CN113087777A

CN113087777A - Protein for resisting SARS-CoV-2 infection and vaccine prepared by using said protein

Info

Publication number: CN113087777A
Application number: CN202110407985.XA
Authority: CN
Inventors: 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
Original assignee: Chengdu Weisk Biomedical Co ltd
Current assignee: Chengdu Weisk Biomedical Co ltd
Priority date: 2020-04-17
Filing date: 2021-04-15
Publication date: 2021-07-09
Anticipated expiration: 2041-04-15
Also published as: CN113087777B

Abstract

The present invention relates to protein for resisting SARS-CoV-2 infection and vaccine prepared with the protein, and belongs to the field of medicine technology. In order to solve the problem that the SARS-CoV-2 infection is lack of effective prevention and treatment medicine, the invention provides a protein for resisting SARS-CoV-2 infection, and its amino acid sequence is shown in SEQ ID No.1, or has more than 99% homology with SEQ ID No.1 and identical 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 the S protein of SARS-CoV-2 and host cell ACE2 receptor by inducing immune reaction such as antibody generation 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 protein for resisting SARS-CoV-2 infection and vaccine prepared with the protein, and belongs to the field of medicine technology.

Background

SARS-CoV-2 is a novel coronavirus of the beta genus named by the world health organization. The virus has an envelope, and the particles are round or oval, usually polymorphic, and have a diameter of 60-140 nm. The gene characteristics of the coronavirus are obviously different from those of SARS-CoV and MERS-CoV, and the coronavirus is a new coronavirus branch which has not been discovered in human before. Bats may be the natural host for SARS-CoV-2 and, in addition, pangolin has been considered by research to be an animal source of the virus. At present, the novel coronavirus SARS-CoV-2 causes infection of tens of thousands of people, no exact effective antiviral drug can be used for prevention and treatment, and the development of a vaccine aiming at the virus is very important for preventing and treating diseases.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the present invention aims to provide a protein for resisting SARS-CoV-2 infection. Another object of the present invention is to provide a vaccine for preventing and/or treating SARS-CoV-2 infection, which contains the protein.

The invention provides a protein for resisting SARS-CoV-2 infection, the amino acid sequence of which is shown in SEQ ID No.1, or has more than 99% homology with SEQ ID No.1 and has the same or similar biological activity.

SEQ ID No.1：

VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

The extracellular domain of the S protein of SARS-CoV-2 virus is shown in FIG. 6, SP, signal peptide; NTD, N-terminal domain; RBD, receptor domain; FP, fusion peptide; IFP, an internal fusion peptide; HR1, heptad repeat region 1; HR2, heptad repeat 2; PTM, membrane proximal region; TM, transmembrane region. The SEQ ID No.1 is a SARS-CoV-2 infection resisting drug designed based on the amino acids Val16-Arg685 in the S protein.

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

Preferably, the protein tag is selected from at least one of: a histidine tag, a thioredoxin tag, a glutathione transferase tag, a ubiquitin-like modified protein tag, a maltose binding protein tag, a c-Myc protein tag, an Avi tag protein tag and a nitrogen source utilization substance A protein tag.

Further, the precursor is connected with a protease recognition area of a protein label of a cutting protein on the protein for resisting SARS-CoV-2 infection.

Preferably, the protease is selected from at least one of: enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase a, rhinovirus 3c protease.

The invention provides the application of the protein and/or the precursor in the preparation of 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 immunological adjuvant.

Preferably, the immunological adjuvant is selected from at least one of: aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoryl lipid A (MPL), muramyl dipeptide, muramyl tripeptide, squalene oil-in-water emulsion (MF59), recombinant cholera toxin (rCTB), GM-CSF cytokine, lipid, cationic liposome material, CpG ODN (nucleotide sequence containing unmethylated cytosine and guanine dinucleotide as core sequences, artificially synthesized CpG).

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

Wherein, the immunological adjuvant is preferably an aluminum hydroxide adjuvant; the protein for resisting SARS-CoV-2 infection: the ratio of the aluminum hydroxide adjuvant is preferably (32-48) mcg/mL: (0.8-1.2) mg/ml, wherein the adjuvant is calculated by the content of aluminum hydroxide; further preferably, the protein against SARS-CoV-2 infection: the ratio of the aluminum hydroxide adjuvant is preferably 40 mcg/mL: 1.0mg/ml, and the adjuvant is calculated by the content of aluminum hydroxide.

