Fatty acid modified coronavirus antigen and application thereof

Abstract

The invention discloses a fatty acid modified coronavirus antigen and application thereof, and belongs to the field of biological medicine. The fatty acid modified coronavirus antigen contains fatty acid modified N-terminal cysteine (Cys) and a coronavirus antigen, and can contain a connecting structural domain; the coronavirus antigen is a coronavirus spike protein (S protein) receptor binding domain (RBD). The fatty acid modified coronavirus S protein RBD can be self-polymerized into nanoparticles, and compared with the RBD which is not subjected to fatty acid modification, the antigen can induce faster and higher humoral immunity through injection immunity, can induce mucosal immunity and humoral immunity through respiratory tract atomization immunity, and is a candidate mucosal vaccine with potential medical value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a fatty acid-modified coronavirus antigen and an application thereof. Background Art

[0002] 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 SARS-CoV-2 virus at the end of 2019, there are five major types of variants (VOCs) identified by the World Health Organization as 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. However, RBD has a small molecular weight and relatively low immunogenicity, which hinders the development of subunit vaccines with RBD as an antigen, and mucosal vaccines with RBD as an antigen are even more difficult.

[0003] 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.

[0004] Displaying antigens on the surface of protein nanocarriers is an effective means to improve the immunogenicity of antigens. The preparation scheme (modular preparation scheme) of expressing and preparing nanoparticles and loaded antigens separately and connecting the carrier and loaded antigens through a universal, efficient and controllable connection method can effectively solve the problems of low expression efficiency, particle assembly failure, structural instability and loss of immunogenicity caused by the fusion expression of carrier and antigen. 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 advantages such as 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, which improves the presentation efficiency and immunogenicity of antigens. This connection system originates from the CnaB2 domain of the fibronectin-binding protein of Streptococcus pyogenes. Lys31 and Asp117 of the CnaB2 domain can form an intramolecular isopeptide bond. After splitting the CnaB2 domain 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 connection 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 connection reaction, the team artificially designed the SpyTag (13aa) / SpyCatcher (116aa) connection system and developed the SpyTag002 / SpyCatcher002 and SpyTag003 / SpyCatcher003 connection systems.

[0005] In the prior art, displaying protective antigens on the surface of protein nanoparticles, and / or physically mixing or embedding adjuvants, and using viral vectors to deliver antigens are effective means to improve the immunogenicity of mucosal vaccines. However, the pre-existing immunity problems caused by the vectors, the imbalance of immune focus, and the safety problems caused by excessive use of adjuvants have plagued the development of mucosal vaccines. Therefore, it is necessary to develop a new strategy that can reduce the immunogenicity of the carrier protein 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

[0006] In order to address the deficiencies of the prior art, the present invention provides a fatty acid-modified coronavirus antigen and application thereof.

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

[0008] The fatty acid-modified N-terminal cysteine ​​(Cys) specifically refers to the thiol group of Cys being modified by diacylglycerol, or the thiol group of Cys being modified by diacylglycerol and the α-amino group being modified by a fatty acyl group.

[0009] The coronavirus includes but is not limited to SARS-CoV-2, SARS-CoV or MERS-CoV.

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

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

[0012] In the SARS-CoV and SARS-CoV-2 coronavirus antigens, the receptor binding domain RBD includes the region where the SARS-CoV-2 and SARS-CoVS proteins bind to ACE2 and their variants, and its amino acid sequence is not limited to any of the sequences described in SEQ ID No. 1-9; In the MERS-CoV coronavirus antigen, the receptor binding domain RBD includes the region where the MERS-CoV S protein binds to DPP4 or a variant thereof; its amino acid sequence includes but is not limited to any sequence described in SEQ ID No.10.

[0013] The fatty acid-modified coronavirus antigen provided by the present invention may further contain a linker domain.

[0014] The connecting domain is an isopeptide bond connector, preferably a SpyCatcher / SpyTag connector.

[0015] The coronavirus antigen is expressed by fusion with the SpyTag polypeptide and covalently linked to the fatty acid-modified SpyCatcher protein via an isopeptide bond; or, the coronavirus antigen is expressed by fusion with the SpyCatcher protein and covalently linked to the fatty acid-modified SpyTag polypeptide via an isopeptide bond.

[0016] The SpyCatcher protein is a polypeptide containing any one of the amino acid sequences of SEQ ID No.11-13, and the amino acid sequence of the SpyTag is shown in any one of SEQ ID No.14-16.

[0017] When the SpyCatcher protein comprises the amino acid sequence shown in SEQ ID NO.11, the SpyTag polypeptide contained in the corresponding fatty acid-modified recombinant RBD protein preferably comprises the amino acid sequence shown in SEQ ID NO.14.

[0018] When the SpyCatcher protein comprises the amino acid sequence shown in SEQ ID NO.12, the SpyTag polypeptide contained in the corresponding fatty acid-modified recombinant RBD protein preferably comprises the amino acid sequence shown in SEQ ID NO.15.

[0019] When the SpyCatcher protein comprises the amino acid sequence shown in SEQ ID NO.13, the SpyTag polypeptide contained in the corresponding fatty acid-modified recombinant RBD protein preferably comprises the amino acid sequence shown in SEQ ID NO.16.

[0020] According to the second aspect of the technical solution of the present invention, a method for preparing the above-mentioned fatty acid-modified coronavirus antigen is provided, and the specific implementation steps are as follows: 1) Introducing a DNA fragment containing a signal peptide coding sequence and a coronavirus antigen coding sequence into Escherichia coli, and culturing the recombinant Escherichia coli to express a recombinant coronavirus antigen with a fatty acid modification at the N-terminus; 2) culturing the recombinant Escherichia coli described in step 1) to express the fatty acid-modified recombinant coronavirus antigen; 3) Lysing the recombinant Escherichia coli, adding a surfactant, and extracting and purifying the fatty acid-modified recombinant coronavirus antigen from the lysate; SARS-CoV-2 RBD JN.1 For example, the fatty acid modified RBD Delta The nucleotide sequence is shown as SEQ ID No.23.

[0021] Alternatively, a method for preparing a fatty acid-modified coronavirus antigen containing a linker is provided, the method comprising: a) introducing a DNA fragment containing a signal peptide coding sequence and a SpyCatcher protein coding sequence into Escherichia coli, culturing the recombinant Escherichia coli to express a recombinant SpyCatcher protein with a fatty acid modification on the N-terminus; lysing the recombinant Escherichia coli, adding a surfactant, and extracting and purifying the fatty acid modified recombinant SpyCatcher protein from the lysate. The nucleotide sequence of the fatty acid modified recombinant SpyCatcher protein is shown in SEQ ID No. 20.

[0022] b) Introducing a DNA fragment containing a signal peptide coding sequence, a SpyTag polypeptide coding sequence, and a coronavirus antigen coding sequence into an engineered cell, culturing a recombinant cell line to express a coronavirus antigen containing a SpyTag polypeptide, and purifying the coronavirus antigen containing a SpyTag polypeptide; the cell line is a mammalian cell, an insect cell, or a yeast cell. Delta For example, the amino acid sequence of the coronavirus antigen containing the SpyTag polypeptide is shown in SEQ ID No. 26.

