CN114456241B - Protein and vaccine for resisting SARS-CoV-2 infection - Google Patents

Protein and vaccine for resisting SARS-CoV-2 infection Download PDF

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CN114456241B
CN114456241B CN202210194653.2A CN202210194653A CN114456241B CN 114456241 B CN114456241 B CN 114456241B CN 202210194653 A CN202210194653 A CN 202210194653A CN 114456241 B CN114456241 B CN 114456241B
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CN114456241A (en
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魏霞蔚
逯光文
王玮
杨金亮
杨莉
李炯
杨静云
魏于全
王震玲
沈国波
赵志伟
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Chengdu Weisk Biomedical Co ltd
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Abstract

The invention relates to a protein and vaccine for resisting SARS-CoV-2 infection, belonging to the field of medicine. In order to solve the problem that the mutant virus infection aiming at SARS-CoV-2 is not provided with effective preventive and therapeutic drugs, the invention provides a protein for resisting SARS-CoV-2 infection, which contains an amino acid sequence which is identical with 320-545 amino acids in S protein of SARS-CoV-2 or has more than 98% homology with 320-545 and has identical or similar biological activity. The invention also provides vaccines for preventing and/or treating SARS-CoV-2 infection. The invention mainly blocks the combination of S protein of SARS-CoV-2 and host cell ACE2 receptor by inducing immune reaction such as antibody and the like in vivo, thereby helping host resist coronavirus infection, and especially has better prevention and treatment effect on mutant viruses.

Description

Protein and vaccine for resisting SARS-CoV-2 infection
Technical Field
The invention relates to a protein and vaccine for resisting SARS-CoV-2 infection, belonging to the field of medicine.
Background
SARS-CoV-2 is a novel class of beta coronaviruses named by the world health organization. The virus is enveloped and the particles are round or oval, often polymorphic, with a diameter of 60-140nm. The gene characteristics of the strain are obviously different from SARS-CoV and MERS-CoV, and the strain is a new coronavirus branch which has not been found in human before. Bats may be the natural host for SARS-CoV-2, and in addition, it has been studied that pangolin scales may also be the animal source of the virus. At present, the novel coronavirus SARS-CoV-2 has caused millions of people to infect, no definite and effective antiviral drug can be prevented and treated, and development of a vaccine against the virus is very important for disease control.
Major structural proteins of SARS-CoV-2 include Spike protein (Spike, S), messenger protein (E), membrane protein (Membrane, M) and Nucleocapsid protein (N), wherein the S protein plays a key role in viral infection and virulence. Angiotensin converting enzyme 2 (ACE 2) is a functional receptor for SARS coronavirus, and recent studies have found that SARS-CoV-2 also enters host cells through binding to ACE2 receptor for viral infection and replication.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention aims to provide proteins against SARS-CoV-2 infection. It is another object of the present invention to provide a vaccine for preventing and/or treating SARS-CoV-2 infection containing the protein.
The invention provides a protein for resisting SARS-CoV-2 infection, which contains an amino acid sequence shown as SEQ ID No.1 or having more than 98% of homology with SEQ ID No.1 and the same or similar biological activity.
Further, the protein contains an amino acid sequence which has more than 98.2% of homology with SEQ ID No.1 and has the same or similar biological activity.
Wherein the homologous amino acid sequence of SEQ ID No.1 satisfies at least one of the following: the 98 th amino acid in SEQ ID No.1 is replaced by N from K; the 165 th amino acid in SEQ ID No.1 is replaced by K; the 182 th amino acid in SEQ ID No.1 is replaced by Y; the 219 th amino acid in SEQ ID No.1 is replaced by S.
Further, the protein also contains an amino acid sequence shown as SEQ ID No.2 or having more than 98% of homology with SEQ ID No.2 and the same or similar biological activity.
Further, the protein also contains an amino acid sequence which has more than 98.8 percent of homology with SEQ ID No.2 and has the same or similar biological activity.
Wherein the homologous amino acid sequence of SEQ ID No.2 satisfies at least one of the following: deletion of amino acid H at position 50 and amino acid V at position 51 in SEQ ID No. 2; the 398 th amino acid in SEQ ID No.2 is replaced by N from K; the 465 th amino acid in SEQ ID No.2 is replaced by K; the 482 th amino acid in SEQ ID No.2 is replaced by Y; the 519 th amino acid in SEQ ID No.2 is replaced by S.
Further, the amino acid sequence of the protein is shown as SEQ ID No.1 or has more than 98% of homology with SEQ ID No.1 and has the same or similar biological activity.
Further, the amino acid sequence of the protein has more than 98.2% homology with SEQ ID No.1 and has the same or similar biological activity.
Wherein the homologous amino acid sequence of SEQ ID No.1 satisfies at least one of the following: the 98 th amino acid in SEQ ID No.1 is replaced by N from K; the 165 th amino acid in SEQ ID No.1 is replaced by K; the 182 th amino acid in SEQ ID No.1 is replaced by Y; the 219 th amino acid in SEQ ID No.1 is replaced by S.
Further, the amino acid sequence of the protein is: the amino acid sequence from the nitrogen end to the carbon end is formed by connecting SEQ ID No.2 or a homologous amino acid sequence thereof with SEQ ID No.1 or a homologous amino acid sequence thereof.
