CN113943373B

CN113943373B - Beta coronavirus polymer antigen, preparation method and application thereof

Info

Publication number: CN113943373B
Application number: CN202011165603.9A
Authority: CN
Inventors: 高福; 戴连攀; 徐坤; 韩雨旋
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2022-09-06
Anticipated expiration: 2040-10-27
Also published as: WO2022089471A1; CN115838433A; CN113943373A

Abstract

The invention relates to a beta coronavirus polymer antigen, a preparation method and application thereof, wherein the amino acid sequence of the beta coronavirus polymer antigen comprises the following components: a plurality of partial amino acid sequences or all amino acid sequences of a receptor binding region of a spike protein of a beta coronavirus, which are connected in series directly or by connecting amino acid sequences, wherein the partial amino acid sequences or all amino acid sequences of the receptor binding region of the spike proteins connected in series are from the same beta coronavirus, and the plurality is an integer of more than or equal to 3. The novel coronavirus RBD polymer can be stably expressed, can strongly induce immune reaction after a mouse is immunized, and has a significant difference with a novel coronavirus RBD dimer in the titer of neutralizing antibodies generated by the mouse induced by the novel coronavirus RBD polymer.

Description

Beta coronavirus polymer antigen, preparation method and application thereof

The background art comprises the following steps:

the family coronaviridae contains 4 genera of coronavirus, which are α, β, γ, and δ, respectively. Severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and novel coronavirus (2019-nCoV, which is hereinafter also referred to as SARS-CoV-2) belong to the genus beta coronavirus. They are both positive-stranded RNA envelope viruses that can infect a wide range of humans and animals. The new coronavirus enters cells through human angiotensin converting enzyme 2(human angiotensin-converting enzyme 2, hACE2), hACE2 receptor is distributed in arteriovenous endothelial cells, arterial smooth muscle cells, intestinal epithelial cells and respiratory system organs such as alveoli and bronchi, and the virus can infect the cells containing hACE2 receptor.

For the treatment of diseases, there are three levels of prevention, one level of prevention, which is a measure taken for the cause of disease before the disease occurs, and a fundamental measure for preventing, controlling and eliminating the disease; secondary prevention is the action taken to prevent or slow the onset of disease during the incubation period, and tertiary prevention: measures to reduce the risk of disease during the clinical phase of disease. Wherein: the primary prevention is the highest-level prevention and is a fundamental measure capable of eliminating diseases, and the beta coronavirus vaccine belongs to the primary prevention, so that the development of the beta coronavirus vaccine is very important.

The envelope of the beta coronavirus is mainly composed of 3 glycoproteins: s protein (Spike protein, S), E protein (Envelope protein, E) protein, and M protein (Membrane protein, M). The S protein is closely related to the process of invading cells by coronavirus, is an important antigen in vaccine development, and can generate neutralizing antibodies. Among them, the Receptor Binding Domain (RBD) of the S protein is the most important antigen target region for the body to induce the production of neutralizing antibodies.

Disclosure of Invention

Object of the Invention

The invention aims to provide a beta coronavirus multimeric antigen, and a preparation method and application thereof. We have previously found that stable expression of a single-chain dimeric RBD protein formed by tandem binding of two RBDs of the S protein of a beta coronavirus, that the dimeric RBD protein is more immunogenic than the monomeric RBD protein and induces higher antibody levels, is a general strategy for subunit vaccine design in beta coronaviruses (PMID: 32645327). The invention focuses on the RBD region of the beta coronavirus S protein as a vaccine antigen and constructs an RBD polymer antigen so as to obtain a better immune effect. The novel coronavirus RBD polymer can be stably expressed, and can strongly induce immune response after being immunized into mice to generate high neutralizing antibodies. And the titer of the neutralizing antibody of the new coronavirus RBD polymer generated by the induced mice of the new coronavirus RBD polymer is remarkably different from that of the new coronavirus RBD dimer, so that the immunogenicity of the new coronavirus RBD polymer is improved relative to that of the RBD dimer, and the new coronavirus RBD polymer is a good candidate vaccine.

A multimeric beta-coronavirus antigen whose amino acid sequence comprises: a plurality of partial amino acid sequences or all amino acid sequences of the receptor binding regions of the spike proteins of the beta coronavirus, which are connected in series directly or by connecting the amino acid sequences in series, wherein the partial amino acid sequences or all amino acid sequences of the receptor binding regions of the spike proteins of the beta coronavirus are derived from the same beta coronavirus, and the plurality is an integer of more than or equal to 3.

In one possible implementation of the above-described multimeric beta-coronavirus antigen, part of the amino acid sequence or the entire amino acid sequence of the receptor binding region of the tandem spike protein is derived from the same beta-coronavirus: part of or all of the amino acid sequences of the receptor binding regions of the tandem spike proteins are from a severe respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a 2019 novel coronavirus.

In one possible implementation, the plurality is 3 or 4.

In one possible implementation, part of the amino acid sequence or all of the amino acid sequences of the receptor binding regions of the spike proteins of the tandem beta coronaviruses are identical sequences. I.e., the sequences in the series are identical to each other and are multiple repeated sequences.