Further, the calcium salt is tricalcium phosphate.

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

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

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

Further, the cationic liposome material is selected from at least one of the following: (2, 3-dioleyloxypropyl) trimethylammonium chloride (DOTAP), N- [1- (2, 3-dioleyl chloride) propyl ] -N, N, N-trimethylamine chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyl-2, 3-dioleyloxypropyl-2- (2-sperminoylamido) 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 protein or said precursor.

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

GTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGT

the SEQ ID No.2 is an original nucleic acid sequence for coding Val16-Arg685, and is obtained according to a sequence of a new crown Wuhan-Hu-1 isolated strain sequenced by a first whole genome.

SEQ ID No.3：

GTGAACCTGACCACCAGGACTCAGCTGCCTCCCGCCTACACCAACTCCTTCACTCGCGGTGTGTACTACCCTGACAAGGTCTTCCGTTCCAGCGTGCTGCACTCTACTCAGGACCTGTTCCTGCCCTTCTTCTCTAACGTCACCTGGTTCCACGCCATCCACGTGTCCGGTACCAACGGCACTAAGCGCTTCGACAACCCAGTGCTGCCTTTCAACGACGGAGTCTACTTCGCTAGCACCGAGAAGTCTAACATCATCCGTGGATGGATCTTCGGTACCACTCTGGACTCAAAGACTCAGTCCCTGCTGATCGTCAACAACGCCACCAACGTGGTCATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCATTCCTGGGCGTCTACTACCACAAGAACAACAAGAGCTGGATGGAGTCTGAGTTCCGCGTCTACTCTTCAGCTAACAACTGCACTTTCGAGTACGTGTCACAGCCTTTCCTGATGGACCTGGAAGGAAAGCAGGGTAACTTCAAGAACCTGAGGGAGTTCGTGTTCAAGAACATCGACGGTTACTTCAAGATCTACTCAAAGCACACCCCAATCAACCTGGTGCGCGACCTGCCTCAGGGATTCTCCGCTCTGGAGCCACTGGTGGACCTGCCTATCGGTATCAACATCACCCGCTTCCAGACTCTGCTGGCTCTGCACCGTAGCTACCTGACTCCTGGCGACTCTTCTTCTGGATGGACTGCTGGAGCTGCTGCTTACTACGTGGGTTACCTGCAGCCTAGGACCTTCCTGCTGAAGTACAACGAAAACGGCACCATCACTGACGCCGTCGACTGCGCTCTGGACCCTCTGAGCGAAACCAAGTGCACTCTGAAGTCTTTCACCGTGGAGAAGGGTATCTACCAGACTAGCAACTTCAGGGTGCAGCCCACCGAATCTATCGTCAGATTCCCTAACATCACTAACCTGTGCCCCTTCGGCGAGGTCTTCAACGCCACCAGATTCGCTTCCGTGTACGCCTGGAACAGGAAGAGAATCAGCAACTGCGTCGCTGACTACTCTGTGCTGTACAACAGCGCCTCTTTCTCAACCTTCAAGTGCTACGGTGTGAGCCCAACTAAGCTGAACGACCTGTGCTTCACCAACGTCTACGCCGACTCTTTCGTGATCAGGGGCGACGAGGTCAGACAGATCGCTCCTGGCCAGACTGGAAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGTTGCGTCATCGCTTGGAACTCAAACAACCTGGACTCCAAAGTGGGTGGCAACTACAACTACCTGTACCGCCTGTTCCGTAAGTCAAACCTGAAGCCATTCGAGAGGGACATCTCAACTGAAATCTACCAGGCTGGCTCCACCCCTTGCAACGGTGTCGAGGGCTTCAACTGCTACTTCCCCCTGCAGTCCTACGGATTCCAGCCAACTAACGGTGTGGGCTACCAGCCTTACAGAGTGGTCGTGCTGTCATTCGAACTGCTCCACGCTCCTGCTACTGTGTGCGGACCAAAGAAGTCCACCAACCTGGTCAAGAACAAGTGCGTGAACTTCAACTTCAACGGTCTGACCGGAACTGGTGTCCTGACCGAGTCAAACAAGAAGTTCCTGCCCTTCCAGCAGTTCGGCAGGGACATCGCTGACACCACTGACGCTGTGCGCGACCCTCAGACCCTGGAAATCCTGGACATCACTCCATGCAGCTTCGGAGGTGTCTCTGTGATCACTCCAGGAACCAACACTTCCAACCAGGTCGCTGTGCTGTACCAGGACGTCAACTGCACCGAGGTCCCTGTGGCCATCCACGCTGACCAGCTGACCCCCACTTGGCGCGTGTACTCTACCGGCTCAAACGTCTTCCAGACTCGTGCTGGTTGCCTGATCGGCGCCGAGCACGTGAACAACTCATACGAATGCGACATCCCCATCGGCGCTGGAATCTGCGCCTCCTACCAGACCCAGACTAACTCACCACGCAGGGCTAGG

