CN114437185B

CN114437185B - Coronavirus trimer subunit vaccine and application thereof

Info

Publication number: CN114437185B
Application number: CN202111485016.2A
Authority: CN
Inventors: 严景华; 史瑞; 黄庆瑞
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-12-31
Filing date: 2021-12-07
Publication date: 2023-06-27
Anticipated expiration: 2041-12-07
Also published as: CN114437185A

Abstract

Subunit novel coronavirus (SARS-CoV-2), severe acute respiratory syndrome (SARS-CoV) and middle east respiratory syncytial virus (MERS-CoV) vaccines are disclosed. The invention uses mammal cell expression vector to express SARS-CoV-2S protein trimer forming region and SARS-CoV-2 RBD region, SARS-CoV RBD region or MERS-CoV RBD region fusion, forms trimer protein SARS-CoV-2 RBDc-trimer, SARS-CoV RBDc-trimer, MERS-CoV RBDc-trimer, and compared with each RBD monomer protein, trimer protein immune effect is better, and it can make mouse produce higher titer specific antibody. The trimeric RBD protein overcomes the defect of insufficient immunogenicity of RBD protein monomers, and improves the level of specific antibodies generated by mice aiming at RBD regions; this design can be used to increase the immunogenicity of the coronavirus RBD, making it a more effective vaccine.

Description

Coronavirus trimer subunit vaccine and application thereof

Technical Field

The invention relates to a coronavirus trimer subunit vaccine and application thereof, belonging to the technical field of medicines.

Background

Coronaviruses (CoVs) are a class of single-stranded RNA viruses with a membrane envelope, spherical or ellipsoidal, with regularly arranged capsular collagen fiber processes, shaped like a crown, and are known. Coronaviruses belong to the family of coronaviruses of the order reticuloviridae, and fall into two subfamilies, coronaviruses and cycloviroids. Among them, the coronaviridae subfamily has 4 genera of α, β, γ and δ, and the β genus coronavirus can be divided into 4 independent subgroups A, B, C and D. The virus envelope is composed of double-layer lipid, penetrating membrane protein and spike protein, and some kinds of coronaviruses also have hemagglutinin. The virus is internally provided with a nucleoprotein core consisting of RNA and capsid protein and has a spiral structure.

The world health organization formally names "2019 novel coronavirus (2019-nCoV)" on

month

1 and 12 of 2020, and thereafter the international committee for classification of viruses (International Committee on Taxonomy of Viruses, ICTV) announced on month 2 and 11-12 of 2020 that the formal classification of 2019 novel coronavirus (2019-nCoV) is named severe acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus 2, sars-CoV-2), and the World Health Organization (WHO) announces on the national tile for global research and innovation forum on the same day that the formal name of the disease caused by this virus is "covd-19".

Until 2003, coronaviruses known to be able to infect humans and pathogenic are generally milder, such as 229E, OC43 and the like. Since the outbreak of SARS-CoV in 2003, several additional novel pathogenic coronaviruses have been discovered in humans. SARS-CoV, MERS-CoV and newly discovered SARS-CoV-2 can cause severe respiratory disease. Numerous virology professionals and teams have now essentially established that SARS-CoV-2 is a subset of the genus beta with SARS-CoV. By analyzing 7 conserved non-structural protein sequences and the whole genome sequence, the SARS-CoV-2 and the SARS-CoV are found to have genome sequence similarity and extremely high homology with bat-derived coronaviruses. Studies have demonstrated that SARS-CoV-2 and SARS-CoV invade cells by binding to the same receptor, angiotensin converting enzyme 2 (ACE 2), ACE2 is present in the respiratory epithelium, molecularly confirming the symptoms of pneumonia in new coronavirus infected individuals, as well as the possibility of transmission.

