Universal trimeric recombinant protein vaccine for prevention of novel coronavirus infection

Abstract

The application discloses a general trimeric recombinant protein vaccine for preventing novel coronavirus infection. Specifically disclosed are a mutant protein RBD4M with an amino acid sequence of SEQ ID No. 1 and a fusion protein of positions 26-277, positions 1-277 and SEQ ID No. 4. The application also discloses a vaccine containing the fusion protein, and further develops a trimeric recombinant protein vaccine containing the fusion protein and a double adjuvant. Experiments show that the vaccine prepared in the application can effectively induce cellular immunity and humoral immunity of the body in animals, a trace amount of the protein can stimulate a very high immune response of mice, has a good protective effect, can produce high-titer neutralizing antibodies against various epidemic strains of the novel coronavirus, is a broad-spectrum effective novel coronavirus vaccine, and has important significance for preventing novel coronavirus infection and a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a universal trimeric recombinant protein vaccine for the prevention of novel coronavirus infection. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) is a single-stranded, positive-sense RNA virus with a genome of approximately 30KB. It consists of internal genetic material (RNA) and proteins including the spike protein (S protein), envelope protein (E protein), membrane protein (M protein), and nucleoprotein (N protein). The spike protein (S protein) is one of the main proteins responsible for the virus's "corona" morphology, mediating SARS-CoV-2 entry into cells. It forms a large, highly glycosylated crown-like projection on the surface of the viral particle, serving as the virus's weapon against host cells. A crucial determinant of coronavirus host specificity is the trimeric spike glycoprotein located on the envelope surface. The S protein can be divided into the N-terminal S1 subunit, the C-terminal S2 region near the viral membrane, a transmembrane region, and a small intracellular region. The receptor-binding domain (RBD) of SARS-CoV-2 is located at the C-terminus of S1 and has about 240 amino acid residues. It can bind to the receptor ACE2 (angiotensin-converting enzyme 2) on mammalian cells and mediate the entry of SARS-CoV-2 virus into cells. After entering the cell, the continuously proliferating SARS-CoV-2 virus particles enter the extracellular fluid through exocytosis and then infect other host cells.

[0003] SARS-CoV-2 is an RNA virus that is prone to errors during replication. Massive replication can lead to various mutations at an extremely rapid rate, frequently resulting in immune evasion and reduced vaccine protection. As the novel coronavirus continues to evolve, the protective efficacy of existing vaccines is affected to varying degrees. Therefore, researching and developing more effective universal vaccines targeting various mutant strains is of great significance and has broad clinical application prospects for preventing novel coronavirus infection. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to effectively prevent infection with the novel coronavirus against various mutant strains. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, the present invention first provides a mutant protein, which may be any of the following:

[0006] A1) The amino acid sequence of this protein is that of SEQ ID No. 1;

[0007] A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1.

[0008] A3) A fusion protein with the same function is obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of A1) or A2).

[0009] The mutant protein may be named RBD4M protein.

[0010] The substitutions described herein can be conserved substitutions (also known as conserved replacements) or non-conserved substitutions in non-core functional regions. As is known to those skilled in the art, conserved substitutions or non-conserved substitutions in non-core functional regions generally do not have a qualitative impact on protein function. The substitutions described herein do not include substitutions of amino acid residues at the four mutation sites described in this invention.

[0011] The tags mentioned in this article include, but are not limited to: GST (glutathione thiotransferase) tag protein, His tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0012] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0013] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0014] The present invention also provides a fusion protein, which may include the mutant protein (RBD4M protein) and a tag, wherein the tag is preferably a trimer tag, more preferably a trimer tag with an amino acid sequence as shown in positions 252-277 of SEQ ID No. 4.

[0015] The trimer tag described therein can fuse any target protein with the trimer tag to form a trimer.

[0016] Furthermore, the trimer tag can trimerize any target protein. The trimer formed by fusing the target protein with the trimer tag can mimic the structure in which it functions physiologically in vivo. When used as an immunogen, neutralizing antibodies generated against this trimer structure are more effective.

[0017] Furthermore, the trimer tag may be any polypeptide or protein capable of causing any of the mutant or fusion proteins described herein to form a trimer.

[0018] The formation of the trimer can be the formation of a biomolecular complex, in which three identical molecules aggregate into a single trimer.

[0019] The trimer tags include, but are not limited to, the T4 phage foldon "folded" trimer domain, the isoleucine zipper and coiled-coil trimer domain derived from yeast transcription activator GCN4, the procollagen C-propeptide domain (Trimer-Tag), the catalytic subunit of Escherichia coli aspartate transcarbamoylase (ATCase), the trimer domain of collagen XV, the trimer domain of collagen XVIII, and the coiled-coil trimer domain of eukaryotic heat shock transcription factors.

[0020] Furthermore, the trimer tag may specifically be the T4 phage fibrin (foldon) "folded" trimer domain (T4Foldon).

[0021] Furthermore, the amino acid sequence of the T4Foldon may be as shown in positions 252-277 of SEQ ID No. 4.

[0022] Furthermore, the trimer tag can be directly or via a linker attached to the N-terminus or C-terminus of the RBD4M protein.

[0023] Furthermore, the trimer tag can be attached to the C-terminus of the RBD4M protein via a connector.

[0024] Furthermore, the linker may be a flexible peptide linker, such as a peptide linker comprising glycine and / or serine residues. The linker may be GGGGS (SEQ ID No. 9), GGSGSSG (SEQ ID No. 10), GGGGSGGGGS (SEQ ID No. 11), or GGSGGSGGGGGSGGGGS (SEQ ID No. 12), but is not limited thereto. Specifically, the linker may be GGSGSSG (SEQ ID No. 10).

[0025] Furthermore, the fusion protein may be any of the following:

[0026] B1) A protein whose amino acid sequence is from position 26 to 277 of SEQ ID No. 4;

[0027] B2) A protein that has more than 80% identity with and has the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in positions 26-277 of SEQ ID No. 4.

[0028] B3) A fusion protein with the same function obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of B1) or B2);

[0029] Preferably, the fusion protein described in B3) can be any of the following:

[0030] C1) The amino acid sequence is the protein of positions 1-277 of SEQ ID No. 4, or a protein that has more than 80% identity and the same function as the protein of positions 1-277 of SEQ ID No. 4 obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in positions 1-277 of SEQ ID No. 4.

[0031] C2) The amino acid sequence is the protein of SEQ ID No. 4 or a protein that has more than 80% identity with and has the same function as the protein of SEQ ID No. 4, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 4.

[0032] The fusion protein described in B1)-B3) can be named RBD4M-T4Foldon trimer fusion protein (abbreviated as RBD4M-T4Foldon protein).

[0033] The RBD4M-T4Foldon protein shown in positions 26-277 of SEQ ID No. 4 is a trimeric fusion protein obtained by fusing the trimeric tag T4Foldon (positions 252-277 of SEQ ID No. 4) to the C-terminus of the RBD4M protein shown in SEQ ID No. 1 via a linker (GSGSGSG).

[0034] The amino acid sequence of the signal peptide described in B3) may be positions 1-25 of SEQ ID No. 4, and the nucleotide sequence of the signal peptide may be positions 1-75 of SEQ ID No. 3.

[0035] The fusion protein shown in positions 1-277 of SEQ ID No. 4 can be named RBD4MFoldon protein. It is a fusion protein obtained by fusing the signal peptide shown in positions 1-25 of SEQ ID No. 4 to the N-terminus of the RBD4M-T4Foldon protein shown in positions 26-277 of SEQ ID No. 4 to facilitate the secretory expression of the protein.

[0036] The fusion protein shown in SEQ ID No. 4 can be named RBD4MFoldon-his protein. This is a fusion protein obtained by fusing a His tag (8-HisTag, HHHHHHHHH, SEQ ID No. 8) to the C-terminus of the RBD4MFoldon protein shown in positions 1-277 of SEQ ID No. 4 for the purpose of facilitating protein purification and detection. Specifically, positions 1-25 of SEQ ID No. 4 are the amino acid sequence of the signal peptide (i.e., positions 1-25 of SEQ ID No. 4), positions 26-244 of SEQ ID No. 4 are the amino acid sequence of the RBD4M protein (i.e., SEQ ID No. 1), positions 245-251 of SEQ ID No. 4 are the linker sequence (i.e., GGSGSSG, SEQ ID No. 10), positions 252-277 of SEQ ID No. 4 are the amino acid sequence of the trimer tag (T4Foldon) (i.e., positions 252-277 of SEQ ID No. 4), and positions 278-285 of SEQ ID No. 4 are the His tag sequence (8-HisTag, i.e., SEQ ID No. 8).

