CN113583096A

CN113583096A - SARS-CoV-2 Spike protein receptor binding domain dimer and its application

Info

Publication number: CN113583096A
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: 2021-11-02
Anticipated expiration: 2040-05-01
Also published as: CN113583096B

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-2RBD dimer) or SARS-CoV-2RBD monomer (SARS-CoV-2RBD monomer) and application thereof in neutralizing SARS-CoV-2, wherein the SARS-CoV-2RBD dimer has more obvious activity. The present invention provides a dimer of SARS-CoV-2 RBD; protein shown in a sequence 1 in a sequence table; protein shown in a sequence 3 in a sequence table; protein shown as amino acid residues from 34 th to 272 th in a sequence 3 of a sequence table. The invention has important value and wide application prospect for developing medicaments, vaccines and the like for treating and preventing SARS-CoV-2.

Description

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

Technical Field

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

Background

The Disease caused by the novel Coronavirus (SARS-CoV-2) is named as new coronary pneumonia (Coronavir Disease 2019, COVID-19).

The novel coronaviruses are manifested by asymptomatic carriage, acute respiratory distress syndrome (ARD) and pneumonia. This disease is of high concern worldwide because it can be transmitted to humans.

The gene sequence alignment shows that SARS-CoV-2 belongs to beta Coronavirus, and has high similarity in gene composition and structure function with other two kinds of high-pathogenicity Coronavirus, which are respectively Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

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

Disclosure of Invention

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

The present invention provides a dimer of SARS-CoV-2 RBD.

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

SARS-CoV-2RBD is (a1), (a2), (a3), (a4), (a5) or (a 6):

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

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

(a3) and (a1) wherein a tag is attached to the N-terminus or/and the C-terminus of the protein.

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

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

(a6) protein shown as amino acid residues from 34 th to 272 th in a sequence 3 of a sequence table.

Illustratively, the labels are shown in table 1. The respective labels and the relationships in table 1 may also be in a relationship of or.

TABLE 1

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 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 (c1) or (c 2)):

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

(c2) a DNA molecule shown as the 910-1728 th nucleotide in the sequence 4 of the sequence table.

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

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

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

The invention also protects a protein which is (b1), (b2), (b3), (b4), (b5) or (b 6):

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

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

(b3) and (b1) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of (b 1).

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

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

(b6) the protein shown as amino acid residues 39-267 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 such 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 (c3) or (c4) as follows:

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

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

Recombinant plasmids having the DNA molecules also belong to the scope of protection of the present invention. The recombinant plasmid can be specifically 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 cell adopted by the Bac-to-Bac system is Sf9 cell. The starting carrier adopted by the Bac-to-Bac system is a pFastBac1 carrier.

The invention also protects the application of any nucleic acid molecule of SARS-CoV-2RBD dimer or SARS-CoV-2RBD monomer or any recombinant plasmid of any recombinant plasmid or any kit of any recombinant plasmid of any nucleic acid molecule in the preparation of products; the application of the product is (e1) or (e 2):

(e1) as a novel coronavirus vaccine;

(e2) can be used as a medicine for preventing and/or treating new coronary pneumonia.

The invention also protects a product, the active component 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 application of the product is (e1) or (e 2):

(e1) as a novel coronavirus vaccine;

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

Drawings

FIG. 1 is a chromatogram and an electrophoretogram in step one of example 1.

FIG. 2 is a chromatogram of step two of example 1.

FIG. 3 is an electrophoretogram in step two of example 1.

FIG. 4 shows the results of the SPR experiment in example 2.

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

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. The SARS-CoV-2 Spike protein receptor binding domain is expressed as SARS-CoV-2 RBD. Insect cell culture medium (Insect-XpressTM Media): lonza Wokingham Ltd., cat # 12-730F. Buffer 1: pH7.2 containing 150mM NaCl, 10mM HEPA buffer.

EXAMPLE 1 preparation of SARS-CoV-2RBD

Preparation of 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, the 910-position 1728 nucleotide constitutes the fusion gene.

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

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

2. The recombinant plasmid obtained in step 1 is transfected into 293T cells growing to 90% density by PEI transfection reagent, and is firstly cultured for 6-8 hours in serum-free DMEM medium, and then is cultured for 72 hours in DMEM medium containing 10% fetal calf serum.

