Proteins and vaccines against infection by SARS-CoV-2 omicron variants and their subtypes.

Abstract

This invention relates to proteins and vaccines against infection by SARS-CoV-2 omicron mutants and their subtypes, and belongs to the pharmaceutical field. To address the problem of the continued lack of effective drugs for preventing and treating infection by SARS-CoV-2 omicron mutants and their subtypes, this invention provides proteins and vaccines against infection by SARS-CoV-2 omicron mutants and their subtypes, which are optimally designed based on the RBD sequences in the S proteins of SARS-CoV-2 omicron mutants and sublines BA.4 / 5, BQ.1.1, and XBB.1.5, and can help the host resist coronavirus infection, and in particular has better preventive and therapeutic effects against cross-infection caused by SARS-CoV-2 omicron mutants and their subtype viruses.

Description

Technical Field

[0001] The present invention relates to proteins and vaccines against infections caused by the SARS-CoV-2 Omicron variant and its subtypes, and belongs to the pharmaceutical field.

Background Art

[0002] The novel coronavirus (SARS-Cov-2) is a new beta coronavirus named by the World Health Organization. This virus has an envelope, its particles are circular or oval, often polymorphic, and have a diameter of 60 nm to 140 nm. Its genetic characteristics are significantly different from those of SARS-CoV and MERS-CoV, meaning it is a novel coronavirus strain that has never been discovered in humans before. Currently, there are five main types of SARS-Cov-2 variants: Alpha, Beta, Gamma, Delta, and Omicron, and the Omicron variant is further classified into several sub-lineages such as BA.1, BA.2, BA.2.12.1, BA.4, BA.5, BQ.1.1, and XBB.1.5.

[0003] The main structural proteins of SARS-CoV-2 include spike (S) protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein. Among them, the S protein plays an important role in virus infection and pathogenicity and is often used as an antigen for vaccines. Since SARS-CoV-2 variants carry multiple mutation sites and the S protein of the virus also has multiple mutation sites, the variants can escape to some extent from the antibodies induced by the vaccines of the SARS-CoV-2 precursor strains, thereby reducing the effectiveness of the SARS-CoV-2 vaccines or reducing their protective immunity, posing a major challenge to the efforts of epidemic suppression. Therefore, the development of vaccines against various variants of the SARS-CoV-2 virus, especially broad-spectrum vaccines against various variants of the SARS-CoV-2 virus, is very important for the prevention and control of SARS-CoV-2.

Summary of the Invention

Means for Solving the Problems

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the object of the present invention is to provide a protein against infection by the SARS-CoV-2 Omicron variant and its subtypes. Another object of the present invention is to provide a vaccine containing the protein and used for the prevention and / or treatment of infection by the SARS-CoV-2 Omicron variant and its subtypes.

[0005] The present invention provides a protein against infection by the SARS-CoV-2 Omicron variant and its subtypes, comprising the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or an amino acid sequence having a homology exceeding 98% and the same or similar biological activity as the amino acid sequence shown in any one of SEQ ID NOs: 1 to SEQ ID NO: 6.

[0006] Furthermore, the homologous amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 satisfy at least one of the following conditions: in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, the 52nd amino acid F is mutated to S, the 54th amino acid P is mutated to S, and the 56th amino acid F is mutated to S.

[0007] Furthermore, the homologous amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 satisfies at least one of the following conditions: in SEQ ID NO: 4 or SEQ ID NO: 351st amino acid sequence F is mutated to S, 353rd amino acid sequence P is mutated to S, and 355th amino acid sequence F is mutated to S. The homologous amino acid sequence shown in SEQ ID NO: 6 satisfies at least one of the following conditions: in SEQ ID NO: 6, 352nd amino acid sequence F is mutated to S, 354th amino acid sequence P is mutated to S, and 356th amino acid sequence F is mutated to S.

[0008] Furthermore, the amino acid sequence of the protein is selected from at least one of sequence numbers 1 through 12.

[0009] The present invention further provides a protein precursor to which a signal peptide and / or protein tag is linked to a protein against infection by SARS-CoV-2 omicron mutants and their subtypes.

[0010] Preferably, the protein tag is selected from at least one of the following: histidine tag (6His tag), thioredoxin tag (Trx tag), glutathione transferase tag, ubiquitin-like modified protein tag, maltose-binding protein tag, c-Myc protein tag, Avi protein tag, and substance A protein tag using a nitrogen source.

[0011] Furthermore, the protease recognition region for cleaving the protein tag is also linked to the protein that protects against infection by the SARS-CoV-2 omicron mutant and its subtypes.

[0012] Preferably, the protease is selected from at least one of the following: enterokinase (EK enzyme), TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

[0013] Furthermore, the amino acid sequence of the precursor is selected from at least one of sequence numbers 13 to 24.

[0014] The present invention further provides polynucleotides that encode proteins or precursors.

[0015] Furthermore, the nucleotide sequence is selected from at least one of sequence numbers 25 to 30.

[0016] The present invention further provides a recombinant vector comprising a polynucleotide.

[0017] Furthermore, the recombinant vector uses at least one of the following: an insect baculovirus expression vector, a mammalian cell expression vector, an E. coli expression vector, and a yeast expression vector.

[0018] Preferably, the insect baculovirus expression vector is pFastBac1.

[0019] Preferably, the E. coli expression vector is pET32a.

[0020] Preferably, the yeast expression vector is pPICZaA.

[0021] Preferably, the mammalian cell expression vector is a CHO cell expression vector.

[0022] More preferably, the CHO cell expression vector is pTT5 or FTP-002.

[0023] The present invention further provides a host cell containing a recombinant vector.

[0024] Furthermore, the host cell used is at least one of insect cells, mammalian cells, Escherichia coli, and yeast. Preferably, the insect cell is selected from at least one of sf9 cells, sf21 cells, and Hi5 cells.

[0025] Preferably, the mammalian cells are CHO cells.

[0026] The present invention further provides a method for preparing a protein or precursor, the method comprising the steps of culturing host cells to express the protein or precursor, and then recovering the protein to obtain the protein.

[0027] The present invention further provides a protein composition for infection by SARS-CoV-2 or its variants, comprising at least two or more combinations, preferably at least two to four proteins, of delta protein, delta full-length S protein, BA.5 protein, BQ.1.1 protein, XBB.1.5 protein, BA.4 / 5 full-length S protein, BQ.1.1 full-length S protein, XBB.1.5 full-length S protein, Mu-BA.5 protein, Mu-BQ.1.1 protein, Mu-XBB.1.5 protein, BA.4 / 5 full-length S protein after amino acid mutation, BQ.1.1 full-length S protein after amino acid mutation, and XBB.1.5 full-length S protein after amino acid mutation.

[0028] More preferably, the protein composition comprises at least two or more combinations of BA.5 protein, BQ.1.1 protein, XBB.1.5 protein, BA.4 / 5 full-length S protein, BQ.1.1 full-length S protein, and XBB.1.5 full-length S protein.

[0029] More preferably, the protein composition comprises at least two combinations of delta protein, Mu-BA.5 protein, Mu-BQ.1.1 protein, and Mu-XBB.1.5 protein.

[0030] More preferably, the protein composition comprises at least two combinations of Mu-BA.5 protein, Mu-BQ.1.1 protein, Mu-XBB.1.5 protein, full-length S protein after BA.4 / 5 amino acid mutation, full-length S protein after BQ.1.1 amino acid mutation, and full-length S protein after XBB.1.5 amino acid mutation.

[0031] Furthermore, the BA.5 protein, BQ.1.1 protein, XBB.1.5 protein, BA.4 / 5 full-length S protein, BQ.1.1 full-length S protein, XBB.1.5 full-length S protein, Mu-BA.5 protein, Mu-BQ.1.1 protein, Mu-XBB.1.5 protein, BA.4 / 5 full-length S protein after amino acid mutation, BQ.1.1 full-length S protein after amino acid mutation, and XBB.1.5 full-length S protein after amino acid mutation include proteins or precursors.