[0037] The present invention also provides a biomaterial, which may be any of the following:

[0038] D1) The nucleic acid molecule encoding the mutant protein (RBD4M protein);

[0039] D2) is a nucleic acid molecule that encodes any of the fusion proteins described herein;

[0040] D3) An expression cassette containing the nucleic acid molecules described in D1) and / or D2);

[0041] D4) A recombinant vector containing the nucleic acid molecules described in D1) and / or D2), or a recombinant vector containing the expression cassette described in D3);

[0042] D5) Recombinant microorganisms containing the nucleic acid molecules described in D1) and / or D2), or recombinant microorganisms containing the expression cassette described in D3), or recombinant microorganisms containing the recombinant vector described in D4);

[0043] D6) Recombinant host cells containing the nucleic acid molecules described in D1) and / or D2), or recombinant host cells containing the expression cassette described in D3), or recombinant host cells containing the recombinant vector described in D4);

[0044] Preferably, the nucleic acid molecule may be any of the following:

[0045] E1) The coding sequence is the 76th-831st position of SEQ ID No. 2, the 1st-831st position of SEQ ID No. 3, or the DNA molecule of SEQ ID No. 3;

[0046] E2) The nucleotide sequence is the DNA molecule of SEQ ID No. 2, SEQ ID No. 3 (positions 76-831), SEQ ID No. 3 (positions 1-831), SEQ ID No. 3, or SEQ ID No. 7.

[0047] Furthermore, the expression cassette described in D3), the recombinant vector described in D4), the recombinant microorganism described in D5), and the recombinant host cell described in D6 can all express the nucleic acid molecules described in D1) and / or D2).

[0048] The DNA molecule shown in SEQ ID No. 2 can be a DNA molecule encoding the RBD4M protein obtained by codon optimization of the nucleotides of the RBD4M protein (SEQ ID No. 1), and its name can be RBD4M gene.

[0049] The DNA molecule shown in positions 76-831 of SEQ ID No. 3 can be a DNA molecule encoding the RBD4M-T4Foldon protein obtained by codon optimization of the nucleotide sequence of the RBD4M-T4Foldon protein (positions 26-277 of SEQ ID No. 4), and its name can be RBD4M-T4Foldon gene.

[0050] The DNA molecule shown in positions 1-831 of SEQ ID No. 3 may be a DNA molecule encoding the RBD4MFoldon protein (positions 1-277 of SEQ ID No. 4), and its name may be the RBD4MFoldon gene.

[0051] The DNA molecule shown in SEQ ID No. 3 may be a DNA molecule encoding the RBD4MFoldon-his protein (SEQ ID No. 4), and its name may be the RBD4MFoldon-his gene.

[0052] The DNA molecule shown in SEQ ID No. 7 is the result of codon optimization of the coding sequence (SEQ ID No. 3) of the trimeric fusion protein RBD4Mfoldon (RBD4MFoldon-his), with Not I and HindIII recognition sites added at both ends. Specifically: positions 1-6 of SEQ ID No. 7 are the HindIII recognition site sequence; positions 7-15 of SEQ ID No. 7 are the Kozak sequence; positions 16-90 of SEQ ID No. 7 are the signal peptide nucleotide sequence (i.e., positions 1-75 of SEQ ID No. 3); positions 91-747 of SEQ ID No. 7 are the nucleotide sequence of the RBD4M gene (i.e., SEQ ID No. 2); positions 748-768 of SEQ ID No. 7 are the linker (GSGSGSG); positions 769-846 of SEQ ID No. 7 are the trimer tag (T4Foldon) nucleotide sequence; positions 847-870 of SEQ ID No. 7 are the His tag (HHHHHHHHH); positions 871-876 of SEQ ID No. 7 are two stop codon sequences; and positions 877-884 of SEQ ID No. 7 are the Not I recognition site sequence.

[0053] Positions 16-870 of SEQ ID No. 7 are the DNA molecule (RBD4MFoldon-his gene) shown in SEQ ID No. 3.

[0054] The nucleic acid molecule may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequences shown in SEQ ID No. 2, SEQ ID No. 3 (positions 76-831), SEQ ID No. 3 (positions 1-831), SEQ ID No. 3, or SEQ ID No. 7. Considering the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression in specific species as needed.

[0055] The vectors described herein refer to vectors capable of delivering exogenous DNA or target genes into host cells for amplification and expression. These vectors can be cloning vectors or expression vectors, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In one or more embodiments of this invention, the vector is the pUC57 vector and / or the pKS001 vector.

[0056] The microorganisms described herein may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. Specifically, the bacteria may originate from, but are not limited to, genera such as *Escherichia sp.*, *Erwinia sp.*, *Agrobacterium sp.*, *Flavobacterium sp.*, *Alcaligenes sp.*, *Pseudomonas sp.*, and *Bacillus sp.*, for example, *Escherichia coli*, *Bacillus subtilis*, or *Bacillus pumilus*. In one or more embodiments of the present invention, the microorganisms are TOP10 competent cells.

[0057] The host cell (also called the recipient cell) described herein may be a plant cell or an animal cell. The term "host cell" can be understood not only to refer to a specific recipient cell, but also to the offspring of such a cell, which may not necessarily be identical to the original parent cell due to natural, accidental, or intentional mutations and / or alterations, but are still included within the scope of the host cell. Suitable host cells are those known in the art, including: plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., but not limited to these; animal cells such as mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frog (Xenopus laevis) cells or giant salamander (Andrias davidianus) cells), fish cells (e.g., grass carp, carp, rainbow trout or catfish cells), insect cells (e.g., Sf21 cells or Sf-9 cells), etc., but not limited to these. In one or more embodiments of the present invention, the host cell is a CHO-K1Q cell.

[0058] The recombinant vectors described in this article refer to recombinant DNA molecules constructed by linking exogenous target genes with vectors in vitro. They can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0059] The recombinant microorganisms (or recombinant host cells) described herein refer to recombinant microorganisms (or recombinant host cells) whose functions have been altered by manipulating and modifying the genes of a target microorganism (or target host cell). Examples include recombinant microorganisms (or recombinant host cells) obtained by introducing exogenous target genes or recombinant vectors into a target microorganism (or target host cell), or recombinant microorganisms (or recombinant host cells) obtained by directly editing the endogenous genes of a target microorganism (or target host cell). The term "recombinant microorganism" (or recombinant host cell) can be understood to refer not only to a specific recombinant microorganism (or recombinant host cell), but also to its offspring. Due to natural, accidental, or intentional mutations and / or alterations, the offspring need not be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms (or recombinant host cells).

[0060] D4) The recombinant vector may be pUC57-RBD4Mfoldon and / or pKS001-RBD4MFoldon-his (also known as PKSRBD4mFoldonhis).

[0061] The recombinant vector pUC57-RBD4Mfoldon is obtained by replacing a small fragment between the HindIII and Not I recognition sites of the pUC57 vector with the DNA fragment whose nucleotide sequence is the DNA fragment of SEQ ID No. 7 in the sequence listing, while keeping the other nucleotide sequences of the pUC57 vector unchanged. The recombinant vector pUC57-RBD4Mfoldon expresses the trimer fusion protein RBD4MFoldon-his, whose amino acid sequence is shown in SEQ ID No. 4.

[0062] The recombinant vector pKS001-RBD4MFoldon-his (also known as PKSRBD4mFoldonhis) has the following vector pattern: Figure 1 As shown, the recombinant vector pKS001-RBD4MFoldon-his expresses the RBD4MFoldon-his protein with the amino acid sequence shown in SEQ ID No. 4.

[0063] D5) The recombinant microorganism may be TOP10 / pKS001-RBD4MFoldon-his. TOP10 / pKS001-RBD4MFoldon-his is a recombinant microorganism obtained by introducing the recombinant vector pKS001-RBD4MFoldon-his into TOP10 competent cells.

[0064] D6) The recombinant host cell may be CHO / pKS001-RBD4MFoldon-his. The CHO / pKS001-RBD4MFoldon-his is a recombinant host cell obtained by introducing the recombinant vector pKS001-RBD4MFoldon-his into CHO-K1Q.

[0065] The importation can be achieved through chemical transformation methods (such as Ca). 2+ The vector carrying the DNA molecule of the present invention can be transformed into host bacteria using any known transformation method, such as induced transformation, polyethylene glycol-mediated transformation, metal cation-mediated transformation, or electroporation transformation; alternatively, the DNA molecule of the present invention can be transduced into the host bacteria via bacteriophage transduction. The introduction can also be achieved by transfecting the host cell with the vector carrying the DNA molecule of the present invention using any known transfection method, such as calcium phosphate co-precipitation, liposome-mediated transformation, electroporation, or viral vector transfection.