To infect a cell, the virus first needs to bind to the host's receptor via the envelope protein. Based on studies on other coronaviruses, especially SARS-CoV and MERS-CoV, an important envelope protein that binds to the receptor is the spike protein (S). S can be further divided into two parts S1 and S2. The role of S2 is to mediate membrane fusion. Both the N-terminus (NTD) and the C-terminus (CTD) of S1 may be Receptor Binding Domains (RBDs). Through studies on SARS-CoV-2, the team found that CTD was the RBD of this coronavirus, binding to the receptor ACE2. Thus, SARS-CoV-2 RBD is used as an immunogen to stimulate the body to produce antibodies specific for SARS-CoV-2 RBD. Subunit vaccines for SARS-CoV-2 RBD may be an effective way to inhibit viral infection. Similarly, subunit vaccines for SARS-CoV RBD and MERS-CoV RBD can be used to prevent infection by the corresponding viruses.

The novel coronavirus (also referred to simply as the novel coronavirus) is a novel virus and there are many uncertainties in vaccine development. At present, inactivated vaccines, nucleic acid vaccines, recombinant protein vaccines, adenovirus vector vaccines and attenuated vaccines of new coronaviruses are mainly developed worldwide. Inactivated vaccine: inactivated vaccines are a classical technical route, i.e. the new coronavirus is cultivated in vitro and then inactivated so that it is non-virulent. The inactivated vaccine has the advantages of simple and quick preparation method and higher safety, but the inactivated vaccine has the defects of large inoculation dosage, short immunization period and most feared defects that antibody dependence enhancement effect (ADE) is caused sometimes, so that virus infection is aggravated. Adenovirus vector vaccine: the adenovirus vector vaccine is prepared by taking modified harmless adenovirus as a vector and filling an S protein gene of a new coronavirus, and has the advantages of safety, high efficiency and less induced adverse reaction, and the adenovirus vector vaccine stimulates a human body to generate antibodies; the disadvantage is that the recombinant viral vector vaccine uses adenovirus type 5 as vector, but most people are infected with adenovirus type 5 in the growth process, antibodies capable of neutralizing the adenovirus vector may exist in the body, and the vaccine effect is reduced. Nucleic acid vaccine: the nucleic acid vaccine includes mRNA vaccine and DNA vaccine, and is prepared through injecting S protein encoding gene, mRNA or DNA directly into human body, synthesizing S protein in human body with human body cell, and stimulating human body to produce antibody. The nucleic acid vaccine has the advantages that no protein or virus is needed to be synthesized during development, the process is simple, and the safety is relatively high; the disadvantage is the lack of success. Attenuated vaccine: the attenuated influenza virus vaccine approved to be marketed is used as a vector to carry S protein of a new coronavirus, and the human body is stimulated to generate antibodies against the two viruses together; since attenuated influenza viruses easily infect the nasal cavity, this vaccine can be vaccinated by just nasal drip. Recombinant protein vaccine: the recombinant subunit vaccine is also called as a genetic engineering recombinant subunit vaccine, which is prepared by a genetic engineering method to produce a large amount of S or RBD proteins which are most likely to be used as antigens of the novel coronavirus, injecting the S or RBD proteins into a human body and stimulating the human body to produce antibodies; the recombinant subunit vaccine has the advantages of safety, high efficiency and large-scale production; the successful genetically engineered subunit vaccine was a hepatitis b surface antigen vaccine. At present, a plurality of new crown vaccines enter a clinical stage and have certain positive clinical results.

Disclosure of Invention

Aiming at coronaviruses, the invention designs coronavirus subunit vaccines in a trimer form according to the natural trimer structure of SARS-CoV-2S, SARS-CoV S and MERS-CoV S proteins and the advantages of subunit vaccines; the trimeric forming region of SARS-CoV-2S protein is fused with RBD region of SARS-CoV-2 protein, RBD region of SARS-CoV protein or RBD region of MERS-CoV to form trimeric proteins SARS-CoV-2 RBDc-trimer, SARS-CoV RBDc-trimer and MERS-CoV RBDc-trimer which can be expressed normally. The trimeric RBD protein overcomes the defect of insufficient immunogenicity of RBD monomers, and improves the specific antibody aiming at the RBD region, which is produced by mice; the design can be used for improving the immunogenicity of the RBD of the coronavirus, so that the trimeric vaccine has excellent immunogenicity and provides a brand new form for the design of the coronavirus vaccine.

