CN113583096B

CN113583096B - SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof

Info

Publication number: CN113583096B
Application number: CN202010369494.6A
Authority: CN
Inventors: 张林琦; 单思思; 史宣玲
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2020-05-01
Filing date: 2020-05-01
Publication date: 2023-06-30
Anticipated expiration: 2040-05-01
Also published as: CN113583096A

Abstract

The invention discloses a SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof, in particular to a SARS-CoV-2RBD dimer (SARS-CoV-2 RBD dimer) or a SARS-CoV-2RBD monomer (SARS-CoV-2 RBD monomer) and application thereof in neutralizing SARS-CoV-2, wherein the SARS-CoV-2RBD dimer has more remarkable activity. The invention provides a dimer of SARS-CoV-2 RBD; a protein shown in a sequence 1 of a sequence table; a protein shown in a sequence 3 of a sequence table; and the 34 th to 272 th amino acid residues in the sequence 3 of the sequence table are shown as proteins. The invention has important value and wide application prospect for developing medicines, vaccines and the like for treating and preventing SARS-CoV-2.

Description

SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof

Technical Field

The invention relates to SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof, in particular to SARS-CoV-2RBD dimer (SARS-CoV-2 RBD dimer) or SARS-CoV-2RBD monomer (SARS-CoV-2 RBD monomer) and application thereof in neutralizing SARS-CoV-2, wherein the SARS-CoV-2RBD dimer has more remarkable activity.

Background

The disease caused by the novel coronavirus (SARS-CoV-2) is designated as new coronapneumonia (Coronavirus Disease 2019, COVID-19).

Novel coronaviruses manifest as asymptomatic carriage, acute respiratory distress syndrome (ARD), and pneumonia. This disease is of great concern worldwide as it can be transmitted to humans.

The comparison of the gene sequences shows that SARS-CoV-2 belongs to beta coronavirus, and has high similarity in gene composition and structural function with other two highly pathogenic coronaviruses, namely acute respiratory syndrome coronavirus (Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV) and middle east respiratory syndrome coronavirus (Middle East Respiratory Syndrome Coronavirus, MERS-CoV), respectively.

SARS-CoV-2 virus is also thought to originate in bats, but there is currently no clear evidence to determine its origin and intermediate host.

Disclosure of Invention

The invention aims to provide SARS-CoV-2 Spike protein receptor binding domain dimer and application thereof, in particular to SARS-CoV-2RBD dimer (SARS-CoV-2 RBD dimer) or SARS-CoV-2RBD monomer (SARS-CoV-2 RBD monomer) and application thereof in neutralizing SARS-CoV-2, wherein the SARS-CoV-2RBD dimer has more remarkable activity.

The present invention provides dimers of SARS-CoV-2 RBD.

The dimer of SARS-CoV-2RBD is also known as SARS-CoV-2RBD dimer.

SARS-CoV-2RBD is as follows (a 1), (a 2), (a 3), (a 4), (a 5) or (a 6):

(a1) A protein shown in a sequence 1 of a sequence table;

(a2) A protein obtained by ligating a signal peptide to the N-terminus of (a 1);

(a3) A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of (a 1).

(a4) A fusion protein obtained by ligating a tag to the C-terminal of (a 2);

(a5) A protein shown in a sequence 3 of a sequence table;

(a6) And the 34 th to 272 th amino acid residues in the sequence 3 of the sequence table are shown as proteins.

For example, the labels are shown in table 1. The relationship of the sum and the label in table 1 may be an or relationship.

TABLE 1

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Nucleic acid molecules encoding the dimers of SARS-CoV-2RBD are also within the scope of the invention.

In particular, the nucleic acid molecule may be a DNA molecule.

The DNA molecule may be (c 1) or (c 2) as follows:

(c1) A DNA molecule shown in a sequence 2 of the sequence table;

(c2) DNA molecules shown as 910 th to 1728 th nucleotides in a sequence 4 of a sequence table.

Recombinant plasmids having said DNA molecules are also within the scope of the invention. The recombinant plasmid can be specifically a recombinant plasmid constructed by taking a pcDNA3.1 (+) vector as a starting vector.

The invention also provides a method for preparing the dimer of SARS-CoV-2RBD, comprising the following steps: the recombinant plasmid is transfected into mammalian cells, which are then subjected to cell culture. The mammalian cells may specifically be 293T cells.

The invention also protects a kit for preparing the dimer of SARS-CoV-2RBD, comprising the recombinant plasmid and mammalian cells. The mammalian cells may specifically be 293T cells.

The invention also protects a protein which is (b 1), (b 2), (b 3), (b 4), (b 5) or (b 6) as follows:

(b1) A protein shown in a sequence 1 of a sequence table;

(b2) A protein obtained by ligating a signal peptide to the N-terminus of (b 1);

(b3) A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of (b 1).

(b4) A fusion protein obtained by ligating a tag to the C-terminal of (b 2);

(b5) A protein shown in a sequence 6 of a sequence table;

(b6) And the 39 th-267 th amino acid residue in the sequence 6 of the sequence table.

The protein is SARS-CoV-2RBD monomer, also called SARS-CoV-2RBD monomer.

Nucleic acid molecules encoding said proteins are also within the scope of the invention.

In particular, the nucleic acid molecule may be a DNA molecule.

The DNA molecule may be (c 3) or (c 4) as follows:

(c3) A DNA molecule shown in a sequence 5 of a sequence table;

(c4) A DNA molecule shown in a sequence 7 of a sequence table.

Recombinant plasmids having said DNA molecules are also within the scope of the invention. The recombinant plasmid can be specifically a recombinant plasmid constructed by taking a pFastBac1 vector as a starting vector.

The invention also provides a method for preparing the protein, which comprises the following steps: the protein was prepared using the Bac-to-Bac system. The cells used in the Bac-to-Bac system are Sf9 cells. The Bac-to-Bac system employs a pFastBac1 vector as the starting vector.