3. After completion of step 2, centrifugation was carried out at 4000rpm for 5min and the supernatant was collected.

4. And (4) filtering the supernatant obtained in the step (3) by using a 0.22 mu m filter membrane, and collecting the filtrate.

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

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

7. Taking all the supernatant obtained in the step 6, carrying out co-incubation with Streptactin magnetic beads, then washing with Buffer 1, then eluting with 10ml of Buffer2, collecting the eluent, and then concentrating by using a10 kD concentration tube to obtain a concentrated solution with the volume of 1.2 ml.

Buffer2(ph 8.0): containing 100mM Tris, 150mM NaCl, 1mM EDTA, 5mM Desthiobiotin and the balance of water.

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

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

The protein electrophoresis pattern of the A9 fraction is shown in the right panel of FIG. 1, where R represents the sample after treatment with the reducing agent DDT and N represents the sample without treatment.

Mixing the components A7-A10 to obtain the SARS-CoV-2RBD dimer solution.

Preparation of monomer

1. Construction of recombinant plasmids

Inserting a double-stranded DNA molecule shown in a sequence 7 of a sequence table between BamH I and HindIII enzyme cutting sites of a pFastBac1 vector to obtain a recombinant plasmid.

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

In the sequence 6 of the sequence table, the 1 st to 38 th amino acid residues form gp67 signal peptide, the 39 th to 261 th amino acid residues form SARS-CoV-2RBD, and the 262 nd and 267 th amino acid residues form His₆And (4) a label.

The gp67 signal peptide is cleaved off, releasing the mature protein. The mature protein is shown as amino acid residues 39-267 in a sequence 6 of a sequence table. The expected molecular weight of the mature protein is 25.9 kD.

2. Preparation of recombinant Bacmid

(1) Adding the recombinant plasmid prepared in the step 1 into a just-thawed escherichia coli DH10 Bac competent cell, and placing for 30min on ice; then thermally shocking for 75s at 42 ℃, and putting back on ice for 2 min; then adding 500 mul LB liquid culture medium, reviving for 5h at 37 ℃; then 10. mu.l of the suspension was applied to 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 cultured in the dark for three days until a clear blue-white spot was formed.

(2) White single colonies were picked, 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) Extracting plasmids from the culture system obtained in the step (2) by using a plasmid Miniprep Kit (QIAprep Spin Miniprep Kit, Qiagen, Inc., having a product number of 27106, wherein the plasmid contains a P1 reagent, a P2 reagent and a P3 reagent), and sequentially carrying out the following steps:

firstly, centrifuging the culture system at 13000rpm for 2min, collecting thalli precipitates, and resuspending thalli by using a P1 reagent;

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

③ adding a P3 reagent, slowly reversing and uniformly mixing for 6-8 times (white precipitates can be seen), centrifuging at 13000rpm for 10min, and taking supernatant;

fourthly, taking 600 mul of supernatant, adding 800 mul of precooled isopropanol, standing for 10min at minus 20 ℃, then centrifuging for 15min at 13000rpm, and collecting the precipitate;

resuspending the precipitate with 500 μ l precooled 70% ethanol aqueous solution, centrifuging at 13000rpm for 5min, collecting the precipitate, drying the ethanol completely, and then using ddH preheated at 65 DEG C₂Dissolving the precipitate with O, centrifuging at 13000rpm for 5min, and sucking the supernatant, namely the solution of the recombinant Bacmid, which is called Bacmid solution for short.

3. Preparation and amplification of recombinant viruses

(1) And (3) adding the well-grown 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) Cellffectin II Reagent 15. mu.l was taken and diluted with 100. mu.l of 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 an insect cell culture medium.

(5) And (3) taking the culture dish which finishes the step (1), discarding the supernatant, slowly and uniformly dripping the liquid phase obtained in the step (4) into the culture dish, standing and culturing at 27 ℃ for 5 hours, then sucking away the supernatant, adding 7ml of fresh insect cell culture medium, sealing by using a sealing film, standing and culturing at 27 ℃ for 8 days, collecting the culture solution, centrifuging for 6min at 600g, taking the supernatant, adding fetal calf serum to ensure that the volume concentration of the fetal calf serum is 2-5%, and storing for a long time to obtain the virus solution of the P0 generation recombinant virus.

