CN113087777B - Protein for resisting SARS-CoV-2 infection and vaccine prepared by using said protein - Google Patents

Abstract

The present invention relates to SARS-CoV-2 infection resisting protein and vaccine prepared with the protein, and belongs to the field of medicine technology. In order to solve the problem that effective preventive and therapeutic medicines are not available for SARS-CoV-2 infection, the invention provides an anti-SARS-CoV-2 infection protein, the amino acid sequence of which is shown as SEQ ID No.1, or which has homology of more than 99% with SEQ ID No.1 and has the same or similar biological activity. In another aspect, the invention also provides a vaccine 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.

Description

Protein for resisting SARS-CoV-2 infection and vaccine prepared by using said protein

Technical Field

The present invention relates to SARS-CoV-2 infection resisting protein and vaccine prepared with the protein, and belongs to the field of medicine technology.

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 tens of thousands of people to be infected, no definite and effective antiviral drug can be prevented and treated, and development of a vaccine against the virus is very important for disease prevention and treatment.

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, the amino acid sequence of which is shown as SEQ ID No.1, or the protein has homology of more than 99% with SEQ ID No.1 and has the same or similar biological activity.

SEQ ID No.1：

VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI

HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVN

NATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVS

QPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP

LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKY

NENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITN

LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL

NDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN

NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPL

QSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFN

GLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVIT

PGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLI

GAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

The extracellular section of S protein of SARS-CoV-2 virus is composed of SP and signal peptide as shown in figure 6; 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. SEQ ID No.1 is an anti-SARS-CoV-2 infection drug designed based on the amino acids Val16-Arg685 of the S protein.

The present invention provides a precursor of the protein, which is connected with a signal peptide and/or a protein label on the SARS-CoV-2 infection resisting 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.

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 (MPL), muramyl dipeptide, muramyl tripeptide, squalene oil-in-water emulsion (MF 59), recombinant cholera toxin (rCTB), GM-CSF cytokines, lipids, cationic liposome materials, cpG ODN (a nucleotide sequence containing unmethylated cytosine and guanine dinucleotides as core sequences, synthetic CpG).

Further, the aluminum salt is at least one selected from aluminum hydroxide and alum.

Wherein, the immune adjuvant is preferably aluminum hydroxide adjuvant; the SARS-CoV-2 infection resistant protein: the proportion of the aluminum hydroxide adjuvant is preferably (32-48) mcg/mL: (0.8-1.2) mg/ml, and the adjuvant is calculated by the content of aluminum hydroxide; further preferred, the anti-SARS-CoV-2 infection protein: the proportion of the aluminum hydroxide adjuvant is preferably 40mcg/mL:1.0mg/ml, the adjuvant being calculated as aluminium hydroxide.

Further, the calcium salt is tricalcium phosphate.

Further, the plant saponin is QS-21 or ISCOM.

Further, the plant polysaccharide is Astragalus Polysaccharide (APS).

Further, the lipid is selected from at least one of the following: phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoyl phosphatidylethanolamine (DOPE).

Further, the cationic liposome material is selected from at least one of the following: (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).

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.

Further, the nucleotide sequence of the polynucleotide is shown as SEQ ID No.2 or SEQ ID No. 3. SEQ ID No.2:

GTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCA

CACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTC

AACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCT

ATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTAC

CATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAG

AGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATT

GTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAA

TGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGA

AAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTC

TCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAAT

CTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAA

GCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTA

GAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTT

TACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTG

GACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTT

CTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCAC

TTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAA

AAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTG

TTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGC

CACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTG

TGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGT

GTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTAT

GCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGG

CAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAG

GCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAA

TTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAG

AGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGT

GTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCA

CTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACT

TCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGT

TAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGT

TCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGA

CATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCT

TGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAAC

AAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGA

AGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTAT

TCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCT

GAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATAT

GCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGT

SEQ ID No.2 is the original nucleic acid sequence encoding Val16-Arg685, and is obtained from the sequence of the novel crown Wuhan-Hu-1 isolate of the whole genome sequence of the first strain.