[0032] Furthermore, the amino acid sequences of the delta protein, delta full-length S protein, BA.5 protein, BQ.1.1 protein, XBB.1.5 protein, BA.4 / 5 full-length S protein, BQ.1.1 full-length S protein, XBB.1.5 full-length S protein, Mu-BA.5 protein, Mu-BQ.1.1 protein, Mu-XBB.1.5 protein, BA.4 / 5 amino acid mutation full-length S protein, BQ.1.1 amino acid mutation full-length S protein, and XBB.1.5 amino acid mutation full-length S protein are constructed based on at least one amino acid sequence shown in SEQ ID NOs. 31, SEQ ID NOs. 33, and SEQ ID NOs. 1 to 24.

[0033] The present invention further provides a vaccine for preventing and / or treating infection by SARS-CoV-2 omicron variants and subtypes, comprising a protein, a precursor and / or a protein composition, and a pharmaceutically acceptable excipient or adjuvant component.

[0034] Furthermore, the adjuvant component is an immunoadjuvant.

[0035] Preferably, the immunoadjuvant is selected from at least one of a squalene-based oil-in-water emulsion, aluminum salts, calcium salts, plant saponins, plant polysaccharides, monophosphate tripeptide A, muramyl dipeptide, muramyl tripeptide, bacterial toxins, GM-CSF cytokines, lipids, and cationic liposome materials.

[0036] Furthermore, the squalene-based oil-in-water emulsion is MF59, the aluminum salt is selected from at least one of aluminum hydroxide and alum, the calcium salt is tricalcium phosphate, the plant saponin is either QS-21 or ISCOM, the plant polysaccharide is astragalus polysaccharide, the bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin, the lipid is selected from at least one of phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine, and the cation The liposome material is selected from at least one of (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloride)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamide)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

[0037] Furthermore, vaccines are available as injections, nasal sprays, and oral preparations. Additionally, vaccines are available as intramuscular injections.

[0038] The present invention further provides the use of proteins, precursors and / or protein compositions or vaccines in preparing drugs for the treatment and / or prevention of SARS-CoV-2 or variant infection or pathogenicity.

[0039] Furthermore, the SARS-CoV-2 variants were selected from one of the alpha, beta, gamma, delta, or omicron strains, and the omicron strain included at least one of BA.1, BA.2, BA.2.12.1, BA.4, BA.5, BQ.1.1, and XBB.1.5.

[0040] The present invention further provides an mRNA vaccine for preventing and / or treating infection by SARS-CoV-2 omicron mutants and their subtypes, comprising a polynucleotide sequence encoding one of the amino acids from SEQ ID NOs: 1 to SEQ ID NOs: 24.

[0041] The present invention further provides an adenovirus vaccine for preventing and / or treating infection by SARS-CoV-2 omicron mutants and their subtypes, comprising a polynucleotide sequence encoding one of the amino acids from SEQ ID NOs: 1 to SEQ ID NOs: 24.

[0042] The following sequences are constructed by the applicant based on the RBD sequences of the S protein in SARS-CoV-2 or its variant delta, the Omicron mutant sublineages BA.4 / 5, BQ.1.1, and XBB.1.5, or after mutating one or more sites.

[0043] Here, recombinant proteins or precursors formed based on the RBD sequences of the S protein in the Omicron mutant sublineages BA.4 / 5, BQ.1.1, and XBB.1.5, as well as the 7-amino acid repeat regions HR1 and HR2 of the SARS-CoV-2 S protein, are defined as the BA.5 protein, BQ.1.1 protein, and XBB.1.5 protein, respectively.

[0044] Proteins or precursors constructed based on the full-length S protein sequences of the Omicron mutant sublines BA.4 / 5, BQ.1.1, and XBB.1.5 are defined as the BA.4 / 5 full-length S protein, the BQ.1.1 full-length S protein, and the XBB.1.5 full-length S protein, respectively.

[0045] Recombinant proteins or precursors formed by introducing three amino acid mutations (F371S, P373S, F375S, residue numbers counted from the full-length sequence of wild-type SARS-CoV-2) into the S protein RBD sequences of the Omicron mutant sublines BA.4 / 5, BQ.1.1, and XBB.1.5, and then combining them with the 7-amino acid repeat regions HR1 and HR2 of the SARS-CoV-2 S protein, are defined as Mu-BA.5 protein, Mu-BQ.1.1 protein, and Mu-XBB.1.5 protein, respectively.

[0046] Full-length S proteins or precursors constructed from the S protein RBD sequences of omicron mutant sublines BA.4 / 5, BQ.1.1, and XBB.1.5, each possessing three amino acid mutations (F371S, P373S, and F375S), are defined as the BA.4 / 5 amino acid mutation full-length S protein, the BQ.1.1 amino acid mutation full-length S protein, and the XBB.1.5 amino acid mutation full-length S protein, respectively.

[0047] Here, Sequence IDs 1 to 3 are the RBD-HR1-HR2 amino acid sequences of the Omicron mutant sublineages BA.4 / 5, BQ.1.1, and XBB.1.5, respectively.

[0048] Sequence IDs 4 through 6 are the full-length amino acid sequences of the S protein from the Omicron mutant sublineages BA.4 / 5, BQ.1.1, and XBB.1.5, respectively.

[0049] Sequence IDs 7 to 9 are the RBD-HR1-HR2 amino acid sequences obtained when the RBD amino acid sequences of the Omicron mutant sublineages BA.4 / 5, BQ.1.1, and XBB.1.5 undergo mutations in three amino acids (F371S, P373S, and F375S), respectively.

[0050] Sequence IDs 10 to 12 represent the full-length amino acid sequences of the S protein obtained when the RBD amino acid sequences of the Omicron mutant sublineages BA.4 / 5, BQ.1.1, and XBB.1.5 undergo mutations in three amino acids (F371S, P373S, and F375S), respectively.

[0051] Sequence IDs 13 to 24 are the RBD-HR1-HR2 amino acid sequences of the Omicron mutant sublineages BA.4 / 5, BQ.1.1, and XBB.1.5, and these amino acid sequences are modified by adding a signal peptide-Trx tag-6His tag-EK enzyme before the mutant RBD-HR1-HR2 amino acid sequences, i.e., Sequence IDs 13 to 15 and Sequence IDs 19 to 21.

[0052] The signal peptide is added before the full-length amino acid sequences of the S protein in the Omicron mutant sublineages BA.4 / 5, BQ.1.1, and XBB.1.5, as well as the full-length amino acid sequences of the mutant S protein, i.e., SEQ ID NOs. 16-18 and SEQ ID NOs. 22-24.

[0053] Sequence IDs 25 to 30 are nucleotide sequences of signal peptide-Trx tag-6His tag-EK enzyme amino acid sequences that are prepended to the RBD-HR1-HR2 amino acid sequence, which encodes the omicron mutant sublineages BA.4 / 5, BQ.1.1, and XBB.1.5, and the mutant RBD-HR1-HR2 amino acid sequence (Sequence IDs 19 to 21), respectively.

[0054] SEQ ID NO: 31 is the precursor amino acid sequence of the delta protein, SEQ ID NO: 32 is the nucleotide sequence encoding SEQ ID NO: 31, SEQ ID NO: 33 is the full-length amino acid sequence of the delta S antigen, SEQ ID NO: 34 is the full-length amino acid sequence of the omicron-BA.1 S antigen, SEQ ID NO: 35 is the full-length amino acid sequence of an amino acid sequence variant of the omicron-BA.1 S antigen, SEQ ID NO: 36 is the amino acid sequence of the delta protein, and SEQ ID NO: 37 is RBD BA.1-The precursor amino acid sequence of the HR protein, SEQ ID NO: 38 is the nucleotide sequence encoding SEQ ID NO: 37, and SEQ ID NO: 39 is RBD BA.1 - This is the amino acid sequence of the HR protein.

[0055] In this invention, three amino acids (F371S, P373S, F375S) can be mutated to the S protein RBD of the Omicron mutant sublines BA.4 / 5, BQ.1.1, and XBB.1.5 in order to enhance the antiviral activity of the vaccine and significantly increase neutralizing antibodies.