[0023] 3) The purified fatty acid-modified recombinant SpyCatcher protein is mixed with a coronavirus antigen containing a SpyTag polypeptide, and the mixture is fully reacted under certain conditions to obtain a fatty acid-modified coronavirus antigen.

[0024] In the above-mentioned preparation method, the signal peptide amino acid sequence is any one of SEQ ID No.17-19.

[0025] According to the third aspect of the technical solution of the present invention, an application of the fatty acid-modified coronavirus antigen as described above is provided, wherein the application is for preparing a vaccine or screening a drug for treating coronavirus.

[0026] According to the fourth aspect of the technical solution of the present invention, a vaccine containing the above-mentioned fatty acid-modified coronavirus antigen is provided. The vaccine is a respiratory mucosal immune vaccine or an injectable vaccine.

[0027] According to the fifth aspect of the technical solution of the present invention, a pharmaceutical composition is provided, which comprises the above-mentioned fatty acid-modified coronavirus antigen.

[0028] According to the sixth aspect of the technical solution of the present invention, a pharmaceutical composition is provided, which comprises a nucleic acid encoding the above-mentioned fatty acid-modified coronavirus antigen.

[0029] According to the seventh aspect of the technical solution of the present invention, a pharmaceutical composition is provided, comprising a vector encoding the above-mentioned fatty acid-modified coronavirus antigen.

[0030] According to an eighth aspect of the technical solution of the present invention, a biomaterial is provided, wherein the biomaterial is any one of the following: C1) a nucleic acid molecule encoding a fatty acid-modified coronavirus antigen according to any one of claims 1 to 14; 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).

[0031] According to the ninth aspect of the technical solution of the present invention, a method for preparing a vaccine using the above-mentioned fatty acid-modified coronavirus antigen is provided.

[0032] According to the tenth aspect of the technical solution of the present invention, a method for preparing a vaccine using the above-mentioned biological material is provided.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The fatty acid-modified coronavirus RBD antigen disclosed in the present invention can produce high-titer IgG binding antibodies and neutralizing antibodies in animals after a single intramuscular injection. In animals vaccinated with respiratory tract vaccines, high-titer RBD-specific IgG, IgA binding antibodies and neutralizing antibodies can be detected in the bronchoalveolar lavage fluid (BALF) after three immunizations. Compared with existing coronavirus protein antigens, this antigen not only has the effect of inducing faster and higher immune protection by conventional intramuscular injection, but also can induce respiratory mucosal immunity, providing a basis for blocking the infection and spread of coronavirus at the infection site and forming all-round immune protection; in addition, respiratory mucosa as a non-invasive vaccination method can improve the compliance of the audience and is suitable for large-scale vaccination during pandemics.

[0034] 2. The fatty acid-modified coronavirus antigen of the present invention is a fatty acid-modified coronavirus RBD antigen, including two structures of directly fatty acid-modified RBD and displaying RBD on a fatty acid-modified SpyCatcher protein through a SpyCatcher / SpyTag connector. The fatty acid-modified coronavirus RBD antigen provided by the present invention can be polymerized into nanoparticles, which can induce a faster and higher RBD-specific antibody response, especially as the main component of an inhaled vaccine, which can induce specific IgG, IgA and sIgA antibodies, significantly improving the mucosal immune effect, and has a very high practical application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the plasmid map of the pET30-MLSARS2RBD expression vector; Figure 2 SDS-PAGE and Western blot expression verification of lipidated MLSARS2RBD protein; Samples 1-6: monoclonal clones 1-6; Figure 3 The lipidated MLSARS2RBD protein was purified by Chelating affinity chromatography column; A: Chromatogram of MLSARS2RBD protein purified by Chelating affinity chromatography column; B: SDS-PAGE of MLSARS2RBD protein purification, lane 1: 1% Triton dissolved sample, 2: dissolved sample loading, 3: through 1, 4: through 2, 5: 2.5% B eluted sample, 6: 15% B eluted sample, 7: 50% B eluted sample, 8: 100% B eluted sample, 9: 0.5M NaOH eluted sample; Figure 4 The lipidated MLSARS2RBD protein was purified by Superdex 200 size exclusion chromatography; Lanes 1-8: elution samples at different time points; Figure 5 It is the MALDI-TOF detection spectrum of lipidated MLSARS2RBD protein; Figure 6 This is the plasmid map of the pET30-MLSARSRBD expression vector; Figure 7 The expression of MLSARSRBD protein was verified by SDS-PAGE and Western blot; A: 1-4 is the SDS-PAGE of 1-4 C43DE3 / pET30-MLSARSRBD monoclonal bacterial solution, B: 1-4 is the Western blot of 1-4 C43DE3 / pET30-MLSARSRBD monoclonal bacterial solution; Figure 8 Purification of lipidated MLSARSRBD protein by Chelating affinity chromatography column; 1: C43DE3 / pET30-MLSARSRBD bacterial solution, 2: bacterial solution ultrasonic sample, 3: bacterial solution ultrasonic precipitation, 4: bacterial solution ultrasonic supernatant, 5: PEG20000 precipitation supernatant, 6: 1% Triton dissolved sample, 7: eluted sample 1, 8: eluted sample 2; Fig. 9 This is the plasmid map of the pET30-MLMERSRBD expression vector; Fig.10The expression verification of MLMERSRBD protein by SDS-PAGE and Western blot; A: 1-4 is SDS-PAGE of 1-4 C43DE3 / pET30-MLSARSRBD monoclonal bacterial solution, 5-7 is SDS-PAGE of 5-7 C43DE3 / pET30-MLMERSRBD monoclonal bacterial solution. B: 1-4 is Western blot of 1-4 C43DE3 / pET30-MLSARSRBD monoclonal bacterial solution, 5-7 is Western blot of 5-7 C43DE3 / pET30-MLMERSRBD monoclonal bacterial solution; Fig.11 Purification of lipidated MLMERSRBD protein by Chelating affinity chromatography column; 1: C43DE3 / pET30-MLMERSRBD bacterial solution, 2: bacterial solution ultrasonic sample, 3: bacterial solution ultrasonic precipitation, 4: bacterial solution ultrasonic supernatant, 5: PEG20000 precipitation supernatant, 6: 1% Triton dissolved sample, 7: eluted sample 1, 8: eluted sample 2; Fig.12 This is the SDS-PAGE identification diagram of the recombinant SpyCatcher protein expression containing different signal peptides; Fig.13 This is the SDS-PAGE identification image of lipidated SpyCatcher (SC) Ni affinity chromatography purification; Fig.14 This is the SDS-PAGE identification diagram of lipidated SpyCatcher (SC) purified by anion exchange chromatography; Fig.15 This is the SDS-PAGE identification diagram of lipidated SpyCatcher (SC) molecular exclusion chromatography purification; Fig.16 The molecular exclusion chromatogram of SpyCatcher (SC) with ML lipidated signal peptide; Fig.17 The molecular exclusion chromatogram of SpyCatcher (SC) with rAg lipidation signal peptide; Fig.18 The molecular exclusion chromatogram of SpyCatcher (SC) with P4 lipidated signal peptide; Fig.19 This is the size exclusion chromatography of human IgG molecules; Fig. 20 Deconvolution plot for lipidated SpyCatcher (SC) mass spectrometry detection; Fig.21 The fusion protein of SARS-CoV-2 Delta mutant RBD and SpyTag (RBD DeltaST) SDS-PAGE identification of purified samples; Fig. 22 LipoSC-RBD Delta ST molecular size exclusion chromatography; Fig.23 LipoSC-RBD Delta ST molecular size exclusion chromatography purification SDS-PAGE identification diagram; Fig.24 LipoSC-RBD Delta Transmission electron microscopy (TEM) identification image of ST; Fig.25 LipoSC-RBD Delta ST dynamic light scattering (DLS) identification diagram; Fig.26 Serum RBD of BALB / c mice before, 2 weeks after, 4 weeks after, and 6 weeks after immunization was delivered to the lungs Delta Specific IgG titer; Fig. 27 Serum RBD of BALB / c mice before, 2 weeks after, 4 weeks after, and 6 weeks after immunization was delivered to the lungs Delta Specific IgA titer; Fig.28 BALB / c mice bronchoalveolar lavage fluid (BALF) RBD was delivered to the lungs 2 weeks after triple immunization Delta Specific IgG titer; Fig.29 BALB / c mice bronchoalveolar lavage fluid (BALF) RBD was delivered to the lungs 2 weeks after triple immunization Delta Specific IgA titer; Fig.30 Serum SARS-CoV-2 Delta pseudovirus neutralizing antibody titers of BALB / c mice before lung delivery, 2 weeks after immunization, 4 weeks after immunization, and 6 weeks after immunization; Fig.31 Neutralizing antibody titers of SARS-CoV-2 Delta pseudovirus in bronchoalveolar lavage fluid (BALF) of BALB / c mice 2 weeks after triple immunization for lung delivery; Fig.32 Serum RBD after intramuscular injection Delta Binding antibody titer; Fig.33 The titer of serum SARS-CoV-2 Delta pseudovirus neutralizing antibodies after intramuscular injection; DETAILED DESCRIPTION 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.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] pET30a was purchased from Novagen.