SEQ ID No.3：

GTGAACCTGACCACCAGGACTCAGCTGCCTCCCGCCTACACCAACTCCTT

CACTCGCGGTGTGTACTACCCTGACAAGGTCTTCCGTTCCAGCGTGCTGCA

CTCTACTCAGGACCTGTTCCTGCCCTTCTTCTCTAACGTCACCTGGTTCCAC

GCCATCCACGTGTCCGGTACCAACGGCACTAAGCGCTTCGACAACCCAGT

GCTGCCTTTCAACGACGGAGTCTACTTCGCTAGCACCGAGAAGTCTAACAT

CATCCGTGGATGGATCTTCGGTACCACTCTGGACTCAAAGACTCAGTCCCT

GCTGATCGTCAACAACGCCACCAACGTGGTCATCAAGGTGTGCGAGTTCC

AGTTCTGCAACGACCCATTCCTGGGCGTCTACTACCACAAGAACAACAAG

AGCTGGATGGAGTCTGAGTTCCGCGTCTACTCTTCAGCTAACAACTGCACT

TTCGAGTACGTGTCACAGCCTTTCCTGATGGACCTGGAAGGAAAGCAGGG

TAACTTCAAGAACCTGAGGGAGTTCGTGTTCAAGAACATCGACGGTTACT

TCAAGATCTACTCAAAGCACACCCCAATCAACCTGGTGCGCGACCTGCCT

CAGGGATTCTCCGCTCTGGAGCCACTGGTGGACCTGCCTATCGGTATCAAC

ATCACCCGCTTCCAGACTCTGCTGGCTCTGCACCGTAGCTACCTGACTCCT

GGCGACTCTTCTTCTGGATGGACTGCTGGAGCTGCTGCTTACTACGTGGGT

TACCTGCAGCCTAGGACCTTCCTGCTGAAGTACAACGAAAACGGCACCAT

CACTGACGCCGTCGACTGCGCTCTGGACCCTCTGAGCGAAACCAAGTGCA

CTCTGAAGTCTTTCACCGTGGAGAAGGGTATCTACCAGACTAGCAACTTCA

GGGTGCAGCCCACCGAATCTATCGTCAGATTCCCTAACATCACTAACCTGT

GCCCCTTCGGCGAGGTCTTCAACGCCACCAGATTCGCTTCCGTGTACGCCT

GGAACAGGAAGAGAATCAGCAACTGCGTCGCTGACTACTCTGTGCTGTAC

AACAGCGCCTCTTTCTCAACCTTCAAGTGCTACGGTGTGAGCCCAACTAA

GCTGAACGACCTGTGCTTCACCAACGTCTACGCCGACTCTTTCGTGATCAG

GGGCGACGAGGTCAGACAGATCGCTCCTGGCCAGACTGGAAAGATCGCC

GACTACAACTACAAGCTGCCCGACGACTTCACCGGTTGCGTCATCGCTTGG

AACTCAAACAACCTGGACTCCAAAGTGGGTGGCAACTACAACTACCTGTA

CCGCCTGTTCCGTAAGTCAAACCTGAAGCCATTCGAGAGGGACATCTCAA

CTGAAATCTACCAGGCTGGCTCCACCCCTTGCAACGGTGTCGAGGGCTTC

AACTGCTACTTCCCCCTGCAGTCCTACGGATTCCAGCCAACTAACGGTGTG

GGCTACCAGCCTTACAGAGTGGTCGTGCTGTCATTCGAACTGCTCCACGCT

CCTGCTACTGTGTGCGGACCAAAGAAGTCCACCAACCTGGTCAAGAACAA

GTGCGTGAACTTCAACTTCAACGGTCTGACCGGAACTGGTGTCCTGACCG

AGTCAAACAAGAAGTTCCTGCCCTTCCAGCAGTTCGGCAGGGACATCGCT

GACACCACTGACGCTGTGCGCGACCCTCAGACCCTGGAAATCCTGGACAT

CACTCCATGCAGCTTCGGAGGTGTCTCTGTGATCACTCCAGGAACCAACA

CTTCCAACCAGGTCGCTGTGCTGTACCAGGACGTCAACTGCACCGAGGTC

CCTGTGGCCATCCACGCTGACCAGCTGACCCCCACTTGGCGCGTGTACTCT

ACCGGCTCAAACGTCTTCCAGACTCGTGCTGGTTGCCTGATCGGCGCCGA

GCACGTGAACAACTCATACGAATGCGACATCCCCATCGGCGCTGGAATCTG

CGCCTCCTACCAGACCCAGACTAACTCACCACGCAGGGCTAGG

SEQ ID No.3 is a nucleic acid sequence encoding Val16-Arg685 obtained by codon optimization of insect cells by the inventors.

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.

The invention provides a protein and vaccine for resisting SARS-CoV-2 infection, which can induce immune reaction such as antibody and the like generated in vivo, and block the combination of S protein of SARS-CoV-2 and host cell ACE2 receptor, thereby helping host resist coronavirus infection. Animal experiments prove that the protein prepared by the invention has good safety, no obvious toxic or side effect, can play a remarkable role in preventing and treating SARS-CoV-2 infection, and has wide application prospect.

Drawings

FIG. 1 is a graph showing the results of measurement of absorbance values of A450 of mouse serum in test example 1;

FIG. 2 is a graph showing the in vitro blocking of S1 protein from ACE2 binding by immune serum in test example 2;