[0066] The present invention also provides the mutant protein, and / or, any of the fusion proteins described herein, and / or, any of the following applications of the biomaterial:

[0067] F1) Use in the preparation of products for the prevention and / or treatment of diseases caused by SARS-CoV-2 infection;

[0068] F2) Application in the preparation of pharmaceutical agents for inducing immune responses to SARS-CoV-2 antigens;

[0069] The application of F3 in the preparation of vaccines to prevent diseases caused by SARS-CoV-2 infection;

[0070] F4) Application in the prevention and / or treatment of diseases caused by SARS-CoV-2 infection;

[0071] The application of F5 in inducing an immune response to SARS-CoV-2 antigen;

[0072] Application of F6 in the prevention of SARS-CoV-2 infection.

[0073] The products described in this article may be reagents or drugs.

[0074] The products described in F1) for the prevention and / or treatment of diseases caused by SARS-CoV-2 infection may be SARS-CoV-2 antibodies, including full-length antibodies or antigen-binding fragments (such as Fab fragments, Fv fragments, Fab′ fragments, F(ab′)2 fragments, single-chain antibodies (ScFv), nanobodies (single-domain antibodies), bispecific antibodies or minimal recognition units (MRUs), etc., but not limited to these).

[0075] Furthermore, the SARS-CoV-2 antibody may be a neutralizing antibody that specifically binds to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein). The neutralizing antibody may be a high-titer neutralizing antibody against multiple circulating strains of the novel coronavirus.

[0076] In the above applications, the diseases caused by SARS-CoV-2 infection may include respiratory infections and / or digestive system infections.

[0077] Preferably, the respiratory infection may include respiratory tract infection and / or lung infection.

[0078] Preferably, the digestive system infection may include intestinal disease, loss of appetite, nausea, vomiting, abdominal pain, and / or diarrhea.

[0079] More preferably, the respiratory infection may include severe acute respiratory syndrome, hypoxic respiratory failure, sepsis, septic shock, nasopharyngitis, rhinitis, pharyngitis, tracheitis and / or bronchitis.

[0080] More preferably, the lung infection may include pneumonia and / or lung injury.

[0081] More preferably, the lung infection may include novel coronavirus infection.

[0082] SARS-CoV-2 can bind to the ACE2 receptor expressed on the mucosal epithelial cells of the digestive tract, thereby affecting the digestive tract (Li Mingsong, Liu Zhanju, Dong Weiguo, Tian De'an. Consensus on effective prevention and treatment of SARS-CoV-2 infection in patients with inflammatory bowel disease. Modern Digestive and Interventional Diagnosis and Treatment 2020, 25(2): 146-149.).

[0083] The present invention also provides a vaccine that may include any of the fusion proteins described herein, or include the mutant proteins. Preferably, the vaccine may also include an adjuvant. More preferably, the vaccine may also include two adjuvants. More preferably, the two adjuvants may be an aluminum adjuvant and a CpG-cjx1 adjuvant.

[0084] The vaccine can be used to prevent infection with the novel coronavirus.

[0085] The active ingredient of the vaccine may include any of the fusion proteins described herein, or may include the mutant proteins.

[0086] Furthermore, the vaccine may include RBD4M-T4Foldon protein (positions 26-277 of SEQ ID No. 4), RBD4MFoldon protein (positions 1-277 of SEQ ID No. 4), and / or RBD4MFoldon-his protein (SEQ ID No. 4).

[0087] The active ingredients of the vaccine may include RBD4M-T4Foldon protein (positions 26-277 of SEQ ID No. 4), RBD4MFoldon protein (positions 1-277 of SEQ ID No. 4) and / or RBD4MFoldon-his protein (SEQ ID No. 4).

[0088] The vaccine, also known as a universal trimeric recombinant protein vaccine for the prevention of novel coronavirus infection (or simply trimeric recombinant protein vaccine), can be used to provide an immune response against SARS-CoV-2. The vaccine may be a gene-engineered subunit vaccine, which is prepared by purifying a genetically engineered protein antigen and combining it with appropriate adjuvants.

[0089] The vaccine may also include an adjuvant and / or a vaccine delivery system.

[0090] The adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response against the co-inoculated antigen. The adjuvants described herein may be those known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic compounds, quinine, saponins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokine and nucleic acid adjuvants (such as monocyte clone stimulating factor, leukocyte cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid carriers, etc.), and emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be pharmaceutically acceptable. In one or more embodiments of the present invention, the adjuvant is at least one of aluminum hydroxide adjuvant (AL(OH)3 adjuvant), CpG1018 adjuvant (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., batch number 0210426), CpG-cjx1 adjuvant (sequence 5'-TGACTGAACGTTTTAACGTCAGACTGA-3', SEQ ID No. 5), and CpG7909 adjuvant (sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3', SEQ ID No. 6).

[0091] The vaccine delivery system described herein can be a substance capable of carrying antigens to the body's immune system, where they can be stored and exert their antigenic effects for an extended period. The vaccine delivery system described herein can be a salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system, or a nano-adjuvant vaccine delivery system.

[0092] As is well known to those skilled in the art, in order to enhance the immunogenicity of antigen proteins, in addition to adding compounds with immunomodulatory effects as adjuvants, gene combinations can be adjusted to express them into particulate structures; or they can be aggregated in vitro and encapsulated in liposomes or microspheres.

[0093] Furthermore, the aluminum adjuvant may be an AL(OH)3 (aluminum hydroxide) adjuvant.

[0094] The CpG-cjx1 adjuvant is a fully thiolated linear CpG ODN adjuvant independently developed and designed by GinoVal, with the nucleotide sequence 5'-TGACTGAACGTTTTAACGTCAGACTGA-3' (SEQ ID No. 5).

[0095] In the dual adjuvant, the mass ratio of the AL(OH)3 (aluminum hydroxide) adjuvant to the CpG-cjx1 adjuvant can be (22-28):1, specifically 22:1, 23:1, 24:1, 25:1, 26:1, 27:1 or 28:1, preferably 25:1.

[0096] The content of antigen protein (RBD4M-T4Foldon protein, RBD4MFoldon protein, or RBD4MFoldon-his protein) in the vaccine can be 10-80 μg / ml, specifically 50 μg / ml.

[0097] In the vaccine, the content of AL(OH)3 (aluminum hydroxide) adjuvant can be 400-600 μg / ml, specifically 500 μg / ml.

[0098] In the vaccine, the content of CpG-cjx1 adjuvant can be 10-30 μg / ml, specifically 20 μg / ml.

[0099] The vaccine for preventing novel coronavirus infection described in this invention can be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection, or a subcutaneous liquid injection.

[0100] The present invention also provides pharmaceutical compositions that may include any of the fusion proteins described herein, or any of the mutant proteins described herein.

[0101] Furthermore, the active ingredient of the pharmaceutical composition may include any of the fusion proteins described herein, or any of the mutant proteins described herein.

[0102] Furthermore, the pharmaceutical composition has at least one of the following uses:

[0103] M1) is used for the prevention and / or treatment of diseases caused by SARS-CoV-2 infection;

[0104] M2) is used to induce an immune response to the SARS-CoV-2 antigen;

[0105] M3 is used to prevent SARS-CoV-2 infection.

[0106] M4 is used to induce an immune response in subjects.

[0107] Furthermore, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers.

[0108] The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, humectant, disintegrant, absorption enhancer, adsorbent, surfactant, or lubricant, but is not limited thereto.

[0109] The present invention also provides a method for preparing the mutant protein or any of the fusion proteins described herein, the method comprising expressing a nucleic acid molecule encoding the mutant protein in a host cell to obtain the mutant protein, or expressing a nucleic acid molecule encoding any of the fusion proteins described herein in a host cell to obtain the fusion protein.

[0110] Preferably, the method may include the following steps:

[0111] H1) Construct a recombinant expression vector containing a nucleic acid molecule encoding the mutant protein;

[0112] H2) The recombinant expression vector was introduced into host cells to obtain recombinant cells;

[0113] H3) The recombinant cells were cultured, and the mutant protein was obtained by isolation and / or purification;

[0114] Preferably, the method may include the following steps:

[0115] G1) Construct a recombinant expression vector containing a nucleic acid molecule encoding any of the fusion proteins described herein;

[0116] G2) The recombinant expression vector is introduced into host cells to obtain recombinant cells;

[0117] G3) The recombinant cells were cultured, and the fusion protein was obtained by isolation and / or purification;

[0118] More preferably, the nucleic acid molecule described in H1) may be the DNA molecule shown in SEQ ID No. 2.

[0119] More preferably, the nucleic acid molecule described in G1) may be the DNA molecule shown in SEQ ID No. 3, positions 76-831, positions 1-831 of SEQ ID No. 3, SEQ ID No. 3, or SEQ ID No. 7.

[0120] The fusion protein described in G1 can be RBD4M-T4Foldon protein, RBD4MFoldon protein, or RBD4MFoldon-his protein.