[0038] Escherichia coli BL21DE3 was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number CB105.

[0039] Escherichia coli C43DEDE3 was purchased from Sigma, catalog number CMC0019.

[0040] Pichia pastoris is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.19488.

[0041] Anti-His tag mouse monoclonal antibody was purchased from Sigma with the catalog number A7058.

[0042] Chelating Sepharose FF chromatography medium is from Cytiva, product catalog number 17057502.

[0043] Sephadex G25 fine chromatography medium was purchased from Cytiva with the product catalog number being 17003202.

[0044] SOURCE30Q chromatography medium was purchased from cytiva, product catalog number 17127503.

[0045] Capto MMC chromatography medium was purchased from Cytiva with catalog number 17531710.

[0046] Phenyl FF low sub chromatography medium was purchased from Cytiva, catalog number 28926988.

[0047] SOURCE 30S chromatography medium was purchased from Cytiva, catalog number 17127302.

[0048] Superdex TM 75 Increase10 / 300 GL prepacked column was purchased from Cytiva, product catalog number is 29148721.

[0049] Superdex TM 200 Increase 10 / 300 GL prepacked column was purchased from Cytiva, catalog number 28990944.

[0050] Balb / c mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0051] SEQ ID No.1: SARS-CoV-2 S-RBD WT Amino acid sequence RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.2: SARS-CoV-2 S-RBD Alpha Amino acid sequence RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.3: SARS-CoV-2 S-RBD Beta Amino acid sequence RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.4: SARS-CoV-2 S-RBD Kappa Amino acid sequence RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGLSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVQGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.5: SARS-CoV-2 S-RBD Delta (R319-V534) Amino acid sequence RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.6: SARS-CoV-2 S-RBD Omicron Amino acid sequence RVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.7: SARS-CoV-2 S-RBD XBB1.5 Amino acid sequence RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.8: SARS-CoV-2 S-RBD JN1 Amino acid sequence RVQPTESIVRFPNVTNLCPFGEVFNATRFASVYAWNRTRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIKGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKHGGNYDYWYRSFRKSNLKPFERDISTEIYQAGSTPCKGKGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV; SEQ ID No.9: Amino acid sequence of SARS-CoV S-RBD RFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLI; SEQ ID No.10: Amino acid sequence of MERS-CoV S-RBD AKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTC LILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL; SEQ ID No.11: Amino acid sequence of SpyCatcher DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG SEQ ID No.12: Amino acid sequence of SpyCatcher002 GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHT; SEQ ID No.13: Amino acid sequence of SpyCatcher003 GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT; SEQ ID No.14: Amino acid sequence of SpyTag AHIVMVDAYKPTK; SEQ ID No.15: Amino acid sequence of SpyTag002 VPTIVMVDAYKRYK; SEQ ID No.16: Amino acid sequence of SpyTag003 RGVPHIVMVDAYKRYK; SEQ ID No.17: Amino acid sequence of ML fatty acid modified signal peptide MKATKLVLGAVILGSTLLAGC; SEQ ID No.18: Amino acid sequence of the rAg signal peptide MKKLLIAAMMAAALAAC; SEQ ID No.19: Amino acid sequence of the P4 signal peptide MKTTLKMTALAALSAFVLAGC; SEQ ID No.20: Nucleotide sequence of ML-SC (NdeⅠ, NotⅠ) CATATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCGGATCCCACCATCATCATCACCACCACCATCACCATGGCGCCATGGTTGATACCTTATCAGGTTTATCAAGTGAGCAAGGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGACAAGTGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACGCTCATATTTGAGCGGCCGC; SEQ ID No.21: Nucleotide sequence of rAg-SC (NdeⅠ, NotⅠ) CATATGAAAAAATTATTGATTGCCGCAATGATGGCGGCTGCCTTGGCAGCTTGCGGATCCCACCATCATCATCACCACCACCATCACCATGGCGCCATGGTTGATACCTTATCAGGTTTATCAAGTGAGCAAGGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGACAAGTGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACGCTCATATTTGAGCGGCGC; SEQ ID No.22: P4-SC nucleotide sequence (NdeⅠ, NotⅠ) CTAATGAAAACCACCCTGAAAATGACCGCTCTGGCTGCTCTGTCTGCTTTCGTTCTGGCTGGTTGCGGATCCCACCATCATCATCACCACCACCATCACCATGGCGCCATGGTTGATACCTTATCAGGTTTATCAAGTGAGCAAGGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGACAAGTGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACGCTCATATTTGAGCGGCCGC; SEQ ID No.23: Nucleotide sequence of MLSARS2RBD ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCGGATCCGGTGGTGGTTCCAGAGTTCAACCAACTGAATCTATTGTTAGATTTCCAAACGTTACTAACTTGTGTCCATTTCATGAAGTTTTTAACGCTACTAGATTTGCTTCTGTTTACGCTTGGAACAGAACTAGAATTTCTAACTGTGTTGCTGATTACTCTGTTTTGTACAACTTTGCTCCATTCTTTGCTTTTAAGTGTTACGGTGTTTCTCCAACTAAGTTGAATGATTTGTGTTTTACTAATGTTTACGCTGATTCTTTTGTTATCAAAGGTAATGAAGTTTCTCAAATTGCTCCAGGTCAAACTGGAAACATTGCTGATTATAACTACAAGTTGCCAGATGATTTTACTGGTTGTGTTATTGCTTGGAATTCAAATAAGTTGGATTCTAAGCATTCTGGTAACTACGATTATTGGTACAGAAGTTTTAGAAAGTCTAAGTTGAAGCCATTTGAAAGAGATATTTCTACTGAAATCTACCAAGCTGGTAATAAGCCTTGTAAGGGTAAAGGTCCAAACTGTTACTTTCCACTTCAATCTTACGGTTTTAGACCTACTTACGGTGTTGGTCATCAACCATACAGAGTTGTTGTTTTGTCTTTTGAATTGTTGCATGCTCCAGCTACTGTTTGTGGTCCAAAGAAGTCTACTAACTTGGTTGGTGGTGGTGGTTCTCTCGAG; SEQ ID No.24: Nucleotide sequence of MLSARSRBD ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCGGATCCGGTGGTGGTTCCAGATTCCCTAATATCACCAACCTTTGCCCTTTCGGAGAAGTTTTCAACGCTACCAAGTTCCCAAGTGTTTACGCTTGGGAAAGAAAGAAAATTTCTAATTGTGTCGCCGATTACTCCGTTTTGTACAACTCAACATTTTTCAGTACCTTTAAGTGTTACGGTGTCTCTGCTACTAAATTGAACGATCTTTGCTTCTCAAACGTTTATGCTGACAGTTTCGTTGTCAAGGGAGATGACGTCAGACAAATTGCACCAGGTCAAACCGGAGTTATCGCTGATTACAACTACAAATTGCCTGATGACTTCATGGGTTGTGTTTTGGCCTGGAATACTAGAAACATTGACGCAACTTCCACAGGAAACTACAACTACAAGTACAGATATTTGAGACATGGAAAACTTAGACCATTTGAAAGAGATATCTCTAACGTTCCATTCTCCCCTGACGGTAAACCATGTACACCACCTGCTTTGAATTGCTATTGGCCTCTTAACGATTACGGATTTTATACTACAACCGGTATTGGATACCAACCTTATAGAGTTGTCGTTTTGTCATTCGAGTTGCTTAATGCTCCAGCCACTGTTTGTGGTCCTAAGTTGAGTACAGACCTTATCGGTTCTGGTGGTGGTGGTTCTCTCGAGCACCACCACCACCACCAC; SEQ ID No.25: Nucleotide sequence of MLMERSRBD ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCGGATCCGGTGGTGGTTCCGCCAAGCCATCCGGATCAGTTGTCGAACAGGCAGAGGGTGTTGAATGTGATTTTAGTCCTTTGCTTTCTGGTACTCCACCTCAAGTTTACAACTTCAAGAGATTGGTCTTCACTAACTGTAACTACAACTTGACAAAGTTGCTTTCTTTGTTTTCTGTTAACGATTTCACTTGTTCACAAATTAGTCCAGCAGCTATCGCTTCAAATTGCTATTCAAGTTTGATCCTTGACTACTTCTCTTACCCATTGTCTATGAAGTCCGATCTTTCAGTTTCTTCCGCAGGTCCTATCTCTCAGTTCAATTACAAGCAAAGTTTTTCTAACCCTACTTGTTTGATCCTTGCTACAGTTCCACACAACTTGACCACTATCACCAAGCCTCTTAAGTACTCTTACATCAACAAGTGTTCCAGATTGCTTTCAGATGACAGAACTGAGGTTCCACAGTTGGTCAATGCTAACCAATACAGTCCTTGTGTTTCTATTGTCCCATCCACTGTTTGGGAGGATGGTGACTACTATAGAAAGCAATTGTCTCCTCTTGAAGGTGGAGGTTGGTTGGTTGCATCCGGATCAACCGTCGCTATGACTGAACAATTGCAGATGGGATTTGGTATTACAGTTCAATACGGTACAGACACCAATTCAGTCTGTCCAAAGTTGGGTGGTGGTGGTTCTCTCGAGCACCACCACCACCACCAC; SEQ ID No.26: SARS-CoV-2 S-RBD Delta -ST nucleotide sequence CTCGAGAAAAGAAGAGTACAACCAACTGAATCCATTGTTAGATTTCCTAATATCACTAACCTGTGCCCATTTGGTGAAGTTTTTAACGCTACTAGATTTGCTTCTGTTTACGCCTGGAACAGAAAGAGAATTTCTAACTGTGTTGCTGATTACTCTGTTCTTTACAACTCTGCCTCTTTTTCTACTTTTAAGTGTTATGGTGTCTCTCCAACCAAGTTGAACGATTTGTGTTTTACCAACGTTTACGCTGATTCTTTTGTTATTAGAGGTGATGAGGTTAGACAAATTGCTCCTGGTCAAACTGGTAAGATTGCTGATTATAACTACAAGTTGCCTGATGATTTTACTGGTTGCGTCATTGCTTGGAACTCTAATAATTTGGATTCTAAGGTTGGTGGAAATTACAACTACAGATACAGATTGTTTAGAAAGAGTAACTTGAAGCCATTTGAAAGAGATATTTCTACTGAAATCTACCAAGCTGGATCTAAGCCTTGTAACGGTGTCGAAGGTTTTAACTGCTACTTTCCTTTGCAGTCTTACGGTTTTCAACCCACTAACGGTGTTGGTTACCAGCCCTACAGAGTTGTTGTTTTGTCTTTTGAGTTGCTTCATGCTCCAGCTACTGTTTGTGGTCCTAAGAAGTCTACTAACTTGGTTGGTGGTGGTGGTTCTGCTCATATTGTTATGGTTGATGCTTACAAGCCAACTAAGTAAGCGGCCGC; Example 1: Preparation of Fatty Acid-Modified SARS-CoV-2 RBD (1.1) Construction of Fatty Acid-Modified SARS-CoV-2 RBD Expression Vector According to the Escherichia coli Murein-lipoprotein signal peptide sequence (EU900370.1) and the sequence of the SARS-CoV-2 S protein JN1 mutant strain published by GenBank (supplementary Genbank number), the signal peptide was fused to the N-terminus of RBDJN1 to form the MLSARS2RBD sequence, and the SEQ ID No. 23 was synthesized by entrusting Sangon Biotech (Shanghai) Co., Ltd., wherein the 1st to 6th nucleotides from the 5' end are the NdeⅠ restriction site sequence, the 4th to 66th nucleotides are the signal peptide coding sequence, the 67th to 84th nucleotides are the linker coding sequence, the 85th to 729th nucleotides are the RBDJN.1 coding sequence, the 730th to 744th nucleotides are the linker coding sequence, and the 745th to 750th nucleotides are the XhoⅠ restriction site sequence; SEQ ID No. 23 was cloned between the NdeⅠ and XhoⅠ restriction sites of the pET30a vector to construct the pET30-MLSARS2RBD expression vector, as shown in FIG. Figure 1 shown.