Specifically, the present invention provides the following embodiments:

1. a coronavirus antigen, wherein the antigen comprises, in order from the 5 'end to the 3' end:

SARS-CoV-2S protein trimer forming region E918-L966, its amino acid sequence is SEQ ID NO. 1,

a linker fragment comprising 7-25 amino acids (e.g.SEQ ID NO: 2),

SARS-CoV-2S protein trimer forming region D1163-L1203, its amino acid sequence is SEQ ID NO. 3,

the RBD region R319-K537 of SARS-CoV-2S protein has the amino acid sequence of SEQ ID NO. 4;

or alternatively, the first and second heat exchangers may be,

the antigen sequentially comprises the following components from the 5 'end to the 3' end:

a linker fragment comprising 7-25 amino acids (e.g.SEQ ID NO: 2),

the RBD region R306-Q523 of SARS-CoV S protein has the amino acid sequence of SEQ ID NO. 8;

or (b)

a linker fragment comprising 7-25 amino acids (e.g.SEQ ID NO: 2),

RBD region E367-N602 of MERS-CoV S protein, the amino acid sequence of which is SEQ ID NO. 12,

preferably the antigen is a trimeric protein.

2. The antigen as described in item 1, which comprises an amino acid sequence as shown in (1), (2), (3) or (4) below:

(1) An amino acid sequence shown as SEQ ID NO. 6;

(2) An amino acid sequence as shown in SEQ ID NO. 10;

(3) An amino acid sequence as shown in SEQ ID NO. 14;

(4) The polypeptide or analogue thereof in which the amino acid sequence in (1), (2) or (3) is substituted, deleted or added with one or more amino acids without changing antigenicity, and is capable of forming a trimer itself.

3. The antigen of

claim

1 or 2, wherein the antigen further comprises a secretion signal peptide (e.g., SARS-CoV-2S protein self signal peptide having the amino acid sequence of SEQ ID NO:16, MERS-CoV S protein self signal peptide having the amino acid sequence of SEQ ID NO:17 or SARS-CoV S protein self signal peptide having the amino acid sequence of SEQ ID NO: 24) added at the 5 'end, a purification TAG (e.g., a 6XHis TAG) and a stop codon (e.g., TAA, TAG or TGA) added at the 3' end.

4. An isolated polynucleotide encoding the polypeptide of any one of claims 1-3, preferably the isolated polynucleotide comprises a sequence as set forth in SEQ ID No. 21, SEQ ID No. 22 or SEQ ID No. 23.

5. A recombinant vector or expression cassette comprising the isolated polynucleotide of item 4.

6. A transgenic cell line or recombinant bacterium comprising the recombinant vector or expression cassette of item 5, respectively.

7. The transgenic cell line of claim 6, which is a mammalian cell.

8. Use of an antigen according to any one of claims 1-3 in the preparation of an anti-coronavirus drug, such as a vaccine.

9. The use of claim 8, wherein the antigen is combined with an aluminum hydroxide adjuvant.

10. A kit comprising the antigen of any one of items 1-3, the isolated polynucleotide of item 4, or the recombinant vector or expression cassette of item 5, the transgenic cell line or recombinant bacterium of item 6, preferably the kit further comprises aluminum hydroxide.

In the coronavirus trimer vaccine design of the present invention, it is particularly important to design the SARS-CoV-2S protein trimer forming region at the 5' end or 3' end of the above three coronavirus RBD regions, because the inventors were unable to express the complete desired protein when trying to design it at the 5' end.

Drawings

Fig. 1: WB identification of SARS-CoV-2 RBDc-primer, SARS-CoV-2 RBD-monomer;

fig. 2: SARS-CoV-2 RBDc-primer molecular sieve chromatography and SDS-PAGE identification;

fig. 3: SARS-CoV-2 RBD-monomer molecular sieve chromatography and SDS-PAGE identification;

fig. 4: analytical ultracentrifugation determination of SARS-CoV-2 RBDc-primer;

fig. 5: determination of serum antibody titer of mice after SARS-CoV-2 RBDc-primer, SARS-CoV-2 RBD-monomer immunization;

fig. 6: comparing the RBD data of SARS-CoV and MERS-CoV with the RBD data of SARS-CoV-2, wherein a is the combined diagram of the RBD data of SARS-CoV and MERS-CoV and SARS-CoV-2, b is the RBD structure of SARS-CoV-2 (PDB data number: 6 LZG), c is the RBD structure of MERS-CoV (PDB data number: 6 WAR), d is the RBD structure of SARS-CoV (PDB data number: 3 BGF).