(6) Adding virus liquid of P0 generation recombinant virus into shake flask culture at a cell concentration of 2 × 10 according to a volume ratio of 1:1000⁶Culturing each cell/mL of Sf9 cell liquid at 27 deg.C and 110rpm for 5 days, collecting culture solution, centrifuging for 6min at 600g, and collecting supernatant to obtain virus liquid of P1 generation recombinant virus, P1 generation virus liquid for short.

4. Expression and purification of proteins

(1) Taking P1-substituted virus solution, adding 1L of 2 × 10 cells at a volume ratio of 1:100⁶cells/mL of Sf9 cell solution were cultured at 27 ℃ for 72 hours at 125rpm, centrifuged at 4000rpm for 15min, and the supernatant was collected.

(2) And (2) carrying out suction filtration on the supernatant obtained in the step (1) by using a double-layer 0.45-micron glass fiber membrane, and collecting filtrate.

(3) The filtrate obtained in step (2) was concentrated by a tangential flow ultrafiltration system (Masterflex PharMed BPT Tubig system, Cole-Parmer Co., Ltd., product number 06508-24) while adding Buffer 1 to displace the protein into Buffer 1, followed by centrifugation at 13000rpm for 30min and collecting the supernatant.

(4) Affinity chromatography

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

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

③ washing the precipitate obtained in the step (c) with 20mL of Buffer 1 containing 300mM imidazole, and collecting the solution.

(5) And (3) concentrating the solution obtained in the step (4) by using a10 kD concentration pipe to obtain a protein concentrated solution.

(6) And (3) taking the protein concentrated solution obtained in the step (5), performing molecular sieve chromatography by using a gel filtration column (Superdex200, GE Healthcare), and eluting by using Buffer 1.

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

Mixing the components B4-C1 to obtain the SARS-CoV-2RBD monomer solution.

The protein electrophoresis pattern of the SARS-CoV-2RBD monomer solution is shown in FIG. 3, wherein R represents the sample after treatment with the reducing agent DDT and N represents the sample without treatment.

Example 2 binding Activity of SARS-CoV-2RBD dimer with ACE2

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

Preparation of ACE2 solution

The preparation of ACE2 was carried out as described in example 1, step two.

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.

Second, SPR test

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

Third, gel filtration experiment

0.5ml of SARS-CoV-2RBD dimer solution (protein concentration: 2mg/ml) and 0.5ml of ACE2 solution (protein concentration: 2mg/ml) were mixed and incubated on ice for 2 hours to give a mixed solution (COMPLEX). Respectively taking the SARS-CoV-2RBD dimer solution, the ACE2 solution and the mixed solution, respectively performing molecular sieve chromatography by using a gel filtration column (Superdex200, GE Healthcare), and eluting by using Buffer 1.

The overlay of the three chromatograms is shown in FIG. 5. COMPLEX showed a peak position earlier than that of SARS-CoV-2RBD dimer and ACE2 alone, and it was confirmed that SARS-CoV-2RBD dimer and ACE2 form a COMPLEX with a larger molecular weight.

Example 3 neutralizing Activity of serum after SARS-CoV-2RBD immunization

One, group immunization

balB/C mice (Wintonlihua) were divided into 6 groups of 5 mice each, immunized separately as follows:

a first group: primary immunization on day 1, 2 immunization on day 15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is intramuscular injection; for the initial immunization, the immunization volume of a single mouse is 40 mul, and the immune substance is ' 20 mul of white emulsion formed by mixing 1mg/ml RBD dimer solution with 20 mul of Freund's complete adjuvant '; the boosting immunization, the volume of the single immunization of a single mouse is 40 mul, and the immune matter is '20 mul of white emulsion formed by mixing 1mg/ml RBD dimer solution with 20 mul of Freund incomplete adjuvant';

second group: primary immunization on day 1, 2 immunization on day 15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is nasal drop, the single immunization volume of a single mouse is 11 mu l, and the immune substance is a uniform mixture of 10 mu l of 2mg/ml RBD dimer solution and 1 mu l C48/80 solution;