The above-mentioned 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 a recombinant vector comprising said polynucleotide.

Furthermore, 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 pFastBac 1.

Preferably, the escherichia coli expression vector is pET32 a.

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 a host cell comprising said recombinant vector.

Furthermore, the host cell adopts at least one of insect cells, mammalian 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 cell is a CHO cell.

The invention provides a preparation method of the protein, which comprises the following steps: culturing the host cell to express the protein or precursor, and recovering the protein.

The invention provides a preparation method of the protein, which comprises the following steps: constructing a recombinant vector containing the polynucleotide, immunizing a human body, and generating the protein.

Further, the carrier is selected from at least one of the following: mRNA, DNA vaccines, adenovirus, vaccinia virus Ankara virus, adeno-associated virus.

The invention provides a protein and a vaccine for resisting SARS-CoV-2 infection, which can induce immune reaction such as antibody generated in vivo, and block the combination of S protein of SARS-CoV-2 and host cell ACE2 receptor, thereby helping a host to 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 significant role in preventing and treating SARS-CoV-2 infection, and has wide application prospect.

Drawings

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

FIG. 2 is a graph showing that immune sera in experiment example 2 blocks the binding of S1 protein to ACE2 in vitro;

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

FIG. 4 is a graph showing the result of the determination of the number of copies of the pneumovirus in the challenged mouse in test example 4;

FIG. 5 is a graph showing HE staining of lung pathology in the virus-challenged mouse in Experimental example 4;

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

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 preparation of proteins of the invention against SARS-CoV-2 infection Using insect baculovirus expression System

Vector construction: the nucleotide sequence shown as SEQ ID No.3 is adopted. An expression vector of an S protein extracellular segment Val16-Arg685 is constructed based on a pFastBac1 vector (ampicillin resistance), and is inserted into a pFast-bacI vector by using BamHI and HindIII enzyme cutting sites and optimized according to insect cell preferred codons.

Amplification of recombinant baculovirus: a Bac-to-Bac expression system is adopted, a bacterial transposon principle is utilized, a Tn7 transposition element generates site-specific transposition, and the construction of recombinant bacmid is completed in escherichia coli (DH10Bac containing bacmid (kanamycin resistance) and helper plasmid (tetracycline resistance)). Successful recombinants of bacmid were extracted and transfected into sf9 insect cells using Cellffectin II to produce recombinant bacmid expressing the gene of interest. The first generation virus was harvested 72h after transfection, followed by amplification of the P2 to P4 generation viruses, and protein expression using P3 or P4 generation viruses.

Protein expression: hi5 insect cells (sf 9 and sf21 cells can also be used) are infected by P3 or P4 generation virus, the multiplicity of infection (MOI is 0.5-10), and supernatants are collected after culturing for 48-72 h. The optimal harvesting time may be different each time according to the virus amount and the cell state of the virus seeds, and is generally suitable for microscopic examination of about 50% of cell diseases.