Further, the amino acid sequence of the protein is at least one of SEQ ID No.3 and SEQ ID No. 4.
The present invention provides precursors of the proteins: a signal peptide and/or a protein tag is attached to the SARS-CoV-2 infection resistant protein.
Preferably, the protein tag is selected from at least one of the following: histidine tag, thioredoxin tag, glutathione transferase tag, ubiquitin-like modifying protein tag, maltose binding protein tag, c-Myc protein tag, avitag protein tag, nitrogen source utilizing substance a protein tag.
Further, the precursor has a protease recognition region for cleaving the protein tag attached to the SARS-CoV-2 infection resistant protein.
Preferably, the protease is selected from at least one of the following: enterokinase, TEV protease, thrombin, factor Xa, carboxypeptidase a, rhinovirus 3c protease.
Further, the amino acid sequence of the precursor is selected from at least one of SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No. 9.
The invention provides the application of the protein and/or the precursor in preparing medicines for preventing and/or treating SARS-CoV-2 infection.
The invention provides a vaccine for preventing and/or treating SARS-CoV-2 infection, which contains the protein and/or the precursor, and pharmaceutically acceptable auxiliary materials or auxiliary components.
Further, the auxiliary component is an immunoadjuvant.
Preferably, the immunoadjuvant is selected from at least one of the following: aluminum salts, calcium salts, plant saponins, plant polysaccharides, monophosphoryl lipid a, muramyl dipeptide, muramyl tripeptide, squalene oil-in-water emulsion, recombinant cholera toxin, GM-CSF cytokines, lipids, cationic liposome materials, cpG ODN.
Further, the aluminum salt is at least one selected from aluminum hydroxide and alum.
Further, the calcium salt is tricalcium phosphate.
Further, the plant saponin is QS-21 or ISCOM.
Further, the plant polysaccharide is astragalus polysaccharide.
Further, the lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, dioleoyl phosphatidylethanolamine.
Further, the cationic liposome material is selected from at least one of the following: (2, 3-dioleoxypropyl) trimethylammonium chloride, N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N, N-trimethylammonium chloride, cationic cholesterol, dimethyl-2, 3-dioleyloxypropyl-2- (2-argininoformylamino) ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide.
Further, the vaccine is an injection preparation.
Preferably, the vaccine is an intramuscular injection preparation.
The present invention provides polynucleotides encoding said proteins or said precursors.
The present invention provides recombinant vectors containing said polynucleotides.
Further, the recombinant vector adopts at least one of insect baculovirus expression vector, mammalian cell expression vector, escherichia coli expression vector and yeast expression vector.
Preferably, the insect baculovirus expression vector is pFastBac1.
Preferably, the E.coli expression vector is pET32a.
Preferably, the yeast expression vector is pPICZaA;
preferably, the mammalian cell expression vector is a CHO cell expression vector.
Further preferably, the CHO cell expression vector is pTT5 or FTP-002.
The present invention provides host cells containing said recombinant vectors.
Further, the host cell adopts at least one of insect cells, mammal cells, escherichia coli and yeast.
Preferably, the insect cell is at least one selected from sf9 cell, sf21 cell and Hi5 cell.
Preferably, the mammalian cells are CHO cells.
The invention provides a preparation method of the protein, which comprises the following steps: culturing said host cell to express said protein or precursor, and recovering said protein.
The invention provides a preparation method of the protein, which comprises the following steps: constructing a recombinant vector containing the polynucleotide, and immunizing a human body to generate the protein.
Further, the carrier is selected from at least one of the following: mRNA, DNA vaccine, adenovirus, vaccinia ankara vaccinia Ankara virus, adeno-associated virus.
SEQ ID No.1：
VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG
SEQ ID No.1 shows the same amino acid sequence as 320-545 of the S protein of SARS-CoV-2.
Examples of homologous amino acids of SEQ ID No. 1:
VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNG
the amino acid sequence replaces 417 th amino acid K with N, 484 th amino acid E with K, 501 th amino acid N with Y, 538 th amino acid C with S in S protein of SARS-CoV-2, and the rest sites are the same as 320-545 amino acids of S protein.
SEQ ID No.2：
TRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG
SEQ ID No.2 is identical to the amino acid sequence of positions 20-545 covering the N-terminal domain in the S protein of SARS-CoV-2.
Examples of homologous amino acids of SEQ ID No. 2:
TRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNG the above amino acid sequence is obtained by deleting 69 th and 70 th amino acids H and V based on 20-545 th amino acid sequence of N-terminal domain contained in S protein of SARS-CoV-2, and substituting 538 th amino acid C with S, and the rest positions are unchanged.