[0056] Sequence ID 1: RBD-HR1-HR2 amino acid sequence of BA.5-Omicron mutant subline BA.4 / 5

[0057] VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCN GVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0058] Sequence ID 2: RBD-HR1-HR2 sequence of BQ.1.1-Omicron mutant sublineage BQ.1.1

[0059] VQPTESIVRFPNITNLCPFDEVFNATTFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSTVGGNYNYRYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0060] Sequence ID 3: RBD-HR1-HR2 sequence of XBB.1.5-Omicron mutant sublineage XBB.1.5

[0061] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0062] Sequence ID 4: Full-length amino acid sequence of the S protein of the Omicron mutant sublineage BA.4 / 5

[0063]

[0064] Sequence ID 5: Full-length amino acid sequence of the S protein of Omicron mutant sublineage BQ.1.1

[0065]

[0066] Sequence ID 6: Full-length amino acid sequence of the S protein of Omicron mutant sublineage XBB.1.5

[0067]

[0068] Sequence ID 7: Mu-BA.5 (Sequence after RBD mutation in the RBD-HR1-HR2 amino acid sequence of the Omicron mutant sublineage BA.4 / 5)

[0069] VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCN GVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0070] Sequence ID 8: Mu-BQ.1.1 (Sequence after RBD mutation in the RBD-HR1-HR2 amino acid sequence of the Omicron mutant sublineage BQ.1.1)

[0071] VQPTESIVRFPNITNLCPFDEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSTVGGNYNYRYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0072] Sequence ID 9: Mu-XBB.1.5 (Sequence after RBD mutation in the RBD-HR1-HR2 amino acid sequence of the Omicron mutant sublineage XBB 1.5)

[0073] VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0074] Sequence ID 10: Mutant sequence of the full-length amino acid sequence of the S protein of the Omicron mutant sublineage BA.4 / 5.

[0075]

[0076] Sequence ID 11: Mutant sequence of the full-length amino acid sequence of the S protein of Omicron mutant sublineage BQ.1.1

[0077]

[0078] Sequence ID 12: Mutant sequence of the full-length amino acid sequence of the S protein of the Omicron mutant sublineage XBB 1.5.

[0079]

[0080] SEQ ID NO: 13:BA.5 signal peptide -Trx-6His-EK-RBD-HR1-HR2 amino acid sequence

[0081] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0082] SEQ ID NO: 14: Signal peptide-Trx-6His-EK-RBD-HR1-HR2 amino acid sequence of BQ.1.1

[0083] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATTFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSTVGGNYNYRYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0084] SEQ ID NO: 15:XBB.1.5 signal peptide -Trx-6His-EK-RBD-HR1-HR2 amino acid sequence

[0085] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0086] Sequence ID 16: Amino acid sequence for adding a signal peptide before the full-length amino acid sequence of the S protein of the Omicron mutant sublineage BA.4 / 5.

[0087]

[0088] Sequence ID 17: Amino acid sequence to add a signal peptide after the full-length amino acid sequence of the S protein of the Omicron mutant sublineage BQ.1.1.

[0089]

[0090] Sequence ID 18: Amino acid sequence to add a signal peptide after the full-length amino acid sequence of the S protein of the Omicron mutant sublineage XBB.1.5

[0091]

[0092] SEQ ID NO: 19: Amino acid sequence of signal peptide Trx-6His-EK-RBD-HR1-HR2 of Mu-BA.5

[0093] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0094] SEQ ID NO: 20: Amino acid sequence of signal peptide Trx-6His-EK-RBD-HR1-HR2 of Mu-BQ.1.1

[0095] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSTVGGNYNYRYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0096] SEQ ID NO: 21: Amino acid sequence of signal peptide Trx-6His-EK-RBD-HR1-HR2 of Mu-XBB.1.5

[0097] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0098] Sequence ID 22: Amino acid sequence for adding a signal peptide before the full-length amino acid sequence of the S protein of mutant omicron sublineage BA.4 / 5.

[0099]

[0100] Sequence ID 23: Amino acid sequence for adding a signal peptide before the full-length amino acid sequence of the S protein of mutant omicron sublineage BQ.1.1.

[0101]

[0102] Sequence ID 24: Amino acid sequence for adding a signal peptide before the full-length amino acid sequence of the S protein of mutant omicron sublineage XBB 1.5.

[0103]

[0104] Sequence ID 25: Nucleotide sequence encoding Sequence ID 13 (construction BA.4 / 5 protein)

[0105]

[0106] Sequence ID 26: Nucleotide sequence encoding Sequence ID 14 (construction BQ.1.1 protein)

[0107]

[0108] Sequence ID 27: Nucleotide sequence encoding Sequence ID 15 (Construction XBB 1.5 protein)

[0109]

[0110] Sequence ID 28: Nucleotide sequence encoding Sequence ID 19 (construction Mu-BA 4 / 5 protein)

[0111]

[0112] Sequence ID 29: Nucleotide sequence encoding Sequence ID 20 (construction Mu-BQ.1.1 protein)

[0113]

[0114] Sequence ID 30: Nucleotide sequence encoding Sequence ID 21 (construction Mu-XBB 1.5 protein)

[0115]

[0116] Sequence ID 31: Amino acid sequence of delta protein precursor

[0117] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYA DSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0118] Proteins formed by amino acid sequences designed based on the SARS-CoV-2 delta strain are defined as delta proteins, and the amino acid sequence is as shown in SEQ ID NO: 36. The protein sequence contains only the RBD and HR sequences, excluding the signal peptide, Trx, 6His tag, and EK amino acid sequence.

[0119] Sequence ID 32: Nucleotide sequence encoding Sequence ID 31

[0120]

[0121] SEQ ID NO: 33: Full-length amino acid sequence of the delta-S antigen

[0122]

[0123] Sequence ID 34: Omicron-BA.1 S antigen full-length amino acid sequence

[0124]

[0125] Sequence ID 35: Full-length amino acid sequence of the omicron-BA.1 S antigen after mutation

[0126]

[0127] Sequence ID 36: Amino acid sequence of the delta protein

[0128] VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0129] Sequence ID 37: RBD BA.1 - Amino acid sequence of HR protein precursor

[0130] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYA DSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0131] Sequence ID 38: Nucleotide sequence encoding Sequence ID 37

[0132]

[0133] Sequence ID 39: RBD BA.1 - Amino acid sequence of HR protein

[0134] VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCN GVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0135] In the aforementioned amino acid sequence, the HR1 and HR2 sequences of the S protein are sequence segments beginning with "LYENQKL" and ending with "IDLQEL".

[0136] Beneficial Effects: Firstly, based on the RBD sequence or mutant RBD sequence and 7-amino acid repeat regions HR1 and HR2 in the S protein of SARS-CoV-2 mutant Delta, Omicron mutant sublineages BA.4 / 5, BQ.1.1, BA.1, and XBB.1.5, the present invention can spontaneously form trimers for protein recombination, and to prepare multiple recombinant proteins, protein construction is carried out based on the full-length S protein sequences of Delta, BA.4 / 5, BQ.1.1, BA.1, and XBB.1.5, which will serve as vaccines to resist infection by different SARS-CoV-2 mutants.

[0137] In this invention, different recombinant proteins are combined and added together with an adjuvant to prepare multivalent vaccines such as bivalent, trivalent, and quaternary vaccines. Based on animal experiments, recombinant proteins prepared based on the RBD sequences of the delta and omicron mutant sublines BA.4 / 5 and XBB 1.5, as well as the 7-amino acid repeat regions HR1 and HR2, were used. Delta -HR, RBD BA.5 -hr and RBD XBB.1.5 -HRs are the delta protein, BA.5 protein, and XBB 1.5 protein, respectively. A trivalent vaccine prepared by combining the delta protein, BA.5 protein, and XBB 1.5 protein and adding an adjuvant was found to significantly enhance the protective efficacy of the vaccine, increase the titer of neutralizing antibodies and the ability to resist viral infection, have a broad spectrum, and in particular have proven to have better preventive and therapeutic effects against cross-infection caused by Omicron and its subtypes. [Brief explanation of the drawing]

[0138] [Figure 1] This is a graph showing the design of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR in Embodiment 1. [Figure 2] These are 1% agarose gel electrophoresis images of PCR products derived from clones 1-3 in Embodiment 1. [Figure 3] This is a 1% agarose gel electrophoresis graph of recombinant bacmid identified by PCR in Embodiment 1. [Figure 4] This is a flowchart for packaging and amplification of recombinant baculovirus in Embodiment 1. [Figure 5] This graph shows the results of WB verification during baculovirus proliferation in Embodiment 1. [Figure 6] This graph shows the results of elution supernatant after baculovirus infection using Ni-affinity chromatography in Embodiment 1. [Figure 7]This graph shows the results of verifying the enzymatic digestion action of the EK enzyme-digested sample in Embodiment 1. [Figure 8] This graph shows the packing purification by Ni affinity chromatography and SDS-PAGE identification after EK enzyme digestion in Embodiment 1. [Figure 9] This graph shows the IgG levels in the serum of mice immunized with different vaccines in Embodiment 3. [Figure 10] This graph shows the results of neutralizing antibodies in mouse serum based on combinations of multiple vaccines in Embodiment 4. [Figure 11] This graph shows the results of neutralizing antibodies in mouse serum for RBDWT monovalent vaccine, RBDXBB.1.5 monovalent vaccine, and bivalent vaccine (RBDWT + RBDXBB.1.5) mixed in different ratios in Embodiment 4. [Figure 12] This graph shows the results of neutralizing antibodies in the serum of mice immunized with RBDDelta-HR, RBDBA.5-HR, RBDXBB.1.5-HR monovalent vaccine and Tri-Vac vaccine mixed in different ratios in Embodiment 4. [Figure 13] This graph shows the results of neutralizing antibodies in the serum of mice immunized with 1.67 μg and 10 μg of RBDDelta-HR, RBDBA.5-HR, RBDXBB.1.5-HR, and 10 μg of Tri-Vac vaccine mixed in a 1:1:4 ratio in Embodiment 4. [Figure 14] This graph shows the results of pseudovirus neutralizing antibodies in the serum of serial mice that received the trivalent vaccine (RBDDelta-HR + RBDBA.5-HR + RBDXBB.1.5-HR) mixed in a 1:1:4 ratio in Embodiment 4. [Figure 15] This graph shows the results of neutralizing antibodies in the serum of serial mice conditioned on bivalent vaccines mixed in different ratios in Embodiment 4. [Figure 16]This graph shows the results of neutralizing antibodies in the serum of mice that were successively immunized with the monovalent vaccine RBDXBB.1.5 and the bivalent vaccine (RBDWT + RBDXBB.1.5, mixing ratio 1:3) in Embodiment 4. [Figure 17] This graph shows the results of neutralizing antibodies in the serum of mice that were sequentially immunized with the monovalent vaccine RBDXBB.1.5- and the trivalent vaccine (mixture ratio 1:1:4) in Embodiment 4. [Figure 18] This graph shows the results of neutralizing antibodies against the true virus in the serum of Tri-Vac mice mixed in a 1:1:4 ratio in Embodiment 5. [Figure 19] This graph shows the results of neutralizing antibodies against true viruses in the serum of Tri-Vac serial mice mixed in a 1:1:4 ratio in Embodiment 5. [Figure 20] This graph shows the results of neutralizing antibodies against the true virus in the serum of serial mice conditioned on a bivalent vaccine mixed in a 1:3 ratio in Embodiment 5. [Figure 21] This graph shows the results of a challenge experiment in mice immunized with different doses of the trivalent vaccine Tri-Vac mixed in a 1:1:4 ratio in Embodiment 6. [Figure 22] This graph shows the results of a live virus challenge experiment in mice that were sequentially immunized with a bivalent vaccine (RBDWT + RBDXBB.1.5, mixing ratio 1:3) in Embodiment 6. [Modes for carrying out the invention]

[0139] Embodiment Terms and abbreviations:

[0140] The present invention mainly induces immune responses such as antibody production in vivo, blocks the binding of the S protein of the SARS-CoV-2 virus to the ACE2 receptor of host cells, particularly the ACE2 receptor binding domain that blocks the S protein, thereby helping the host resist the infection of the coronavirus. In particular, it has a better preventive and therapeutic effect against cross-infection caused by SARS-CoV-2 or its mutant viruses, such as cross-infection caused by SARS-CoV-2 or the Delta variant, Omicron variant sub-lineages BA.5, BQ.1.1, XBB.1.5, etc., and provides proteins, monovalent vaccines and multivalent vaccines against infections caused by SARS-CoV-2 Omicron variants and their subtypes.

[0141] The solution of the present invention will be described with reference to embodiments. It will be understood by those skilled in the art that the following examples are not intended to limit the scope of the present invention, but merely to illustrate the present invention. When specific technologies or conditions are not specified in the embodiments, the technologies or conditions described in the literature or product specifications of the relevant technical field should be prioritized. When the manufacturer of the reagents or equipment used is not indicated, they are all conventional products that can be obtained by market purchase.

[0142] Embodiment 1: Preparation of recombinant protein through systematic expression of insect baculovirus (taking S-RBD XBB.1.5 -HR as an example)

[0143] 1. S-RBD XBB.1.5 -HR construction and design

[0144] Since the SARS-CoV-2 S protein is a membrane-located protein, to mimic its secretion process, the GP67 signal peptide sequence was added to the N-terminus of the S-RBD(omicron_XBB.1.5)-HR protein during construction to support protein secretion and expression, although this signal peptide would be spontaneously removed by insect cells during the protein secretion process. On the other hand, the thioredoxin (Trx) tag from Spodoptera fulgiperda (S. fulgiperda) was added after the GP67 signal peptide to support the folding of S-RBD(omicron_XBB.1.5)-HR, the 6×his tag was added to support subsequent purification, and the EK enzyme digestion site was added to remove both the Trx and 6×his tags. Protein expression construction and design can be performed by removing all non-S-RBD(omicron_XBB.1.5)-HR redundant amino acids by EK enzyme digestion. The modes constructed and designed through expression were as shown in Figure 1; namely, the amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR is as shown in SEQ ID NO: 15, and the nucleotide sequence encoding SEQ ID NO: 15 is as shown in SEQ ID NO: 27.

[0145] The above sequence was constructed based on sites 320–545 of the RBD sequence of the S protein in the SARS-CoV-2 omicron mutant XBB.1.5, where the amino acids at sites 52, 54, and 56 were F, P, and F, respectively.

[0146] 2. Identification for Recombinant Plasmid Construction

[0147] The signal peptide-Trx-6His-EK-RBD-HR1-HR2 amino acid sequence of XBB 1.5, encoded by SEQ ID NO: 15, was cloned into the pFastBac1 vector plasmid as a designed coding fragment and subjected to PCR identification of bacterial solution. Bacterial solution PCR identification, as shown in Figure 2, demonstrated that clones 2 and 3 successfully amplified the GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR fragment in three selected clones.

[0148] 3. Identification of recombinant bacmids

[0149] We selected precisely identified pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR recombinant clones, extracted the recombinant plasmids, transformed DH10B competent cells, and performed identification by bacterial suspension PCR using blue-white spots. The bacterial suspension PCR products were detected by 1% agarose gel electrophoresis, and the identification results are shown in Figure 3. White spots indicated bacmid clones that underwent recombination, while blue spots indicated bacmid clones that did not undergo recombination.

[0150] 4. Packaging of recombinant baculoviruses

[0151] Recombinant bacmid was transfected into sf9 insect cells, and P0 passaged recombinant baculovirus was recovered after 5 days. The flowchart for packaging and amplification of the recombinant baculovirus is shown in Figure 4.

[0152] 5. Expression and validation of target proteins

[0153] The expression of the target protein was performed simultaneously with the amplification of the baculovirus described above. The His tag was included before removing the target protein tag, and therefore we verified the expression of the recombinant protein using anti-His WB experiments. The verification results showed clear stripes between the 40KD–55KD marker stripes, indicating that the size was consistent with that of the Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR protein, demonstrating successful baculovirus amplification and target protein expression. The test results are shown in Figure 5.