The invention also protects the application of the SARS-CoV-2RBD dimer or the SARS-CoV-2RBD monomer or any one of the above nucleic acid molecules or any one of the above recombinant plasmids or any one of the above kits in the preparation of products; the use of the product is as follows (e 1) or (e 2):

(e1) As a novel coronavirus vaccine;

(e2) As a medicament for preventing and/or treating new coronaries pneumonia.

The invention also protects a product, the active ingredient of which is SARS-CoV-2RBD dimer or SARS-CoV-2RBD monomer or any one of the above nucleic acid molecules or any one of the above recombinant plasmids or any one of the above kits;

the use of the product is as follows (e 1) or (e 2):

(e1) As a novel coronavirus vaccine;

(e2) As a medicament for preventing and/or treating new coronaries pneumonia.

SARS-CoV-2RBD dimer or SARS-CoV-2RBD monomer has the ability to induce the production of neutralizing antibodies. Animal serum immunized with SARS-CoV-2RBD dimer has better neutralizing activity than animal serum immunized with SARS-CoV-2RBD monomer. The invention has important value and wide application prospect for developing medicines, vaccines and the like for treating and preventing SARS-CoV-2.

Drawings

Fig. 1 is a chromatogram and an electrophoresis chart in step one of example 1.

Fig. 2 is a chromatogram in step two of example 1.

FIG. 3 is an electrophoresis chart in the second step of example 1.

FIG. 4 shows the SPR test results in example 2.

FIG. 5 shows the results of the gel filtration test in example 2.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged. The SARS-CoV-2 Spike protein receptor binding domain is represented by SARS-CoV-2 RBD. Insect cell culture medium (instruction-XpressTM Media): lonza Wokingham ltd, cat No. 12-730F. Buffer 1: pH7.2 containing 150mM NaCl, 10mM HEPES buffer.

EXAMPLE 1 preparation of SARS-CoV-2RBD

1. Preparation of dimer (dimer)

1. The pcDNA3.1 (+) vector is used as a starting vector to construct a recombinant plasmid.

The recombinant plasmid is shown as a sequence 4 in a sequence table. In the sequence 4 of the sequence table, 910 th to 1728 th nucleotides form a fusion gene.

And the fusion gene expresses a fusion protein shown in a sequence 3 of a sequence table. In the sequence 3 of the sequence table, amino acid residues 1-33 form a signal peptide, amino acid residues 34-256 form SARS-CoV-2RBD, amino acid residues 257-264 form Strep-tag II tag, and amino acid residues 265-272 form FLAG tag.

The signal peptide in the fusion protein is recognized by and bound to a receptor on the endoplasmic reticulum membrane, then the fusion protein reaches the lumen of the endoplasmic reticulum through a channel formed by the protein in the endoplasmic reticulum membrane, then the signal peptide is cleaved by the signal peptidase to form a mature protein, and then the mature protein is secreted extracellularly. The mature protein is shown as 34 th-272 th amino acid residues in sequence 3 of a sequence table. The expected molecular weight of the mature protein monomer is 27KD. The expected molecular weight of the mature protein dimer was 54KD.

2. The recombinant plasmid obtained in step 1 was transfected into 293T cells grown to 90% density with the aid of PEI transfection reagent, and cultured in serum-free DMEM medium for 6-8 hours, followed by culturing in DMEM medium containing 10% fetal bovine serum for 72 hours.

3. After completion of step 2, the mixture was centrifuged at 4000rpm for 5min, and the supernatant was collected.

4. The supernatant obtained in the step 3 was filtered through a 0.22 μm filter membrane, and the filtrate was collected.

5. 200ml of the filtrate obtained in the step 4 was subjected to system displacement (using a concentration pump, a10 KD filter membrane and Buffer 1) to obtain 200ml of a product solution.

6. Taking 200ml of the product solution obtained in the step 5, centrifuging at 13000rpm for 30min at 4 ℃, and collecting supernatant.

7. Taking all the supernatant obtained in the step 6, incubating with streppTactin magnetic beads, washing with Buffer 1, eluting with 10ml of Buffer2, collecting the eluent, and concentrating with 10kD concentration tube to obtain a concentrated solution with the volume of 1.2 ml.

Buffer2 (ph 8.0): contains 100mM Tris, 150mM NaCl, 1mM EDTA, 5mM Desthiobiotics, the balance being water.

8. And (3) taking the concentrated solution obtained in the step (7), carrying out molecular sieve chromatography by using a Hiload superdex75 column, and adopting Buffer 1 as eluent.

The chromatogram of the elution process is shown on the left of figure 1 (retention volume on the abscissa).

The protein electrophoresis diagram of the A9 component is shown in the right diagram of FIG. 1, R represents a sample after the DDT treatment of the reducing agent, and N represents a sample which is not treated.

The A7 to A10 components are mixed to obtain SARS-CoV-2RBD dimer solution.

2. Preparation of monomers (Monomer)

1. Construction of recombinant plasmids

The double-stranded DNA molecule shown in the sequence 7 of the sequence table is inserted between BamHI and HindIII cleavage sites of the pFastBac1 vector to obtain a recombinant plasmid.

The double-stranded DNA molecule shown in the sequence 7 in the sequence table expresses the fusion protein shown in the sequence 6 in the sequence table.

In the sequence 6 of the sequence table, amino acid residues 1-38 form gp67 signal peptide, amino acid residues 39-261 form SARS-CoV-2RBD, and amino acid residues 262-267 form His ₆ And (5) a label.

After cleavage of the gp67 signal peptide, the mature protein is released. The mature protein is shown as 39 th-267 th amino acid residues in a sequence 6 of a sequence table. The expected molecular weight of the mature protein is 25.9KD.