FIG. 3 is a graph showing the results of measurement of neutralizing antibody titer against the immune serum virus in test example 3;

FIG. 4 is a graph showing the results of determining the copy number of the virus of the challenge mouse in test example 4;

FIG. 5 is a graph of the lung pathology HE staining of the challenged mice in test example 4;

FIG. 6 is a schematic diagram showing the constitution of the extracellular phase of S protein of SARS-CoV-2 virus.

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 nucleotide sequence shown as SEQ ID No.3 is adopted. An expression vector for the extracellular segment Val16-Arg685 of the S protein was constructed based on the pFastBac1 vector (ampicillin resistance), and the BamHI, hindIII cleavage site was inserted into the pFast-bacI vector, 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 Cellfectin II 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 and sf21 cells may also be used) 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 sequence of the finally obtained recombinant protein is shown as SEQ ID No.1, and can be used for subsequent researches such as animal immunization and the like.

EXAMPLE 2 preparation of the vaccine against SARS-CoV-2 infection of the invention

Antigen formulation was performed under sterile conditions. The purified recombinant protein antigen (prepared in example 1) was diluted to a concentration of 80mcg/ml with 5mmol/L phosphate buffer (pH 7.2). The adjuvant was prepared under aseptic conditions, and 5mmol/L phosphate buffer (pH 7.2) diluted the aluminum hydroxide adjuvant stock solution (aluminum hydroxide content: 14.55 mg/ml) to a concentration of 2.0mg/ml. Antigen-adjuvant adsorption is carried out under the aseptic condition, and diluted protein antigen liquid is dripped into diluted aluminum hydroxide adjuvant working solution according to the speed of 20ml/min, wherein the volume ratio is (V/V) 1:1, the final concentration of the recombinant protein antigen in the mixed solution is 40mcg/mL, and the final concentration of the aluminum adjuvant is 1.0mg/mL. The reaction temperature was kept at 25℃and the stirring speed was 800rpm. After the completion of the dropwise addition, the mixture was kept at 25℃and adsorbed by stirring at 800rpm for 60 minutes. The pH of the mixed solution was adjusted to 7.2. And storing in dark at 4 ℃. Characterization of the adsorbed vaccine formulation includes particle size, spot location, antigen content, adjuvant content, adsorption rate, pH, endotoxin, adjuvant-antigen adsorption rate, adsorption strength and its retention state, post-adsorption antigen integrity and stability, etc. And (5) filling. The vaccine preparation which is qualified by detection is filled into 1mL sterile penicillin/ampoule bottle and 1 mL/bottle. The stirring is kept continuously during filling, so that the filling liquid is uniform. Sealing the container immediately after canning, attaching a serial number label, and storing in a dark place at 4 ℃.