third group: primary immunization on day 1, 2 immunization on day 15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is intramuscular injection; for the initial immunization, the immunization volume of a single mouse is 40 mul, and the immune substance is ' 20 mul of white emulsion formed by mixing 1mg/ml RBD monomer solution with 20 mul of Freund's complete adjuvant '; the boosting immunization, the single immunization volume of a single mouse is 40 mul, and the immune matter is '20 mul of white emulsion formed by mixing 1mg/ml RBD monomer solution with 20 mul of Freund incomplete adjuvant';

and a fourth group: primary immunization on day 1, 2 immunization on day 15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is nasal drop, the single immunization volume of a single mouse is 11 mu l, and the immune substance is a uniform mixture of 10 mu l of 2mg/ml RBD monomer solution and 1 mu l C48/80 solution;

and a fifth group: primary immunization on day 1, 2 immunization on day 15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is intramuscular injection; for the initial immunization, the immunization volume of a single mouse is 40 mul, and the immune substance is ' 20 mul Buffer 1 and 20 mul Freund's complete adjuvant are mixed to form white emulsion '; the boosting immunization, the volume of a single immunization of a single mouse is 40 mul, and the immune substance is ' 20 mul Buffer 1 mixed with 20 mul Freund's incomplete adjuvant to form white emulsion ';

a sixth group: primary immunization on day 1, 2 immunization on day 15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is nasal drop, the single immunization volume of a single mouse is 11 mu l, and the immune substance is a mixture of 10 mu l Buffer 1 and 1 mu l C48/80 solution;

the SARS-CoV-2RBD dimer solution prepared in example 1 was concentrated with PBS buffer to a protein concentration of 1mg/ml, i.e., a 1mg/ml RBD dimer solution. The SARS-CoV-2RBD dimer solution prepared in example 1 was concentrated with PBS buffer to a protein concentration of 2mg/ml, that is, a 2mg/ml RBD dimer solution. The SARS-CoV-2RBD monomer solution prepared in example 1 was taken and adjusted to a protein concentration of 1mg/ml in PBS buffer, to give a 1mg/ml RBD monomer solution. The SARS-CoV-2RBD monomer solution prepared in example 1 was taken and adjusted to a protein concentration of 2mg/ml with PBS buffer, to give a 2mg/ml RBD monomer solution. C48/80(Compound 48/80): sigma Co, lot C2313. The C48/80 is taken and dissolved by water to make the concentration of the C48/80 to be 10mg/ml, namely the C48/80 solution.

The 2 nd, 3 rd and 4 th immunizations are collectively referred to as booster immunizations.

Blood was collected on day 22, day 36 and day 50 (retrobulbar venous plexus blood collection).

Preparation of SARS-CoV-2 pseudovirus

The plasmid expressing the full-length SARS-CoV-2 Spike protein (named as SARS-CoV-2 plasmid) and the skeleton plasmid pNL4-3R-E-luciferase transfect 293T cells together, after incubation, SARS-CoV-2 pseudotyped virus with infectivity but without replication capacity can be obtained, and the infectivity is similar to that of live virus. Backbone plasmid pNL4-3R-E-Luciferase, i.e.a backbone plasmid pNL4-3R-E containing Luciferase (i.e.vector with the Luciferase conjugation backbone pNL4-3R-E in the literature): wang Q, Liu L, Ren W, Gettie a, Wang H, Liang Q, Shi X, Montefiori DC, Zhou T, Zhang l.cell rep.2019.

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

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

Thirdly, detecting the neutralizing activity of the antibody

Solution to be tested: and (4) taking the blood sample obtained in the step one, and separating to obtain serum.

1. And (3) adopting a DMEM medium containing 10% FBS to dilute the solution to be detected in a multiple ratio manner, and sequentially obtaining diluents 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 TCID50) prepared in step two, and the mixture was incubated at 37 ℃ for 1 hour. A blank control was set up with 100 μ l DMEM medium containing 10% FBS instead of 100 μ l of diluent.

3. After completion of step 2, 50. mu.l of cell fluid of Huh7 cells (about 2X 10 cells) was added⁴Huh7 cells), and standing and incubating for 48 hours at 37 ℃ (in practical application, 48-72 hours can be used).