Protein purification: the obtained culture supernatant is subjected to high-speed centrifugation at 4 ℃, 0.22um filter membrane filtration, the recombinant protein is preliminarily purified by adopting an affinity purification method (Histrap nickel column), and then the protein purity is identified by utilizing a MonoQ ion column and a Superdex 20010/300 GL molecular sieve, and the purity requirement reaches more than 95 percent by SDS-PAGE. The amino acid sequence of the finally obtained recombinant protein is shown as SEQ ID No.1, and the recombinant protein can be used for subsequent researches such as animal immunity and the like.

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

Antigen formulation was performed under sterile conditions. The purified recombinant protein antigen (prepared in example 1) was diluted with 5mmol/L phosphate buffer (pH7.2) to a concentration of 80 mcg/ml. The adjuvant was prepared under aseptic conditions, and the aluminum hydroxide adjuvant stock solution (the content of aluminum hydroxide was 14.55mg/ml) was diluted to a concentration of 2.0mg/ml with 5mmol/L phosphate buffer solution (pH 7.2). And (3) performing antigen-adjuvant adsorption under aseptic conditions, and dripping the diluted protein antigen liquid into the diluted aluminum hydroxide adjuvant working solution at the speed of 20ml/min, wherein the volume ratio of (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 1.0 mg/mL. The reaction temperature was kept at 25 ℃ and the stirring speed was 800 rpm. After the dropwise addition, the temperature is kept at 25 ℃ and the stirring and adsorption are carried out at 800rpm for 60 min. The pH of the mixed solution was adjusted to 7.2. Storing at 4 ℃ in the dark. Characterizing the vaccine preparation after adsorption, including particle size, point position, antigen content, adjuvant content, adsorption rate, pH value, endotoxin, adjuvant-antigen adsorption rate, adsorption strength and its retention state, antigen integrity and stability after adsorption, etc. And (6) filling. And filling the vaccine preparation qualified by detection into a 1mL sterile penicillin/ampoule bottle, wherein the volume of the sterile penicillin/ampoule bottle is 1 mL. Stirring continuously during filling to make the filling liquid uniform. Immediately sealing the can, labeling with number label, and storing at 4 deg.C in dark place.

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

Test example 1 in mice vaccinated with the vaccine of the present invention, specific antibodies against extracellular domain Val16-Arg685 (abbreviated as S1) of S protein were induced

And (3) immune animal experiment: BALB/C mice or C57BL/6 mice were used, grouped as shown in FIG. 1, with 5 to 10 mice per group. The amino acid sequence of the extracellular section aa16-685 of the used recombinant protein is shown in SEQ ID No.1, the dosage is 1.0 mu g to 20.0 mu g/cell, and the specific dosage is shown in figure 1. Experimental groups mice were immunized with the vaccine (prepared according to example 2) injected into each mouse in a volume of 50 μ L, and injected intramuscularly (im) in the right hind leg of the mice, with the immunization program on days 1, 7 and 21 (3 total immunizations).

ELISA (enzyme-linked immunosorbent assay) assay of mouse serum antibodies: on day 7 after the completion of the mouse immunization program (day 28 after the first immunization), the mouse plasma was collected by capillary orbital bleeding, and 6 mice were collected per group. Standing at room temperature for 1-2h, coagulating, centrifuging at 4 deg.C and 3000rpm for 10min, collecting upper layer serum, and storing at-20 deg.C.

Preparation of 50mM carbonate coating buffer (pH 9.6): 0.293g NaHCO was weighed₃And 0.15g of Na₂CO₃Dissolving with double distilled water, adjusting pH to 9.6, diluting to 100ml, and storing at 4 deg.C.

1M H₂SO₄Preparation of a stop solution: to 47.3mL of double distilled water was added dropwise 2.7mL of concentrated sulfuric acid (98%).

ELISA method for determination of serum IgG: a1. mu.g/ml solution of recombinant protein S1 was prepared in 50mM carbonate coating buffer and added to a 96-well coated plate (Thermo Scientific, NUNC-MaxiSorp) at 100. mu.l/well and coated overnight at 4 ℃. The next day, after washing 3 times with PBS solution (PBST) containing 0.1% Tween20, the PBST was washed 1 time after blocking for 1h at room temperature with blocking solution (prepared in PBST) containing 1% BSA or 5% skim milk. After diluting the mouse serum with blocking solution to different ratios, the mouse serum was loaded at a loading 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. mu.l/well and after incubation for 1h at 37 ℃ PBST was washed 5 times. Finally, 100 mul/hole of 3,3',5,5' -tetramethyl biphenyl diamine (TMB) is added, after the light shielding and color development is carried out for 10-15min, 50 mul/hole of 1M H2SO4 stop solution is added, and the mixture is stirred and mixed evenly without stopping stirring. Readings were taken on a microplate reader at a wavelength of 450 nm.