[0154] 6. Purification and identification of target proteins

[0155] P0 passaged recombinant baculovirus was used to infect sf9 cells, and the cell culture medium was collected after 3 days. Protein purification was verified by Ni affinity chromatography. The results are shown in Figure 6. The target protein was mainly eluted under 40 mM and 250 mM imidazole, and it is possible that high-purity target protein was obtained after elution.

[0156] 7. Verification of tag removal by EK enzyme digestion

[0157] The target protein eluate was concentrated to a concentration of 1 mg / mL, EK protease was added, and the protein was digested enzymatically at 18°C ​​for 14 hours. Identification was then performed by SDS-PAGE gel electrophoresis. The results are shown in Figure 7. These results indicate that the EK enzyme can remove the Trx-His-EK (amino acid sequence of the EK enzyme digestion site) tag from the target protein.

[0158] 8. Verification of the removal of removed tags

[0159] The sample after EK enzyme digestion was back-suspended for 10 minutes using Ni-affinity chromatography packing, and then eluted using 250 mM imidazole. The Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR protein (the complete, uncleaved protein) and S-RBD(Omicron_XBB.1.5)-HR protein were separated from the cleaved Trx-His-EK tag. The results are shown in Figure 8. These results indicate that the Trx-His-EK tag can be removed after cleavage by the back-suspension purification method using Ni-affinity chromatography packing. Therefore, an S-RBD(Omicron_XBB.1.5)-HR protein without either tag was obtained, resulting in a recombinant RBD protein with the protein amino acid sequence shown in SEQ ID NO: 3. XBB.1.5 -HR was defined as the XBB.1.5 protein.

[0160] Similarly, recombinant ProteinRBD XBB.1.5 - Refer to the above method for building HR.

[0161] Recombinant protein RBD XBB.1.5 We constructed a protein and removed the HR1 and HR2 sequences from the amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB 1.5) as shown in SEQ ID NO: 15, and removed the HR1 and HR2 sequences from the amino acid sequence of the recombinant protein as shown in SEQ ID NO: 3.

[0162] Recombinant protein RBD BA.5 -HR, i.e., the BA.5 protein, was constructed, and the amino acid sequence of GP67-Trx-His-EK-S-RBD(OmicronBA.5)-HR is as shown in SEQ ID NO: 13, the nucleotide sequence encoding that amino acid sequence is shown in SEQ ID NO: 25, and the amino acid sequence of the recombinant protein is as shown in SEQ ID NO: 1. RBD BA.5-HR construction was based on sites 320–545 of the RBD sequence of the S protein in the SARS-CoV-2 omicron mutant BA.4 / 5, where amino acids at sites 52, 54, and 56 were F, P, and F, respectively.

[0163] Recombinant protein RBD Delta -HR, i.e., the delta protein, was constructed. The amino acid sequence of GP67-Trx-His-EK-S-RBD(delta)-HR is shown in SEQ ID NO: 31, the nucleotide sequence encoding that amino acid sequence is shown in SEQ ID NO: 32, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 36.

[0164] Recombinant protein RBD BA.1 -HR was constructed, and the amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_BA.1)-HR is as shown in SEQ ID NO: 37, the nucleotide sequence encoding that amino acid sequence is shown in SEQ ID NO: 38, and the amino acid sequence of the recombinant protein is as shown in SEQ ID NO: 39.

[0165] Recombinant protein RBD WT -HR was constructed. In the amino acid sequence of GP67-Trx-His-EK-S-RBD(original strain)-HR, RBD is the amino acid sequence spanning 320-545 sites of the SARS-CoV-2 original strain S protein, and the remaining amino acid sequence of the protein and its construction method are RBD XBB.1.5 -It matched HR's.

[0166] Similarly, recombinant ProteinRBD WT It is constructed using the RBD sequence of the protein consisting of amino acid sites 320-545 of the original SARS-CoV-2 strain's S protein, but the remaining protein sequence and construction method are RBD XBB.1.5 It matches that of [the other one].

[0167] By the method described above, the applicant has developed recombinant protein RBD XBB.1.5-HR (having the amino acid sequence shown in SEQ ID NO: 3), RBD XBB.1.5 , RBD Delta -HR (having the amino acid sequence shown in SEQ ID NO: 36), RBD BA.5 -HR (having the amino acid sequence shown in SEQ ID NO: 1), RBD BA.1 -HR (having the amino acid sequence shown in SEQ ID NO: 39), RBD WT -hr, and RBD WT We successfully expressed and constructed the recombinant protein. For subsequent research such as animal immunology, we diluted the expressed recombinant protein with PBS buffer and added an adjuvant separately to prepare a monovalent vaccine, or we prepared a multivalent vaccine using the combined recombinant protein.

[0168] Embodiment 2: Animal preparation, mouse immunization, and sample collection

[0169] 1. Immunization procedure for monovalent vaccine: Female NIH mice aged 6-8 weeks were purchased from Charles River Laboratories and reared in a pathogen-free environment at the National Key Laboratory for Biotherapy (Sichuan University). Recombinant protein was mixed with MF59 adjuvant in an isovolume ratio, and 10ug of protein was administered to each mouse three times at days 0, 14, and 28 to perform intramuscular immunization. Seven days after the three immunizations, the mice's blood was collected and neutralizing antibodies were examined to evaluate the immunizing effect of the vaccine.

[0170] 2. Immunization procedure for bivalent vaccine: Female NIH mice aged 6-8 weeks were purchased from Charles River Laboratories and raised in a specific pathogen-free environment at the National Key Laboratory for Biotherapy, Sichuan University. Two different recombinant proteins were mixed with an isovolume adjuvant MF59 in a specific mass ratio. 10ug of the protein was administered to each mouse three times at days 0, 14, and 28 to immunize the mice intramuscularly. Seven days after the three immunizations, the mice's blood was collected and neutralizing antibodies were examined to evaluate the vaccine's immune effect.

[0171] 3. Immunization Procedure for Trivalent Vaccine: Female NIH mice aged 6-8 weeks were purchased from Charles River Laboratories and raised in a specific pathogen-free environment at the National Key Laboratory for Biotherapy, Sichuan University. Three different recombinant proteins were mixed in a specific mass ratio and then mixed with an isovolume ratio of the adjuvant MF59. 10ug of the protein was administered to each mouse at three time points (days 0, 14, and 28) to perform intramuscular immunization. Seven days after the three immunizations, the mice's blood was collected to examine neutralizing antibodies and evaluate the vaccine's immune effect. Here, RBD Delta -HR+RBD BA.5 -HR+RBD XBB.1.5 - The HR trivalent vaccine was abbreviated as "Tri-Vac".

[0172] 4. Basic immunization procedure for the quadrivalent vaccine: Female NIH mice aged 6-8 weeks were purchased from Charles River Laboratories and raised in an environment free of specific pathogens at the National Key Laboratory for Biotherapy, Sichuan University. Four different recombinant proteins were mixed in specific mass ratios and then mixed with an isovolume ratio of the adjuvant MF59. 10ug of the protein was administered to each mouse three times at days 0, 14, and 28 to perform intramuscular immunization. Seven days after the three immunizations, the mice's blood was collected and neutralizing antibodies were examined to evaluate the vaccine's immune effect.

[0173] 5. Sequential immunization procedure: Female NIH mice aged 6-8 weeks were purchased from Charles River Laboratories and reared in an environment free of specific pathogens at the National Key Laboratory of Biotherapy, Sichuan University. Each mouse was immunized three times with 25U inactivated vaccine (abbreviated as IV, China National Pharmaceutical Group Co., Ltd.) on days 0, 14, and 42. Sequential reinforcement and intramuscular injection of inactivated vaccine and recombinant protein vaccine were performed separately after the final immunization, with an immunization dose of 10ug per mouse. Serum was collected 14 days after reinforcement to detect pseudovirus neutralizing antibodies.