[0052] (1.2) Construction of fatty acid-expressing SARS-CoV-2 RBD strain The expression vector pET30-MLSARS2RBD constructed above was introduced into the host cells of Escherichia coli C43DE3 (purchased from Sigma, catalog number CMC0019), and LB solid culture medium containing kanamycin with a final concentration of 50 μg / mL was coated. The positive clone was the expression strain C43DE3 / pET30-MLSARS2RBD.

[0053] (1.3) Expression and purification of fatty acid-modified SARS-CoV-2 RBD The recombinant bacteria C43DE3 / pET30-MLSARS2RBD single clone was picked and inoculated into 3 mL of LB medium containing kanamycin with a final concentration of 50 μg / mL. It was cultured at 37°C until OD600 was about 0.6, IPTG with a final concentration of 0.5 mM was added, and the temperature was lowered to 25°C for induction for 6 h. Take 1 mL of the above-mentioned bacterial solution induced at 25°C for 6 h, centrifuge to obtain the bacteria, suspend the bacteria with distilled water at a ratio of 1:30 (W / V), disrupt the bacteria with ultrasound, centrifuge at 12000 rpm for 5 min, aspirate the supernatant with a pipette, resuspend the precipitate with the original volume, prepare the sample with 5X reducing buffer (250 mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500 mM DTT), boil in water bath for 10 min, perform 12% 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) (e.g. Figure 2 as shown).

[0054] Figure 2 The left figure shows the SDS-PAGE electrophoresis results of the supernatant and precipitate after the 6 screened monoclonal clones were induced to express MLSARS2RBD. The right figure shows the corresponding Western blot results. The results show that all 6 monoclonal clones successfully expressed MLSARS2RBD protein, and the content of target protein in the supernatant was higher.

[0055] After expression was confirmed by SDS-PAGE and WB, the culture volume was expanded and the recombinant fatty acid-expressed SARS-CoV-2 RBD was purified.

[0056] The culture volume was expanded to 1.6L, and the C43DE3 / pET30-MLSARS2RBD bacterial precipitate was collected by centrifugation at 8000rpm for 20min, resuspended in 100mL water, ultrasonically broken, centrifuged at 8000rpm for 10min, and the supernatant was collected. 1% PEG 20000, 0.9% NaCl, 50 mM PB 7.0 were added, and stirred at room temperature for 2 hours; the precipitate was collected by centrifugation at 8000rpm for 10min, and the precipitate was resuspended in 100mL water, 1% Triton was added, and stirred at room temperature for 2 hours. 20 mM Tris Hcl pH 7.5, 0.25 M NaCl and 5 mM imidazole were added, and the supernatant was collected by centrifugation at 8000rpm for 10min. The sample was purified using a Chelating affinity chromatography column (Φ1.6cm*15cm).

[0057] Prepare buffer A (20 mM Tris Hcl 7.5 + 0.5 M NaCl + 5 mM imidazole + 1% Triton) and buffer B (20 mM Tris Hcl 7.5 + 0.5 M NaCl + 1 M imidazole + 1% Triton). First, rinse the column bed with 0.5M NaOH aqueous solution for 3 column volumes, then balance with deionized water to neutral pH, then balance with 0.2M NiSO4 aqueous solution for 3 column volumes, then balance with buffer B for one column volume, and finally balance with buffer A for 3 column volumes. Load the dissolved solution containing the recombinant MLSARS2RBD protein onto the Chelating affinity chromatography column, wash away the unbound proteins with buffer A, balance for 2 column volumes, and then elute with 2.5%, 15%, 50% and 100% buffer B; collect the 15% buffer B elution sample to obtain a preliminary purified sample, such as Figure 3 shown.

[0058] Figure 3The left figure is a chromatogram of MLSARS2RBD protein purified by Chelating affinity chromatography column; the results show that a UV absorption peak appears when eluted with 15% B, indicating that the target protein is eluted at 15% B (150 mM imidazole).

[0059] Figure 3 The right picture shows the purified SDS-PAGE of MLSARS2RBD protein. Lane 6 is the 15% B eluted protein. According to the previous Western blot results, the target protein band is at 25kDa.

[0060] A: Chromatogram of MLSARS2RBD protein purified by Chelating affinity chromatography column; B: SDS-PAGE of MLSARS2RBD protein purification, lane 1: 1% Triton dissolved sample, 2: dissolved sample loading, 3: through 1, 4: through 2, 5: 2.5% B eluted sample, 6: 15% B eluted sample, 7: 50% B eluted sample, 8: 100% B eluted sample, 9: 0.5M NaOH eluted sample The sample was purified using Superdex 200. A Superdex 200 column (φ1×30 cm, cytiva, 28990944) was equilibrated with SEC (5mM pH7.4 PB + 0.9% NaCl + 0.5% Tween) buffer, and 1 mL of the 15% B purified sample was loaded for purification. SDS-PAGE showed that a highly pure MLSARS2RBD protein could be obtained, as shown in FIG. Figure 4 shown.

[0061] (1.4) Mass spectrometry characterization and detection In order to further confirm whether the N-terminus of the purified recombinant MLSARS2RBD protein was modified by fatty acids, MLSARS2RBD was used as the research object, and Shanghai Zhongke New Life Biotechnology Co., Ltd. was commissioned to use the MALDI-TOF method to detect the relative molecular mass. The relative molecular mass of the sample was measured to be 27089.86Da. It is known that the theoretical molecular weight of unmodified SARS2RBD is 26187.48 Da, and the molecular weight of Pam3Cys is 910.46 Da. It is inferred that the recombinant MLSARS2RBD with a molecular weight of 27089.86 Da has added one molecule of Pam3Cys (26187.48 + 910.46 - 18) (error 9.92 Da). The recombinant MLSARS2RBD protein prepared by this method was named LipoSARS2RBD. Figure 5 As shown, the MALDI-TOF detection spectrum of lipidated MLSARS2RBD protein.

[0062] Example 2: Preparation of fatty acid-modified SARS-CoV RBD (2.1) Construction of fatty acid SARS-CoV RBD expression vector The signal peptide sequence (EU900370.1) was fused to the N-terminus of SARS RBD to form the MLSARSRBD sequence, and the sequence SEQ ID No. 24 was synthesized by entrusting Sangon Biotech (Shanghai) Co., Ltd., wherein the 1st to 6th nucleotides from the 5' end were NdeⅠ restriction site sequences, the 4th to 66th nucleotides were signal peptide coding sequences, the 67th to 84th nucleotides were linker coding sequences, the 85th to 708th nucleotides were SARSRBD coding sequences, the 709th to 723rd nucleotides were linker coding sequences, and the 724th to 729th nucleotides were XhoⅠ restriction site sequences; SEQ ID No. 24 was cloned between the NdeⅠ and XhoⅠ restriction sites of the pET30a vector to construct the pET30-MLSARSRBD expression vector, as shown in FIG. Figure 6 shown.