[0121] Furthermore, the nucleic acid molecule encoding the RBD4M-T4Foldon protein (positions 26-277 of SEQ ID No. 4) can be the RBD4M-T4Foldon gene shown in positions 76-831 of SEQ ID No. 3; the nucleic acid molecule encoding the RBD4MFoldon protein (positions 1-277 of SEQ ID No. 4) can be the RBD4MFoldon gene shown in positions 1-831 of SEQ ID No. 3; and the nucleic acid molecule encoding the RBD4MFoldon-his protein (SEQ ID No. 4) can be the RBD4MFoldon-his gene shown in SEQ ID No. 3.

[0122] Furthermore, the host cell may be a CHO-K1Q cell.

[0123] Furthermore, the import can be performed electronically.

[0124] The present invention also provides a method for generating an immune response, the method comprising administering the pharmaceutical composition or the vaccine to a subject.

[0125] In the above method, administering the pharmaceutical composition or the vaccine to a subject can induce an immune response against SARS-CoV-2 in the subject. The immune response may be a cellular immune response, a humoral immune response, or a combination of both.

[0126] The cellular immune response may include B cell immune response and T cell immune response.

[0127] The subjects described in this article may be humans or non-human animals.

[0128] Furthermore, the non-human animal may be a non-human mammal.

[0129] The non-human mammal may be any one of the following, but is not limited to: mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow, horse.

[0130] The subjects mentioned in this article include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after SARS-CoV-2 infection).

[0131] The administration methods described herein include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral or nasal spray, or nebulized inhalation.

[0132] The present invention also provides a method for preparing a trimeric recombinant protein vaccine for the prevention of novel coronavirus infection, the method comprising the following steps:

[0133] 1) Prepare fusion proteins (such as RBD4M-T4Foldon protein, RBD4MFoldon protein, or RBD4MFoldon-his protein);

[0134] 2) Using the fusion protein prepared in step 1) as an immunogen, it was diluted with a buffer (pH 6.0) containing 20 mM histidine hydrochloride, 140 mM arginine hydrochloride and 0.02% polysorbate 80 by volume, and mixed with adjuvant at room temperature to prepare a vaccine solution.

[0135] In the above method, the adjuvant may be at least one of aluminum adjuvant, CpG1018 adjuvant (purchased from Guangzhou Ruibo Biotechnology Co., Ltd., batch number 0210426), CpG-cjx1 adjuvant (sequence 5'-TGACTGAACGTTTTAACGTCAGACTGA-3', SEQ ID No. 5), or CpG7909 adjuvant (sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3', SEQ ID No. 6).

[0136] Specifically, the adjuvant may be a dual adjuvant, which may be 500 μg / ml of AL(OH)3 (aluminum hydroxide) adjuvant and 20 μg / ml of CpG-cjx1 adjuvant.

[0137] The present invention also provides methods for preventing and / or treating diseases caused by SARS-CoV-2 infection, the methods including administering the pharmaceutical composition or the vaccine to a subject.

[0138] In the above method, the diseases caused by SARS-CoV-2 infection may include respiratory system infections and / or digestive system infections.

[0139] In the above methods, the respiratory infection may include respiratory tract infection and / or lung infection.

[0140] In the above methods, the respiratory tract infection may include severe acute respiratory syndrome, hypoxic respiratory failure, sepsis, septic shock, nasopharyngitis, rhinitis, pharyngitis, tracheitis and / or bronchitis, and the lung infection may include pneumonia and / or lung injury.

[0141] In the above method, the lung infection may include novel coronavirus infection.

[0142] In the above methods, the digestive system infection may include intestinal diseases, loss of appetite, nausea, vomiting, abdominal pain and / or diarrhea.

[0143] The present invention also provides a method for preventing or inhibiting SARS-CoV-2 infection, the method comprising administering the pharmaceutical composition or the vaccine to a subject.

[0144] In the above method, administering the pharmaceutical composition or the vaccine to a subject can induce an immune response against SARS-CoV-2 in the subject. The immune response may be a cellular immune response, a humoral immune response, or a combination of both.

[0145] The cellular immune response may include B cell immune response and T cell immune response.

[0146] The subjects described in this article may be humans or non-human animals.

[0147] Furthermore, the non-human animal may be a non-human mammal.

[0148] The non-human mammal may be any one of the following, but is not limited to: mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow, horse.

[0149] The subjects mentioned in this article include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after SARS-CoV-2 infection).

[0150] The administration methods described herein include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral or nasal spray, or nebulized inhalation.

[0151] The vaccine design of this invention is based on the antigenic epitope sequence of the RBD region of the original novel coronavirus. Simultaneously, it analyzes key antigenic epitopes in the RBD region of later-discovered variant viruses. By comparing gene sequences of different SARS-CoV-2 subtypes and referring to previously reported studies on the trimer structure of the SARS-CoV RBD protein, the SARS-CoV-2 RBD gene was further modified. Mutations at four key sites were designed to obtain the mutant protein (RBD4M protein, SEQ ID No. 1). Based on this, the trimer fusion protein RBD4M-T4Foldon (SEQ ID No. 4, positions 252-277) was obtained by introducing the trimer tag T4Foldon (SEQ ID No. 4, positions 26-277). Furthermore, to facilitate protein secretion and expression, a signal peptide (SEQ ID No. 4, positions 1-25) was fused to the N-terminus of the RBD4M-T4Foldon protein (SEQ ID No. 4, positions 26-277), resulting in the RBD4MFoldon protein (SEQ ID No. 1). The RBD4MFoldon protein (SEQ ID No. 4, positions 1-277) was further fused with an 8-HisTag at its C-terminus to obtain the RBD4MFoldon-his protein (SEQ ID No. 4) for purification and detection.

[0152] Although the present invention designs and constructs trimeric fusion proteins RBD4M-T4Foldon, RBD4MFoldon, and RBD4MFoldon-his, the present invention is not limited to this specific fusion protein sequence. Those skilled in the art can replace the trimeric tag, linker, signal peptide, and / or purification tag in the trimeric fusion protein. For example, other trimer tags known in the art (such as the isoleucine zipper and coiled-coil trimer domains derived from yeast transcription activator GCN4, the procollagen C-propeptide domain (Trimer-Tag), the catalytic subunit of Escherichia coli aspartate transcarbamate (ATCase), the trimer domain of collagen XV, the trimer domain of collagen XVIII, or the coiled-coil trimer domain of eukaryotic heat shock transcription factors, etc.) can be used. As long as the trimer tag can enable the mutant protein (RBD4M protein) described herein to form a trimer, and the resulting trimer complex has the same function as the fusion protein described in this invention, it can be regarded as a fusion protein equivalent to the fusion protein of this invention. These equivalent fusion proteins have not departed from the protection scope of this invention.

[0153] This invention utilizes a CHO eukaryotic cell expression system to express and purify the RBD4mFoldon trimer protein, and the protein expression was successfully confirmed by SDS-PAGE. The purified RBD4mFoldon trimer protein was used as an antigen, combined with an adjuvant, to immunize mice and obtain antibody serum. The antibody serum was then validated using ELISA, Elispot assays, and a pseudovirus neutralization test. The results indicate that the vaccine prepared in this invention has excellent protective efficacy in animals, effectively blocking viral binding and neutralizing viral infection.

[0154] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0155] (1) Introduce the trimer tag T4Foldon to form a trimer 3D protein.

[0156] (2) Experiments show that the trimeric protein constructed and expressed in this invention can induce the production of specific antibodies in mice with very small doses, and has a specific neutralizing effect on the novel coronavirus RBD protein. This provides an experimental basis for the later development of recombinant protein vaccines against the novel coronavirus.

[0157] (3) Broad-spectrum antigen, which can simultaneously produce high-titer neutralizing antibodies against multiple circulating strains of the novel coronavirus, effectively neutralizing the original strain of the novel coronavirus and various variant strains.

[0158] (4) By selectively selecting key antigenic epitopes as immunogens, antibodies that are neutralizing viral infection can be precisely generated, avoiding the generation of other non-neutralizing antibodies, thereby avoiding the potential risk of ADE, and avoiding excessive “immune consumption” caused by the generation of a large number of non-neutralizing antibodies.

[0159] (5) The adjuvants include aluminum adjuvant and fully thiolated linear CpG ODN adjuvant independently developed and designed by GinoVal, which can simultaneously stimulate B cell and T cell immunity. A small amount of protein can stimulate a very high immune response in mice and achieve good protective effect in a short time.

[0160] The universal trimeric recombinant protein vaccine for the prevention of novel coronavirus infection developed in this invention can effectively induce cellular and humoral immunity in the body. It is a broad-spectrum (universal) and effective COVID-19 vaccine, which is of great significance and has broad clinical application prospects for the prevention of novel coronavirus infection. Attached Figure Description

[0161] Figure 1 The structure diagram of the carrier PKSRBD4mFoldonhis.