[0174] Embodiment 3: Detection of antibodies by enzyme-linked immunosorbent assay (ELISA)

[0175] To detect anti-SARS-CoV-2 RBD-specific IgG and IgA, 96-well plates (NUNC-MaxiSorp, Thermo Fisher Scientific) were coated with 1 μg / mL recombinant RBD protein in carbonic acid-bicarbonate buffer and incubated overnight at 4°C. The following day, the plates were washed three times with 1×PBS (PBST) containing 0.1% Tween-20, and then sealed with PBST containing 1% BSA for 1 hour. Serially diluted serum, bronchopulmonary lavage, or nasal swabs in dilution buffer were added to the wells (100 μl / well). After incubation at 37°C for 1 hour, the plates were washed three times, and then diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10000, Southern Biotech, catalog number: 0107-05), HRP-conjugated goat anti-mouse IgG (1:5000), HRP-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-human IgG (Southern Biotech, catalog number: 62-8420), or HRP-conjugated goat anti-human IgA (Southern Biotech, catalog number: 2050-05) were added. After incubation at 37°C for 1 hour, the plates were washed three more times and then developed with 3,3',5,5'-tetramethylbenzidine (TMB) at room temperature for 10 minutes. Such incubations were stopped with 1 M H2SO4. Finally, absorbance at 450 nm was measured using a microwell plate reader (Spectramax ABS, Molecular Devices). Final titer was defined as the highest inverse dilution of serum with an absorbance of 2.1 times or more the negative control serum value.

[0176] The results are shown in Figure 9, and all monovalent RBDs were detected after completing three immunization doses. Delta -HR, RBD BA.5 -hr and RBD XBB.1.5 -HR, recombinant protein vaccines and trivalent vaccines TriVac(RBD) Delta-HR, RBD BA.5 -hard and RBDX XBB.1.5 - Mixing different ratios in order of HR was able to induce high levels of IgG. Separate RBD XBB.1.5 - HR vaccine and trivalent vaccine TriVac for RBD Delta -hr and RBD XBB.1.5 - The IgG generated for HR is clearly RBD BA.5 - Higher than HR, the trivalent vaccine TriVac is RBD XBB.1.5 - This demonstrated that it may provide stronger humoral immune protection against HR.

[0177] Embodiment 4: Pseudovirus neutralization test for SARS-CoV-2

[0178] To detect the titer of neutralizing antibodies in serum and bronchoalveolar lavage (BALF) samples, pseudovirus neutralization tests were performed as described above. Pseudoviruses expressing luciferase, including precursors, δ, and omicron sublineages (BA.2.75, BA.5, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.16, etc.), purchased from Genomeditech Company, were used. In short, the inactivated serum and BALF sample (30 minutes, 56°C) are diluted threefold in the range of 30 to 65610, and then the diluted inactivated serum and BALF sample are used.

[0179] The cells were incubated with pseudoviruses of equal volume and different dilutions at 37°C for 1 hour. Subsequently, 1.2 × 10⁶ HEK-293T cells expressing the human ACE2 receptor (293T / ACE2) were placed in each well. 4Cells were added one by one and incubated at 37°C for 48 hours to express luciferase. Finally, the supernatant was removed, and then the lysis reagent (Beyotime, RG005) was added along with the luciferase substrate. The luminescence of 293T / ACE2 cells was measured using a multimode microwell plate reader (PerkinElmer, USA). The 50% neutralization rate of pseudoviruses was measured and calculated using GraphPad Prism 8.0.2. The positive control group contained cells and viruses, the negative control group contained cells only, and the sample group contained cells, samples, and viruses. The neutralization percentage was calculated using the following formula. Neutralization (%) = (Positive sample - Sample being measured / Positive sample - Negative sample) 100%

[0180] RBD Delta -HR, RBD BA.5 -HR, RBD BA.1 -hr and RBD WT -HR proteins constitute monovalent, bivalent (mixing ratio 1:1), trivalent (mixing ratio 1:1:1), and quadrivalent (mixing ratio 1:1:1:1) vaccines for immunizing mice, respectively. Serum was collected and neutralizing antibody levels against WT, Delta, BA.1, BA.2, BA.2.12.1, BA.3, and BA.4 / 5 viruses were measured. As shown in Figure 10, pure RBD Delta -HR and the bivalent, trivalent, and quaternary vaccines prepared from it all produced stronger neutralizing antibodies, and the antibody levels of the multivalent vaccines were significantly higher than those of the pure protein vaccines. This suggests that multivalent vaccines may provide better protection against the original strain and omicron variant viruses.

[0181] RBD WT , RBD XBB.1.5 Monovalent vaccines and RBDs composed of proteins WT +RBD XBB.1.5Mice were immunized with a bivalent protein vaccine (different mixing ratios), and serum was collected to evaluate the levels of neutralizing antibodies against WT, Delta, BA.2.75, BA.5, BF.7, BQ.1, BQ.1.1, XBB, and XBB.1.5 viruses. As shown in Figure 11, pure RBD WT The protein vaccine showed strong protection against the original and delta strain viruses, but weak protection against the omicron variant virus. RBD XBB.1.5 The vaccine showed strong protection against the Omicron variant virus, but weak protection against the original and Delta strain viruses. However, no matter how high the mixing ratio, RBD WT +RBD XBB.1.5 The bivalent vaccine provides robust protection against the original strain, delta strain, and omicron variant virus, and RBD WT +RBD XBB.1.5 The unique advantages of the bivalent vaccine were demonstrated.

[0182] RBD Delta -HR, RBD BA.5 -hr and RBD XBB.1.5 -Mice were immunized with monovalent and Tri-Vac trivalent vaccines (different mixing ratios) using HR protein, serum was collected, and neutralizing antibody levels against WT, Delta, BA.2.75, BA.5, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, and XBB.1.16 viruses were measured. As shown in Figure 12, pure RBD Delta - The HR protein vaccine showed strong protection against the original strain and the delta variant virus, but weak protection against the omicron variant virus. Pure RBD BA.5 -HR showed strong protection only against BA.2.75, BA.5, BF.7, BQ.1, and BQ.1.1 variant viruses, but weak protection against other viruses, particularly the currently circulating XBB virus and its subtypes. Individual RBD XBB.1.5The vaccine shows weak protection against the original strain and the delta variant virus, but strong protection against the omicron variant virus. However, regardless of the mixing ratio, the Tri-Vac trivalent vaccine (different mixing ratios) shows strong protection against the original strain, the delta strain, and the omicron variant virus, and demonstrates clear protective effects against XBB and its subtypes. In the Tri-Vac trivalent vaccine, RBD Delta -HR, RBD BA.5 -hr and RBD XBB.1.5 -HR showed better efficacy when the mixing ratio was 1:1:4. When the protective effect of the trivalent vaccine with a 1:1:4 ratio was examined, as shown in Figure 13, the trivalent vaccine showed stronger protection against Delta, BA.5, and XBB.1.5 mutant viruses compared to the single component, compared to equal amounts of 10 μg of individual RBD. XBB.1.5 - It was clearly stronger than the HR protein vaccine.

[0183] To verify the continuous immunization effect of Tri-Vac (1:1:4 ratio), each mouse was first immunized intramuscularly with 25U inactivated vaccine (IV, China National Pharmaceutical Group Co., Ltd.) three times on days 0, 14, and 42. Inactivated vaccine reinforcement and recombinant protein vaccine reinforcement were then performed separately on day 70 after the final immunization, administered intramuscularly by injection, with an immunization dose of 10U per mouse. Serum was collected 14 days after reinforcement to detect pseudovirus neutralizing antibodies. As shown in Figure 14, homologous booster immunization provided protection only against WT and delta variant viruses, showing little to no protection against omicron variant viruses, particularly the currently circulating XBB and subtype variant viruses. However, the Tri-Vac trivalent vaccine with a 1:1:4 mixing ratio exhibited broad-spectrum protection against all SARS-CoV-2 and produced over 1000 neutralizing antibodies.