2. Preparation of recombinant Bacmid

(1) Adding the recombinant plasmid prepared in the step 1 into the E.coli DH10 Bac competent cells which are just melted, and standing on ice for 30min; then heat shock is carried out for 75s at 42 ℃, and the ice is put back on the ice for 2min; then adding 500 μl of LB liquid medium, resuscitating at 37deg.C for 5h; then 10. Mu.l of the mixture was plated on LB solid medium plates containing 50. Mu.g/ml kanamycin, 7. Mu.g/ml gentamicin, 10. Mu.g/ml tetracycline, 40. Mu.g/ml IPTG and 100. Mu.g/ml X-gal, and incubated in the dark for three days until clear bluish-white spots developed.

(2) White single colonies were picked and inoculated into 5mL of LB liquid medium containing 50. Mu.g/mL kanamycin, 7. Mu.g/mL gentamicin, 10. Mu.g/mL tetracycline, and cultured at 37℃for 12 hours with shaking at 220 rpm.

(3) Taking the culture system obtained in the step (2), extracting plasmids by using a plasmid small extraction kit (QIAprep Spin Miniprep Kit, qiagen company, product number is 27106, wherein the plasmid small extraction kit contains a P1 reagent, a P2 reagent and a P3 reagent), and the specific steps are as follows:

(1) centrifuging the culture system at 13000rpm for 2min, collecting bacterial precipitate, and re-suspending the bacterial with P1 reagent;

(2) adding a P2 reagent, slowly reversing and uniformly mixing for 6-8 times;

(3) adding P3 reagent, slowly reversing and mixing for 6-8 times (white precipitate is visible), centrifuging at 13000rpm for 10min, and collecting supernatant;

(4) taking 600 μl of supernatant, adding 800 μl of pre-cooled isopropanol, standing at-20deg.C for 10min, centrifuging at 13000rpm for 15min, and collecting precipitate;

(5) re-suspending the precipitate with 500 μl of precooled 70% ethanol aqueous solution, centrifuging at 13000rpm for 5min, collecting the precipitate, drying the alcohol completely, and preheating with ddH at 65deg.C ₂ O dissolves the precipitate, centrifugate for 5min at 13000rpm, suck the supernatant, namely the solution of recombinant Bacmid, abbreviated as Bacmid solution.

3. Preparation and amplification of recombinant viruses

(1) And (3) taking the Sf9 cells with good growth, adding the Sf9 cells into a10 cm culture dish, standing for 10min, adhering the cells to the wall, and observing under a microscope to ensure that about 70% -80% of the bottom of the culture dish is covered by the cells.

(2) Cellfectin II Reagent 15 μl was taken and diluted with 100 μl insect cell culture medium.

(3) 15-20. Mu.l of Bacmid solution was taken and diluted with 100. Mu.l of insect cell culture medium.

(4) Slowly adding the liquid phase obtained in the step (2) into the liquid phase obtained in the step (3), slowly and uniformly blowing, standing at room temperature for 30min, and diluting to 2ml with insect cell culture medium.

(5) And (3) taking a culture dish after the step (1), discarding the supernatant, slowly and uniformly dripping the liquid phase obtained in the step (4) into the culture dish, standing at 27 ℃ for 5 hours, absorbing and discarding the supernatant, adding 7ml of fresh insect cell culture medium, sealing by a sealing film, standing at 27 ℃ for 8 days, collecting the culture solution, centrifuging for 6 minutes at 600g, taking the supernatant, adding fetal bovine serum, keeping the volume concentration of the supernatant to be 2-5%, and preserving for a long time to obtain the virus solution of the P0 generation recombinant virus.

(6) Taking virus liquid of P0 generation recombinant virus, adding into shake flask according to the volume ratio of 1:1000 to culture cell concentration of 2×10 ⁶ And (3) culturing the cells/mL of Sf9 cell fluid at 27 ℃ for 5 days at 110rpm, collecting culture fluid, centrifuging at 600g for 6min, and taking supernatant, namely the P1 generation recombinant virus liquid, namely the P1 generation virus liquid for short.

4. Expression and purification of proteins

(1) Taking P1 generation virus liquid, adding 1L cell concentration into the solution according to the volume ratio of 1:100 to obtain a solution with the concentration of 2X 10 ⁶ The cells/mL of Sf9 cell fluid were incubated at 27℃and 125rpm for 72 hours, centrifuged at 4000rpm for 15 minutes, and the supernatant was collected.

(2) Filtering the supernatant obtained in the step (1) by using a glass fiber membrane with a double layer of 0.45 mu m, and collecting filtrate.

(3) Concentrating the filtrate obtained in step (2) by tangential flow ultrafiltration system (Masterflex PharMed BPT Tubing system, cole-Parmer, cat# 06508-24), simultaneously adding Buffer 1 for continuous dilution, substituting protein into Buffer 1, centrifuging at 13000rpm for 30min, and collecting supernatant.

(4) Affinity chromatography

(1) Adding Ni-NTA purifying medium into the supernatant obtained in the step (3), incubating for 3 hours at 4 ℃, centrifuging at 400rpm for 5min, and taking a precipitate.

(2) The precipitate obtained in step (1) was washed with 100mL Buffer 1 containing 20mM imidazole to remove the contaminating proteins.

(3) The precipitate obtained in step (2) was washed with 20mL Buffer 1 containing 300mM imidazole and the solution was collected.

(5) Concentrating the solution obtained in the step (4) by using a10 kD concentration tube to obtain a protein concentrate.

(6) And (3) taking the protein concentrate obtained in the step (5), performing molecular sieve chromatography by using a gel filtration column (Superdex 200, GE Healthcare), and eluting by using Buffer 1.

The chromatogram of the elution with Buffer 1 is shown in FIG. 2.

The components B4 to C1 are mixed to obtain SARS-CoV-2RBD monomer solution.

A protein electrophoresis diagram of SARS-CoV-2RBD monomer solution is shown in FIG. 3, R represents a sample after DDT treatment with a reducing agent, and N represents a sample not subjected to treatment.