[0162] Figure 2This is an agarose gel electrophoresis result of the recombinant plasmid identification (colony PCR identification) in Example 1. Marker: Nucleic acid molecular standard; bacterial cultures 1-10 are all positive clones.

[0163] Figure 3 This is an electrophoresis image of the recombinant vector pKS001-RBD4MFoldon-his identified by enzyme digestion in Example 1.

[0164] Figure 4 The image shows the SDS-PAGE detection results of the RBD4MFoldon trimer fusion protein in Example 1.

[0165] Figure 5 The results are ELISA titer assays for different vaccine combinations targeting the original strain of COVID-19 in Example 2.

[0166] Figure 6 The results are ELISA titer assays for different vaccine combinations against the COVID-19 Delta strain in Example 2.

[0167] Figure 7 The results are ELISA titer assays for different vaccine combinations against the Omicron strain of COVID-19 in Example 2.

[0168] Figure 8 The results are ELISA titer assays for different vaccine combinations against the Omicron Ba.4 / 5 strain of COVID-19 in Example 2.

[0169] Figure 9 The results of ELISpot testing for different vaccine prescriptions in Example 2 are shown. Detailed Implementation

[0170] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0171] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0172] The pUC57 vector used in the following examples was provided by Nanjing Genscript Biotech Co., Ltd.

[0173] The Balb / c female mice used in the following examples were all purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0174] Example 1: Preparation of RBD4MFoldon trimer fusion protein

[0175] The vaccine design of this invention is based on the antigenic epitope sequence of the RBD region of the original novel coronavirus, while also analyzing key antigenic epitopes in the RBD region of later-discovered variant viruses. By comparing the gene sequences of different SARS-CoV-2 subtypes and referring to previously reported studies on the trimer structure of the SARS-CoV RBD protein, the SARS-CoV-2 RBD gene was further modified by designing mutations at four important sites.

[0176] The mutant protein designed was named RBD4M protein. RBD4M protein was obtained by modifying the RBD sequence of the original novel coronavirus strain. Its amino acid sequence is shown in SEQ ID No. 1. Among them, the aspartic acid (Asn, N) at position 99 of SEQ ID No. 1 is derived from the mutation of lysine (Lys, K), the serine (Ser, S) at position 128 of SEQ ID No. 1 is derived from the mutation of glycine (Gly, G), the arginine (Arg, R) at position 134 of SEQ ID No. 1 is derived from the mutation of leucine (Leu, L), and the alanine (Ala, A) at position 166 of SEQ ID No. 1 is derived from the mutation of glutamic acid (Glu, E).

[0177] Further codon optimization of the nucleotides of the RBD4M protein yielded the DNA molecule encoding the RBD4M protein, named the RBD4M gene. The nucleotide sequence of the RBD4M gene is shown in SEQ ID No. 2.

[0178] Based on this, a trimeric fusion protein, named RBD4M-T4Foldon, was obtained by introducing the trimeric tag T4Foldon (its amino acid sequence is shown in positions 252-277 of SEQ ID No. 4), with its amino acid sequence shown in positions 26-277 of SEQ ID No. 4. The RBD4M-T4Foldon protein shown in positions 26-277 of SEQ ID No. 4 is a trimeric fusion protein obtained by fusing the trimeric tag T4Foldon (positions 252-277 of SEQ ID No. 4) to the C-terminus of the RBD4M protein shown in SEQ ID No. 1 via a linker (GSGSGSG, SEQ ID No. 10).

[0179] Further codon optimization was performed on the nucleotide sequence of the RBD4M-T4Foldon trimer fusion protein (abbreviated as RBD4M-T4Foldon protein) to obtain the DNA molecule encoding the RBD4M-T4Foldon protein, which was named the RBD4M-T4Foldon gene. The nucleotide sequence of the RBD4M-T4Foldon gene is shown in positions 76-831 of SEQ ID No. 3.

[0180] Simultaneously, to facilitate protein secretion and expression, a signal peptide was added (fused) to the N-terminus of the RBD4M-T4Foldon protein. The amino acid sequence of this signal peptide is shown in positions 1-25 of SEQ ID No. 4, and the nucleotide sequence is shown in positions 1-75 of SEQ ID No. 3. The RBD4M-T4Foldon protein with the signal peptide fused to its N-terminus was named RBD4MFoldon protein. The amino acid sequence of the trimer fusion protein RBD4MFoldon is shown in positions 1-277 of SEQ ID No. 4, and its encoding gene was named the RBD4MFoldon gene. The nucleotide sequence of the RBD4MFoldon gene is shown in positions 1-831 of SEQ ID No. 3.

[0181] Furthermore, to facilitate protein purification and detection, a His tag (HHHHHHHH, SEQ ID No. 8) was fused to the C-terminus of the RBD4MFoldon protein (positions 1-277 of SEQ ID No. 4), resulting in a trimeric fusion protein named RBD4MFoldon-his (abbreviated as RBD4MFoldon-his protein). The amino acid sequence of the RBD4MFoldon-his protein is shown in SEQ ID No. 4, and its encoding gene is named the RBD4MFoldon-his gene. The nucleotide sequence of the RBD4MFoldon-his gene is shown in SEQ ID No. 3.

[0182] The preparation method of RBD4MFoldon trimer fusion protein is as follows:

[0183] 1. Construction of RBD4MFoldon trimeric fusion protein expression vector

[0184] 1.1 Obtaining a vector containing the target gene (RBD4MFoldon-his gene)

[0185] Template synthesis: The coding sequence (SEQ ID No. 3) of the trimeric fusion protein RBD4Mfoldon (RBD4MFoldon-his) was codon-optimized, and Not I and HindIII recognition sites were added to both ends, ultimately yielding a DNA molecule (884 bp) with the nucleotide sequence SEQ ID No. 7, wherein:

[0186] The first six positions of SEQ ID No. 7 are the HindIII recognition site sequence; the seventh to fifteenth positions of SEQ ID No. 7 are the Kozak sequence; the 16th to 90th positions of SEQ ID No. 7 are the signal peptide nucleotide sequence (i.e., the first to seventh 75th positions of SEQ ID No. 3); the 91st to 747th positions of SEQ ID No. 7 are the RBD4M gene nucleotide sequence (i.e., SEQ ID No. 2); the 748th to 768th positions of SEQ ID No. 7 are the linker (GSGSGSG); the 769th to 846th positions of SEQ ID No. 7 are the trimer tag (T4Foldon); the 847th to 870th positions of SEQ ID No. 7 are the His tag (HHHHHHHHH); the 871st to 876th positions of SEQ ID No. 7 are the two stop codon sequences; and the 877th to 884th positions of SEQ ID No. 7 are the NotI recognition site sequence. Positions 16-870 of SEQ ID No. 7 are the DNA molecule (RBD4MFoldon-his gene) shown in SEQ ID No. 3.

[0187] The DNA molecule shown in SEQ ID No. 7 was inserted into the pUC57 plasmid through the Not I and HindIII recognition sites to obtain the recombinant vector pUC57-RBD4Mfoldon (provided by Nanjing Genscript Biotech Co., Ltd.).

[0188] The recombinant vector pUC57-RBD4Mfoldon is obtained by replacing the fragment (small fragment) between the Not I and HindIII recognition sites of the pUC57 vector with the DNA fragment whose nucleotide sequence is the DNA fragment of SEQ ID No. 7 in the sequence listing, while keeping the other nucleotide sequences of the pUC57 vector unchanged. The recombinant vector pUC57-RBD4Mfoldon expresses the trimer fusion protein RBD4MFoldon-his with the amino acid sequence shown in SEQ ID No. 4.

[0189] In the fusion protein RBD4MFoldon-his, positions 1-25 of SEQ ID No. 4 are the amino acid sequence of the signal peptide (i.e., positions 1-25 of SEQ ID No. 4), positions 26-244 of SEQ ID No. 4 are the amino acid sequence of the RBD4M protein (i.e., SEQ ID No. 1), positions 245-251 of SEQ ID No. 4 are the linker sequence (i.e., GGSGSSG, SEQ ID No. 10), positions 252-277 of SEQ ID No. 4 are the amino acid sequence of the trimer tag (T4Foldon) (i.e., positions 252-277 of SEQ ID No. 4), and positions 278-285 of SEQ ID No. 4 are the His tag sequence (8-HisTag, i.e., SEQ ID No. 8).

[0190] 1.2 Double enzyme digestion of the target gene vector and the basic vector plasmid

[0191] The pKS001 vector (purchased from Zhongshan Kangcheng Biotechnology, catalog number A13201) and the recombinant vector pUC57-RBD4Mfoldon were digested with Not I (purchased from NEB, catalog number R3189L) and Hind III restriction endonuclease (purchased from NEB, catalog number R3104V), respectively. The digestion system is shown in Table 1.