[0184] Similarly, bivalent vaccines (RBD) composed of different ratios WT +RBD XBB.1.5The following immunization procedure was used: NIH mice were immunized three times with an inactivated vaccine on days 0, 14, and 42, followed by sequential immunization with different ratios of bivalent protein vaccine on postimmunization day 128, and serum was collected on post-sequential immunization day 14 (day 142 of the immunization procedure) to detect pseudovirus neutralizing antibodies. The immunization dose was 10 μg of protein per mouse. As shown in Figure 15, sequential immunization also showed broad protection against the original WT strain, delta variants, and omicron variant viruses, including XBB and its subtypes, whereas immunization with the allogeneic inactivated vaccine provided protection only against the original WT strain and delta variant viruses.

[0185] As shown in Figures 16 and 17, NIH mice were intramuscularly immunized with 25U inactivated vaccine three times on days 0, 14, and 42 according to the immunization procedure, and 84 days after the last immunization, the inactivated vaccine was administered to RBD. XBB.1.5 / RBD XBB.1.5 -HR, RBD WT +RBD XBB.1.5 Bivalent vaccine (mixture ratio 1:3) / RBD Delta -HR+RBD BA.5 -HR+RBD XBB.1.5 - HR trivalent vaccine (mixture ratio 1:1:4) was administered intramuscularly as a continuous intensification, with an immunizing dose of 10 ug per mouse. Results for pseudovirus neutralizing antibodies showed broad protective efficacy of the bivalent and trivalent vaccines against the original WT strain, BA.5 omicron mutant, and omicron mutant viruses including XBB and its subtypes. However, immunization with homologous inactivated vaccine provided protection only against the original WT mutant virus. The protective efficacy of the monovalent vaccine was equivalent to that of the bivalent vaccine.

[0186] Embodiment 5: SARS-CoV-2 true virus neutralization test

[0187] Neutralizing antibodies against live mutant SARS-CoV-2 in serial mouse serum samples from the trivalent vaccine Tri-Vac (mixture ratio 1:1:4) were tested through actual viral neutralization tests. Diluted serum from each group was mixed with live SARS-CoV-2 virus at a 50% tissue culture infectious dose (TCID50). After incubation at 37°C for 1 hour, the mixture was tested on Vero E6 cells (5 × 10⁶). 4 The sample was added to a 96-well microwell plate lined with (1 / well) and incubated for 72 hours. Cellular pathogenicity (CPE) was measured under a microscope, and the titer of the neutralizing antibody that resulted in EC50 inhibition (50% neutralization) in immunoserum was calculated.

[0188] Three different doses of the Tri-Vac vaccine (mixture ratio 1:1:4) were administered to the muscles three times on days 0, 14, and 28. Blood was collected seven days after the three immunizations, and the neutralizing ability against live viruses in the serum was detected. The results are shown in Figure 18. Mouse serum immunized with the trivalent vaccine showed stronger neutralizing ability against live viruses of Delta, BA.2, BA.5.2.48, and XBB.1.16 than the control group. In particular, the titer of the neutralizing antibody against XBB.1.16, a currently circulating strain, reached over 1000, and the titer in the high-dose group was higher than that in the low-dose group, showing a dose-dependent relationship.

[0189] Each mouse was immunized intramuscularly with 25U of inactivated vaccine three times on days 0, 14, and 42. On day 84 after the final immunization, the mice were continuously strengthened with the inactivated vaccine and Tri-Vac (mixed in a 1:1:4 ratio) via intramuscular injection, with an immunizing dose of 10ug per mouse. Serum was collected on day 14 after strengthening to detect neutralizing antibodies against the true virus. The results are shown in Figure 19. The neutralizing ability of the trivalent vaccine Tri-Vac continuous serum against live Delta, BA.2.75, BA.5.2.48, and XBB.1.16 viruses was clearly stronger than that of the inactivated vaccine sequence. In particular, the titer of the neutralizing antibody against the currently circulating XBB.1.16 strain reached over 1000.

[0190] After immunizing mice with an inactivated vaccine three times on days 0, 14, and 42, they are given an inactivated vaccine or a bivalent vaccine (RBD) on day 128. WT +RBD XBB.1.5 Sequential immunization was performed with either (a mixture in a 1:3 ratio), blood was collected 7 days after the fourth immunization for live virus neutralization assay, the immunization dose was 10 μg of protein per mouse, and serum was collected 14 days after the booster for detection of live virus neutralizing antibodies. The results are shown in Figure 20, and the neutralizing ability of bivalent vaccine sequential serum against wild-type, Delta, BA.2, BA.5.2, BA.5.2.48 and XBB live viruses was clearly stronger than that of inactivated vaccine sequences, and in particular, the neutralizing antibody titer for the currently circulating XBB.1.16 strain reached over 1000.

[0191] Embodiment 6: Attack by SARS-CoV-2 XBB.1.16 variant

[0192] BALB / c mice were immunized in the muscles with low-dose (5 μg) and high-dose (10 μg) trivalent vaccine Tri-Vac (mixed in a 1:1:4 ratio) on days 0, 28, and 56. Mice immunized with a separate adjuvant served as the control group, with n=6 mice in each group. Subsequently, 21 days after the last vaccination, live SARS-CoV-2 XBB.1.16 omicron variant (1 × 10⁻¹⁶) was administered. 6All mice were challenged intranasally with PFU. The mice were euthanized, and their tissues were collected 4 days post-infection. Pathological changes in lung tissue were observed by hematoxylin-eosin staining. The body weight of the mice was observed, and viral load in turbinate, bronchial, and lung tissue samples was detected by measuring the reverse transcription quantitative polymerase chain reaction (RT-qPCR) of the viral genome (gRNA). The primer sequences were 5'-GACCCCAAAATCAGCGAAAT-3' (forward) (SEQ ID NO: 40) and 5'-TCTGGTTACTGCCCAGTTGAATCTG-3' (reverse) (SEQ ID NO: 41), and the probe sequence was 5'-FAM-ACGCCGCATTACGTTTGGTGGGACC-BHQ1-3' (SEQ ID NO: 42). All procedures related to the attack using SARS-CoV-2 omicron mutant mice were reviewed and approved by the Animal Experimentation Committee of the Institute of Medical and Biological Sciences, Chinese Academy of Medical Sciences, and were carried out at the ABSL-4 facility of the Kunming National High-level Biosafety Primate Research Center.

[0193] As shown in Figure 21, the change in mouse body weight after viral infection was significantly weaker in the Tri-Vac trivalent vaccine group (treated with a 1:1:4 mixture) than in the adjuvant control group. Pharyngeal swab viruses were virtually eliminated in mice treated with Tri-Vac, no viral RNA was detected in the nasal turbinates, trachea, and lung tissue, lung histology was normal, alveolar structure was complete, and there was no apparent inflammation. However, in the adjuvant control group, pharyngeal swab samples still contained a higher viral load, viral RNA levels in the nasal turbinates, trachea, and lung tissue remained high, and severe pathological changes, including multiple consolidation areas, thickening of the alveolar septa, and alveolar congestion, could be observed through pathological sections. Small inflammatory lesions consisting of macrophages, neutrophils, and lymphocytes were sometimes observed around small blood vessels. The trivalent vaccine Tri-Vac, mixed in a 1:1:4 ratio, demonstrated complete protection against infection by the XBB.1.16 omicron variant and showed superior safety.

[0194] As shown in Figure 22, the bivalent vaccine (RBD) was mixed in a 1:3 ratio. WT +RBD XBB.1.5 In mice treated with the ) consecutive immunization group, viral RNA was not detected in the nasal turbinates, trachea, and lung tissue, and was essentially eliminated. However, viral RNA levels in the nasal turbinates, trachea, and lung tissue were higher in the adjuvant control group. Pathological histoscoring was also significantly lower in the adjuvant control group than in the control group, and in consecutive immunization, the bivalent vaccine (RBD) mixed in a 1:3 ratio was significantly lower. WT +RBD XBB.1.5 ) demonstrated complete protection against infection by the XBB.1.16 omicron variant, indicating better safety.

Claims

1. A protein for infection by SARS-CoV-2 omicron mutant strains and their subtypes, comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 6, or an amino acid sequence having more than 98% homology and identical or similar biological activity to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6.

2. The protein according to claim 1, wherein the homologous amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 satisfies at least one of the following conditions: in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, the 52nd amino acid F is mutated to S, the 54th amino acid P is mutated to S, and the 56th amino acid F is mutated to S.