The beneficial effects of the invention are demonstrated by biological experiments below.

Test example 1 in vivo induction of specific antibodies to the extracellular segment Val16-Arg685 (abbreviated as S1) of the S protein in mice vaccinated with the vaccine of the present invention

Immune animal experiments: BALB/C mice or C57BL/6 mice were used, and groups of 5 to 10 mice each were as shown in FIG. 1. The amino acid sequence of the extracellular segment aa16-685 of the recombinant protein is shown as SEQ ID No.1, and the dosage is 1.0 mug to 20.0 mug per dose, and the specific dosage is shown in figure 1. The mice of the experimental group were each injected with vaccine (prepared according to example 2) in a volume of 50 μl and were intramuscular injected (im) at the right hind leg of the mice, and immunized with the immunization program 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 hr, solidifying, centrifuging at 4deg.C and 3000rpm for 10min, and collecting upper serum layer and storing at-20deg.C.

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 Co., 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 a second 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 (1:5000 diluted in blocking solution) was then added at 100 μl/well and after incubation for 1h at 37deg.C PBST was washed 5 times. Finally, 100 μl/well of 3,3', 5' -tetramethyl biphenyl diamine (TMB) is added, after developing for 10-15min in dark, 50 μl/well of 1M H2SO4 stop solution is added, and stirring and mixing are continued. Read at 450nm wavelength on a microplate reader.

The serum was serially diluted at different fold and the absorbance of a450 was measured by titration to determine the titer of the recombinant protein-induced S1-specific antibodies. The absorbance at 450nm is plotted on the ordinate and the dilution is plotted on the abscissa. As can be seen from fig. 1, the optical density values of a450 of the control group inoculated with physiological saline and the control group inoculated with aluminum hydroxide adjuvant are lower, and the optical density values of a450 of other inoculated protein groups are obviously increased compared with the control group, so that the recombinant protein of the invention is proved to excite obvious S1 specific antibody. Further, the aluminum hydroxide adjuvant significantly improves the antibody titer of the protein vaccine and is in recombinant protein dose-dependent relationship. 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 binding activity by flow cytometry. The specific operation is as follows:

digesting and collecting in vitro cultured ACE 2-highly expressed cell strain (lung cancer A549) into flow tube, 10 ⁶ Cells/tube were 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 no antiserum was added to the positive control tube, and normal serum from the non-immunized mice in test example 1 was added to the physiological saline tube. 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 the mixture was put on a flow-type machineAnd (5) detecting.

As shown in fig. 2, 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 S1 protein immune serum

The immune 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 as virus control VC, and columns 4 to 11 as sample wells), and 42.5. Mu.l/well of DMEM complete medium was added to wells B4 to B11.

TABLE 1

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.

The multichannel pipettor was adjusted to 50. Mu.l, the liquid in the wells B4 to B11 was gently and repeatedly blown and sucked 6 to 8 times and thoroughly mixed, then 50. Mu.l of the liquid was transferred to the corresponding wells C4 to C11, and after 6 to 8 times of the soft and repeated blown and sucked, the liquid was transferred to the wells D4 to D11, and so on, and finally 50. Mu.l of the liquid was sucked from the wells G4 to G11, and the sample adding sequence was referred to Table 1.

Pseudoviruses were diluted to 1.3X10 with DMEM complete medium ⁴ (1×10 ⁴ ～2×10 ⁴ ) TCID50/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. The pseudovirus used in the experiment is detected by Chinese food and medicineThe established institute provides a Vesicular Stomatitis Virus (VSV) based pseudovirus detection system that expresses the full-length S protein, which enters cells in the same manner as live viruses, and can be used for detection and quantitative analysis of SARS-CoV-2 neutralizing antibodies.