The titer of recombinant protein-induced S1-specific antibodies was determined by titrating the sera to measure a450 absorbance values after serial dilutions of different fold. The absorbance value at 450nm was plotted on the ordinate and the dilution factor 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 significantly higher than that of the control group, which proves that the recombinant protein of the invention stimulates the obvious specific antibody of S1. Further, the aluminum hydroxide adjuvant remarkably improves the antibody titer of the protein vaccine and is in a recombinant protein dose-dependent relationship. The results show that the recombinant S1 protein vaccine has high immunogenicity in mice.

Test example 2 blocking test of binding of the S1 protein of the present invention to ACE2 receptor

This experiment used cell-expressed ACE2, a protein that is believed 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 high expression ACE2 cell strain (lung cancer A549) into flow tube, 10⁶Cells/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 the immunized anti-S1 mouse (serum from the mouse immunized at 10. mu.g/dose in test example 1 was diluted 50-fold) was added thereto, and the mixture was incubated at room temperature for 30 min. The positive control tube was not added with antiserum, and the normal serum of the non-immunized mouse in test example 1 was added to the physiological saline tube. After washing with PBS/HBSS several times, Anti-Human IgG (Fc specific) -FITC (SIGMA) fluorescent secondary antibody (1:100-1:200) was added, and the mixture was incubated at room temperature for 30min in the absence of light. After washing with PBS/HBSS several times, 500. mu.l of 1% paraformaldehyde-containing PBS was added for fixation, and then the detection was performed by flow-on machine.

The result is shown in figure 2, the S1-Fc protein can be combined with the cells expressing ACE2, and the percentage of positive cells in a positive control group is more 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 cells expressing ACE2, and the percentage of positive cells is less than 25%; while the same dilution of non-or pre-immune serum showed no blocking effect, the percentage of positive cells was greater than 80%.

Test example 3 pseudovirus neutralization test of S1 protein immune serum

Inactivating immune serum (or blood plasma) to be detected in 56 deg.C water bath for 30min, centrifuging at 6000g for 3min, and transferring the supernatant to 1.5ml centrifuge tube for use.

A96-well plate was used, and 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 is virus control VC, columns 4 to 11 are 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. mu.l) … … was added to wells B4 and B5 and so on, and plasma sample 4 (7.5. mu.l) was added to wells B10 and B11.

And adjusting a multi-channel pipette to 50 mu l, gently and repeatedly blowing and sucking the liquid in the B4-B11 holes for 6-8 times, fully and uniformly mixing, then transferring 50 mu l of liquid to the corresponding C4-C11 holes, gently and repeatedly blowing and sucking for 6-8 times, transferring to the D4-D11 holes, repeating the operation, and finally, sucking and discarding 50 mu l of liquid from G4-G11, wherein the sample adding sequence refers to Table 1.

Pseudovirus was diluted to 1.3X 10 with DMEM complete medium⁴(1×10⁴～2×10⁴) TCID50/ml (diluted by the dilution factor given) was added to each well in columns 3-11 in an amount of 50. mu.l per well such that the amount of pseudovirus contained in each well was 650(500- & 1000)/well. The pseudovirus used in this experiment was provided by the Chinese food and drug assay institute, and was a Vesicular Stomatitis Virus (VSV) -based pseudovirus detection system that expressed the full-length S protein, in the same manner as the live virusCan enter cells in the mode of (1), 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 ℃ C., 5% CO)₂) Incubate for 1 hour.