Fig. 7: WB identification of SARS-CoV RBDc-primer, SARS-CoV RBD-monomer;

fig. 8: SARS-CoV RBDc-oligomer molecular sieve chromatography and SDS-PAGE identification;

fig. 9: SARS-CoV RBD-monomer molecular sieve chromatography and SDS-PAGE identification;

fig. 10: analytical ultracentrifugation determination of SARS-CoV RBDc-trimer;

fig. 11: determination of serum antibody titer of mice after SARS-CoV RBDc-trimer and SARS-CoV RBD-monomer immunization;

fig. 12: WB identification of MERS-CoV RBDc-primer, MERS-CoV RBD-monomer;

fig. 13: MERS-CoV RBDc-primer molecular sieve chromatography and SDS-PAGE identification;

fig. 14: MERS-CoV RBD-monomer molecular sieve chromatography and SDS-PAGE identification;

fig. 15: analytical ultracentrifugation determination of MERS-CoV RBDc-trimers;

fig. 16: mouse serum antibody titer determination after MERS-CoV RBDc-trimer, MERS-CoV RBD-monomer immunization.

Detailed Description

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

Example 1: construction of SARS-CoV-2 RBDc-primer and SARS-CoV-2 RBD-monomer protein recombinant expression plasmid

Two SARS-CoV-2 RBD recombinant expression fragments were designed:

SARS-CoV-2 RBDc-trimer:5'-sp+hr1+ linker +hr2+rbd+6×his+ stop codon-3';

SARS-CoV-2 RBD-monomer:5 '-SP+RBD+6XHis+stop codon-3'.

Wherein the method comprises the steps of

SP is SARS-CoV-2S protein self signal peptide, amino acid sequence is SEQ ID NO. 16;

HR1 is SARS-CoV-2S protein trimer forming region E918-L966, the amino acid sequence is SEQ ID NO. 1;

the amino acid sequence of the connecting fragment is SEQ ID NO. 2;

HR2 is SARS-CoV-2S protein trimer forming region D1163-L1203, amino acid sequence is SEQ ID NO 3;

RBD is RBD region R319-K573 of SARS-CoV-2S protein, and amino acid sequence is SEQ ID NO. 4.

According to the preference of mammalian cell codons, the nucleic acid sequences encoding the above amino acid sequences are optimized to obtain optimized nucleic acid sequences SEQ ID NO. 20 and SEQ ID NO. 18 respectively, wherein the optimized nucleic acid sequences except for the rest fragments of SP and 6XHis are SEQ ID NO. 21 and SEQ ID NO. 19, and the optimized nucleic acid sequences are connected to a pCAGGS (purchased from Addgene) vector through two restriction sites of EcoRI and XhoI to form a SARS-CoV-2 RBD recombinant expression plasmid.

Example 2: SARS-CoV-2 RBDc-trimer, SARS-CoV-2 RBD-monomer protein test expression and identification

HEK293T cells were cultured in DMEM containing 10% fbs. HEK293T was transfected (in duplicate) with SARS-CoV-2 RBD recombinant expression plasmid harboring the antigen encoding gene obtained in example 1. Cell culture broth was replaced with serum-free DMEM 4-6 hours after transfection, and culture was continued for 3 days, and cell culture supernatants were collected for Western Blot (WB) detection using HRP-labeled Anti-His antibody (purchased from MBL).

Two repeated samples of SARS-CoV-2 RBDc-trimer and SARS-CoV-2 RBD-trimer can be normally expressed by WB detection, and the expression level of SARS-CoV-2 RBDc-trimer is obviously higher than that of SARS-CoV-2 RBD-trimer (shown in figure 1), so that we select SARS-CoV-2 RBDc-trimer construction as a preferred scheme and SARS-CoV-2 RBD-trimer as a control construction to continue the subsequent experiment.