In one possible implementation of the above-mentioned multimeric beta-coronavirus antigen, the linking amino acid sequences at different positions are independently selected from the following sequences: (GGS) n connecting sequences, wherein n represents the number of GGS, and n is an integer more than or equal to 1; optionally, n is an integer selected from 1-10; further optionally, n is an integer selected from 1-5. GGS three letters represent amino acids G, G, S, respectively. The different positions of the connecting amino acid sequence are independent of each other, and the following means that: the linking amino acid sequence linking the first and second tandem sequences from the N-terminus can be different from the linking amino acid sequence linking the second and third tandem sequences from the N-terminus, and so on.

In one possible implementation of the above-described multimeric antigen of a beta coronavirus, the partial amino acid sequence of the receptor binding region of the spike protein of the beta coronavirus is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% of the entire amino acid sequence of the receptor binding region of the spike protein of the beta coronavirus.

In one possible implementation of the multimeric antigen of the β -coronavirus, when the β -coronavirus is a 2019 novel coronavirus, a part of or the entire amino acid sequence of the receptor binding region of the spike protein of the β -coronavirus is selected from any one of the following amino acid sequences:

(1) 319-537 region from the receptor binding region of the 2019-nCoV spike protein;

(2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence in (1), wherein the encoded protein of the amino acid sequence has the same or basically the same immunogenicity as the encoded protein of (1).

Wherein: the 319-537 region of the receptor binding region of the 2019-nCoV spike protein is derived from the R319-K537 region of the WH01 strain spike protein sequence of 2019-nCoV (GenBank: QHR63250 at NCBI).

In one possible implementation of the above-described multimeric beta-coronavirus antigen, when the beta-coronavirus is a 2019 novel coronavirus, the amino acid sequence of the beta-coronavirus antigen includes any one selected from the following amino acid sequences:

the 319-537 region of the receptor binding region of 3 directly connected in series from 2019-nCoV spike protein has the sequence shown in SEQ ID NO.1, namely R319-K537-R319-K537-R319-K537;

the 319-537 region of the receptor binding region of the 2019-nCoV spike protein is directly connected in series, and the sequence is shown as SEQ ID NO.2, namely R319-K537-R319-K537-R319-K537.

The invention also provides a method for preparing the beta coronavirus multimeric antigen, which comprises the following steps: adding a sequence for coding a signal peptide at the 5 'end of the nucleotide sequence for coding the beta coronavirus antigen, adding a sequence for coding a histidine tag and a stop codon at the 3' end, carrying out cloning expression, screening a correct recombinant, then transfecting cells of an expression system for expression, collecting cell supernatant after expression, and purifying to obtain the beta coronavirus antigen.

In one possible implementation of the above method, the cell of the expression system comprises a cell that is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell, optionally; the mammalian cells include 293T cells or CHO cells, and the bacterial cells include E.coli cells.

The invention also provides a polynucleotide for coding the beta coronavirus multimeric antigen, a recombinant vector comprising the polynucleotide and an expression system cell comprising the recombinant vector.

The invention also provides an application of the beta coronavirus multimeric antigen, the polynucleotide for coding the beta coronavirus multimeric antigen, the recombinant vector comprising the polynucleotide or the expression system cell comprising the recombinant vector in preparing the beta coronavirus vaccine.

The invention also provides a beta coronavirus vaccine, which comprises the beta coronavirus multimeric antigen and an adjuvant.

In one possible implementation of the above-mentioned beta coronavirus vaccine, the adjuvant is selected from the group consisting of an aluminum adjuvant, an MF59 adjuvant, an MF 59-like adjuvant, or an AddaVax ^TM An adjuvant.

The invention also provides a beta coronavirus DNA vaccine, which comprises: a recombinant vector comprising a DNA sequence encoding a multimeric antigen of the above-mentioned beta coronavirus.

The invention also provides a beta coronavirus mRNA vaccine, which comprises: a recombinant vector comprising an mRNA sequence encoding the above-described beta coronavirus multimeric antigen.

The invention also provides a beta coronavirus viral vector vaccine, which comprises: a recombinant viral vector comprising a nucleotide sequence encoding the beta coronavirus multimeric antigen described above; optionally, the viral vector is selected from one or more of the following: adenoviral vectors, poxvirus vectors, influenza viral vectors, adeno-associated viral vectors, Vesicular Stomatitis viral vectors (VSV).

Description of the drawings:

FIG. 1 is a Western Blot of the novel coronavirus RBD monomer (nCoV-RBD-monomer), dimer (nCoV-RBD-dimer), trimer (nCoV-RBD-trimer) and tetramer (nCoV-RBD-tetramer) in example 1 of the present invention.

FIG. 2 shows the molecular sieve analysis and gel electrophoresis analysis of nCoV-RBD-trimer neo-corona RBD trimer protein in example 2 of the present invention.

FIG. 3 shows the molecular sieve analysis and gel electrophoresis analysis of nCoV-RBD-tetramer neo-corona RBD tetramer protein in example 2 of the present invention.

FIG. 4 shows the affinity assay of hACE2 and nCoV-RBD monomers in example 3 of the present invention, with 400 abscissa time (S) and high-to-low ordinate responses for samples (different concentrations of hACE2 protein) of 800nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.156nM, 0.78nM, respectively.