[0063] (2.2) Construction of fatty acid-expressing SARS-CoV RBD strain The expression vector PET30-MLSARSRBD constructed above was introduced into the host cells of Escherichia coli C43DE3 (purchased from Sigma, catalog number CMC0019), and LB solid culture medium containing kanamycin with a final concentration of 50 μg / mL was coated. The positive clone was the expression strain C43DE3 / pET30-MLSARSRBD.

[0064] (2.3) Expression and purification of fatty acid-modified SARS-CoV RBD The recombinant bacteria C43DE3 / pET30-MLSARSRBD monoclone was selected and inoculated into 3 mL of LB medium containing a final concentration of 50 μg / mL kanamycin. The culture was cultured at 37°C until OD600 was about 0.6, and IPTG was added at a final concentration of 0.5 mM, and the temperature was lowered to 25°C for induction for 6 hours. Take 1 mL of the above bacterial solution induced at 25°C for 6 hours, centrifuge to obtain the bacteria, suspend the bacteria in distilled water at a ratio of 1:30 (W / V), prepare the sample with 5X reducing buffer (250mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500 mM DTT), boil in water bath for 10 minutes, perform 12% SDS-PAGE electrophoresis, transfer to PVDF membrane after electrophoresis, transfer at 20V constant voltage for 1 hour, and detect with mouse Anti-His tag monoclonal antibody (Sigma, A7058), as shown in Figure 2. Figure 7 shown.

[0065] in Figure 7In the left picture, 1-4 is SDS-PAGE of C43DE3 / pET30-MLSARSRBD monoclonal bacterial solution No. 1-4. Figure 7 In the right picture, 1-4 are Western blots of C43DE3 / pET30-MLSARSRBD monoclonal bacterial solutions No. 1-4.

[0066] After the expression was confirmed by SDS-PAGE and WB, the culture volume was expanded and the recombinant fatty acid-modified SARS-CoV RBD was purified.

[0067] The culture volume was expanded to 1.6L, and the C43DE3 / pET30-MLSARSRBD bacterial precipitate was collected by centrifugation at 8000rpm for 20min, resuspended in 100mL water, ultrasonically broken, and centrifuged at 8000rpm for 10min to collect the supernatant, added 1% Triton, stirred at room temperature for 2h, added 20mM Tris Hcl pH 7.5, 0.25M NaCl and 5mM imidazole, adjusted to 8000rpm for centrifugation for 10min to collect the supernatant. The sample was purified using a Chelating affinity chromatography column (Φ1.6cm*15cm).

[0068] Prepare buffer A (20 mM Tris Hcl 7.5 + 0.5 M NaCl + 5 mM imidazole + 1% Triton) and buffer B (20 mM Tris Hcl 7.5 + 0.5 M NaCl + 1 M imidazole + 1% Triton). First, rinse the column bed with 0.5 M NaOH aqueous solution for 3 column volumes, then balance to neutral pH with deionized water, then balance with 0.2 M NiSO4 aqueous solution for 3 column volumes, then balance with buffer B for one column volume, and finally balance with buffer A for 3 column volumes. Load the recombinant MLSARSRBD onto the Chelating affinity chromatography column, wash away unbound proteins with buffer A, balance for 2 column volumes, and then elute with 100% buffer B to obtain a preliminarily purified sample. Figure 8 The lipidated MLSARSRBD protein was purified by Chelating affinity chromatography column, wherein 1: C43DE3 / pET30-MLSARSRBD bacterial solution, 2: bacterial solution ultrasonic sample, 3: bacterial solution ultrasonic precipitation, 4: bacterial solution ultrasonic supernatant, 5: PEG20000 precipitation supernatant, 6: 1% Triton dissolved sample, 7: eluted sample 1, 8: eluted sample 2.

[0069] Example 3: Preparation of fatty acid-modified MERS-CoV RBD (3.1) Construction of fatty acid-expressed MERS-CoV RBD expression vector The signal peptide sequence (EU900370.1) was fused to the N-terminus of MERS RBD to form the MLSARSRBD sequence, and the sequence SEQ ID No.25 was synthesized by entrusting Sangon Biotech (Shanghai) Co., Ltd., wherein the 1st to 6th nucleotides from the 5' end were NdeⅠ restriction site sequences, the 4th to 66th nucleotides were signal peptide coding sequences, the 67th to 84th nucleotides were linker coding sequences, the 85th to 747th nucleotides were SARSRBD coding sequences, the 748th to 762th nucleotides were linker coding sequences, and the 763th to 768th nucleotides were XhoⅠ restriction site sequences; SEQ ID No.25 was cloned between the NdeⅠ and XhoⅠ restriction sites of the pET30a vector to construct the pET30-MLMERSRBD expression vector, as shown in FIG. Fig. 9 shown.

[0070] (3.2) Construction of fatty acid-expressing MERS-CoV RBD strain The above-constructed expression vector PET30-MLMERSRBD was introduced into the host cells of Escherichia coli C43DE3 (purchased from Sigma, catalog number CMC0019), and LB solid culture medium containing kanamycin with a final concentration of 50 μg / mL was coated. The positive clone was the expression strain C43DE3 / pET30-MLMERSRBD.

[0071] (3.3) Expression and purification of fatty acid-modified MERS-CoV RBD Pick the recombinant bacteria C43DE3 / pET30-MLMERSRBD monoclone and inoculate it into 3mL LB medium containing a final concentration of 50μg / mL kanamycin. Cultivate at 37℃ until OD600 is about 0.6, add IPTG with a final concentration of 0.5 mM, and cool to 25℃ for induction for 6h. Take 1mL of the above bacterial solution induced at 25℃ for 6h, centrifuge to obtain the bacteria, suspend the bacteria in distilled water at a ratio of 1:30 (W / V), prepare the sample with 5X reducing buffer (250mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500mMDTT), boil in water bath for 10min, perform 12% SDS-PAGE electrophoresis, transfer to PVDF membrane after electrophoresis, transfer at 20V constant voltage for 1h, and detect with mouse Anti-His tag monoclonal antibody (Sigma, A7058). Fig.10 The MLMERSRBD protein expression was verified as shown, Fig.10 In the left figure, 1-4 is SDS-PAGE of C43DE3 / pET30-MLSARSRBD monoclonal bacterial solution No. 1-4, and 5-7 is SDS-PAGE of C43DE3 / pET30-MLMERSRBD monoclonal bacterial solution No. 5-7; Fig.10 In the right figure, 1-4 is the Western blot of the monoclonal bacterial solution of C43DE3 / pET30-MLSARSRBD No. 1-4, and 5-7 is the Western blot of the monoclonal bacterial solution of C43DE3 / pET30-MLMERSRBD No. 5-7.