[0192] Table 1 Double enzyme digestion system

[0193]

[0194]

[0195] Prepare the double enzyme digestion system according to Table 1, mix well, digest at 37℃ for 1.5 hours, perform nucleic acid electrophoresis identification, and after the bands are shown to be correct, perform gel recovery, determine the DNA concentration, and proceed to the next experiment.

[0196] The pKS001 vector was double-digested to obtain the pKS001 vector fragment (large fragment), and the recombinant vector pUC57-RBD4Mfoldon was double-digested to obtain DNA fragment 1 containing the RBD4MFoldon-his gene (SEQ ID No. 3).

[0197] 1.3 Connection and Transformation

[0198] (1) Connection

[0199] The DNA fragment 1 obtained in step 1.2 was ligated with the pKS001 vector fragment (large fragment). The ligation system (20 μL) is shown in Table 2.

[0200] Table 2 Connection Reaction System

[0201]

[0202] The mixed reaction solution was placed at room temperature (25℃) for 5-10 minutes to obtain the ligation product.

[0203] (2) Transformation of the linker products

[0204] Add the ligation product to 50–100 μL of TOP10 competent cells (purchased from TransGen Biotech, Beijing, catalog number CD101) thawed on ice. Incubate on ice for 30 min to ensure thorough mixing of the competent cells and ligation product. Heat shock the product at 42°C for 90 s, then quickly transfer it to ice for 2 min. Add 400 μL of sterile LB medium (antibiotic-free) to the product, mix well, and incubate at 37°C and 220 rpm for 1 hr. Spread the entire bacterial culture evenly onto ampicillin-resistant LB agar plates. Invert the plates and incubate overnight at 37°C.

[0205] (3) Identification of recombinant plasmids

[0206] The recombinant plasmid was detected and identified using PCR. The primers used were the specific primers PLHS, suitable for identifying the positive pKS001 vector. The constructed plasmid served as the template, and the PCR amplification region was approximately 900 bp in size. PCR identification results showed that the fragment size was approximately 900 bp, consistent with the theoretical value, indicating successful construction of the eukaryotic expression plasmid. The results of agarose gel electrophoresis of the PCR amplification products are shown below. Figure 2 As shown.

[0207] (4) Select strains that amplify the target band and send them for sequencing.

[0208] (5) The sequenced strains were cultured in large quantities in LB medium containing ampicillin resistance. Expression plasmids were extracted in large quantities using an endotoxin-free plasmid extraction kit and identified by enzyme digestion. The results are as follows: Figure 3 As shown.

[0209] The plasmid (recombinant vector) with correct sequencing results is named pKS001-RBD4MFoldon-his or PKSRBD4mFoldonhis (e.g., Figure 1 (As shown).

[0210] The recombinant bacteria containing the above-mentioned recombinant vector pKS001-RBD4MFoldon-his (PKSRBD4mFoldonhis) were named TOP10 / pKS001-RBD4MFoldon-his. TOP10 / pKS001-RBD4MFoldon-his is a recombinant microorganism obtained by introducing the recombinant vector pKS001-RBD4MFoldon-his into TOP10 competent cells.

[0211] 2. Expression of RBD4MFoldon trimer fusion protein

[0212] (1) Maintenance and passage of cell lines: CHO-K1Q cells, passage P3 (purchased from Zhongshan Kangtianshenghe Biotechnology Co., Ltd., catalog number A14101), were revived and placed in 125 mL flat-bottomed conical flasks containing 30 mL of CD04 medium (Zhongshan Kangtianshenghe Biotechnology Co., Ltd., catalog number A11004). The cells were incubated at 37°C and 5% CO2 with shaking at 125 rpm for 3–4 days. Subsequently, a small amount of cells was aspirated daily, and trypan blue staining solution was added. Cell counts were performed under a microscope to assess cell viability. Cell viability was considered positive when it exceeded 95% and the cell density reached 2 × 10⁶ cells / year. 6 ~4×10 6 Cells were passaged and expanded at a concentration of 0.5 × 10⁶ cells / ml, and then diluted to 0.5 × 10⁶ cells / ml. 6 Cells / ml were cultured under the same conditions for 3–4 days.

[0213] (2) Cell transfection:

[0214] 1) Introduce viable CHO-K1Q cells in the logarithmic growth phase at a concentration of 2×10⁻⁶. 6 Cells / ml were passaged into 500ml Erlenmeyer flasks containing 100ml of CD04 medium (Zhongshan Kangcheng Biotechnology Co., Ltd., catalog number A11004) and cultured overnight at 37℃ with shaking at 125rpm in a 5% CO2 incubator. The next day, cells were collected and the culture medium was removed. Cells were gently washed once with PBS, centrifuged, and the supernatant was discarded. The cell density was adjusted to 1×10⁶ cells / ml using electroporation reagent. 7 Cells / ml, gently pipette the cells to form a single-cell suspension. Mix the plasmid (pKS001-RBD4MFoldon-his) with the electroporation reagent, let stand at room temperature for 5 min, add the mixed liquid to 200 μL of cells, and immediately mix gently. Transfer the mixture to an electroporation cuvette, avoiding the formation of air bubbles.

[0215] 2) Select the appropriate electroporation scheme and press the Start button on the touchscreen. After the electrical pulse is released, the touchscreen will display "Completed," indicating that the electroporation is complete.

[0216] 3) Immediately transfer the pulsed sample to the prepared container containing preheated, non-pressurized reagent. Cells were cultured in T75 flasks containing CHOCD04 serum-free medium (purchased from QuaCell, catalog number A11004). Cells were then incubated at 37°C in a 5% CO2 incubator for 16–48 hours.

[0217] (3) Medium change: Collect transfected cells cultured for 16-48 h, centrifuge, remove supernatant, add 30 ml of serum-free pressurized medium containing 25 uM MSX (L-Methionine Sulfoximine, purchased from Sigma, catalog number M5379-1G), transfer cells to 125 ml shake flasks, and incubate at 37°C with 5% CO2. Observe cell growth status, and change pressurized medium every 5-7 days until cells begin to expand and grow.

[0218] (4) Cell passage: Observe cell growth regularly, and pass the cells until the cell density is greater than 2 × 10⁻⁶. 6 Cells can be passaged or preserved at a concentration of / ml. Passage is typically performed every 3-5 days, with cell dilution at a 1:4 ratio, until CD04 medium (Zhongshan Kangcheng Biotechnology Co., Ltd., catalog number A11004) is added to a final volume of 1L.

[0219] (5) Add supplements: It is recommended to add supplements according to the following: Feeding was performed according to the FEED instructions (purchased from QuaCell, catalog numbers A11952 and A11902A). Daily sampling and counting were conducted to determine the feeding strategy. Cells were harvested when viability was less than 60%. The obtained cells were recombinant cells, named CHO / pKS001-RBD4MFoldon-his.

[0220] The recombinant cell CHO / pKS001-RBD4MFoldon-his is a recombinant cell obtained by introducing the recombinant vector pKS001-RBD4MFoldon-his into CHO-K1Q cells. The recombinant cell CHO / pKS001-RBD4MFoldon-his contains the DNA molecule shown in SEQ ID No. 3 and expresses the RBD4MFoldon-his protein with the amino acid sequence of SEQ ID No. 4.

[0221] 3. Purification and detection of RBD4MFoldon trimer fusion protein

[0222] The recombinant antigen contains an 8×His tag at its C-terminus, and the antigen is purified using Ni ion affinity purification.

[0223] Cultivate the recombinant cell CHO / pKS001-RBD4MFoldon-his, centrifuge to collect the supernatant, filter it through a 0.45 μm filter membrane, and after affinity purification with a nickel column, perform SDS-PAGE detection and analysis on the eluted protein. After concentrating the eluted protein, perform SDS-PAGE detection( Figure 4 ), where M is the Marker, "Red" is the RBD4MFoldon trimer fusion protein (reduced), and "Non-Red" is the RBD4MFoldon trimer fusion protein (non-reduced).

[0224] Example 2: Screening and immunogenicity study of the vaccine composition

[0225] The universal trimeric recombinant protein vaccine for preventing novel coronavirus infection in the present invention uses the RBD4MFoldon trimer fusion protein (positions 1-277 of SEQ ID No. 4) as the vaccine antigen. In this example, the histidine-tagged RBD4MFoldon trimer fusion protein prepared in Example 1 (i.e., the RBD4MFoldon-his protein expressed and purified in Example 1) is selected to prepare the trimeric recombinant protein vaccine.