FIG. 5 shows the affinity assay of hACE2 and nCoV-RBD-trimers in example 3 of the present invention, with a 400 abscissa time (S) value and high to low ordinate response values, corresponding to 800nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.156nM, 0.78nM, respectively, of the hACE2 protein at different concentrations.

FIG. 6 shows the affinity assay of hACE2 and nCoV-RBD tetramer in example 3 of the present invention, with a 400 abscissa time (S) value and high to low ordinate response values, corresponding to 800nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.156nM, 0.78nM samples (different concentrations of hACE2 protein).

FIG. 7 is a flowchart of the immunization of a mouse in example 4 of the present invention.

FIG. 8 is a graph showing the results of neutralizing antibody titers against the novel coronavirus pseudovirus induced after day 35 in example 5 of the present invention.

Detailed Description

In previous studies, we found that subunit vaccines prepared based on single-chain dimeric RBD proteins of the novel coronaviruses are more immunogenic than monomeric RBD protein vaccines and induce better antibody levels (PMID: 32645327). In order to further improve the effect of the vaccine, more RBD proteins are connected in series, and whether the obtained proteins can induce stronger immune response than the dimer RBD proteins can induce after animals are immunized is detected and used for vaccine development.

Example 1: design of novel coronavirus RBD homotrimers and tetramers

Three or four new coronavirus RBD proteins are connected in series by design to obtain new coronavirus RBD tripolymers and RBD tetramers, and expression purification and immunogenicity detection are attempted.

The construction of trimers and tetramers we designed was: (1) three new coronavirus RBD sequences (319-537) are connected in series (SEQ ID NO.1), the N end is connected with a signal peptide (MIHSVFLLMFLLTPTES), the C end is added with 6 histidines (HHHHHHH) and a stop codon to obtain a construction named nCoV-RBD-polymer; (2) four novel coronavirus RBD sequences (319-537) are connected in series (SEQ ID NO.2), the N end is connected with a signal peptide (MIHSVFLLMFLLTPTES), the C end is added with 6 histidines (HHHHHH) and a stop codon, and the obtained construction is named nCoV-RBD-tetramer.

We also prepared the construction of monomers and dimers as controls, respectively, as follows: (1) the construction obtained by connecting a new coronavirus RBD sequence (319-541) (SEQ ID NO.3) with a signal peptide (MFVFLVLLPLVSSQC) at the N terminal and adding 6 histidines (HHHHHH) and a stop codon at the C terminal is named nCoV-RBD-monomer; (2) two new coronavirus RBD sequences (319-537) are connected in series (SEQ ID NO.4), the N end is connected with a signal peptide (MIHSVFLLMFLLTPTES), the C end is added with 6 histidines (HHHHHH) and a stop codon, and the obtained construction is named nCoV-RBD-dimer.

Optimizing an open reading frame (comprising a signal peptide, a His tag and a stop codon) for coding nCoV-RBD-trimer according to a human source codon to obtain a DNA sequence (SEQ ID NO.5), optimizing an open reading frame (comprising the signal peptide, the His tag and the stop codon) for coding nCoV-RBD-tetramer according to the human source codon to obtain a DNA sequence (SEQ ID NO.6), optimizing an open reading frame (comprising the signal peptide, the His tag and the stop codon) for coding nCoV-RBD-monomer according to the human source codon to obtain a DNA sequence (SEQ ID NO.7), optimizing an open reading frame (comprising the signal peptide, the His tag and the stop codon) for coding nCoV-RBD-dimer according to the human source codon to obtain a DNA sequence (SEQ ID NO.8), wherein the upstream of ORF of the coding gene comprises a Kozak sequence gccgcacc, and then synthesizing and cloning the four genes into a pCAGGS vector, plasmids pCAGGS-nCoV-RBD-trimer, pCAGGS-nCoV-RBD-tetramer expressing trimer RBD and tetramer RBD, and plasmids pCAGGS-nCoV-monomer, pCAGGS-nCoV-dimer expressing monomer RBD and dimer RBD were obtained.

HEK293T cells were transfected with the four plasmids pCAGGS-nCoV-trimer, pCAGGS-nCoV-tetramer, pCAGGS-nCoV-dimer, pCAGGS-nCoV-monomer and pCAGGS-nCoV-dimer, and after 72 hours, cell supernatants were collected and the expression of the target protein was detected by Western blotting (Western Blot), and as a result, the cells stably expressed the neocoronavirus RBD trimer, RBD tetramer, RBD monomer and RBD dimer protein, as shown in FIG. 1.