EXAMPLE 2 binding Activity of SARS-CoV-2RBD dimer to ACE2

Angiotensin converting enzyme 2 (angiotensin I converting enzyme, ACE 2) is a receptor for SARS-CoV-2. ACE2 is shown as a sequence 8 in the sequence table.

1. Preparation of ACE2 solution

The preparation of ACE2 is described in step two of example 1.

The difference is that the DNA molecule encoding SARS-CoV-2RBD is replaced by a DNA molecule encoding ACE 2.

An ACE2 solution was obtained.

2. SPR test

ACE2 was coated on a CM5 chip, and then SARS-CoV-2RBD dimer solution was fed in, and the binding activity of SARS-CoV-2RBD dimer to ACE2 was detected. The results are shown in FIG. 4.

3. Gel filtration experiments

0.5ml of SARS-CoV-2RBD dimer solution (protein concentration: 2 mg/ml) and 0.5ml of ACE2 solution (protein concentration: 2 mg/ml) were mixed, and incubated on ice for 2 hours to obtain a mixed solution (COMPLEX). The SARS-CoV-2RBD dimer solution, ACE2 solution and mixed solution are respectively taken, and a gel filtration column (Superdex 200, GE Healthcare) is respectively adopted for molecular sieve chromatography, and elution is carried out by using Buffer 1.

The superimposed image of the three chromatograms is shown in fig. 5. The peak position of COMPLEX is earlier than the peak positions of SARS-CoV-2RBD dimer and ACE2 alone, which proves that the SARS-CoV-2RBD dimer and ACE2 form a compound with larger molecular weight.

EXAMPLE 3 neutralization Activity of serum after SARS-CoV-2RBD immunization

1. Group immunization

balB/C mice (Vetong Lihua Co.) were divided into 6 groups of 5 animals each, immunized as follows:

a first group: primary immunization on day 1, immunization on day 2, immunization on day 29, immunization on day 3, immunization on day 43, immunization on day 4; the immune mode is intramuscular injection; for primary immunization, the immunization volume of a single mouse is 40 μl, and the immunity is a white emulsion formed by mixing 20 μl of 1mg/ml RBD dimer solution and 20 μl of Freund's complete adjuvant; boosting, wherein the single immunization volume of a single mouse is 40 μl, and the immunity is a white emulsion formed by mixing 20 μl of 1mg/ml RBD dimer solution and 20 μl of Freund's incomplete adjuvant;

second group: primary immunization on day 1, immunization on day 2, immunization on day 29, immunization on day 3, immunization on day 43, immunization on day 4; the immunization mode is nasal drip, the single immunization volume of a single mouse is 11 mul, and the immunity is a mixture of 10 mul of 2mg/ml RBD dimer solution and 1 mul l C/80 solution;

third group: primary immunization on day 1, immunization on day 2, immunization on day 29, immunization on day 3, immunization on day 43, immunization on day 4; the immune mode is intramuscular injection; for primary immunization, the immunization volume of a single mouse is 40 μl, and the immunity is a white emulsion formed by mixing 20 μl of 1mg/ml RBD monomer solution and 20 μl of Freund's complete adjuvant; boosting, wherein the single immunization volume of the single mouse is 40 μl, and the immune substance is a white emulsion formed by mixing 20 μl of 1mg/ml RBD monomer solution and 20 μl of Freund's incomplete adjuvant;

fourth group: primary immunization on day 1, immunization on day 2, immunization on day 29, immunization on day 3, immunization on day 43, immunization on day 4; the immunization mode is nasal drop, the single immunization volume of a single mouse is 11 mul, and the immunity is a mixture of 10 mul of 2mg/ml RBD monomer solution and 1 mul l C/80 solution;

fifth group: primary immunization on day 1, immunization on day 2, immunization on day 29, immunization on day 3, immunization on day 43, immunization on day 4; the immune mode is intramuscular injection; for primary immunization, the immune volume of a single mouse is 40 mu l, and the immune substance is a white emulsion formed by mixing 20 mu l of Buffer 1 and 20 mu l of Freund's complete adjuvant; boosting, wherein the single immunization volume of a single mouse is 40 μl, and the immune substance is a white emulsion formed by mixing 20 μl Buffer 1 and 20 μl Freund's incomplete adjuvant;

sixth group: primary immunization on day 1, immunization on day 2, immunization on day 29, immunization on day 3, immunization on day 43, immunization on day 4; the immunization mode is nasal drop, the single immunization volume of a single mouse is 11 mu l, and the immunity is a mixture of 10 mu l Buffer 1 and 1 mu l C/80 solution;

the SARS-CoV-2RBD dimer solution prepared in example 1 was taken and the concentration was adjusted with PBS buffer to give a protein concentration of 1mg/ml, i.e., 1mg/ml RBD dimer solution. The SARS-CoV-2RBD dimer solution prepared in example 1 was taken and the concentration was adjusted to 2mg/ml with PBS buffer, i.e., 2mg/ml RBD dimer solution. The SARS-CoV-2RBD monomer solution prepared in example 1 was used to adjust the concentration to 1mg/ml with PBS buffer, i.e., 1mg/ml RBD monomer solution. The SARS-CoV-2RBD monomer solution prepared in example 1 was used to adjust the concentration to 2mg/ml with PBS buffer, i.e., 2mg/ml RBD monomer solution. C48/80 (Compound 48/80): sigma, lot C2313. Dissolving C48/80 in water to obtain C48/80 solution with concentration of 10 mg/ml.

Immunization 2, immunization 3, and immunization 4 are collectively referred to as boost.

Blood was taken on day 22, day 36, and day 50, respectively (retroocular vein Cong Caixie).