Example 3: SARS-CoV-2 RBDc-trimer and Monomer protein expression, purification and identification

HEK293T cells were cultured in DMEM containing 10% fbs. HEK293T was transfected with the plasmid containing the antigen-encoding gene obtained in example 1. After 4-6 hours of transfection, the cell culture broth was replaced with serum-free DMEM and the culture was continued for 3 days, after which the supernatant was collected, DMEM was further added and the culture was continued for 4 days, and the supernatant was collected again.

The two supernatants were mixed, centrifuged at 5000rpm for 30 min, filtered through a 0.22 μm filter, bound to a HisTrap excel column (5mL,GE Healthcare), and the nonspecifically bound proteins were eluted with 20mM Tris, 150mM NaCl, pH8.0, 20mM imidazole, and the target proteins were eluted with 20mM Tris, 150mM NaCl, pH8.0, 100mM imidazole. The protein of interest was collected and concentrated and then subjected to molecular sieve chromatography Superdex 200 Increate 10/300GL or Superdex 200 Hiload 16/60 (GE Healthcare). The peak of the order was determined by SDS-PAGE (reducing), and the results are shown in FIG. 2 and FIG. 3. Purified SARS-CoV-2 RBDc-primer and SARS-CoV-2 RBD-monomer antigen are obtained. As a result of the ultracentrifugation, the molecular weight of SARS-CoV-2 RBDc-oligomer was found to be 151kDa, which corresponds to the theoretical molecular weight of trimer, as shown in FIG. 4, and it was determined to be trimer.

Example 4: mouse immunity experiment

SARS-CoV-2 RBDc-primer antigen obtained in example 3 was diluted in physiological saline and emulsified in groups with an equal volume of adjuvant according to the method of Table 1 below, followed by group immunization of 6-week-old Balb/c mice (purchased from Vetong rituximab). Immunization strategy was by intramuscular injection of the thigh, with each mouse being vaccinated 1 time on days 0 and 14, respectively, with an inoculation volume of 100 μl each. Mice were bled tail on day 28. The serum of the mice is separated out after standing for a period of time, the serum is obtained by centrifugation at 3000rpm for 10 minutes, and the serum is frozen in a refrigerator at-80 ℃ after being inactivated at 56 ℃ for 30 minutes for ELISA binding detection.

Table 1: animal immune grouping situation

Example 5: serum ELISA detection experiment after mouse immunization

Will lastThe mouse serum prepared in example 4 was subjected to ELISA to determine RBD specific antibody levels. 200ng of the purified SARS-CoV-2 RBD-Monomer protein obtained in example 3 was added to each well of ELISA plate, and each well was then coated with ELISA coating solution (50 mM Na ₂ CO ₃ 、NaHCO ₃ Buffer, ph 9.6) coating overnight at 4 ℃; after the coating solution was removed, 150. Mu.l of 5% nonfat dry milk was added to each well and the mixture was blocked at room temperature for 1 hour. After blocking, ELISA plates were washed 2 times with PBS containing 0.05% Tween 20, and each well was incubated with serum after immunization with antigen or 100. Mu.L of serum after immunization with PBS at room temperature for 1 hour; after discarding the supernatant, the ELISA plate was washed 5 times with PBS containing 0.05% Tween 20; 100 μl of horseradish peroxidase-labeled goat anti-mouse IgG antibody (secondary antibody, purchased from Zhongshan gold bridge) was added to each well at a ratio of 1:3000, and incubated for 1 hour at room temperature; after discarding the secondary antibody, the ELISA plate was washed 5 times with PBS containing 0.05