Example 2: nCoV-RBD-trimer and nCoV-RBD-tetramer expression purification

HEK293T cells were used to express nCoV-RBD-trimers and nCoV-RBD-tetramers. Plasmids pCAGGS-nCoV-trimer and pCAGGS-nCoV-tetramer were transfected into HEK293T cells, respectively, and after 72 hours, the supernatants were collected, centrifuged to remove the precipitate and filtered through a 0.22 μm filter to further remove impurities. The cell supernatant was adsorbed by a nickel affinity column (Histrap, GE Healthcare) at 4 ℃. Non-specific binding proteins were removed by washing with buffer A (20mM Tris,150mM NaCl, pH 8.0). The protein of interest is then eluted from Histrap with buffer B (20mM Tris,150mM NaCl, pH 8.0,300mM imidazole) and the eluate is concentrated by more than 30 fold using a 30kD concentration tube to buffer A with a final volume of less than 1 ml. Then passes through Superdex ^TM The 200 Increate 10/300GL column (GE Healthcare) was subjected to molecular sieve chromatography to further purify the protein of interest. The molecular sieve chromatography buffer solution is PBS buffer solution (8mM Na) ₂ HPO4,136mM NaCl,2mM KH ₂ PO ₄ 2.6mM KCl, pH 7.2). After molecular sieve chromatography, the nCoV-RBD-trimer has an elution peak at about 13-14ml (figure 2), and SDS-PAGE analysis shows that the protein is about 75KD (figure 2) under non-reduction (without DTT) and reduction (with DTT) conditions, and is in the size of trimer. The nCoV-RBD-tetramer had an elution peak at about 12-13ml (FIG. 3), and SDS-PAGE analysis showed thatThe protein was around 100kD under both non-reducing (without DTT) and reducing (with DTT) conditions (FIG. 3), and was tetrameric in size.

The monomer and dimer expression purification was also performed as described above.

Example 3: interaction affinity experiment of RBD trimer protein and RBD tetramer protein of novel coronavirus and hACE2 protein

To examine the affinity of the RBD trimer protein and the RBD tetramer protein of the novel coronavirus to the hACE2 protein, we examined the binding affinity thereof by a surface plasmon resonance experiment, and the affinity of the RBD monomer protein of the novel coronavirus and the hACE2 protein was used as a control.

The protein used in the experiment was changed to (10mM Na) by concentration and centrifugation ₂ HPO ₄ ；2mM KH ₂ PO ₄ pH 7.4; 137mM NaCl; 2.7mM KCl; 0.005% (v/v) Tween-20), the instrument used in this experiment was BIAcore 3000, CM5 chip (GE Healthcare), the monomeric, trimeric and tetrameric proteins of the new coronavirus RBD were immobilized on the chip with 1000-fold response values, hACE2 protein was used as mobile phase, the mobile phase protein was diluted at 800nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.156nM, 0.78nM fold ratio, and the mobile phase proteins were sequentially flowed over the chip with different concentrations of mobile phase protein, and the real-time response values were recorded. The data are processed by BIAevaluation Version 4.1(GE Healthcare) software, and the affinity of the RBD monomer, trimer and tetramer proteins of the new coronavirus to the hACE2 protein is calculated.

The results of the surface plasmon resonance experiments for detecting the binding affinity are shown in FIGS. 4, 5 and 6. Binding affinity of the new coronavirus RBD monomer and hACE2 was 2.83 + -0.32 nM (FIG. 4), that of nCoV-RBD-trimer and hACE2 was 4.31 + -0.97 nM (FIG. 5), and that of nCoV-RBD-tetramer and hACE2 was 7.37 + -0.96 nM (FIG. 6). The affinities of the nCoV-RBD-trimer and the nCoV-RBD-tetramer to hACE2 are equivalent to those of the new coronavirus RBD monomer and hACE2, which indicates that the nCoV-RBD-trimer and the nCoV-RBD-tetramer can well expose Receptor Binding Motifs (RBMs).

Example 4: experiment of mice immunized by RBD trimer protein and RBD tetramer protein of new coronavirus

To further test the immunogenicity of the vaccine, we immunized BALB/c mice with purified RBD trimer, RBD tetramer, RBD monomer, RBD dimer protein. The BALB/c mice used were purchased from Witongli, Inc., and were all female, 7 weeks old. Groups of mice (6 per group) and vaccine doses are shown in table 1, and the immunization scheme is shown in fig. 7. The immunization groups of the mouse immunization experiment set up used the RBD trimer protein and the RBD tetramer protein (obtained in example 2) of the novel coronavirus as immunogens, and the control groups were the monomers of nCoV-RBD, the RBD dimer protein and PBS, respectively, as negative controls.

TABLE 1 grouping and dosing of coronavirus RBD homotrimer and tetramer vaccine immunized mice

The immunogen was diluted to 0.2mg/ml with PBS, AddaVax ^TM The adjuvant and the immunogen are mixed and emulsified according to the volume ratio of 1:1 to prepare the vaccine. The mixed vaccine was used to immunize BALB/c mice, 6 in each group. Mouse experimental protocol As shown in FIG. 7, all mice were immunized first and second on days 0 and 21, respectively, with 100. mu.l each of the antigen adjuvant mixture (in which 50. mu.l of immunogen + 50. mu.l of adjuvant were mixed) injected intramuscularly. Orbital bleeds were performed on day 35 and serum was collected by centrifugation and stored at-80 ℃ in a freezer before use to detect pseudovirus neutralizing antibody titers.

Example 5: neutralization assay to detect the level of neutralizing antibodies raised after immunization with vaccines against the novel coronavirus

The preparation of the novel coronavirus pseudovirus is described in the paper assessment of a pseudo viral infection for SARS-CoV-2(PMID:32207377), which is the same as the method reported in the paper.