When the incubation time is half an hour, taking hACE2-293T cells prepared in advance in an incubator (the confluence rate is 80% -90%), taking a T75 culture bottle as an example, absorbing the culture medium in the bottle, adding 5ml of PBS buffer solution to clean the cells, pouring off the PBS, adding 3ml of 0.25% pancreatin-EDTA to immerse the cells for digestion for 1 minute, pouring off the pancreatin, placing the cells in the cell incubator for digestion for 5 minutes, slightly beating the side wall of the culture bottle to enable the cells to fall off, adding 10ml of culture medium to neutralize the pancreatin, blowing for several times, transferring the cells to a centrifuge tube, centrifuging for 5 minutes at 210g, pouring off supernatant, completely culturing the cells by using 10ml of DMEM, counting the cells, diluting the cells to 2 x 10 by using the DMEM complete culture medium, and transferring the cells to a⁵One per ml.

Incubate for 1 hour, add 100. mu.l of cells per well in 96-well plate to make 2X 10 cells per well⁴And (4) respectively.

Gently shaking the 96-well plate to disperse the cells uniformly in the wells, placing the 96-well plate in a cell culture box at 37 deg.C and 5% CO₂Culturing for 20-28 hours.

And after 20-28 hours, taking out the 96-well plate from the cell culture box, sucking 150 mu l of supernatant from each sample loading hole by using a multi-channel pipette, then adding 100 mu l of luciferase detection reagent, and reacting for 2min at room temperature in a dark place.

After the reaction is finished, repeatedly blowing and sucking the liquid in the reaction hole for 6-8 times by using a multi-channel pipette to fully lyse the cells, sucking 150 mu l of liquid from each hole, adding the liquid into a corresponding 96-hole chemiluminescence detection plate, and placing the plate in a chemiluminescence detector to read the luminescence value.

Calculating the neutralization inhibition rate: the inhibition rate was [1- (mean value of luminescence intensity of sample group-CC mean value of blank control)/(mean value of luminescence intensity of negative group VC-CC mean value of blank control value) ] × 100%.

From the results of the neutralization inhibition ratio, IC50 was calculated by the Reed-Muench method.

The serum neutralizing antibody EC50 titers of the mice injected with the saline and the S1 vaccine were counted, respectively, and the results are shown in FIG. 3, in which only a very low EC50 neutralizing antibody titer was detected in the serum of the mice injected with the saline, and a higher EC50 neutralizing antibody titer was detected in the serum of the mice immunized with the S1 vaccine.

Test example 4 mouse SARS-CoV-2 Virus infection challenge test

Mice were immunized, 6 to 8 week old, hACE2 transgenic C57BL/6 mice, and the recombinant S1 protein vaccine (prepared according to example 2) was injected intramuscularly at a dose of 10 μ g per dose. For example, mice received one vaccine injection on days 1, 14, and 21, and control mice were injected with aluminum hydroxide immunoadjuvant or physiological saline only. Serum was collected again 7 days after immunization. 7 days after immunization, SARS-CoV-2 virus challenge (intranasal infection at a dose of 10)⁵TCID 50). In addition, control mice were injected with 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 lungs and other organs of the mice were excised. Lung tissue is used to detect viral replication. A real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) reaction was performed using the PowerUp SYBG Green Master Mix kit (Applied Biosystems, USA), and the viral RNA copy number in lung tissue of mice challenged with SARS-COV-2 was determined and expressed as RNA copy number of lung tissue/ml. The primer sequence for qRT-PCR was the envelope (E) gene for SARS-cov-2 as follows:

forward: 5'-TCGTTTCGGAAGAGACAGGT-3' (SEQ ID No. 4);

and (3) 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 mouse lung tissue was collected 5 days after virus challenge and measured for virus replication in either vaccine or control. As shown in FIG. 4, after mice were immunized with the protein vaccine of the present invention, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) detected little viral replication, whereas the level of viral replication was higher in lung tissue of control mice.

Portions of lung tissue were collected and fixed with 10% neutral formalin, embedded in paraffin, sectioned at a thickness of 5 μm, and stained with Hematoxylin and Eosin (HE). Histopathological changes were observed with a light microscope. As shown in FIG. 5, the lung tissues of the control mice (saline-aluminum hydroxide combined group) showed significant histopathological changes of interstitial pneumonia, including significant thickening of alveolar walls, congestion, and interstitial mass mononuclear cell infiltration. In contrast, the recombinant S1 protein vaccine immunized mice showed no histopathological changes.

