain in the MERS-CoV S protein prototype and its various variants that can bind to the DPP4 receptor.

18. The immune composition according to any one of claims 15 to 17, characterized in that: The amino acid sequence of RBD is shown in any one of SEQ ID No. 13-21.

19. The immune composition according to any one of claims 6 to 9, characterized in that: The antigen fused with the SpyTag polypeptide for expression is a mycoplasma protein antigen.

20. The immune composition according to claim 19, characterized in that The mycoplasma protein antigen is specifically Mycoplasma pneumoniae P1 protein and partial fragments thereof, or P40 / P90 protein and partial fragments thereof.

21. The immune composition according to claim 20, characterized in that The amino acid sequence of the Mycoplasma pneumoniae P1 protein is shown in SEQ ID No.

31.

22. The immune composition according to claim 20, characterized in that The amino acid sequence of the Mycoplasma pneumoniae P40 / P90 protein is shown in SEQ ID No.

32.

23. A method for preparing a fatty acid-modified SpyCatcher protein according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: 1) Introducing a DNA fragment containing a signal peptide coding sequence and a SpyCatcher protein coding sequence into Escherichia coli, and culturing the recombinant Escherichia coli to express the fatty acid-modified SpyCatcher protein; 2) Cultivating the recombinant E. coli described in step 1), harvesting and lysing the cells, and adding an appropriate surfactant to extract and purify the fatty acid-modified SpyCatcher protein from the lysate.

24. The preparation method according to claim 23, characterized in that: The signal peptide has an amino acid sequence as shown in any one of SEQ ID No. 4-6.

25. The preparation method according to claim 23, characterized in that: The amino acid sequence of the fatty acid-modified SpyCatcher protein is shown in SEQ ID No.

36.

26. Use of the fatty acid modified SpyCatcher protein according to any one of claims 1 to 4 in the preparation of a product capable of inducing animals to produce antigen-specific antibodies, wherein the immune composition can be administered by intramuscular injection, subcutaneous injection, nasal drops, or aerosol immunization.

27. The use according to claim 26, characterized in that The product is a vaccine.

28. Use of the immune composition according to any one of claims 5 to 22 in the preparation of a product capable of inducing an animal to produce antigen-specific antibodies, wherein the immune composition can be administered by intramuscular injection, subcutaneous injection, nasal drops, or aerosol injection.

29. The use according to claim 28, characterized in that The product is a vaccine.

30. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the fatty acid-modified SpyCatcher protein according to any one of claims 1 to 4.

31. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a nucleic acid encoding the fatty acid-modified SpyCatcher protein according to any one of claims 1 to 4.

32. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a vector encoding the fatty acid-modified SpyCatcher protein according to any one of claims 1 to 4.

33. Biomaterial, characterized in that The biological material is any of the following: C1) a nucleic acid molecule encoding the fatty acid-modified SpyCatcher protein according to any one of claims 1 to 4; 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) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) A recombinant cell containing the nucleic acid molecule described in C1), or a recombinant cell containing the expression cassette described in C2), or a recombinant cell containing the recombinant vector described in C3).

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