Neutralization test method:

(1) one day before the experiment, Huh7 cells in the logarithmic growth phase were harvested by trypsinization, counted, re-seeded in 96-well plates, and a microneutralization experiment was performed when the cell density reached 80-100% at 18-24 hours.

(2) Serum was diluted and the mice serum to be tested was diluted with complete medium (DMEM with 10% FBS) starting with 20-fold serum samples and diluted sequentially with a 2-fold gradient.

(3) Diluting pseudovirus, melting pseudovirus on ice in advance, diluting virus with complete culture medium, mixing virus and serum dilution, and adding pseudovirus 100TCID per well ₅₀ Is placed at 37 ℃ in 5% CO ₂ The cells were incubated for 1 hour. Blank controls containing medium alone and controls containing an equivalent amount of pseudovirus without mouse serum were set separately.

(4) Discarding the cell supernatant of the 96-well plate plated on the previous day, adding the virus and serum mixture, and standing at 37 deg.C with 5% CO ₂ And culturing for 24 hours.

(5) Discarding cell supernatant, adding 50 μ L of cell lysate, lysing for 10min on ice, taking 10 μ L of cell lysate supernatant, adding to a detection plate, using Luciferase Assay Systemm (Promega, E4550), operating according to the instructions provided by the kit, and detecting Luciferase activity value using GloMax 96Microplate Luminometer (Promega).

(6) Data analysis, antibody titer value was defined as the highest dilution of serum with a response value less than 10% of the negative control value, i.e. neutralizing antibody NT 90. The titer of this sample was defined as half the lowest dilution (limit of detection) when the response value was still greater than 10% of the negative control value.

And (4) analyzing results:

the results of the neutralizing antibody titer of the mouse sera against the new coronavirus pseudovirus after the second immunization, as detected by the microneutralization experiment, are shown in fig. 8. nCoV-RBD-trimer and nCoV-RBD-tetramer can induce higher neutralizing antibody against new coronavirus pseudovirus after immunization.

The results of pseudovirus neutralization of mouse sera against the new coronavirus after immunization of trimeric nCoV-RBD-trimer are shown in FIG. 8. Trimer nCoV-RBD-trimer induced a 1:10 approach ⁴ Has a level of neutralizing antibodies against the pseudovirus of the new coronavirus which is obviously higher than that of neutralizing antibodies induced by the dimer nCoV-RBD-dimer (P represents the value of X)<0.01) compared to the level of specific antibodies induced by the monomer nCoV-RBD-monomer (P is represented by X)<0.0001), significantly increased levels of neutralizing antibodies induced compared to PBS control immunised group (indicated by P)<0.0001)。

In addition, the results of pseudovirus neutralization of mouse sera against the new corona virus after immunization of the tetrameric nCoV-RBD-tetramer are shown in fig. 8. The tetramer nCoV-RBD-tetramer induced an induction of about 1:10 ⁴ Compared with the neutralizing antibody level induced by a tripolymer nCoV-RBD-trimer immune group, the neutralizing antibody level of the new coronavirus pseudovirus is not obviously improved (ns represents P)>0.05), more significant level of neutralizing antibodies (P is represented by x) than induced by the dimer nCoV-RBD-dimer<0.001), significantly higher levels of neutralizing antibodies (P is represented by x) than induced by monomer nCoV-RBD-monomer<0.0001), significantly increased levels of neutralizing antibodies induced compared to PBS control immunised group (indicated by P)<0.0001)。

The results show that the new coronavirus RBD trimer and the new coronavirus RBD tetramer can be stably expressed, and after mice are immunized, the immune response can be strongly induced, so that high new coronavirus neutralizing antibodies are generated. And the titer of neutralizing antibodies generated by mice induced by the new coronavirus RBD trimer and the new coronavirus RBD tetramer is remarkably different from that of the new coronavirus RBD dimer, so that the immunogenicity of the new coronavirus RBD trimer and the new coronavirus RBD tetramer is improved relative to that of the RBD dimer, and the new coronavirus RBD trimer and the new coronavirus RBD tetramer are good candidate vaccines. We have previously found that the two RBDs of a coronavirus can be concatenated together to form a single-chain dimeric RBD protein which can be stably expressed, the dimeric RBD protein is more immunogenic than the monomeric RBD protein and can induce higher antibody levels, and this subunit vaccine design strategy is universal in beta coronavirus (PMID:32645327), so we have found here that the neutralizing antibody levels induced by mice immunized with neocoronavirus RBD trimers and tetramers are higher (significantly different) than the dimeric RBD, and this RBD trimer and tetramer strategy is likely to be universal in subunit vaccine design of other beta coronaviruses (such as MERS virus, SARS virus, etc.).