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, 100. Mu.l of cells were added to each well of a 96-well plate 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 ℃ with 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 neutralizing antibodies in the sera of mice injected with physiological saline and S1 vaccine were counted, respectively, and as shown in fig. 3, only extremely low EC50 neutralizing antibody titers were detected in the sera of mice injected with physiological saline, while higher EC50 neutralizing antibody titers were detected in the sera of mice immunized with S1 vaccine.

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 mice in the control group were injected with an aluminum hydroxide immunoadjuvant or physiological saline alone. Serum was collected again 7 days after immunization. At 7 days post immunization, SARS-CoV-2 virus challenge (intranasal infection, dose 10 ⁵ TCID 50). In addition, mice of the control group were injected with an aluminum hydroxide immunoadjuvant or mice infected with the virus only with physiological saline as a control. 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. 4);

reversing: 5'-GCGCAGTAAGGATGGCTAGT-3' (SEQ ID No. 5).

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. 4, 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), while 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. 5, the lung tissue of the control group (normal saline combined with aluminum hydroxide) showed obvious histopathological changes of interstitial pneumonia, including obvious thickening of alveolar wall, congestion, and massive infiltration of single nuclear cells. 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> university of Sichuan

<120> protein against SARS-CoV-2 infection and vaccine prepared using the same

<130> A210224K

<150> 202010304546.1

<151> 2020-04-17

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 670

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser

1 5 10 15

Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val

20 25 30

Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr

35 40 45

Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe

50 55 60

Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr

65 70 75 80

Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp

85 90 95

Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val

100 105 110

Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val

115 120 125

Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val

130 135 140

Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe

145 150 155 160

Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu

165 170 175

Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His

180 185 190

Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu

195 200 205

Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln

210 215 220

Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser

225 230 235 240

Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln

245 250 255

Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp

260 265 270

Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu

275 280 285

Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg

290 295 300

Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

305 310 315 320

Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr

325 330 335

Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val

340 345 350

Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser

355 360 365

Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser

370 375 380

Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr

385 390 395 400

Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly

405 410 415

Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly

420 425 430

Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro

435 440 445

Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro

450 455 460

Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr

465 470 475 480

Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val

485 490 495

Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro

500 505 510

Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe

515 520 525

Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe

530 535 540

Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala

545 550 555 560

Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser

565 570 575

Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln

580 585 590

Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala

595 600 605

Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly

610 615 620

Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His

625 630 635 640

Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys

645 650 655

Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg

660 665 670

<210> 2

<211> 2010

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

gttaatctta caaccagaac tcaattaccc cctgcataca ctaattcttt cacacgtggt 60

gtttattacc ctgacaaagt tttcagatcc tcagttttac attcaactca ggacttgttc 120

ttacctttct tttccaatgt tacttggttc catgctatac atgtctctgg gaccaatggt 180

actaagaggt ttgataaccc tgtcctacca tttaatgatg gtgtttattt tgcttccact 240

gagaagtcta acataataag aggctggatt tttggtacta ctttagattc gaagacccag 300

tccctactta ttgttaataa cgctactaat gttgttatta aagtctgtga atttcaattt 360

tgtaatgatc catttttggg tgtttattac cacaaaaaca acaaaagttg gatggaaagt 420

gagttcagag tttattctag tgcgaataat tgcacttttg aatatgtctc tcagcctttt 480

cttatggacc ttgaaggaaa acagggtaat ttcaaaaatc ttagggaatt tgtgtttaag 540

aatattgatg gttattttaa aatatattct aagcacacgc ctattaattt agtgcgtgat 600

ctccctcagg gtttttcggc tttagaacca ttggtagatt tgccaatagg tattaacatc 660

actaggtttc aaactttact tgctttacat agaagttatt tgactcctgg tgattcttct 720

tcaggttgga cagctggtgc tgcagcttat tatgtgggtt atcttcaacc taggactttt 780

ctattaaaat ataatgaaaa tggaaccatt acagatgctg tagactgtgc acttgaccct 840

ctctcagaaa caaagtgtac gttgaaatcc ttcactgtag aaaaaggaat ctatcaaact 900

tctaacttta gagtccaacc aacagaatct attgttagat ttcctaatat tacaaacttg 960

tgcccttttg gtgaagtttt taacgccacc agatttgcat ctgtttatgc ttggaacagg 1020

aagagaatca gcaactgtgt tgctgattat tctgtcctat ataattccgc atcattttcc 1080

acttttaagt gttatggagt gtctcctact aaattaaatg atctctgctt tactaatgtc 1140

tatgcagatt catttgtaat tagaggtgat gaagtcagac aaatcgctcc agggcaaact 1200

ggaaagattg ctgattataa ttataaatta ccagatgatt ttacaggctg cgttatagct 1260

tggaattcta acaatcttga ttctaaggtt ggtggtaatt ataattacct gtatagattg 1320

tttaggaagt ctaatctcaa accttttgag agagatattt caactgaaat ctatcaggcc 1380

ggtagcacac cttgtaatgg tgttgaaggt tttaattgtt actttccttt acaatcatat 1440

ggtttccaac ccactaatgg tgttggttac caaccataca gagtagtagt actttctttt 1500

gaacttctac atgcaccagc aactgtttgt ggacctaaaa agtctactaa tttggttaaa 1560

aacaaatgtg tcaatttcaa cttcaatggt ttaacaggca caggtgttct tactgagtct 1620

aacaaaaagt ttctgccttt ccaacaattt ggcagagaca ttgctgacac tactgatgct 1680

gtccgtgatc cacagacact tgagattctt gacattacac catgttcttt tggtggtgtc 1740

agtgttataa caccaggaac aaatacttct aaccaggttg ctgttcttta tcaggatgtt 1800

aactgcacag aagtccctgt tgctattcat gcagatcaac ttactcctac ttggcgtgtt 1860

tattctacag gttctaatgt ttttcaaaca cgtgcaggct gtttaatagg ggctgaacat 1920

gtcaacaact catatgagtg tgacataccc attggtgcag gtatatgcgc tagttatcag 1980

actcagacta attctcctcg gcgggcacgt 2010

<210> 3

<211> 2010

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gtgaacctga ccaccaggac tcagctgcct cccgcctaca ccaactcctt cactcgcggt 60