2. Preparation of SARS-CoV-2 pseudovirus

The plasmid expressing full-length SARS-CoV-2 Spike protein (named SARS-CoV-2 plasmid) and skeleton plasmid pNL4-3R-E-luciferase are co-transfected into 293T cells, and after incubation, SARS-CoV-2 pseudotyped virus with infectivity but no replication ability can be obtained, and the infectivity is similar to that of live virus. Backbone plasmid pNL 4-3R-E-luciferases, i.e.backbone plasmid pNL4-3R-E containing luciferases (i.e. vector with the Luciferase gene containing backbone pNL4-3R-E in the literature): wang Q, liu L, ren W, getlie a, wang H, liang Q, shi X, montefiori DC, zhou T, zhang l.cell rep.2019.

The double-stranded DNA molecule shown in the sequence 9 of the sequence table is inserted between the BamHI and EcoRI cleavage sites of the pcDNA3.1 (+) vector to obtain SARS-CoV-2 plasmid.

The SARS-CoV-2 plasmid and the skeleton plasmid pNL4-3R-E-luciferase are co-transfected into 293T cells, the cells are kept stand and incubated at 37 ℃ for 48 hours (DMEM culture medium containing 10% fetal calf serum is adopted), and cell culture supernatant is collected after transfection, thus obtaining virus liquid containing SARS-CoV-2 pseudovirus (called SARS-CoV-2 virus liquid for short).

3. Antibody neutralization activity assay

Solution to be measured: taking the blood sample obtained in the first step, and separating to obtain serum.

1. And (3) performing multiple ratio dilution on the solution to be tested by adopting a DMEM culture medium containing 10% FBS, and sequentially obtaining the dilutions with different serum concentrations.

2. 100 microliters of the dilution obtained in step 1 was mixed with 50 microliters of SARS-CoV-2 virus solution (virus content 100 TCID 50) prepared in step two, and incubated at 37℃for 1 hour. A blank was set up with 100 μl of DMEM medium containing 10% fbs instead of 100 μl of diluent.

3. After completion of step 2, 50. Mu.l of Huh7 cell broth (approximately 2X 10 in volume) was added ⁴ Huh7 cells), and incubating at 37℃for 48 hours (in practical applications, 48-72 hours may be used).

4. After completion of step 3, 100. Mu.l of PBS buffer and 50. Mu.l of cell lysate (Bright-Glo ^TM Luciferase Assay System, promega, E2650), left for 2min, and then luciferase activity was detected with a chemiluminescent instrument.

3 duplicate wells were set for each treatment and the results averaged.

Neutralization activity= (fluorescence intensity of blank control-fluorescence intensity of experimental group added with diluent)/fluorescence intensity of blank control x 100%.

The corresponding dilution of serum at 50% neutralization activity was position ID50.

The ID50 values are shown in Table 2. The serum of mice immunized with SARS-CoV-2RBD dimer has stronger neutralization effect than that of mice immunized with SARS-CoV-2RBD monomer, and can inhibit SARS-CoV-2 infection susceptible cells.

TABLE 2

	Day 22 serum	Day 36 serum	Day	50 serum
					First group of	＜45	＜45	16756
Second group of	＜45	＜45	1049
				Third group of	＜45	＜45	6450
Fourth group	＜45	＜45	1390
				Fifth group of	＜45	＜45	＜45
Sixth group of	＜45	＜45	＜45

4. Preparation of SARS-CoV-2RBD protein

1. The double-stranded DNA molecule shown in the sequence 10 of the sequence table is inserted into NheI and HindIII cleavage sites of the pcDNA3.1 (+) vector to obtain a recombinant plasmid. The recombinant plasmid expresses the fusion protein and the signal peptide is excised to form the mature protein. The mature protein consists of the following elements in sequence from the N-terminus to the C-terminus: SARS-CoV-2RBD, strep-tag II tag, FLAG tag.

3. After the step 2 is completed, collecting the supernatant, carrying out affinity purification by using streptactin, and then collecting the purified protein solution.

4. Concentrating and system replacement are carried out on the protein solution obtained in the step 3, and the protein system is replaced by PBS buffer solution with pH of 7.2, so as to obtain SARS-CoV-2RBD protein solution.

5. Vaccine-induced detection of total antibodies

Taking the blood sample obtained in the first step, separating serum, and detecting the total IgG by ELISA. In the detection of total IgG, the SARS-CoV-2RBD protein prepared in the fourth step is used for coating an ELISA plate (100 ng/hole), the ELISA plate is diluted by serum multiple ratio (PBS buffer with pH7.2 for dilution), and the secondary antibody is Anti-mouse IgG HRP.

ED50 value (half maximal effect dilution): a dilution factor that causes 50% of the maximum effect.

ED50 values are shown in Table 3.

TABLE 3 Table 3

	Day 22 serum	Day 36 serum	Day	50 serum
					First group of	＜300	882.7	227703.6
Second group of	＜300	＜300	50294.6
				Third group of	＜300	1228.95	80034.8
Fourth group	＜300	＜300	46554.6
				Fifth group of	＜300	＜300	＜300
Sixth group of	＜300	＜300	＜300

SEQUENCE LISTING

<110> university of Qinghua

<120> SARS-CoV-2 Spike protein receptor binding domain dimer and use thereof

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agcttcgtga tccgcggcga cgaggtgcgc cagatcgccc ccggccagac cggcaagatc 300

gccgactaca actacaagct gcccgacgac ttcaccggct gcgtgatcgc ctggaacagc 360

aacaacctgg acagcaaggt gggcggcaac tacaactacc tgtaccgcct gttccgcaag 420

agcaacctga agcccttcga gcgcgacatc agcaccgaga tctaccaggc cggcagcacc 480

ccctgcaacg gcgtggaggg cttcaactgc tacttccccc tgcagagcta cggcttccag 540

cccaccaacg gcgtgggcta ccagccctac cgcgtggtgg tgctgagctt cgagctgctg 600

cacgcccccg ccaccgtgtg cggccccaag aagagcacca acctggtgaa gaacaagtgc 660

gtgaacttc 669

<210> 3

<211> 272

<212> PRT

<213> Artificial sequence

<400> 3

Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val Phe

1 5 10 15

Val Ser Pro Ser Gln Glu Ile His Ala Arg Phe Arg Arg Gly Ala Arg

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Trp Ser His Pro Gln Phe Glu Lys Asp Tyr Lys Asp Asp Asp Asp Lys