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

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr

210 215 220

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

225 230 235 240

Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875

<210> 3

<211> 223

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210> 4

<211> 438

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr

210 215 220

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

225 230 235 240

Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Asn Leu Val Lys Asn Lys

435

<210> 5

<211> 2043

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

atgatccaca gcgtgtttct actgatgttc ctgcttaccc caaccgaaag ccgtgttcag 60

ccaaccgagt cgatcgtaag gttccctaac attaccaact tatgcccctt cggtgaggtt 120

ttcaacgcca cgagattcgc atccgtgtat gcctggaatc gtaagcgtat ctcaaactgc 180

gttgcggact actccgtgct ctacaatagt gccagcttta gcaccttcaa atgctacggt 240

gtcagcccca cgaagctgaa cgatttatgt tttacaaatg tctatgccga tagctttgtt 300

attcgcggcg atgaggttag acaaatagcg ccaggacaaa ctggaaagat agccgactac 360

aattacaaac ttcccgatga ctttacgggt tgcgtcatag cctggaacag caataacttg 420

gactccaagg ttgggggaaa ttacaattat ctctaccggc tattcagaaa gtcaaatctg 480

aagccgtttg agagagacat cagtacagaa atataccagg ccggtagcac tccatgtaac 540

ggggtggaag ggttcaattg ttacttcccc ctccagagtt atggtttcca acccacgaac 600

ggagtgggct accaacctta cagagtagta gtactgagct tcgagttatt gcatgctccg 660

gcgacagtct gtggcccaaa gaagagcaca aacctggtaa agaacaaaag agttcaaccc 720

actgagagta ttgtaagatt ccccaatatt accaacttgt gtcctttcgg ggaggtattt 780

aatgccacca gatttgcctc tgtgtacgca tggaatcgca aaagaatcag caattgtgtg 840

gccgactata gcgtcctgta taacagcgcc tctttctcaa ccttcaagtg ttacggggta 900

agccccacta agctcaacga tctatgcttc accaatgtct acgccgattc ttttgtgatc 960

cgcggcgatg aagttagaca gatcgcccct gggcaaaccg gaaagatcgc cgactataac 1020

tacaaactgc cggacgactt cactggctgc gttatcgcct ggaactcgaa caatcttgac 1080

agcaaggtgg gaggcaacta caattatctg tatcggctgt tcaggaaatc taacctcaag 1140

cccttcgaaa gagatatctc taccgaaatc tatcaagcgg gtagcacgcc gtgcaatggc 1200

gtcgagggtt ttaactgcta ttttcccctg cagagctacg ggtttcaacc cactaatggt 1260

gtgggatatc agccctaccg cgttgtggtg ttgagcttcg aactgctgca cgcgccagcg 1320

acagtatgcg gtcccaagaa gtccacgaat ttggttaaaa acaagagagt acagcccaca 1380

gagagcatag tgcggttccc caacattacg aacctgtgtc cgttcggcga ggtgttcaac 1440

gccactagat ttgcaagtgt atatgcttgg aaccgcaaga gaatctcgaa ctgcgttgct 1500

gactacagcg tactctataa ctcggcctca ttttcgacat tcaagtgcta cggcgtgagc 1560

cccaccaagc tgaacgacct gtgtttcacc aacgtctacg ctgactcgtt tgtgattaga 1620

ggcgatgaag tgcggcagat cgcacccggg caaacaggca aaatcgcaga ctacaactac 1680

aagttgccag acgacttcac gggctgcgtg atcgcttgga actctaacaa cctggattca 1740

aaggtggggg gcaactataa ttacctgtac cgactgttcc gtaagagcaa cttgaagccc 1800

tttgagaggg acattagcac cgaaatctac caggccggca gcacaccctg taatggcgtc 1860

gaaggtttca attgctactt tcctctccaa agctacggct ttcagcccac caacggggtg 1920

ggctaccagc cttaccgcgt ggtggtgcta tcgttcgagc tgctgcatgc ccccgctacc 1980

gtgtgtgggc ccaagaagag cactaatctg gtgaagaaca aacatcatca ccaccaccac 2040

tga 2043

<210> 6

<211> 2700

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

atgatccaca gcgtgtttct actgatgttc ctcctgaccc ctaccgagtc tagggtacag 60

cccaccgaga gtatcgtgcg ttttccaaac ataaccaacc tgtgcccgtt cggcgaagtg 120

ttcaatgcca cgagatttgc tagcgtgtac gcgtggaata gaaaaagaat ctcaaactgt 180

gttgccgatt acagcgtgct gtacaatagc gcctctttta gcacattcaa atgttacggt 240

gttagcccca ccaaactaaa cgacctgtgc ttcactaacg tgtatgcaga cagcttcgtg 300

atcagaggtg atgaagtaag gcagatagcg cctggacaga ccgggaagat tgccgattac 360

aattataaac tgcccgacga ctttacgggc tgtgtgatcg cgtggaactc caacaacctg 420

gacagcaagg taggcggtaa ctataactac ctatacagac ttttcagaaa gagcaacctt 480

aaaccctttg agagagatat cagcactgag atctatcaag ctggttctac cccctgcaat 540

ggcgtggagg gattcaactg ctatttcccg ttacagtctt acggctttca gccgactaac 600

ggcgtaggct accagcctta cagagtggtc gtgctgagct ttgagctgct gcacgcccca 660

gccaccgtat gcggccccaa aaagagcacg aacctggtta agaacaaacg tgttcagcca 720