gtgtactacc ctgacaaggt cttccgttcc agcgtgctgc actctactca ggacctgttc 120

ctgcccttct tctctaacgt cacctggttc cacgccatcc acgtgtccgg taccaacggc 180

actaagcgct tcgacaaccc agtgctgcct ttcaacgacg gagtctactt cgctagcacc 240

gagaagtcta acatcatccg tggatggatc ttcggtacca ctctggactc aaagactcag 300

tccctgctga tcgtcaacaa cgccaccaac gtggtcatca aggtgtgcga gttccagttc 360

tgcaacgacc cattcctggg cgtctactac cacaagaaca acaagagctg gatggagtct 420

gagttccgcg tctactcttc agctaacaac tgcactttcg agtacgtgtc acagcctttc 480

ctgatggacc tggaaggaaa gcagggtaac ttcaagaacc tgagggagtt cgtgttcaag 540

aacatcgacg gttacttcaa gatctactca aagcacaccc caatcaacct ggtgcgcgac 600

ctgcctcagg gattctccgc tctggagcca ctggtggacc tgcctatcgg tatcaacatc 660

acccgcttcc agactctgct ggctctgcac cgtagctacc tgactcctgg cgactcttct 720

tctggatgga ctgctggagc tgctgcttac tacgtgggtt acctgcagcc taggaccttc 780

ctgctgaagt acaacgaaaa cggcaccatc actgacgccg tcgactgcgc tctggaccct 840

ctgagcgaaa ccaagtgcac tctgaagtct ttcaccgtgg agaagggtat ctaccagact 900

agcaacttca gggtgcagcc caccgaatct atcgtcagat tccctaacat cactaacctg 960

tgccccttcg gcgaggtctt caacgccacc agattcgctt ccgtgtacgc ctggaacagg 1020

aagagaatca gcaactgcgt cgctgactac tctgtgctgt acaacagcgc ctctttctca 1080

accttcaagt gctacggtgt gagcccaact aagctgaacg acctgtgctt caccaacgtc 1140

tacgccgact ctttcgtgat caggggcgac gaggtcagac agatcgctcc tggccagact 1200

ggaaagatcg ccgactacaa ctacaagctg cccgacgact tcaccggttg cgtcatcgct 1260

tggaactcaa acaacctgga ctccaaagtg ggtggcaact acaactacct gtaccgcctg 1320

ttccgtaagt caaacctgaa gccattcgag agggacatct caactgaaat ctaccaggct 1380

ggctccaccc cttgcaacgg tgtcgagggc ttcaactgct acttccccct gcagtcctac 1440

ggattccagc caactaacgg tgtgggctac cagccttaca gagtggtcgt gctgtcattc 1500

gaactgctcc acgctcctgc tactgtgtgc ggaccaaaga agtccaccaa cctggtcaag 1560

aacaagtgcg tgaacttcaa cttcaacggt ctgaccggaa ctggtgtcct gaccgagtca 1620

aacaagaagt tcctgccctt ccagcagttc ggcagggaca tcgctgacac cactgacgct 1680

gtgcgcgacc ctcagaccct ggaaatcctg gacatcactc catgcagctt cggaggtgtc 1740

tctgtgatca ctccaggaac caacacttcc aaccaggtcg ctgtgctgta ccaggacgtc 1800

aactgcaccg aggtccctgt ggccatccac gctgaccagc tgacccccac ttggcgcgtg 1860

tactctaccg gctcaaacgt cttccagact cgtgctggtt gcctgatcgg cgccgagcac 1920

gtgaacaact catacgaatg cgacatcccc atcggcgctg gaatctgcgc ctcctaccag 1980

acccagacta actcaccacg cagggctagg 2010

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tcgtttcgga agagacaggt 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gcgcagtaag gatggctagt 20

Claims (13)

1. Use of a protein against SARS-CoV-2 infection or a protein precursor against SARS-CoV-2 infection in the manufacture of a medicament for the prevention and/or treatment of SARS-CoV-2 infection; wherein the amino acid sequence of the protein for resisting SARS-CoV-2 infection is shown as SEQ ID No.1, and the protein precursor for resisting SARS-CoV-2 infection is obtained by connecting signal peptide and/or protein label on the protein for resisting SARS-CoV-2 infection.

2. The use according to claim 1, characterized in that: 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.

3. The use according to claim 1, characterized in that: the protein precursor is a protease recognition region with a cleavage protein tag connected to the SARS-CoV-2 infection resisting protein.

4. A use according to claim 3, wherein: the protease is at least one selected from the following: enterokinase, TEV protease, thrombin, factor Xa, carboxypeptidase a, rhinovirus 3c protease.

5. A vaccine for preventing and/or treating SARS-CoV-2 infection, characterized in that: contains proteins or protein precursors for resisting SARS-CoV-2 infection and pharmaceutically acceptable auxiliary materials or auxiliary components; wherein the amino acid sequence of the protein for resisting SARS-CoV-2 infection is shown as SEQ ID No.1, and the protein precursor for resisting SARS-CoV-2 infection is obtained by connecting signal peptide and/or protein label on the protein for resisting SARS-CoV-2 infection.

6. The vaccine of claim 5, wherein: 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.

7. The vaccine of claim 5, wherein: the protein precursor is a protease recognition region with a cleavage protein tag connected to the SARS-CoV-2 infection resisting protein.

8. The vaccine of claim 7, wherein: the protease is at least one selected from the following: enterokinase, TEV protease, thrombin, factor Xa, carboxypeptidase a, rhinovirus 3c protease.

9. The vaccine of claim 5, wherein: the auxiliary component is an immunoadjuvant.

10. The vaccine of claim 9, 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.

11. The vaccine of claim 10, 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.

12. The vaccine of any one of claims 5-11, characterized in that: the vaccine is an injection preparation.

13. The vaccine of claim 12, wherein: the vaccine is an intramuscular injection preparation.

Images