SEQ ID No.3, the amino acid sequence of the anti-SARS-CoV-2 infection protein of the present invention is exemplified by RBD (320-545+K417N+E484K+N501Y):
VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG
SEQ ID No.4, an example of the amino acid sequence of the anti-SARS-CoV-2 infection protein of the present invention, NTD_RBD (20-545+69-70del+C538S) -RBD (320-545+K417N+E484K+N501Y+C538S):
TRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNG
SEQ ID No.5, an example of the amino acid sequence of the precursor anti-SARS-CoV-2 infection protein of the present invention, GP67-Trx (wt) -His-EK-RBD (320-545+K417N+E484K+N501Y):
MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDCIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG
SEQ ID No.6, an example of the amino acid sequence of the precursor anti-SARS-CoV-2 infection protein of the present invention, GP67-Trx (Cys-mut) -His-EK-RBD (320-545+K417N+E484K+N 501Y):
MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG
SEQ ID No.7, an example of the amino acid sequence of the precursor anti-SARS-CoV-2 infection protein of the present invention, GP67-RBD (320-545+K417N+E484K+N 501Y) -6his:
MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGHHHHHH
SEQ ID No.8, an example of the amino acid sequence of the anti-SARS-CoV-2 infection protein precursor of the present invention, GP67-NTD_RBD (20-545+69-70del+C538S) -RBD (320-545+K417N+E484K+N501Y+C538S) -6His:
MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGHHHHHH
SEQ ID No.9, an example of the amino acid sequence of the anti-SARS-CoV-2 infection protein precursor of the present invention, GP67-trx (wt) -His-EK-NTD_RBD (20-545+69-70del+C538S) -RBD (320-545+K417 N+E484K+N501Y+C538S):
MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDCIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNG
the extracellular section of S protein of SARS-CoV-2 virus has the structure shown in figure 1, SP and signal peptide; NTD, N-terminal domain; RBD, receptor domain; FP, fusion peptide; IFP, inner fusion peptide; HR1, heptad repeat region 1; HR2, heptad repeat region 2; PTM, near film zone; TM, transmembrane region.
The protein for resisting SARS-CoV-2 infection is designed based on amino acids 320-545 and amino acids 20-545 of S protein.
Abbreviations for terms:
monophosphoryl lipid A (MPL)
Squalene oil-in-water emulsion (MF 59), recombinant cholera toxin (rCTB)
Astragalus Polysaccharides (APS)
Phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoyl phosphatidylethanolamine (DOPE)
(2, 3-Dioleoxypropyl) trimethylammonium chloride (DOTAP), N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N, N-trimethylammonium chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyl-2, 3-dioleyloxypropyl-2- (2-sperminecarboxamido) ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), cpG ODN (a nucleotide sequence containing unmethylated cytosine and guanine dinucleotides as core sequences), synthetic CpG
The beneficial effects are that: the invention provides a protein and vaccine for resisting SARS-CoV-2 infection, which mainly aims at S protein of SARS-CoV-2 virus, especially by blocking ACE2 receptor binding region of S protein, inducing immune reaction such as antibody in vivo, blocking the binding of S protein of SARS-CoV-2 and host cell ACE2 receptor, thus helping host resist coronavirus infection, especially having better preventing and treating effect on mutant virus.
Drawings
FIG. 1 is a diagram showing the construction of an extracellular segment of S protein of SARS-CoV-2 virus of the present invention;
FIG. 2 is a graph showing the results of protein preparation of protein 1 (corresponding to SEQ ID No. 3) against SARS-CoV-2 infection in example 1;
FIG. 3 is a graph showing the results of the preparation of protein 2 (corresponding to SEQ ID No. 4) against SARS-CoV-2 infection in example 1;
FIG. 4 is a graph showing the results of measurement of absorbance values A450 to A630 of the serum of the mice in test example 1;
FIG. 5 is a graph showing the in vitro blocking of S1 protein from ACE2 binding by immune serum in test example 2;
FIG. 6 is a graph showing the results of measurement of neutralizing antibody titer against the immune serum virus in test example 3;
FIG. 7 is a graph showing the results of determining the copy number of the virus of the challenge mouse in test example 4;
FIG. 8 is a graph of the lung pathology HE staining of the challenged mice in test example 4.
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 preparation of proteins of the invention against SARS-CoV-2 infection Using insect baculovirus expression System
And (3) constructing a carrier: the amino acid sequences shown as SEQ ID No. 1-SEQ ID No.9 are adopted. An expression vector for the S protein was constructed based on the pFastBac1 vector (ampicillin resistance) and was inserted into the pFast-bacI vector using BamHI, hindIII cleavage sites, optimized for the preferred codons of insect cells.
Amplification of recombinant baculovirus: the construction of recombinant bacmid was completed in E.coli (DH 10Bac, containing bacmid (kanamycin resistance) and helper plasmid (tetracyclomycin resistance)) using the bacterial transposon principle and generating site-specific transposition by Tn7 transposable elements. The recombinant bacmid was extracted and transfected into sf9 insect cells with the transfection reagent lipofect from Biyun to produce recombinant baculovirus expressing the gene of interest. The first generation virus was harvested 72h after transfection, and then P2 to P4 generation virus amplification was performed, using either P3 or P4 generation virus to express the protein.
Protein expression: hi5 insect cells (sf 9 cells) were infected with P3 or P4 generation virus at a multiplicity of infection (MOI 0.5-10), and the supernatant was collected after 48-72h of culture. The optimal harvest time may vary each time depending on the virus amount and cell status of the virus species, and it is generally preferred to examine about 50% of the cytopathic effects by microscopy.