[0072] After expression was identified by SDS-PAGE and WB, the culture volume was expanded and the recombinant fatty acid-modified MERS-CoV RBD was purified.

[0073] The culture volume was expanded to 1.6L, and the C43DE3 / pET30-MLMERSRBD bacterial precipitate was collected by centrifugation at 8000rpm for 20min, resuspended in 100mL water, ultrasonically disrupted, and centrifuged at 8000rpm for 10min to collect the supernatant, added with 1% Triton, stirred at room temperature for 2h, added with 20mM Tris Hcl pH 7.5, 0.25M NaCl and 5mM imidazole, adjusted to 8000rpm for centrifugation for 10min to collect the supernatant. The sample was purified using a Chelating affinity chromatography column (Φ1.6cm*15cm).

[0074] Prepare buffer A (20 mM Tris Hcl 7.5 + 0.5 M NaCl + 5 mM imidazole + 1% Triton) and buffer B (20 mM Tris Hcl 7.5 + 0.5 M NaCl + 1 M imidazole + 1% Triton). First, wash the column bed with 0.5 M NaOH aqueous solution for 3 column volumes, then balance to neutral pH with deionized water, then balance with 0.2 M NiSO4 aqueous solution for 3 column volumes, then balance with buffer B for one column volume, and finally balance with buffer A for 3 column volumes. Load the recombinant MLMERSRBD onto the Chelating affinity chromatography column, wash away unbound proteins with buffer A, balance for 2 column volumes, and then elute with 100% buffer B to obtain a preliminary purified sample, such as Fig.11 The lipidated MLMERSRBD protein shown was purified by Chelating affinity chromatography, wherein 1: C43DE3 / pET30-MLMERSRBD bacterial solution, 2: bacterial solution ultrasonic sample, 3: bacterial solution ultrasonic precipitation, 4: bacterial solution ultrasonic supernatant, 5: PEG20000 precipitation supernatant, 6: 1% Triton dissolved sample, 7: eluted sample 1, 8: eluted sample 2.

[0075] Example 4: Preparation of recombinant SpyCatcher protein (4.1) Construction of fatty acid modified SpyCatcher protein expression vector According to the Escherichia coli Murein-lipoprotein signal peptide sequence (EU900370.1) (SEQID No.17), Neisseria meningitidis Ag473 signal peptide sequence (AY566590.1) (SEQ ID No.18), and Haemophilus influenzae P4 signal peptide sequence (M68502.1) (SEQ ID No.19) published in GenBank, they were fused to the N-terminus of the SpyCatcher protein (amino acid sequence as shown in SEQ ID No.11), and a His tag was added for easy purification. They were named MLSC, rAgSC, and P4SC, respectively, and the DNA sequences were optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd. according to the preferred codons of Escherichia coli.

[0076] The optimized MLSC nucleotide sequence is shown in SEQ ID No. 20, wherein the nucleotides 1 to 6 from the 5' end are NdeⅠ restriction site sequences, the nucleotides 4 to 66 are Murein-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. 21, wherein the nucleotides 1 to 6 from the 5' end are NdeⅠ restriction site sequences, the nucleotides 4 to 54 are Ag473 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. 22, wherein the nucleotides 1 to 6 from the 5' end are NdeⅠ restriction site sequences, the nucleotides 4 to 66 are P4 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.

[0077] (4.2) 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 culture medium 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.

[0078] (4.3) 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 20 h.

[0079] The next day, 1 mL of each bacterial solution induced at 25°C for 20 h 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 centrifuged to separate the precipitate and supernatant. The precipitate was resuspended in an equal volume of water and prepared with 5X reducing buffer (250 mM pH 6.8 Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 500 mM DTT). The cells were boiled in a water bath for 10 min and subjected to 15% SDS-PAGE electrophoresis. After electrophoresis, the cells were transferred to a PVDF membrane and transferred at a constant voltage of 20 V for 1 h. The cells were detected with a mouse Anti-His tag monoclonal antibody (Sigma, A7058). The results are shown in Table 2. Fig.12 shown.

[0080] 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.

[0081] 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.

[0082] 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 preliminarily purified sample, such as Fig.13 The lipidated SpyCatcher (SC) was purified by Ni affinity chromatography and SDS-PAGE was shown.

[0083] 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.

[0084] The sample was purified using a SOURCE30Q anion exchange chromatography column (Φ1.6cm*15cm). First, the column bed was washed 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 was collected, as shown in FIG. Fig.14 The lipidated SpyCatcher (SC) was purified by anion exchange chromatography and identified by SDS-PAGE.

[0085] 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-mentioned SOURCE30Q purified sample was loaded for purification, and 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, indicating that the molecular weights of the three recombinant proteins MLSC, rAgSC, and P4SC are greater than 150 kDa, which is much greater than the 16 kDa of the SC monomer. Therefore, it is judged that the three recombinant proteins MLSC, rAgSC, and P4SC exist in the form of polymers (see Fig.15 , 16 , 17, 18). Fig.15 This is the SDS-PAGE identification diagram of lipidated SpyCatcher (SC) molecular exclusion chromatography purification. Fig.16 This is the size exclusion chromatogram of SpyCatcher (SC) with ML lipidated signal peptide. Fig.17 This is the size exclusion chromatogram of SpyCatcher (SC) with rAg lipidated signal peptide. Fig.18 This is the size exclusion chromatogram of SpyCatcher (SC) with P4 lipidated signal peptide. Fig.19It is the size exclusion chromatography diagram of human IgG molecules. The retention volume is 13.1 mL, which is larger than the retention volumes of MLSC, rAgSC, and P4SC. This indicates that the molecular weights of the three recombinant proteins MLSC, rAgSC, and P4SC are larger than 150 kDa, and the three proteins exist in the form of polymers.

[0086] (4.4) Characterization of MLSC In order to further confirm whether the N-terminus of the purified recombinant SpyCatcher protein was modified by fatty acids, 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.

[0087] Beijing Mingde Zhengkang Technology Co., Ltd. was commissioned to use mass spectrometry (LC / Q-TOF-MS) (such as Fig. 20 The lipidated SpyCatcher (SC) mass spectrometry detection deconvolution diagram shown in the figure) 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 the 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, the molecular weight of the recombinant SpyCatcher is inferred to be 14810.1 The recombinant SpyCatcher with a molecular weight of 14824.2 Da was Pam3C-SpyCatcher, i.e., SpyCatcher with fatty acid modification at the N-terminus, in which the thiol group of the N-terminal Cys was 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).

[0088] 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.

[0089] Example 5: Preparation of recombinant SARS-CoV-2 virus S protein RBD The present invention takes the SARS-CoV-2 S protein delta mutant RBD as an example to illustrate the antigen containing the SpyTag polypeptide.

[0090] (5.1) Construction of SARS-CoV-2 S protein RBD-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 (amino acid sequence is sequence 12) 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. 26 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.