260 265 270

<210> 4

<211> 6246

<212> DNA

<213> Artificial sequence

<400> 4

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

atggccacca tgctgcgcgg actgtgctgc gtgctgctac tgtgcggcgc cgtgttcgtg 960

agccccagcc aggagatcca cgcccgattc aggagaggag ccagaggacg cgtgcagccc 1020

accgagagca tcgtgcgctt ccccaacatc accaacctgt gccccttcgg cgaggtgttc 1080

aacgccaccc gcttcgccag cgtgtacgcc tggaaccgca agcgcatcag caactgcgtg 1140

gccgactaca gcgtgctgta caacagcgcc agcttcagca ccttcaagtg ctacggcgtg 1200

agccccacca agctgaacga cctgtgcttc accaacgtgt acgccgacag cttcgtgatc 1260

cgcggcgacg aggtgcgcca gatcgccccc ggccagaccg gcaagatcgc cgactacaac 1320

tacaagctgc ccgacgactt caccggctgc gtgatcgcct ggaacagcaa caacctggac 1380

agcaaggtgg gcggcaacta caactacctg taccgcctgt tccgcaagag caacctgaag 1440

cccttcgagc gcgacatcag caccgagatc taccaggccg gcagcacccc ctgcaacggc 1500

gtggagggct tcaactgcta cttccccctg cagagctacg gcttccagcc caccaacggc 1560

gtgggctacc agccctaccg cgtggtggtg ctgagcttcg agctgctgca cgcccccgcc 1620

accgtgtgcg gccccaagaa gagcaccaac ctggtgaaga acaagtgcgt gaacttctgg 1680

agccaccccc agttcgagaa ggactacaag gacgacgacg acaagtaaaa gcttggtacc 1740

gagctcggat ccactagtcc agtgtggtgg aattctgcag atatccagca cagtggcggc 1800

cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt 1860

gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 1920

ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 1980

ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 2040

ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 2100

ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 2160

cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 2220

cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 2280

tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 2340

gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 2400

cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 2460

attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 2520

tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa 2580

gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 2640

caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 2700

ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 2760

ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 2820

cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 2880

ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg 2940

aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt 3000

ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt 3060

gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc 3120

cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc 3180

ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga 3240

agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat 3300

ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca 3360

agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga 3420

tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc 3480

gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat 3540

catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga 3600

ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg 3660

ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt 3720

ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa 3780

gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg 3840

ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 3900

ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc 3960

aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 4020

tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg 4080

gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 4140

aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 4200

acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 4260

cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 4320

tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 4380

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 4440

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 4500

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 4560

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 4620

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4680

ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4740

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4800

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 4860

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 4920

ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 4980

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tttttttgtt 5040

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 5100

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 5160

tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 5220

agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 5280

tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 5340

acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 5400

tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 5460

ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 5520

agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 5580

tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 5640

acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 5700

agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 5760

actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 5820

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 5880

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 5940

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 6000

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 6060