Protein purification: the obtained culture supernatant is centrifuged at a high speed at 4 ℃ and filtered by a 0.22um filter membrane, the recombinant protein is primarily purified by an affinity purification method (Histrap nickel column), and then the pure recombinant protein is purified by a MonoQ ion column and Superdex 200/300 GL molecular sieve, and the purity of the protein is identified by SDS-PAGE, so that the purity requirement is more than 95%. The amino acid sequences of the finally obtained recombinant proteins are shown as SEQ ID No.3 and SEQ ID No.4, the expression and purification patterns of the obtained proteins are shown as figures 2 and 3 respectively, and the protein antigen with better purity and capable of being used for the subsequent immune protection research is obtained after purification by means of size exclusion chromatography and the like.
EXAMPLE 2 preparation of the vaccine against SARS-CoV-2 infection of the invention
The formulation is carried out under aseptic conditions. The purified recombinant protein antigen (prepared in example 1) was diluted to a concentration of 240. Mu.g/mL with 20mmol/L phosphate buffer, 250mmol/L sodium chloride solution (pH 7.4-7.6).
WGa01 adjuvant (squalene content is 3.12-4.68%) is added into the antigen solution, the volume ratio is (V/V) 1:1, and the final concentration of recombinant protein antigen in the mixed solution is 120 mug/mL. Stirring at 80-150 rpm is started to generate slight vortex, and stirring at room temperature is uniform. Characterization of the adsorbed vaccine formulation included particle size and size distribution, large milk particle, viscosity, sterility testing, and bacterial endotoxin testing. And (5) filling. The vaccine formulation is filled, for example, into 2mL sterile penicillin bottles, 0.5 mL/bottle. The stirring is kept continuously during filling, so that the filling liquid is uniform. Sealing the container after canning, attaching a serial number label, and storing the container at 2-8 ℃ in a dark place.
The beneficial effects of the invention are demonstrated by biological experiments below.
Test example 1 specific antibodies to the S proteins Va20-Arg545 (abbreviated as S1) were induced in mice vaccinated with the vaccine of the present invention
Immune animal experiments: BALB/C mice, C57BL/6 mice or NIH mice were used, and groups of 5 to 10 mice per group were as shown in FIG. 4. The amino acid sequence of the extracellular segment Va20-Arg545 of the recombinant protein is shown as SEQ ID No.4, and the dosage is 0.1 mug to 20.0 mug per dose, and the specific dosage is shown in FIG. 4. Mice of the experimental group were each injected with vaccine (prepared according to example 2) in a volume of 50 μl or in a volume converted according to the administered dose, and were immunized on the right hind leg intramuscular injection (im) of the mice by immunization procedures on days 1, 7 and 21 (total immunization 3 times).
ELISA (enzyme-Linked immunosorbent assay) assay of mouse serum antibodies: on day 7 after the end of the mouse immunization program (day 28 after the first immunization), mouse plasma was collected by capillary orbital blood collection, 6 per group. Standing at room temperature for 1-2 h, centrifuging at 3000rpm for 10min at 4 ℃ after solidification, and taking upper serum for storage at-20 ℃ for later use.
Preparation of 50mM carbonate coating buffer (pH 9.6): weigh 0.293g NaHCO 3 And 0.15g Na 2 CO 3 After dissolution with double distilled water, the pH was adjusted to 9.6, and then the volume was set to 100mL and stored at 4℃for further use.
1M H 2 SO 4 Preparing a stop solution: to 47.3mL of double distilled water was added dropwise 2.7mL of concentrated sulfuric acid (98%).
Method for ELISA determination of serum IgG: a1. Mu.g/mL solution of recombinant protein S1 was prepared with 50mM carbonate coating buffer, added to a 96-well coating plate (Thermo Scientific, NUNC-MaxiSorp) at 100. Mu.L/well, and coated overnight at 4 ℃. After 3 times washing with PBS solution (PBST) containing 0.1% Tween20 for the next time, blocking with blocking solution (formulated in PBST) containing 1% BSA or 5% skimmed milk was performed for 1 hour at room temperature, and then PBST was washed 1 time. After dilution of the mouse serum with blocking solution in various proportions, the sample was loaded in a loading amount of 100. Mu.L/well, incubated at 37℃for 1h-2h (or overnight at 4 ℃) and then washed 3 times with PBST. HRP-goat anti-mouse IgG antibody (1:10000 diluted in blocking solution) was then added at 100 μl/well and after incubation for 1h at 37 ℃ PBST was washed 5 times. Finally, 100. Mu.l/well of 3,3', 5' -tetramethyl biphenyldiamine (TMB) was added, and after development in the dark for 10-15min, 50. Mu.l/well of 1M H was added 2 SO 4 Stopping liquid and stirring and mixing uniformly. Read at 450nm wavelength on a microplate reader.
Serum was serially diluted at different fold and the absorbance values of a450 and a630 were measured by titration to determine the titer of the recombinant protein-induced S1-specific antibodies. The absorbance values from 450nm to 630nm are plotted on the ordinate and the dilution factors are plotted on the abscissa. As can be seen from fig. 4, the optical density values of a450 of the PBS-inoculated control group and the MF59 adjuvant-inoculated control group are lower, and the optical density values of a450 of the other inoculated protein groups are significantly increased compared with the control group, which proves that the recombinant protein of the present invention stimulates an obvious S1 specific antibody. Further, MF59 adjuvant significantly increased the antibody titer of the protein vaccine and was recombinant protein dose dependent. The above results indicate that the recombinant S1 protein vaccine is highly immunogenic in mice.