4. After completion of step 3, 100. mu.l of PBS buffer and 50. mu.l of cell lysate (Bright-Glo) were added^TMLuciferase Assay System, Promega, E2650), left for 2min, and then Luciferase activity was detected using a chemiluminescence apparatus.

Each treatment was set up with 3 replicates and the results averaged.

Neutralization activity ═ (fluorescence intensity of blank-fluorescence intensity of experimental group to which diluent was added)/fluorescence intensity of blank × 100%.

The serum dilution at 50% neutralization activity corresponds to position ID 50.

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

TABLE 2

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

Preparation of SARS-CoV-2RBD protein

1. Inserting the double-stranded DNA molecule shown in the sequence 10 of the sequence table into the NheI and HindIII enzyme cutting sites of the pcDNA3.1(+) vector to obtain the recombinant plasmid. The recombinant plasmid expresses the fusion protein, and the signal peptide is cut off to form mature protein. The mature protein sequentially consists of the following elements from N end to C end: SARS-CoV-2RBD, Strep-tag II tag, FLAG tag.

3. After step 2 is completed, the supernatant is collected, affinity purification is performed using streptavidin, and then the purified protein solution is collected.

4. And (3) concentrating the protein solution obtained in the step (3) and replacing the system, wherein the protein system is replaced by PBS (phosphate buffer solution) with pH7.2 to obtain the SARS-CoV-2RBD protein solution.

Fifth, detection of vaccine-induced Total antibodies

And (4) taking the blood sample obtained in the step one, separating serum, and detecting the total IgG by adopting ELISA. In the total IgG detection, an ELISA plate (100 ng/hole) is coated with SARS-CoV-2RBD protein prepared in the fourth step, the serum is diluted in a fold ratio (the solvent for dilution is PBS buffer solution with pH7.2), and the secondary antibody is Anti-mouse IgG HRP.

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

The ED50 values are shown in Table 3.

TABLE 3

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

SEQUENCE LISTING

<110> Qinghua university

<120> SARS-CoV-2 Spike protein receptor binding domain dimer and its application

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<170> PatentIn version 3.5

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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

Dimers of SARS-CoV-2 RBD;

SARS-CoV-2RBD is (a1), (a2), (a3), (a4), (a5) or (a 6):

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

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

(a3) and (a1) wherein a tag is attached to the N-terminus or/and the C-terminus of the protein.

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

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

(a6) protein shown as amino acid residues from 34 th to 272 th in a sequence 3 of a sequence table.
2. A nucleic acid molecule encoding the dimer of claim 1.
3. A recombinant plasmid having the nucleic acid molecule of claim 2; the nucleic acid molecule is a DNA molecule.
4. A method of making a dimer of SARS-CoV-2RBD according to claim 1, comprising the steps of: mammalian cells are transfected with the recombinant plasmid of claim 3 and then cultured.
5. A kit for preparing the dimer of SARS-CoV-2RBD of claim 1, comprising the recombinant plasmid of claim 3 and mammalian cells.
6. The protein is (b1), (b2), (b3), (b4), (b5) or (b 6):

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

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

(b3) and (b1) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of (b 1).

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

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

(b6) the protein shown as amino acid residues 39-267 in the sequence 6 of the sequence table.
7. A nucleic acid molecule encoding the protein of claim 6.
8. A method for producing the protein of claim 6, comprising the steps of: the protein was prepared using the Bac-to-Bac system.
9. Use of a dimer of SARS-CoV-2RBD according to claim 1 or a protein according to claim 6 or a nucleic acid molecule according to claim 2 or a recombinant plasmid according to claim 3 or a kit according to claim 5 or a nucleic acid molecule according to claim 7 for the preparation of a product; the application of the product is (e1) or (e 2):

(e1) as a novel coronavirus vaccine;

(e2) can be used as a medicine for preventing and/or treating new coronary pneumonia.
10. A product, the active ingredient of which is the dimer of SARS-CoV-2RBD according to claim 1 or the protein according to claim 6 or the nucleic acid molecule according to claim 2 or the recombinant plasmid according to claim 3 or the kit according to claim 5 or the nucleic acid molecule according to claim 7;

the application of the product is (e1) or (e 2):

(e1) as a novel coronavirus vaccine;

(e2) can be used as a medicine for preventing and/or treating new coronary pneumonia.