The above experimental results further confirm that the S1 protein vaccine of the invention can block the infection of SARS-CoV-2 virus.

It should be appreciated 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 and features of the various embodiments described in this specification can be combined and combined by one skilled in the art without contradiction.

Sequence listing

<110> Sichuan university

<120> protein for anti SARS-CoV-2 infection and vaccine prepared by the protein

<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

1. Protein for resisting SARS-CoV-2 infection, which is characterized in that: the amino acid sequence is shown as SEQ ID No.1, or has more than 99 percent of homology with SEQ ID No.1 and has the same or similar biological activity.

2. A precursor of the protein of claim 1, characterized by: a signal peptide and/or a protein tag are connected to the protein for resisting SARS-CoV-2 infection; preferably, the protein tag is selected from at least one of: a histidine tag, a thioredoxin tag, a glutathione transferase tag, a ubiquitin-like modified protein tag, a maltose binding protein tag, a c-Myc protein tag, an Avi tag protein tag and a nitrogen source utilization substance A protein tag.

3. The precursor as set forth in claim 2, wherein: a protease recognition area of a protein cutting label is also connected to the protein for resisting SARS-CoV-2 infection; preferably, the protease is selected from at least one of: enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase a, rhinovirus 3c protease.

4. Use of the protein of claim 1, the precursor of claim 2 or 3 for the preparation of a medicament for the prevention and/or treatment of SARS-CoV-2 infection.

5. Vaccine for the prevention and/or treatment of SARS-CoV-2 infection, characterised in that: comprising the protein of claim 1, the precursor of claim 2 or 3, and a pharmaceutically acceptable adjuvant or auxiliary ingredient.

6. The vaccine of claim 5, which is characterized by: the auxiliary component is an immunologic adjuvant; preferably, the immunological adjuvant is selected from at least one of: aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoryl lipid A, muramyl dipeptide, muramyl tripeptide, squalene oil-in-water emulsion, bacterial toxin, GM-CSF cytokine, lipid, cationic liposome material, CpG ODN.

7. The vaccine of claim 6, which is characterized by: 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 angelica polysaccharide; the squalene oil-in-water emulsion is MF 59; 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, dioleoylphosphatidylethanolamine; the cationic liposome material is selected from at least one of the following: (2, 3-dioleyloxypropyl) trimethylammonium chloride, N- [1- (2, 3-dioleyl chloride) propyl ] -N, N, N-trimethylamine chloride, cationic cholesterol, dimethyl-2, 3-dioleyloxypropyl-2- (2-sperminocarboxamido) ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide.

8. The vaccine of any one of claims 5 to 7, which is characterized by: the vaccine is an injection preparation; preferably, the vaccine is an intramuscular injection preparation.

9. A polynucleotide characterized by: encoding the protein of claim 1, the precursor of claim 2 or 3.

10. The polynucleotide of claim 9, wherein: the nucleotide sequence is shown as SEQ ID No.2 or SEQ ID No. 3.

11. A recombinant vector characterized by: comprising the polynucleotide of claim 9 or 10.

12. The recombinant vector of claim 11, wherein: adopting at least one of insect baculovirus expression vector, mammal cell expression vector, colibacillus expression vector and yeast expression vector; preferably, the insect baculovirus expression vector is pfastBac 1; preferably, the Escherichia coli expression vector is pET32 a; 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.

13. A host cell characterized by: comprising the recombinant vector of claim 11 or 12.

14. The host cell of claim 13, wherein: adopting at least one of insect cell, mammalian cell, Escherichia coli, and yeast; preferably, the insect cell is at least one selected from sf9 cell, sf21 cell and Hi5 cell; preferably, the mammalian cell is a CHO cell.

15. The method for producing the protein of claim 1, wherein: the method comprises the following steps: culturing the host cell of claim 13 or 14 to express said protein or precursor, and recovering said protein.

16. The method for producing the protein of claim 1, wherein: the method comprises the following steps: constructing a recombinant vector containing the polynucleotide of claim 9 or 10, immunizing a human body, and producing the protein.

17. The method of claim 16, wherein: the carrier is selected from at least one of the following: mRNA, DNA vaccines, adenovirus, vaccinia virus Ankara virus, adeno-associated virus.