Test example 2 blocking test of binding of the S1 protein of the present invention to ACE2 receptor
The present experiment uses cell expressed ACE2, a protein that is thought to retain its native conformation for detection of S1-Fc protein binding activity by flow cytometry. The specific operation is as follows:
the in vitro cultured high-expression ACE2 cell strain (lung cancer a 549) was digested and collected into flow tubes, 106 cells/tube, washed several times with PBS/HBSS. Adding recombinant S1-Fc protein to each tube of cells at a final concentration of 1. Mu.g/mL; serum from immunized anti-S1 mice (10. Mu.g/dose of immunized mice in test example 1 was diluted 50-fold) was added and incubated at room temperature for 30min. Wherein positive control tubes were not antiserum and PBS tubes were added to PBS-immunized negative serum of test example 1. After washing several times with PBS/HBSS, anti-Human IgG (Fc specific) -FITC (SIGMA) fluorescent secondary antibody (1:100-1:200) was added and incubated at room temperature for 30min in the absence of light. After washing several times with PBS/HBSS, 500. Mu.l of PBS containing 1% paraformaldehyde was added for fixation and detection was performed on-stream.
As shown in fig. 5, the added S1-Fc protein can bind to ACE 2-expressing cells, and the positive control group positive cell percentage is greater than 80%; only background signal was detected without addition of S1-Fc protein (negative control); the mouse antiserum effectively blocks the combination of the S1-Fc protein and ACE 2-expressing cells, and the percentage of positive cells is less than 25%; while the same dilution of serum without or before immunization showed no blocking, the percentage of positive cells was greater than 80%.
Test example 3 pseudo-virus neutralization assay of protein immune serum
The serum (or plasma) to be tested was inactivated in a 56℃water bath for 30min, and 6000g was centrifuged for 3min, and the supernatant was transferred to a 1.5ml centrifuge tube for use.
A96-well plate was used, 150. Mu.l/well of DMEM complete medium (1% double antibody, 25mM HEPES,10%FBS) was added to column 2 (cell control CC, see Table 1), 100. Mu.l/well of DMEM complete medium was added to columns 3 to 11 (column 3 was virus control VV, and columns 4 to 11 were sample wells), and 42.5. Mu.l/well of DMEM complete medium was added to wells B4 to B11.
Plasma sample 1 (7.5 μl) … … was added to wells B4 and B5, and plasma sample 4 (7.5 μl) was added to wells B10 and B11.
Regulating the multi-channel liquid transfer device to 50 mul, and repeatedly blowing and sucking the liquid in the holes B4-B11 for 6-8 times
And (3) uniformly mixing, transferring 50 μl of liquid to the corresponding C4-C11 hole, gently and repeatedly blowing and sucking for 6-8 times, transferring to the D4-D11 hole, and so on, finally sucking 50 μl of liquid from G4-G11, and the sample adding sequence is shown in Table 1.
Pseudoviruses were diluted to 1.3X10 with DMEM complete medium 4 (1×10 4 ～2×10 4 ) TCID 50/ml (diluted at the dilution provided) was added to each well in columns 3-11 at 50. Mu.l, so that the pseudovirus content per well was 650 (500-1000)/well.
Table 1 test sample distribution table
1CC 2CV 3 4 5 6 7 8 9 10 11 12
A 150
B 150 100 100 100 100 100 100 100 100 100 100 100
C 150 100 100 100 100 100 100 100 100 100 100 100
D 150 100 100 100 100 100 100 100 100 100 100 100
E 150 100 100 100 100 100 100 100 100 100 100 100
F 150 100 100 100 100 100 100 100 100 100 100 100
G 150 100 100 100 100 100 100 100 100 100 100 100
H 150 100 100 100 100 100 100 100 100 100 100 100
The 96-well plate was placed in a cell incubator (37 ℃,5% co) 2 ) Incubate for 1 hour.
When the incubation time reaches half an hour, hACE2-293T cells prepared in advance in an incubator (the confluence rate reaches 80% -90%), taking a T75 flask as an example, sucking and discarding the culture medium in the flask, adding 5ml of PBS buffer to clean the cells, pouring out the PBS, adding 3ml of 0.25% pancreatin-EDTA to submerge the cells for digestion for 1 minute, pouring out pancreatin, placing the cells in the cell incubator for digestion for 5 minutes, gently beating the side wall of the flask to detach the cells, adding 10ml of culture medium to neutralize pancreatin, transferring the cells into a centrifuge tube after several times of blowing, centrifuging for 5 minutes, pouring out the supernatant, re-suspending the cells with 10ml of DMEM complete medium, counting the cells, diluting the cells to 2X 10 with the DMEM complete medium 5 And each ml.
Incubation to 1 hour, every time into 96-well platesMu.l of cells were added to the wells to give 2X 10 cells per well 4 And each.
Slightly shaking the 96-well plate back and forth and left and right to uniformly disperse cells in the well, placing the 96-well plate into a cell incubator at 37 ℃ and 5% CO 2 Culturing for 20-28 hours.
After 20 to 28 hours, the 96-well plate is taken out of the cell incubator, 150 μl of supernatant is sucked from each loading well by a multi-channel pipette, 100 μl of luciferase detection reagent is added, and the reaction is carried out at room temperature in a dark place for 2min.