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 6120

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 6180

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 6240

gacgtc 6246

<210> 5

<211> 669

<212> DNA

<213> Artificial sequence

<400> 5

agagttcagc cgacggagag catagtgagg ttccccaaca ttactaatct ctgtccattt 60

ggcgaagtgt tcaacgcgac gagattcgcc agtgtttatg cgtggaaccg caaacgcatt 120

tcaaattgtg tggccgatta ctccgtcctt tacaactccg cttccttctc aacatttaaa 180

tgttatggcg tttccccaac aaagctgaac gacttgtgct tcactaatgt ttatgctgat 240

agttttgtga tccgtggaga tgaggtacgt caaatagctc caggtcaaac cggtaagatt 300

gcggattata actataaatt gcctgatgac ttcacaggct gtgtgatagc ctggaatagc 360

aacaacctcg atagtaaggt tggaggtaat tataactatt tgtataggct tttcagaaag 420

tccaacttga aaccatttga gcgtgacatc tctacggaaa tataccaagc aggtagcact 480

ccttgtaatg gtgtcgaggg atttaattgc tatttcccac tccagagtta tggattccaa 540

cccactaacg gagtgggtta tcagccctac cgcgtagtag tgctgtcttt cgagctgttg 600

cacgctcccg ctacagtgtg cggtccaaaa aaaagtacga acctggttaa gaacaagtgc 660

gtcaatttc 669

<210> 6

<211> 267

<212> PRT

<213> Artificial sequence

<400> 6

Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr

1 5 10 15

Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala

20 25 30

Ala His Ser Ala Phe Ala Arg Val Gln Pro Thr Glu Ser Ile Val Arg

35 40 45

Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala

50 55 60

Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn

65 70 75 80

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

85 90 95

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

100 105 110

Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg

115 120 125

Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys

130 135 140

Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn

145 150 155 160

Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe

165 170 175

Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile

180 185 190

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

195 200 205

Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly

210 215 220

Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala

225 230 235 240

Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn

245 250 255

Lys Cys Val Asn Phe His His His His His His

260 265

<210> 7

<211> 804

<212> DNA

<213> Artificial sequence

<400> 7

atgctactag taaatcagtc acaccaaggc ttcaataagg aacacacaag caagatggta 60

agcgctattg ttttatatgt gcttttggcg gcggcggcgc attctgcctt tgcgagagtt 120

cagccgacgg agagcatagt gaggttcccc aacattacta atctctgtcc atttggcgaa 180

gtgttcaacg cgacgagatt cgccagtgtt tatgcgtgga accgcaaacg catttcaaat 240

tgtgtggccg attactccgt cctttacaac tccgcttcct tctcaacatt taaatgttat 300

ggcgtttccc caacaaagct gaacgacttg tgcttcacta atgtttatgc tgatagtttt 360

gtgatccgtg gagatgaggt acgtcaaata gctccaggtc aaaccggtaa gattgcggat 420

tataactata aattgcctga tgacttcaca ggctgtgtga tagcctggaa tagcaacaac 480

ctcgatagta aggttggagg taattataac tatttgtata ggcttttcag aaagtccaac 540

ttgaaaccat ttgagcgtga catctctacg gaaatatacc aagcaggtag cactccttgt 600

aatggtgtcg agggatttaa ttgctatttc ccactccaga gttatggatt ccaacccact 660

aacggagtgg gttatcagcc ctaccgcgta gtagtgctgt ctttcgagct gttgcacgct 720

cccgctacag tgtgcggtcc aaaaaaaagt acgaacctgg ttaagaacaa gtgcgtcaat 780

ttccatcatc atcatcatca ctaa 804

<210> 8

<211> 597

<212> PRT

<213> Homo sapiens

<400> 8

Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn

305 310 315 320

Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala Asp

595

<210> 9

<211> 3822

<212> DNA

<213> SARS-CoV-2

<400> 9

atgttcgtgt tcctggtgct gctgcctctg gtgagcagcc agtgcgtgaa tctgaccacc 60

agaacccagc tgcctcctgc ctacaccaat agcttcacca gaggagttta ttatcccgat 120

aaggtgttca gaagtagtgt attacatagt acccaggacc tgttcctacc tttcttcagt 180

aacgtgacct ggttccacgc catccacgtg agcggcacca atggcaccaa gagattcgac 240

aatcctgtgc tgcctttcaa tgacggcgtg tacttcgcca gcaccgagaa gagcaatatc 300

atcagaggct ggatcttcgg caccaccttg gattccaaga ctcagagcct gctgattgta 360

aacaacgcta caaatgtggt gatcaaggtg tgcgagttcc agttctgcaa tgaccctttc 420

ctgggtgttt attatcataa gaacaacaag agctggatgg agagcgagtt ccgcgtatat 480

tcgtcggcta ataattgcac cttcgagtac gtgagccagc ctttcctgat ggacctggag 540

ggcaagcagg gcaatttcaa gaatctgaga gagttcgtgt tcaagaatat cgacggctac 600

ttcaagatct acagcaagca cacacccatt aatctggtga gagacctgcc tcagggcttc 660

agcgccctgg agcctctggt ggacctgcct atcggcatca atatcaccag attccagacc 720

ctgctggccc tgcacagatc atatcttaca ccaggcgatt cgtcaagcgg ttggaccgct 780

ggagctgcgg catattacgt gggctacctg cagcctagaa ccttcctgct gaagtacaat 840

gagaatggta cgataaccga cgcagttgat tgtgccctgg accctctgag cgagaccaag 900

tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa tttcagagtg 960

cagcctaccg agagcatcgt gagattccct aatatcacca atctgtgccc tttcggcgag 1020

gtgttcaatg ccaccagatt cgccagcgtg tacgcatgga accgcaagcg gataagcaat 1080

tgcgtggccg actacagcgt gctgtacaat agcgccagct tcagcacctt caaatgttat 1140

ggtgtttcgc caacaaagct gaatgacctg tgcttcacca atgtgtacgc cgacagcttc 1200

gtgatcagag gcgacgaggt gagacagatc gcgccagggc agaccggcaa gatcgccgac 1260

tacaattaca agctgcctga cgacttcacc ggctgcgtga tcgcgtggaa ctctaacaat 1320

ctagattcga aagttggagg caattacaat tacctgtaca gactgttcag aaagagcaat 1380

ctgaagcctt tcgagagaga catcagcacc gagatctacc aggccggcag cacaccgtgt 1440

aatggcgtgg agggcttcaa ttgctacttc cctctgcaga gctacggctt ccagcctacc 1500

aatggcgtgg gctaccagcc ttacagagtg gtggtgctga gcttcgagct gctgcacgct 1560

cccgctaccg tgtgcggccc taagaagagc accaatctgg tgaagaataa gtgcgtgaat 1620

ttcaatttca atggtctaac tggaacgggc gtgctgaccg agagcaataa gaagtttctt 1680