accgagtcga tcgtaaggtt ccctaacatt accaacttat gccccttcgg tgaggttttc 780

aacgccacga gattcgcatc cgtgtatgcc tggaatcgta agcgtatctc aaactgcgtt 840

gcggactact ccgtgctcta caatagtgcc agctttagca ccttcaaatg ctacggtgtc 900

agccccacga agctgaacga tttatgtttt accaatgtct atgccgatag ctttgttatt 960

cgcggcgatg aggttagaca aatagcgcca ggacaaactg gaaagatagc cgactacaat 1020

tacaaacttc ccgatgactt tacgggttgc gtcatagcct ggaacagcaa taacttggac 1080

tccaaggttg ggggaaatta caattatctc taccggctat tcagaaagtc aaatctgaag 1140

ccgtttgaga gagacatcag tacagaaata taccaggccg gtagcactcc atgtaacggg 1200

gtggaagggt tcaattgtta cttccccctc cagagttatg gtttccaacc cacgaacgga 1260

gtgggctacc aaccttacag agtagtagta ctgagcttcg agttattgca tgctccggcg 1320

acagtctgtg gcccaaagaa gagcacaaac ctggtaaaga acaaaagagt tcaacccact 1380

gagagtattg taagattccc caatattacc aacttgtgtc ctttcgggga ggtatttaat 1440

gccaccagat ttgcctctgt gtacgcatgg aatcgcaaaa gaatcagcaa ttgtgtggcc 1500

gactatagcg tcctgtataa cagcgcctct ttctcaacct tcaagtgtta cggggtaagc 1560

cccactaagc tcaacgatct atgcttcacc aatgtctatg ccgattcttt tgtgatccgc 1620

ggcgatgaag ttagacagat cgcccctggg caaaccggaa agatcgccga ctataactac 1680

aaactgccgg acgacttcac tggctgcgtt atcgcctgga actcgaacaa tcttgacagc 1740

aaggtgggag gcaactacaa ttatctgtat cggctgttca ggaaatctaa cctcaagccc 1800

ttcgaaagag atatctctac cgaaatctat caagcgggta gcacgccgtg caatggcgtc 1860

gagggtttta actgctattt tcccctgcag agctacgggt ttcaacccac taatggtgtg 1920

ggataccagc cctaccgcgt tgtggtgttg agcttcgaac tgctgcacgc gccagcgaca 1980

gtatgcggtc ccaagaagtc cacgaatttg gttaaaaaca agagagtaca gcccacagag 2040

agcatagtgc ggttccccaa cattacgaac ctgtgtccgt tcggcgaggt gttcaacgcc 2100

actagatttg caagtgtata tgcttggaac cgcaagagaa tctcgaactg cgttgctgac 2160

tacagcgtac tctataactc ggcctcattt tcgacattca agtgctacgg cgtgagcccc 2220

accaagctga acgacctgtg tttcaccaac gtctacgctg actcgtttgt gattagaggc 2280

gatgaagtgc ggcagatcgc acccgggcaa acaggcaaaa tcgcagacta caactacaag 2340

ttgccagacg acttcacggg ctgcgtgatc gcttggaact ctaacaacct ggattcaaag 2400

gtggggggca actataatta cctgtaccga ctgttccgta agagcaactt gaagcccttt 2460

gagagggaca ttagcaccga aatctaccag gccggcagca caccctgtaa tggcgtcgaa 2520

ggtttcaatt gctactttcc tctccaaagc tacggctttc agcccaccaa cggggtgggc 2580

taccagccct accgcgtggt ggtgctatcg ttcgagctgc tgcatgcccc cgctaccgtg 2640

tgtgggccca agaagagcac taatctggtg aagaacaaac atcatcacca ccaccactga 2700

<210> 7

<211> 735

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

atgtttgtgt ttcttgtgct tcttcctctt gtgtcatcac aatgcagagt gcaacctaca 60

gaatcaatcg tgagatttcc taacatcaca aacctttgcc ctttcggcga ggtgtttaac 120

gcaacaagat ttgcatcagt gtacgcatgg aacagaaagc gtatatcaaa ctgcgtggca 180

gattactcag tgctttacaa ctcagcatca ttcagtacgt ttaaatgcta cggagtgtca 240

cctacaaagc taaatgatct ttgctttaca aacgtgtacg cagattcatt tgtgatcaga 300

ggagatgaag tgagacaaat cgcacctgga caaacaggaa agattgccga ttacaactac 360

aaacttcctg atgatttcac cggctgcgtg atcgcatgga actcaaacaa ccttgattca 420

aaggtaggtg gtaattataa ttatttgtat aggctctttc gtaagagcaa cttaaagcca 480

tttgagcgag atatctcaac agaaatctac caagcaggat caacaccttg caacggagtg 540

gaaggattta actgctactt tcctcttcaa tcatacggat ttcaacctac aaacggagtg 600

ggataccaac cttacagagt ggtggtgctt tcatttgaac ttcttcacgc acctgcaaca 660

gtgtgcggac ctaagaagag cacgaacctt gtgaagaata agtgcgtgaa ctttcaccac 720

caccaccacc actga 735

<210> 8

<211> 1386

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

atgatccaca gcgtgtttct actgatgttc ctcctgaccc ctaccgagtc tagagtgcaa 60

cctacagaat caatcgtgag atttcctaac atcacaaacc tttgcccttt cggcgaggtg 120

tttaacgcaa caagatttgc atcagtgtac gcatggaaca gaaagcgtat atcaaactgc 180

gtggcagatt actcagtgct ttacaactca gcatcattca gtacgtttaa atgctacgga 240

gtgtcaccta caaagctaaa tgatctttgc tttacaaacg tgtacgcaga ttcatttgtg 300

atcagaggag atgaagtgag acaaatcgca cctggacaaa caggaaagat tgccgattac 360

aactacaaac ttcctgatga tttcaccggc tgcgtgatcg catggaactc aaacaacctt 420

gattcaaagg taggtggtaa ttataattat ttgtataggc tctttcgtaa gagcaactta 480

aagccatttg agcgagatat ctcaacagaa atctaccaag caggatcaac accttgcaac 540