After the reaction is finished, the liquid in the reaction holes is repeatedly blown and sucked for 6 to 8 times by using a multi-channel liquid-transferer, so that cells are fully cracked, 150 mu l of liquid is sucked out of each hole, and the liquid is added into a corresponding 96-hole chemiluminescent detection plate and placed into a chemiluminescent detector to read a luminescent value. And (3) calculating the neutralization inhibition rate: inhibition ratio = [1- (mean of luminous intensity of sample group-mean of control CC)/(mean of luminous intensity of negative group-mean of control CC) ] ×100%.
Based on the neutralization inhibition result, IC50 was calculated using Reed-Muench method.
The EC50 titers of the neutralizing antibodies of the mice injected with PBS and S1 vaccine were counted, and as shown in fig. 6, only extremely low EC50 neutralizing antibody titers were detected in the sera of the mice injected with PBS, while higher EC50 neutralizing antibody titers were detected in the sera of mice immunized with S1 vaccine, and the EC50 value was close to 700, thus having a good neutralizing activity function.
Test example 4 test for toxicity attack by SARS-CoV-2 Virus infection in mice
Mice were immunized and 6 to 8 week old hACE2 transgenic C57BL/6 mice were intramuscular injected with recombinant S1 protein vaccine (prepared according to example 2) at a dose of 10. Mu.g each. For example, mice received one vaccine injection on days 1, 14, 21, and control mice were injected with MF59 immunoadjuvant or PBS only. Serum was collected again 7 days after immunization. At 7 days after the end of the immunization = immunization,
SARS-CoV-2 virus challenge (intranasal infection at a dose of 105TCID 50). In addition, control mice were injected with MF59 immunoadjuvant or PBS-only infected mice with the virus as controls. Mice were sacrificed 5 days after virus challenge and their lungs and other organs were excised. Lung tissue was used to detect viral replication. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using a PowerUp SYBG Green Master Mix kit (Applied Biosystems, USA), and viral RNA copy number in lung tissue of mice challenged with SARS-COV-2 was determined and expressed as RNA copy number per ml of lung tissue. The primer sequences used for qRT-PCR are envelope (E) genes for SARS-cov-2 as follows:
forward direction: 5'-TCGTTTCGGAAGAGACAGGT-3' (SEQ ID No. 10);
reversing: 5'-GCGCAGTAAGGATGGCTAGT-3' (SEQ ID No. 11).
This experiment tested whether vaccination could prevent mice from being infected with SARS-CoV-2 virus. Human ACE-2 transgenic mice were challenged with SARS-CoV-2 virus, and lung tissue was collected 5 days after virus challenge and tested for replication of the virus in the vaccine or control. As shown in FIG. 7, after mice are immunized with the protein vaccine of the present invention, very little viral replication is detected by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), whereas the level of viral replication in lung tissue of mice in the control group is higher.
Portions of lung tissue were harvested and fixed with 10% neutral formalin, embedded in paraffin, sectioned at 5 μm thickness, and stained with Hematoxylin and Eosin (HE). The histopathological changes were observed with a light microscope. As shown in fig. 8, the lung tissues of the control group (PBS group and MF59 group) showed significant histopathological changes of interstitial pneumonia, including significant thickening of alveolar wall, congestion, and massive single nuclear cell infiltration of interstitium. In contrast, the recombinant S1 protein vaccine immunized mice did not see histopathological changes.
The experimental results further prove that the S1 protein vaccine of the invention can block infection of SARS-CoV-2 virus.
It is to be noted that the particular features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in any one or more embodiments. Furthermore, the various embodiments described in this specification, as well as the features of the various embodiments, can be combined and combined by one skilled in the art without contradiction.
Sequence listing
<110> Chengdu Wilson biological medicine Co., ltd
<120> protein and vaccine against SARS-CoV-2 infection
<130> A220067K (order)
<141> 2022-03-01
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Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile
165 170 175
Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val
180 185 190
Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu
195 200 205
Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His
210 215 220
Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly
225 230 235 240
Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu
245 250 255
Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu
260 265 270
Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu
275 280 285
Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser
290 295 300
Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val
305 310 315 320
Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg
325 330 335
Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser
340 345 350
Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp
355 360 365
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp
370 375 380
Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr
385 390 395 400
Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn
405 410 415
Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr
420 425 430
Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser
435 440 445
Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly
450 455 460
Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn
465 470 475 480
Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu
485 490 495
Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu
500 505 510
Val Lys Asn Lys Ser Val Asn Phe Asn Phe Asn Gly Val Gln Pro Thr
515 520 525
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
530 535 540
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
545 550 555 560
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
565 570 575
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
580 585 590
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
595 600 605
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
610 615 620
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
625 630 635 640
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
645 650 655
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
660 665 670
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val
675 680 685
Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
690 695 700
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
705 710 715 720
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
725 730 735
Asn Leu Val Lys Asn Lys Ser Val Asn Phe Asn Phe Asn Gly
740 745 750
<210> 5
<211> 394
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr
1 5 10 15
Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala
20 25 30
Ala His Ser Ala Phe Ala Ala Asp Ser Ile His Ile Lys Asp Ser Asp
35 40 45
Asp Leu Lys Asn Arg Leu Ala Glu Ala Gly Asp Lys Leu Val Val Ile
50 55 60
Asp Phe Met Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Gly Pro Lys
65 70 75 80
Leu Asp Glu Met Ala Asn Glu Met Ser Asp Cys Ile Val Val Leu Lys
85 90 95
Val Asp Val Asp Glu Cys Glu Asp Ile Ala Thr Glu Tyr Asn Ile Asn
100 105 110
Ser Met Pro Thr Phe Val Phe Val Lys Asn Ser Lys Lys Ile Glu Glu
115 120 125
Phe Ser Gly Ala Asn Val Asp Lys Leu Arg Asn Thr Ile Ile Lys Leu