ccctttcaac aattcggcag agacatcgcc gacaccacag atgctgtaag agaccctcag 1740

accctggaga tcctggacat cactccgtgt agcttcggcg gcgtgagcgt gatcacaccg 1800

ggtaccaata ccagcaatca ggtggccgtg ctgtaccagg acgtgaattg caccgaggtg 1860

cctgtggcca tccacgccga ccagctgact cccacttgga gggtatattc cacgggaagc 1920

aatgtgttcc agaccagagc cggctgcctg atcggcgccg agcacgtgaa taatagctac 1980

gagtgcgaca tccctatcgg cgccggcatc tgcgccagct accagaccca gaccaatagc 2040

cctagaagag ccagaagcgt ggccagccag agcatcatcg cctacaccat gagcctgggc 2100

gccgagaata gcgtggccta cagcaataat agcatcgcca tccctaccaa tttcaccatc 2160

agcgtgacca ccgaaatatt accagtctcc atgaccaaga ccagcgtgga ctgcaccatg 2220

tacatctgcg gcgacagcac cgagtgcagc aatctgctgc tgcagtacgg cagcttctgc 2280

acccagctga atagagccct gaccggcatc gccgtggagc aggacaagaa tacccaggag 2340

gtgttcgccc aggtgaagca gatctacaag actccgccga tcaaggactt cggcggcttc 2400

aatttcagcc aaatactccc agatccaagc aagcctagca agaggagctt catcgaggac 2460

ctgctgttca ataaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520

ctaggtgata ttgcggcaag agacctgatc tgcgcccaga agtttaacgg tttgacagta 2580

ctacctcctc tgctgaccga cgagatgata gcacaatata cgtcggcatt gctcgctggc 2640

acgatcacat cgggctggac tttcggcgcc ggagcagcgt tgcaaatccc tttcgccatg 2700

cagatggcct acagattcaa tggcatcggc gtgacccaga atgtgctgta cgagaatcag 2760

aagctgatcg ccaatcagtt caatagcgcc atcggcaaga tccaggacag cctgagcagc 2820

accgccagcg ccctgggcaa gctgcaggac gtggtgaatc agaatgccca ggccctgaat 2880

accctggtga agcagctgag cagcaatttc ggcgccatca gtagtgtact caacgatatc 2940

ctgagcagac tggacaaggt ggaggccgag gtgcaaattg atcgtcttat tactggcaga 3000

ctgcagagcc tgcagaccta cgtgacccag cagctgatca gagccgccga gatcagagcc 3060

agcgccaatc tggccgccac caagatgagc gagtgcgtgc tgggccagag caagagagtg 3120

gacttctgcg gcaagggcta ccacctgatg agcttccctc agagcgctcc acatggcgtg 3180

gtgttcctgc acgtgaccta cgtgcctgcc caggagaaga atttcaccac cgcacccgca 3240

atctgccacg acggcaaggc ccacttccct agagagggcg tgttcgtgag caatggcacc 3300

cactggttcg tgacccagag aaatttctac gagcctcaga tcatcaccac cgacaatacc 3360

ttcgtgagcg gcaattgcga cgtggtgatc gggatagtca ataatactgt ctacgaccct 3420

ctgcagcctg agctggacag cttcaaggag gagctggaca agtacttcaa gaatcacacc 3480

agccctgacg tggacctcgg tgatatttcg ggaatcaatg ccagcgtggt gaatatccag 3540

aaggaaattg atcggctcaa cgaagtggcc aagaatctga atgagagcct gatcgacctg 3600

caggagctgg gcaagtacga gcagtacatc aagtggcctt ggtacatctg gctgggcttc 3660

atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gacctcctgt 3720

tgttcctgtt tgaaagggtg ttgttcgtgt gggtcctgct gcaagttcga cgaggacgac 3780

agcgagcctg tgctgaaggg cgtgaagctg cactacacct ag 3822

<210> 10

<211> 825

<212> DNA

<213> Artificial sequence

<400> 10

gccaccatgc tgcgcggact gtgctgcgtg ctgctactgt gcggcgccgt gttcgtgagc 60

cccagccagg agatccacgc ccgattcagg agaggagcca gaggacgcgt gcagcccacc 120

gagagcatcg tgcgcttccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 180

gccacccgct tcgccagcgt gtacgcctgg aaccgcaagc gcatcagcaa ctgcgtggcc 240

gactacagcg tgctgtacaa cagcgccagc ttcagcacct tcaagtgcta cggcgtgagc 300

cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatccgc 360

ggcgacgagg tgcgccagat cgcccccggc cagaccggca agatcgccga ctacaactac 420

aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggacagc 480

aaggtgggcg gcaactacaa ctacctgtac cgcctgttcc gcaagagcaa cctgaagccc 540

ttcgagcgcg acatcagcac cgagatctac caggccggca gcaccccctg caacggcgtg 600

gagggcttca actgctactt ccccctgcag agctacggct tccagcccac caacggcgtg 660

ggctaccagc cctaccgcgt ggtggtgctg agcttcgagc tgctgcacgc ccccgccacc 720

gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttctggagc 780

cacccccagt tcgagaagga ctacaaggac gacgacgaca agtaa 825

Claims

Application of a dimer of SARS-CoV-2RBD or a nucleic acid molecule encoding the dimer of SARS-CoV-2RBD or a recombinant plasmid having a DNA molecule encoding the dimer of SARS-CoV-2RBD or a kit for preparing the dimer of SARS-CoV-2RBD in preparing a novel coronavirus vaccine;

the SARS-CoV-2RBD is (a 1), (a 2), (a 3), (a 4), (a 5) or (a 6) as follows:

(a1) A protein shown in a sequence 1 of a sequence table;

(a2) A protein obtained by ligating a signal peptide to the N-terminus of (a 1);

(a3) A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of (a 1);

(a4) A fusion protein obtained by ligating a tag to the C-terminal of (a 2);

(a5) A protein shown in a sequence 3 of a sequence table;

(a6) Protein shown as 34 th-272 th amino acid residues in sequence 3 of a sequence table;

the kit for preparing the dimer of SARS-CoV-2RBD comprises a recombinant plasmid and mammalian cells, wherein the recombinant plasmid comprises a DNA molecule encoding the dimer of SARS-CoV-2 RBD.
Use of a dimer of SARS-CoV-2RBD or a nucleic acid molecule encoding a dimer of SARS-CoV-2RBD or a recombinant plasmid having a DNA molecule encoding a dimer of SARS-CoV-2RBD or a kit for preparing a dimer of SARS-CoV-2RBD in the preparation of a medicament for preventing new coronaries;

the SARS-CoV-2RBD is (a 1), (a 2), (a 3), (a 4), (a 5) or (a 6) as follows:

(a1) A protein shown in a sequence 1 of a sequence table;

(a2) A protein obtained by ligating a signal peptide to the N-terminus of (a 1);

(a3) A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of (a 1);

(a4) A fusion protein obtained by ligating a tag to the C-terminal of (a 2);

(a5) A protein shown in a sequence 3 of a sequence table;

(a6) Protein shown as 34 th-272 th amino acid residues in sequence 3 of a sequence table;

the kit for preparing the dimer of SARS-CoV-2RBD comprises a recombinant plasmid and mammalian cells, wherein the recombinant plasmid comprises a DNA molecule encoding the dimer of SARS-CoV-2 RBD.