[0226] Specifically, in this example, the RBD4MFoldon-his protein prepared in Example 1 is used as the immunogen and diluted to a final protein concentration of 50 μg / ml with a buffer solution (pH 6.0) containing 20 mM histidine hydrochloride, 140 mM arginine hydrochloride, and 0.02% (v / v) polysorbate 80. At room temperature, it is respectively mixed evenly with an aluminum hydroxide adjuvant suspension (aluminum content 500 μg / ml), CpG1018 adjuvant (purchased from Guangzhou Ribobio Co., Ltd., batch number 0210426), the fully thiolated modified linear CpG ODN adjuvant - CpG-cjx1 adjuvant independently developed and designed by GenoWay (sequence: 5'-TGACTGAACGTTTTAACGTCAGACTGA-3', SEQ ID No. 5), or CpG7909 adjuvant (sequence: 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3', SEQ ID No. 6) (the above two adjuvants are synthesized by Sangon Biotech). Then, an aluminum adjuvant-containing vaccine solution, a CpG adjuvant-containing vaccine solution, and a dual adjuvant vaccine solution containing aluminum adjuvant and CpG adjuvant are respectively prepared and placed at 2-8°C.

[0227] Under aseptic conditions, the above vaccine solutions at 2-8°C are filled into 2 ml vials (or pre-filled glass syringes), 0.5 ml (or 1.0 ml) per vial, sealed, and stored in the dark at 2-8°C.

[0228] The above-mentioned vaccine solution (i.e., vaccine composition) was taken out and immunogenicity studies were conducted using Balb / c mice (purchased from Spifor (Beijing) Biotechnology Co., Ltd.) as an animal model.

[0229] The specific method is as follows: Female Balb / c mice aged 6-8 weeks were randomly divided into 3 groups of 5 mice each. The above-mentioned vaccine composition was injected intramuscularly into the mice, and three groups were designated: a vaccine group, a protein group, and an adjuvant group. Immunization was performed at weeks 0 and 3 (0.1 ml dose per immunization), blood was collected at weeks 3 and 5, and spleens were harvested at week 5. The antibody titers (i.e., total IgG) against the original SARS-CoV-2 strain RBD, Delta RBD, Omicron RBD, and Omicron4 / 5 RBD proteins in serum were detected using ELISA. A pseudovirus neutralization assay was used to determine the neutralizing titers against the original SARS-CoV-2 strain, Delta strain, Omicron strain, and Omicron4 / 5 strain. The cellular immunity level in spleen cells, primarily the expression of IFN-γ, was detected using the ELISPOT method. The results showed that the vaccine composition prepared from the RBD4M protein obtained through the technical solution provided by this invention has excellent immunogenicity and can be used as a potential recombinant SARS-CoV-2 vaccine antigen. The specific operation is as follows:

[0230] I. ELISA Method

[0231] 1. Reagent preparation

[0232] 1.1 Preparation of ELISA coating solution (1×): Take ELISA coating solution (10×) and dilute it to 1× with sterile distilled water.

[0233] 1.2 Preparation of PBS: Take out PBS powder (Solepro product, catalog number G0002) and dissolve each bag in 2L of sterile distilled water.

[0234] 1.3 Washing solution: PBST (PBS containing 0.05% Tween-20)

[0235] Measure 1L of filtered PBS into a blue-capped bottle, add 500μL of Tween-20, mix thoroughly, and store at 2–8℃ for later use. Prepare the required volume of PBST according to the actual situation (prepare fresh for immediate use on the same day).

[0236] 1.4 Blocking buffer and sample dilution buffer (PBS containing 5% skim milk powder)

[0237] Weigh out 5% of the volume of skim milk powder in PBS (milk powder mass / g = PBS volume / mL × 5%). Equilibrate the PBS taken from 2–8℃ to room temperature. Measure the required volume of PBS solution into a centrifuge tube containing the added milk powder, and then dissolve thoroughly. Prepare the blocking buffer and sample diluent fresh each time they are used.

[0238] 1.5. Dilute the goat anti-mouse secondary antibody

[0239] Equilibrate the 1mL secondary antibody (named Anti-Mouse IgG, HRP-Linked Antibody, CST, catalog number 7076S) to room temperature, then aliquot and store at -20℃±5℃. Before each use, dilute 1:4000 with PBS. Use the diluted secondary antibody on the same day.

[0240] 1.6 Termination solution (purchased from Solarbio)

[0241] 2. ELISA detection of serum binding antibody titer:

[0242] 2.1 Coating: Take the original strains RBD-his, Delta RBD-his, Omicron RBD-his, and Omicron Ba.4 / 5RBD-his protein antigen stock solutions (all proteins were purchased from Sinocare, catalog numbers: 40592-V08H, 40592-V08H90, 40592-V08H121 and 40592-V08H130 respectively) and dilute them to 1000 ng / mL with ELISA coating buffer (1×). Coat the enzyme-linked immunosorbent assay (ELISA) plate with 100 μl / well and incubate overnight at 4°C.

[0243] 2.2 Sealing: After removing the coated plate from 2-8℃, wash the plate 3 times, each time with a washing solution volume of 300μl / well. If there is residual washing solution in the well after washing, pat it dry on absorbent paper. Then add the pre-prepared sealing solution to the coated well, 300μl / well, cover with the sealing film, and seal at 37℃ for 60-90min.

[0244] 2.3 Serum dilution: Dilute the serum to be tested to an appropriate concentration in a centrifuge tube using sample diluent.

[0245] 2.4. Sample Addition: Wash the sealed coated plate three times, with a washing buffer volume of 300 μl / well each time. If there is residual washing buffer in the wells after washing, pat them dry on absorbent paper. Add the diluted test samples (i.e., the serum to be tested) of each concentration sequentially to the sample wells, 100 μl / well; add 100 μl of sample diluent as a blank control (Blk), set up 5 replicates, cover with sealing film, and incubate at 37°C for 60 min.

[0246] 2.5 Add secondary antibody: Discard the sample, wash the plate 3 times, each time with a washing buffer volume of 300 μl / well. If there is any residual washing buffer in the well after washing, pat it dry on absorbent paper. Add the diluted secondary antibody, 100 μl / well, cover with the sealing film, and incubate at 37°C for 60 min.

[0247] 2.6. Color development: Wash the 96-well plate three times, with a washing solution volume of 300 μl / well each time. If there is any residual washing solution in the well after washing, pat it dry on absorbent paper, add 100 μl / well of single-component TMB color development solution 1 (taken out of 2-8℃ in advance and equilibrated to room temperature), and develop the color at 25℃ in the dark for 15 min.

[0248] 2.7 Termination: Immediately after color development, add the stop solution to terminate the reaction, 50 μl / well, and gently shake to mix.

[0249] 2.8 Detection: Place the ELISA plate into the ELISA reader and measure the absorbance at a wavelength of 450 nm.

[0250] 2.9 Judgment: A result greater than 2.1 times the OD value of a negative mouse is considered positive.

[0251] The following ELISA methods for detecting antibody titers are all the same as above.

[0252] II. ELISPOT detection method for mouse spleen:

[0253] The spleen lymphocytes of 14-day-old second-immunized mice were detected according to the instructions of the Murine IFN-γ Single-Color Enzymatic ELISPOT Assay (purchased from CTL (Cellular Technology Ltd.), catalog number mIFNgp-2M / 2). The specific steps are as follows:

[0254] 1. Add 5ml of mouse lymphocyte separation medium to spleen tissue, grind gently, filter through a 200-mesh nylon mesh into a clean centrifuge tube, gently add 1ml of PBS above the liquid surface, centrifuge at 800g for 30min with a fast rise and slow fall rate, and collect the intermediate lymphocyte layer.

[0255] 2. Add 5ml of PBS to wash the cells, centrifuge at 500g for 5min, and discard the supernatant.

[0256] 3. Resuspend cells in CTL-Test Medium (supplemented with L-glutamine to a final concentration of 3 mM), count cells (using flow cytometry), and then adjust the cell concentration to 5 × 10⁶ cells / mL. 6 / ml.

[0257] 4. Prepare CTL-Test Medium at a final concentration of 2x: For groups 1-9, add 100 μL / well of the original RBD-his, Delta RBD-his, Omicron RBD-his, and Omicron Ba.4 / 5RBD-his mixture (2x final concentration 10 μg / ml) to the sample tubes. Plate the medium into the corresponding ELISPOT plates. Also reserve negative control wells (without any stimulants) and positive control wells (containing Cell Stimulation Cocktail cell activators (PMA and Ionomycin) 500X, purchased from eBioscience, catalog number 00-4970-93. Dilute to 1X according to the prepared volume). Incubate at 37°C, 5% CO2 for 20 min.

[0258] 5. Add 100 μL of cells per well, equivalent to 5 × 10⁶ cells / well. 5 / well, incubated at 37℃ and 5% CO2 for 48h.

[0259] 6. Wash the plate twice with 200 μL / well PBS, and then wash the plate three times with 200 μL / well 0.05% Tween-PBS.

[0260] 7. Add 80 μL of anti-murine IFN-γ detection solution to each well and incubate at room temperature for 2 hours.

[0261] 8. Wash the plate three times with 200 μL / well of 0.05% Tween-PBS.