ggagtggaag gatttaactg ctactttcct cttcaatcat acggatttca acctacaaac 600

ggagtgggat accaacctta cagagtggtg gtgctttcat ttgaacttct tcacgcacct 660

gcaacagtgt gcggacctaa gaagagcacg aaccttgtga agaataagag agtgcaacct 720

acagaatcaa tcgtgagatt tcctaacatc acaaaccttt gccctttcgg cgaggtgttt 780

aacgcaacaa gatttgcatc agtgtacgca tggaacagaa agcgtatatc aaactgcgtg 840

gcagattact cagtgcttta caactcagca tcattcagta cgtttaaatg ctacggagtg 900

tcacctacaa agctaaatga tctttgcttt acaaacgtgt acgcagattc atttgtgatc 960

agaggagatg aagtgagaca aatcgcacct ggacaaacag gaaagattgc cgattacaac 1020

tacaaacttc ctgatgattt caccggctgc gtgatcgcat ggaactcaaa caaccttgat 1080

tcaaaggtag gtggtaatta taattatttg tataggctct ttcgtaagag caacttaaag 1140

ccatttgagc gagatatctc aacagaaatc taccaagcag gatcaacacc ttgcaacgga 1200

gtggaaggat ttaactgcta ctttcctctt caatcatacg gatttcaacc tacaaacgga 1260

gtgggatacc aaccttacag agtggtggtg ctttcatttg aacttcttca cgcacctgca 1320

acagtgtgcg gacctaagaa gagcacgaac cttgtgaaga ataagcatca tcaccaccac 1380

cactga 1386

Claims

1. A multimeric beta-coronavirus antigen, characterized by: the amino acid sequence of the beta coronavirus multimeric antigen is any one of the following amino acid sequences:

3 319-537 regions from the receptor binding region of 2019-nCoV spike protein which are directly connected in series, and the sequence is shown as SEQ ID NO. 1;

the 319-537 region of the receptor binding region of the 2019-nCoV spike protein is directly connected in series with 4, and the sequence is shown as SEQ ID NO. 2.

2. A method of preparing the multimeric beta-coronavirus antigen of claim 1, characterized by: the method comprises the following steps: adding a sequence coding a signal peptide to the 5 'end of the nucleotide sequence coding the beta coronavirus multimeric antigen of claim 1, adding a sequence coding a histidine tag to the 3' end of the nucleotide sequence and a stop codon, performing cloning expression, screening correct recombinants, transfecting cells of an expression system for expression, collecting cell supernatants after expression, and purifying to obtain the beta coronavirus multimeric antigen.

3. The method of claim 2, wherein: the cells of the expression system include mammalian cells, insect cells, yeast cells, or bacterial cells.

4. The method of claim 3, wherein: the mammalian cells include 293T cells or CHO cells, and the bacterial cells include E.coli cells.

5. A polynucleotide encoding the beta coronavirus multimeric antigen of claim 1.

6. A recombinant vector comprising the polynucleotide of claim 5.

7. An expression system cell comprising the recombinant vector of claim 6.

8. Use of the beta coronavirus multimeric antigen of claim 1, the method of any one of claims 2-4, the polynucleotide of claim 5, the recombinant vector of claim 6, or the expression system cell of claim 7 for the preparation of a beta coronavirus vaccine.

9. A beta coronavirus vaccine, characterized by: comprising the beta coronavirus multimeric antigen of claim 1 and an adjuvant.

10. The beta coronavirus vaccine of claim 9, characterized in that: the adjuvant is selected from aluminum adjuvant, MF59 adjuvant, MF 59-like adjuvant or AddaVax ^TM An adjuvant.

11. A beta coronavirus DNA vaccine characterized by: comprises the following steps: a recombinant vector comprising a DNA sequence encoding the beta coronavirus multimeric antigen of claim 1.

12. A beta coronavirus mRNA vaccine, characterized by: comprises the following steps: a recombinant vector comprising an mRNA sequence encoding the beta coronavirus multimeric antigen of claim 1.

13. A beta coronavirus viral vector vaccine characterized by: comprises the following steps: a recombinant viral vector comprising a nucleotide sequence encoding the beta coronavirus multimeric antigen of claim 1.

14. The beta coronavirus viral vector vaccine of claim 13, characterized in that: the viral vector is selected from one or more of the following: adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors, vesicular stomatitis virus vectors.