3. The protein according to claim 1, wherein the homologous amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 satisfies at least one of the following conditions: in SEQ ID NO: 4 or SEQ ID NO: 351st amino acid sequence F is mutated to S, 353rd amino acid sequence P is mutated to S, and 355th amino acid sequence F is mutated to S, and the homologous amino acid sequence shown in SEQ ID NO: 6 satisfies at least one of the following conditions: in SEQ ID NO: 6, 352nd amino acid sequence F is mutated to S, 354th amino acid sequence P is mutated to S, and 356th amino acid sequence F is mutated to S.

4. The protein according to any one of claims 1 to 3, wherein the amino acid sequence of the protein is selected from at least one of SEQ ID NOs: 1 to 12.

5. A signal peptide and / or protein tag is linked to the protein against infection by the SARS-CoV-2 omicron mutant and its subtypes, preferably the protein tag is selected from at least one of a histidine tag, a thioredoxin tag, a glutathione transferase tag, a ubiquitin-like protein modification tag, a maltose-binding protein tag, a c-Myc protein tag, an Avi-tagged protein tag, and a substance A protein tag using a nitrogen source, the precursor of the protein according to any one of claims 1 to 4.

6. The precursor according to claim 5, wherein the protease recognition region for cleaving the protein tag is also linked to the protein for infection by the SARS-CoV-2 omicron mutant and its subtypes, and preferably the protease is selected from at least one of enterokinase, TEV protease, thrombin, blood coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

7. The precursor according to claim 5 or 6, wherein the amino acid sequence of the precursor is selected from at least one of SEQ ID NOs: 13 to 24.

8. A polynucleotide encoding a protein according to any one of claims 1 to 4 or a precursor according to any one of claims 5 to 7.

9. The polynucleotide according to claim 8, wherein the nucleotide sequence is selected from at least one of SEQ ID NOs: 25 to 30.

10. A recombinant vector comprising the polynucleotide described in claim 8 or 9.

11. The recombinant vector according to claim 10, wherein at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector, and a yeast expression vector is used, preferably the insect baculovirus expression vector is pFastBac1, preferably the Escherichia coli expression vector is pET32a, preferably the yeast expression vector is pPICZaA, preferably the mammalian cell expression vector is a CHO cell expression vector, and more preferably the CHO cell expression vector is pTT5 or FTP-002.

12. A host cell comprising the recombinant vector according to claim 10 or 11.

13. The host cell according to claim 12, wherein at least one of insect cells, mammalian cells, Escherichia coli, and yeast is used, preferably the insect cell is selected from at least one of sf9 cells, sf21 cells, and Hi5 cells, and preferably the mammalian cell is a CHO cell.

14. A method for preparing a protein according to any one of claims 1 to 4 or a precursor according to any one of claims 5 to 7, comprising the steps of: culturing a host cell according to claim 12 or 13 such that the host cell expresses a protein or precursor; then recovering the protein to obtain the protein.

15. A protein composition for infection by SARS-CoV-2 or its variants, comprising at least two combinations of delta protein, delta full-length S protein, BA.5 protein, BQ.1.1 protein, XBB.1.5 protein, BA.4 / 5 full-length S protein, BQ.1.1 full-length S protein, XBB.1.5 full-length S protein, Mu-BA.5 protein, Mu-BQ.1.1 protein, Mu-XBB.1.5 protein, BA.4 / 5 full-length S protein after amino acid mutation, BQ.1.1 full-length S protein after amino acid mutation, and XBB.1.5 full-length S protein after amino acid mutation, preferably a combination of at least two to four proteins.

16. The composition according to claim 15, comprising at least two combinations of the BA.5 protein, the BQ.1.1 protein, the XBB.1.5 protein, the BA.4 / 5 full-length S protein, the BQ.1.1 full-length S protein, and the XBB.1.5 full-length S protein.

17. The composition according to claim 15, comprising at least two combinations of the delta protein, the Mu-BA.5 protein, the Mu-BQ.1.1 protein, and the Mu-XBB.1.5 protein.

18. The composition according to claim 15, comprising at least two combinations of the Mu-BA.5 protein, the Mu-BQ.1.1 protein, the Mu-XBB.1.5 protein, the full-length S protein after the BA.4 / 5 amino acid mutation, the full-length S protein after the BQ.1.1 amino acid mutation, and the full-length S protein after the XBB.1.5 amino acid mutation.

19. The composition according to claim 15, wherein the BA.5 protein, the BQ.1.1 protein, the XBB.1.5 protein, the BA.4 / 5 full-length S protein, the BQ.1.1 full-length S protein, the XBB.1.5 full-length S protein, the Mu-BA.5 protein, the Mu-BQ.1.1 protein, the Mu-XBB.1.5 protein, the BA.4 / 5 full-length S protein after amino acid mutation, the BQ.1.1 full-length S protein after amino acid mutation, and the XBB.1.5 full-length S protein after amino acid mutation each contain the protein according to any one of claims 1 to 4 or the precursor according to any one of claims 5 to 7.

20. The composition according to claim 19, wherein the amino acid sequences of the delta protein, the delta full-length S protein, the BA.5 protein, the BQ.1.1 protein, the XBB.1.5 protein, the BA.4 / 5 full-length S protein, the BQ.1.1 full-length S protein, the XBB.1.5 full-length S protein, the Mu-BA.5 protein, the Mu-BQ.1.1 protein, the Mu-XBB.1.5 protein, the BA.4 / 5 full-length S protein after amino acid mutation, the BQ.1.1 full-length S protein after amino acid mutation, and the XBB.1.5 full-length S protein after amino acid mutation are constructed based on at least one amino acid sequence shown in SEQ ID NOs: 31, SEQ ID NOs: 33, and SEQ ID NOs: 1 to 24.

21. A vaccine for preventing and / or treating infections caused by SARS-CoV-2 omicron variants and their subtypes, comprising a protein according to any one of claims 1 to 4, a precursor according to any one of claims 5 to 7 and / or a protein composition according to any one of claims 15 to 20, and a pharmaceutically acceptable excipient or adjuvant component.

22. The vaccine according to claim 21, wherein the adjuvant component is an immunoadjuvant, preferably selected from at least one of a squalene-based oil-in-water emulsion, an aluminum salt, a calcium salt, a plant saponin, a plant polysaccharide, a monophosphate tripeptide A, a muramyl dipeptide, a muramyl tripeptide, a bacterial toxin, a GM-CSF cytokine, a lipid, and a cationic liposome material.

23. The squalene-based oil-in-water emulsion is MF59, the aluminum salt is selected from at least one of aluminum hydroxide and alum, the calcium salt is tricalcium phosphate, the plant saponin is either QS-21 or ISCOM, the plant polysaccharide is astragalus polysaccharide, the bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin, and the lipid is selected from at least one of phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine. The vaccine according to claim 22, wherein the cationic liposome material satisfies at least one of the following: (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloride)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamide)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

24. The vaccine according to any one of claims 20 to 23, wherein the vaccine is an injectable, nasal spray, or oral preparation, and preferably the vaccine is an intramuscular injection.

25. Use of the protein according to any one of claims 1 to 4, the precursor according to any one of claims 5 to 7 and / or the protein composition according to any one of claims 15 to 30 or the vaccine according to any one of claims 21 to 24 in preparing a drug for treating and / or preventing infection or pathogenicity of SARS-CoV-2 or its variants.

26. The use according to claim 25, wherein the SARS-CoV-2 mutant is selected from at least one of alpha, beta, gamma, delta, or omicron strains, and the omicron strain comprises at least one of BA.1, BA.2, BA.2.12.1, BA.4, BA.5, BQ.1.1, and XBB.1.

5.

27. An mRNA vaccine for preventing and / or treating infection by SARS-CoV-2 omicron mutant strains and subtypes, comprising the polynucleotide sequence described in claim 8.

28. An adenovirus vaccine for preventing and / or treating infection by SARS-CoV-2 omicron mutant strains and subtypes, comprising the polynucleotide sequence described in claim 8.

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