130 135 140
Lys Leu Ala Gly Ser Gly Ser Gly His Met His His His His His His
145 150 155 160
Ser Ser Gly Asp Asp Asp Asp Lys Val Gln Pro Thr Glu Ser Ile Val
165 170 175
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
180 185 190
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
195 200 205
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
210 215 220
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
225 230 235 240
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
245 250 255
Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr
260 265 270
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
275 280 285
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
290 295 300
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
305 310 315 320
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn
325 330 335
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val
340 345 350
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
355 360 365
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
370 375 380
Asn Lys Cys Val Asn Phe Asn Phe Asn Gly
385 390
<210> 6
<211> 394
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr
1 5 10 15
Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala
20 25 30
Ala His Ser Ala Phe Ala Ala Asp Ser Ile His Ile Lys Asp Ser Asp
35 40 45
Asp Leu Lys Asn Arg Leu Ala Glu Ala Gly Asp Lys Leu Val Val Ile
50 55 60
Asp Phe Met Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Gly Pro Lys
65 70 75 80
Leu Asp Glu Met Ala Asn Glu Met Ser Asp Ser Ile Val Val Leu Lys
85 90 95
Val Asp Val Asp Glu Cys Glu Asp Ile Ala Thr Glu Tyr Asn Ile Asn
100 105 110
Ser Met Pro Thr Phe Val Phe Val Lys Asn Ser Lys Lys Ile Glu Glu
115 120 125
Phe Ser Gly Ala Asn Val Asp Lys Leu Arg Asn Thr Ile Ile Lys Leu
130 135 140
Lys Leu Ala Gly Ser Gly Ser Gly His Met His His His His His His
145 150 155 160
Ser Ser Gly Asp Asp Asp Asp Lys Val Gln Pro Thr Glu Ser Ile Val
165 170 175
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
180 185 190
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
195 200 205
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
210 215 220
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
225 230 235 240
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
245 250 255
Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr
260 265 270
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
275 280 285
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
290 295 300
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
305 310 315 320
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn
325 330 335
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val
340 345 350
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
355 360 365
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
370 375 380
Asn Lys Cys Val Asn Phe Asn Phe Asn Gly
385 390
<210> 7
<211> 272
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr
1 5 10 15
Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala
20 25 30
Ala His Ser Ala Phe Ala Ala Asp Val Gln Pro Thr Glu Ser Ile Val
35 40 45
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
50 55 60
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
65 70 75 80
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
85 90 95
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
100 105 110
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
115 120 125
Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr
130 135 140
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
145 150 155 160
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
165 170 175
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
180 185 190
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn
195 200 205
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val
210 215 220
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
225 230 235 240
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
245 250 255
Asn Lys Cys Val Asn Phe Asn Phe Asn Gly His His His His His His
260 265 270
<210> 8
<211> 796
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr
1 5 10 15
Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala
20 25 30
Ala His Ser Ala Phe Ala Ala Asp Thr Arg Thr Gln Leu Pro Pro Ala
35 40 45
Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe
50 55 60
Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe
65 70 75 80
Ser Asn Val Thr Trp Phe His Ala Ile Ser Gly Thr Asn Gly Thr Lys
85 90 95
Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala
100 105 110
Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr
115 120 125
Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn
130 135 140
Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu
145 150 155 160
Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe
165 170 175
Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln
180 185 190
Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu
195 200 205
Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser
210 215 220
Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser
225 230 235 240
Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg
245 250 255
Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp
260 265 270
Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr
275 280 285
Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile
290 295 300
Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys
305 310 315 320
Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn
325 330 335
Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
340 345 350
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
355 360 365
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
370 375 380
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
385 390 395 400
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
405 410 415
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
420 425 430
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
435 440 445
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
450 455 460
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
465 470 475 480
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
485 490 495
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
500 505 510
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
515 520 525
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
530 535 540
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val Asn Phe
545 550 555 560
Asn Phe Asn Gly Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn
565 570 575
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
580 585 590
Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala
595 600 605
Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys
610 615 620
Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val
625 630 635 640
Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala
645 650 655
Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp
660 665 670
Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser
675 680 685
Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
690 695 700
Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
705 710 715 720
Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro
725 730 735
Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro
740 745 750
Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr
755 760 765
Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val
770 775 780
Asn Phe Asn Phe Asn Gly His His His His His His
785 790 795
<210> 9
<211> 918
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr
1 5 10 15
Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala
20 25 30
Ala His Ser Ala Phe Ala Ala Asp Ser Ile His Ile Lys Asp Ser Asp
35 40 45
Asp Leu Lys Asn Arg Leu Ala Glu Ala Gly Asp Lys Leu Val Val Ile
50 55 60
Asp Phe Met Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Gly Pro Lys
65 70 75 80
Leu Asp Glu Met Ala Asn Glu Met Ser Asp Cys Ile Val Val Leu Lys
85 90 95
Val Asp Val Asp Glu Cys Glu Asp Ile Ala Thr Glu Tyr Asn Ile Asn
100 105 110
Ser Met Pro Thr Phe Val Phe Val Lys Asn Ser Lys Lys Ile Glu Glu
115 120 125
Phe Ser Gly Ala Asn Val Asp Lys Leu Arg Asn Thr Ile Ile Lys Leu
130 135 140
Lys Leu Ala Gly Ser Gly Ser Gly His Met His His His His His His
145 150 155 160
Ser Ser Gly Asp Asp Asp Asp Lys Thr Arg Thr Gln Leu Pro Pro Ala
165 170 175
Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe
180 185 190
Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe
195 200 205
Ser Asn Val Thr Trp Phe His Ala Ile Ser Gly Thr Asn Gly Thr Lys
210 215 220
Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala
225 230 235 240
Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr
245 250 255
Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn
260 265 270
Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu
275 280 285
Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe
290 295 300
Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln
305 310 315 320
Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu
325 330 335
Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser
340 345 350
Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser
355 360 365
Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg
370 375 380
Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp
385 390 395 400
Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr
405 410 415
Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile
420 425 430
Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys
435 440 445
Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn
450 455 460
Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
465 470 475 480
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
485 490 495
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
500 505 510
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
515 520 525
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
530 535 540
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
545 550 555 560
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
565 570 575
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
580 585 590
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
595 600 605
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
610 615 620
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
625 630 635 640
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
645 650 655
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
660 665 670
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val Asn Phe
675 680 685
Asn Phe Asn Gly Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn
690 695 700
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
705 710 715 720
Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala
725 730 735
Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys
740 745 750
Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val
755 760 765
Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala
770 775 780
Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp
785 790 795 800
Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser
805 810 815
Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
820 825 830
Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
835 840 845
Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro
850 855 860
Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro
865 870 875 880
Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr
885 890 895
Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val
900 905 910
Asn Phe Asn Phe Asn Gly
915
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
tcgtttcgga agagacaggt 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
gcgcagtaag gatggctagt 20

Claims (17)

1. A protein against SARS-CoV-2 infection, characterized by: the amino acid sequence of the protein is shown as SEQ ID No. 4.
2. A precursor of a protein against SARS-CoV-2 infection, characterized by: the amino acid sequence of the precursor is shown as SEQ ID No.8 or SEQ ID No. 9.
3. A vaccine for preventing SARS-CoV-2 infection, characterized by: comprising the protein of claim 1 and/or the precursor of claim 2, and a pharmaceutically acceptable auxiliary ingredient.
4. A vaccine as claimed in claim 3, characterised in that: the auxiliary component is an immunoadjuvant.
5. The vaccine of claim 4, wherein: the immune adjuvant is selected from at least one of the following: aluminum salts, calcium salts, plant saponins, plant polysaccharides, monophosphoryl lipid a, muramyl dipeptide, muramyl tripeptide, squalene oil-in-water emulsions, bacterial toxins, GM-CSF cytokines, lipids, cationic liposome materials, cpG ODN.
6. The vaccine of claim 5, wherein: at least one of the following is satisfied: the aluminum salt is at least one selected from aluminum hydroxide and alum; the calcium salt is tricalcium phosphate; the plant saponin is QS-21 or ISCOM; the plant polysaccharide is astragalus polysaccharide; the squalene oil-in-water emulsion is MF59; the bacterial toxin is at least one of recombinant cholera toxin and diphtheria toxin; the lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, dioleoyl phosphatidylethanolamine; the cationic liposome material is selected from at least one of the following materials: (2, 3-dioleoxypropyl) trimethylammonium chloride, N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N, N-trimethylammonium chloride, cationic cholesterol, dimethyl-2, 3-dioleyloxypropyl-2- (2-argininoformylamino) ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide.
7. The vaccine of any one of claims 3-6, characterized in that: the vaccine is an injection preparation.
8. The vaccine of claim 7, wherein: the vaccine is an intramuscular injection preparation.
9. A polynucleotide, characterized in that: encoding the protein of claim 1 or the precursor of claim 2.
10. The recombinant vector is characterized in that: comprising the polynucleotide according to claim 9.
11. The recombinant vector of claim 10, wherein: at least one of insect baculovirus expression vector, mammalian cell expression vector, escherichia coli expression vector and yeast expression vector is adopted.
12. The recombinant vector of claim 11, wherein: the insect baculovirus expression vector is pFastBac1; the escherichia coli expression vector is pET32a; the yeast expression vector is pPICZaA; the mammalian cell expression vector is a CHO cell expression vector.
13. The recombinant vector of claim 12, wherein: the CHO cell expression vector is pTT5 or FTP-002.
14. A host cell characterized by: a recombinant vector according to any one of claims 10 to 13.
15. The host cell of claim 14, wherein: at least one of insect cells, mammalian cells, escherichia coli and yeast is used.
16. The host cell of claim 15, wherein: the insect cells are selected from at least one of sf9 cells, sf21 cells and Hi5 cells; the mammalian cells are CHO cells.
17. The method for preparing the protein according to claim 1, wherein: the method comprises the following steps: culturing the host cell according to any one of claims 14 to 16 to express the protein or precursor, and recovering the protein.
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