[0262] 9. Add 80 μL of tertiary solution per well and incubate at room temperature for 30 minutes.

[0263] 10. Wash the plate twice with 200 μL / well of 0.05% Tween-PBS, and then wash the plate twice with 200 μL / well of pure water.

[0264] 11. Add 80ul of Blue Developer Solution per well and incubate at room temperature for 15 minutes.

[0265] 12. Discard the liquid and rinse the well plate three times with pure water to terminate the reaction.

[0266] 13. ELISPOT board readings.

[0267] III. Neutralization Experiment

[0268] The fake virus test kits were purchased from Nanjing Novizan Biotechnology Co., Ltd. Detailed information is shown in Table 3.

[0269] Table 3. Detailed information on pseudovirus test kits

[0270] fake virus name reagent kit name Item number Original strain pseudovirus SARS-CoV-2-Fluc WT DD1746-01 / 02 / 03 Delta strain pseudovirus SARS-CoV-2-Fluc B.1.617.2 DD1754-01 / 02 / 03 Omicron strain pseudovirus SARS-CoV-2-Fluc B.1.1.529 DD1768-01 / 02 / 03 Omicron BA.4 / BA.5 fake virus SARS-CoV-2-Fluc BA.4 / BA.5 DD1776-01 / 02 / 03

[0271] 1. Select the best vaccine prescription

[0272] Forty-five 6-8 week old female Balb / c mice were randomly divided into nine groups and immunized according to the immunization protocol designed in Table 4. The mice were immunized twice, on days 0 and 21. The optimal vaccine prescription was screened by detecting the ELISA titer of the second immunization 14 days after the second immunization in groups 1-9 and by detecting the expression of IFN-γ factor in spleen lymphocytes of groups 1-9 14 days after the second immunization using ELISPOT.

[0273] Table 4. Immunization regimens for mice immunized with different formulations of recombinant RBD4MFoldon vaccine

[0274]

[0275] In Table 4, RBD4MFoldon is the RBD4MFoldon-his protein prepared in Example 1, and im indicates intramuscular injection.

[0276] See results Figures 5-8 ,from Figure 5-8 The ELISA results showed that the vaccine combination of RBD4MFoldon 5μg + 50ug AL(OH)3 + 2μg CpG-cjx1 (group 7) exhibited the best antibody titers against the original strain, Delta strain, Omicron strain, and Omicron Ba.4 / 5 strain, superior to the single antigen group and the single adjuvant group. Among these, the aluminum adjuvant group (groups 7 / 8 / 9) was superior to the non-aluminum adjuvant group (groups 4 / 5 / 6), indicating that aluminum adjuvant primarily plays a role in enhancing humoral immunity. Figure 9 The ELISpot results showed that the RBD4MFoldon 5μg+50ug AL(OH)3+2μgCpG-cjx1 vaccine produced the highest number of cells expressing IFN-γ, superior to the single antigen group and the single adjuvant group. Three CpGs were screened, and CpG-cjx1 was superior to CpG1018 and CpG7909. There was no significant difference between the two CpG adjuvants, CpG1018 and CpG7909. The CpG adjuvant group (7 / 8 / 9) was superior to the group without CpG adjuvant (2), thus confirming that CpG adjuvants mainly play a role in enhancing cellular immunity.

[0277] 2. Detection of neutralizing antibodies against pseudoviruses in dual-adjuvant recombinant universal COVID-19 vaccines

[0278] Fifteen 6-8 week old female Balb / c mice were randomly divided into three groups of five each. The animals were immunized twice, on days 0 and 21. The neutralizing antibody titers against the original strain, Delta strain, Omicron strain, and Omicron Ba.4 / 5 were measured in the mouse serum 14 days after the second immunization using a pseudovirus neutralization assay.

[0279] Table 5. Immunization regimens for the dual-adjuvant recombinant universal COVID-19 vaccine in mice.

[0280] Group Immunogen adjuvant immune volume Immunization methods Number of immunizations Animal numbers 10 PBS - 50ul im 2 5 11 - <![CDATA[AL(OH)3+CpG-cjx1]]> 50ul im 2 5 12 RBD4MFoldon <![CDATA[AL(OH)3+CpG-cjx1]]> 50ul im 2 5

[0281] In Table 5, RBD4MFoldon refers to the RBD4MFoldon-his protein prepared in Example 1, and im indicates intramuscular injection. The amounts of immunogen and adjuvant in Table 5 are the same as those in group 7 in Table 4.

[0282] The results of the pseudovirus neutralization test are shown in Table 6.

[0283] Table 6. Neutralizing antibody titers against different SARS-CoV-2 strains of pseudoviruses

[0284]

[0285] The results showed that the antibodies induced by the trimeric recombinant protein vaccine with AL(OH)3+CpG-cjx1 dual adjuvants could effectively neutralize different SARS-CoV-2 virus strains and enhance humoral immunity, making it a broad-spectrum (universal) and effective vaccine for the prevention of novel coronavirus infection.

[0286] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A fusion protein, characterized in that, The fusion protein consists of a mutant protein with an amino acid sequence as shown in SEQ ID No. 1 and a trimer tag with an amino acid sequence as shown in positions 252-277 of SEQ ID No. 4, which is attached to the C-terminus of the mutant protein via a flexible peptide linker.

2. A fusion protein, characterized in that, The fusion protein is any one of the following: B1) A protein whose amino acid sequence is from position 26 to 277 of SEQ ID No. 4; B2) A fusion protein with the same function is obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of B1).

3. The fusion protein according to claim 2, characterized in that, B3) The fusion protein is any one of the following: C1) The amino acid sequence is the protein consisting of positions 1-277 of SEQ ID No. 4; C2) The amino acid sequence of the protein is SEQ ID No.

4.

4. A biomaterial, characterized in that, The biomaterial is any one of the following: D1) A nucleic acid molecule encoding any of the fusion proteins described in claims 1-3; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) A recombinant host cell containing the nucleic acid molecule described in D1), or a recombinant host cell containing the expression cassette described in D2), or a recombinant host cell containing the recombinant vector described in D3).

5. The biomaterial according to claim 4, characterized in that, The nucleic acid molecule is the DNA molecule shown in SEQ ID No. 3, positions 76-831, positions 1-831 of SEQ ID No. 3, or SEQ ID No.

7.

6. The fusion protein according to any one of claims 1-3, and / or, any of the following applications of the biomaterial according to claim 4 or 5: F1) Application in the preparation of products for the prevention of disease caused by SARS-CoV-2 infection; F2) Application in the preparation of pharmaceutical agents for inducing immune responses to SARS-CoV-2 antigens.

7. The application according to claim 6, characterized in that, The product in question is a vaccine.

8. The application according to claim 6, characterized in that, The diseases caused by SARS-CoV-2 infection include respiratory and / or digestive system infections.

9. The application according to claim 8, characterized in that, The digestive system infections include intestinal diseases, loss of appetite, nausea, vomiting, abdominal pain, and / or diarrhea.

10. The application according to claim 8, characterized in that, The respiratory infections include respiratory tract infections and / or lung infections.

11. The application according to claim 10, characterized in that, The respiratory infections include severe acute respiratory syndrome, hypoxic respiratory failure, sepsis, septic shock, nasopharyngitis, rhinitis, pharyngitis, tracheitis and / or bronchitis.

12. The application according to claim 10, characterized in that, The lung infections include pneumonia and / or lung injury.

13. The application according to claim 12, characterized in that, The pneumonia mentioned includes novel coronavirus pneumonia.

14. A vaccine, characterized in that, The vaccine comprises the fusion protein described in any one of claims 1-3.

15. The vaccine according to claim 14, characterized in that, The vaccine also includes adjuvants.

16. The vaccine according to claim 15, characterized in that, The adjuvant is a dual adjuvant.

17. The vaccine according to claim 16, characterized in that, The dual adjuvants are an aluminum adjuvant and a CpG-cjx1 adjuvant.

18. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fusion protein according to any one of claims 1-3.

19. A method for preparing the fusion protein according to any one of claims 1-3, characterized in that, The preparation method includes expressing the fusion protein in a host cell by expressing a nucleic acid molecule encoding any of the fusion proteins of claims 1-3.

20. The preparation method according to claim 19, characterized in that, The preparation method includes the following steps: G1) Construct a recombinant expression vector containing a nucleic acid molecule encoding any of the fusion proteins described in claims 1-3; G2) The recombinant expression vector is introduced into host cells to obtain recombinant cells; G3) The recombinant cells were cultured, and the fusion protein was obtained by isolation and / or purification.

21. The preparation method according to claim 20, characterized in that, The nucleic acid molecule described in G1) is the DNA molecule at positions 76-831 of SEQ ID No. 3, positions 1-831 of SEQ ID No. 3, or SEQ ID No. 7.

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