[0091] (5.2) 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, centrifuged at 4°C 1500g×5min, resuspended the bacteria with an equal volume of pre-cooled distilled water, and centrifuged at 4°C 1500g×5min, discarded the supernatant, and repeated this step 3 times; then resuspended with an equal volume of pre-cooled 1M sorbitol, and centrifuged at 4°C 1500g×5min, discarded the supernatant, and repeated this step 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.

[0092] 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.

[0093] 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 the YPD plate for screening resistance to Zeocin, and incubated in a 25℃ incubator for 60-72h.

[0094] (5.3) 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.

[0095] 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.

[0096] (5.4) 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.

[0097] (5.5) 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.

[0098] After loading, use MMC-A for equilibration and then use MMC-B for elution.

[0099] 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.

[0100] First, the target protein was eluted with 40% Phenyl FF-B, and then the impurity proteins were eluted with 40-100% Phenyl FF-B.

[0101] 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.

[0102] 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.

[0103] After loading, balance with 30Q-A and then elute with 30Q-B. The target protein is in the flow-through.

[0104] 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.

[0105] After loading, balance with A, and then elute the target protein with 50% B.

[0106] 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.

[0107] After the above purification steps, the RBD DeltaST sample was obtained. The purification process was identified by SDS-PAGE. Fig.21 , Fig.21 This is the SDS-PAGE identification diagram of the purified samples of SARS-CoV-2 Delta mutant RBD and SpyTag fusion protein (RBDDeltaST).

[0108] Example 6: 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 (see Fig. 22 ); SDS-PAGE reduction electrophoresis detection showed (see Fig.23 ), RBDST is connected to LipoSC, with a molecular weight of about 43 kDa; TEM analysis shows that MLSC-RBDST is a nanoparticle (see Fig.24 ); DLS test showed (see Fig.25 ), LipoSC-RBD Delta The diameter of ST is about 13~20 nm (Number (Percent)). The purified LipoSC-RBD was purified by BCA method. Delta Protein quantification was performed using ST.

[0109] Example 7: Immunogenicity study of LipoSC-RBDST (7.1) Lung delivery immunization protocol The LipoSC-RBD obtained in Example 6 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. Fig.26 .

[0110] (7.2) 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.

[0111] The results are as follows Fig.26 As shown: the titer of anti-RBDDelta specific IgG antibody in the serum of mice immunized for two weeks was 1:371; the titer of RBD in the serum of mice immunized for two weeks was 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.

[0112] RBD in serum Delta The specific IgA antibody titer was tested. Fig. 27 As shown, RBD in the serum of mice immunized for two weeks Delta The specific IgA antibody titer was 1:417, and the RBD Delta-specific IgA antibody titer in the serum of mice immunized for two weeks was 1:4677.

[0113] 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 (see Fig.28 ), IgA titer (see Fig.29 ). RBD Delta The specific IgG antibody titers were 1:6026 and the specific IgA titers were 1:3162.

[0114] (7.3) 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, count the cells and dilute the cells to 2×105 cells / mL, add 100 μL of cells to each well to make the number of cells in each well 2×104 cells / well, and culture overnight.

[0115] 2) Sample preparation: 10 μL of mouse serum from each group was inactivated in a 56°C water bath for 30 min.

[0116] 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.

[0117] 4) Dilute the pseudovirus to 1.34×104 TCID50 / mL and add 50 μL / well except for the cell control. After the mixture is shaken for 30 seconds, the 96-well plate is placed in a cell culture incubator (37°C, 5% CO2) and incubated for 1 hour.

[0118] 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% CO2.

[0119] 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.

[0120] 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).

[0121] 8) The luminescence value (RLU) was read using a PerkinElmer EnSight multi-function imaging microplate reader.

[0122] 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.

[0123] The results are as follows Fig.30 , 31 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.

[0124] (7.4) Evaluation of the immunogenicity and protective effect of lipidated RBD by intramuscular immunization The RBD obtained in Examples 5 and 6 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 immunization and two weeks after the second immunization. Fig.30 , Fig.31 shown.

[0125] 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 was used Delta ST(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 When ST (LipoSC-RBDDeltaST) was combined with Al(OH)3 (100 μg) 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.32 ,33 shown.

[0126] 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.

[0127] 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 coronavirus antigen.

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

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

4. The fatty acid-modified coronavirus antigen according to any one of claims 1 to 3, characterized in that: The coronaviruses include but are not limited to SARS-CoV-2, SARS-CoV, and MERS-CoV.

5. The fatty acid-modified coronavirus antigen according to any one of claims 1 to 4, characterized in that: The SARS-CoV-2, SARS-CoV or MERS-CoV virus antigen is a spike protein or a partial functional fragment thereof.

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

7. The fatty acid-modified coronavirus antigen according to claim 4, characterized in that: In the SARS-CoV and SARS-CoV-2 coronavirus antigens, the receptor binding domain RBD includes a region capable of binding to ACE2 and its variants.

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

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

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

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

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

11.

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

14.

14. A method for preparing a fatty acid-modified coronavirus antigen according to any one of claims 1 to 9, the method comprising: 1) Introducing a DNA fragment containing a signal peptide coding sequence and a coronavirus antigen coding sequence into Escherichia coli, and culturing the recombinant Escherichia coli to express a recombinant coronavirus antigen with a fatty acid modification at the N-terminus; 2) culturing the recombinant Escherichia coli described in step 1) to express the fatty acid-modified recombinant coronavirus antigen; 3) Lysing the recombinant Escherichia coli, adding a surfactant, and extracting and purifying the fatty acid-modified recombinant coronavirus antigen from the lysate.

15. A method for preparing a fatty acid-modified coronavirus antigen as claimed in any one of claims 10 to 13, the method comprising: 1) introducing a DNA fragment containing a signal peptide coding sequence and a SpyCatcher protein coding sequence into Escherichia coli, culturing the recombinant Escherichia coli to express a recombinant SpyCatcher protein with the N-terminus modified by fatty acid; lysing the recombinant Escherichia coli, adding a surfactant, and extracting and purifying the recombinant SpyCatcher protein modified by fatty acid from the lysate. 2) Introducing a DNA fragment containing a signal peptide coding sequence, a SpyTag polypeptide coding sequence, and a coronavirus antigen coding sequence into an engineered cell, culturing the recombinant cell line to express the coronavirus antigen containing the SpyTag polypeptide, and purifying the coronavirus antigen containing the SpyTag polypeptide. 3) The purified fatty acid-modified recombinant SpyCatcher protein is mixed with a coronavirus antigen containing a SpyTag polypeptide, and the mixture is fully reacted under certain conditions to obtain a fatty acid-modified coronavirus antigen.

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

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

18. The use of a fatty acid modified coronavirus antigen according to any one of claims 1 to 13, characterized in that: The application is to prepare vaccines or screen drugs for treating coronavirus.

19. A vaccine comprising the fatty acid-modified coronavirus antigen according to any one of claims 1 to 13.

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

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

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

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

24. Biomaterial, characterized in that The biological material is any of the following: C1) a nucleic acid molecule encoding a fatty acid-modified coronavirus antigen according to any one of claims 1 to 13; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) 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).

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

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

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