% Tween

20, 50. Mu.L of ELISA color development solution was added to each well for 15 minutes, and 50. Mu.L of 2M H was added to each well ₂ SO ₄ Terminating the reaction, and reading OD by using an ELISA reader ₄₅₀ A numerical value; the antibody Titer (Titer) was calculated by taking the lg value of the lowest dilution factor higher than the negative control value x 2.1 times of the non-added serogroup, i.e., calculating the serum antibody Titer of each group. The results showed that the antibody titres (T2 group 4.4654, T5 group 4.7914) binding to SARS-CoV-2 RBD antigen were significantly higher in the serum of SARS-CoV-2 RBDc-trimer immunized mice than in SARS-CoV-2 RBD-monomer (M2 group 1.6193, M5 group 3.9383) and PBS control group (1.0000) (FIG. 5), 2 μg group T2 titres were higher than M2 (P)<0.0001 Group T5 titres of 5 μg were also higher than M5 (p=0.0059).

SARS-CoV and MERS-CoV and SARS-CoV-2 belong to the same class of beta coronaviruses, and we compared the structure of RBDs of the three by PDB data (PDB: 3BGF, 6WAR, 6 LZG) and found that they have very high similarity (FIG. 6), so that SARS-CoV RBD and MERS-CoV RBD can also be prepared into trimeric protein vaccines as in SARS-CoV-2 RBD by the method of examples 1-5. The above point is demonstrated experimentally hereinafter.

Example 6: construction of SARS-CoV RBDc-trimer and SARS-CoV RBD-monomer protein recombinant expression plasmid

Two SARS-CoV RBD recombinant expression fragments were designed:

SARS-CoV RBDc-trimer:5'-sp+hr1+ linker +hr2+rbd+6×his+ stop codon-3';

SARS-CoV RBD-monomer:5 '-SP+RBD+6XHis+stop codon-3'.

Wherein the method comprises the steps of

SP is SARS-CoV-2S protein signal peptide, amino acid sequence is SEQ ID NO. 17;

the amino acid sequence of the connecting fragment is SEQ ID NO. 2;

RBD is RBD region R306-Q523 of SARS-CoV S protein, and amino acid sequence is SEQ ID NO. 8.

According to the preference of mammalian cell codon, the nucleic acid sequence of the above amino acid sequence is optimized to obtain SARS-CoV RBDc-primer encoding nucleic acid sequence with SP and 6XHis fragments removed optimally as SEQ ID NO. 22, and then the SARS-CoV-2 RBD recombinant expression plasmid is formed by connecting the nucleic acid sequence to pCAGGS (purchased from Addgene) vector through two restriction sites of EcoRI and XhoI.

Example 7: SARS-CoV RBDc-trimer, SARS-CoV RBD-monomer protein test expression and identification

HEK293T cells were cultured in DMEM containing 10% fbs. HEK293T was transfected with SARS-CoV RBD recombinant expression plasmid harboring the antigen-encoding gene obtained in example 6. Cell culture broth was replaced with serum-free DMEM 4-6 hours after transfection, and culture was continued for 3 days, and cell culture supernatants were collected for Western Blot (WB) detection using HRP-labeled Anti-His antibody (purchased from MBL). Judging whether the sample of SARS-CoV RBDc-oligomer can be expressed normally or not and comparing the expression level of the sample and the sample. We selected the SARS-CoV RBDc-primer construct as the preferred protocol and the SARS-CoV RBD-monomer as the control construct to continue the subsequent experiments.

Detection of SARS-CoV RBDc-trimer and SARS-CoV RBD-monomer by WB the SARS-CoV RBDc-trimer was expressed normally (as shown in FIG. 7), we continued the subsequent experiments with the SARS-CoV RBDc-trimer construction as the preferred protocol and the SARS-CoV RBD-monomer as the control construction.

Example 8: SARS-CoV RBDc-trimer and Monomer protein expression, purification and identification

HEK293T cells were cultured in DMEM containing 10% fbs. HEK293T was transfected with the plasmid containing the antigen-encoding gene obtained in example 6. After 4-6 hours of transfection, the cell culture broth was replaced with serum-free DMEM and the culture was continued for 3 days, after which the supernatant was collected, DMEM was further added and the culture was continued for 4 days, and the supernatant was collected again.

The two supernatants were mixed, centrifuged at 5000rpm for 30 min, filtered through a 0.22 μm filter, bound to a HisTrap excel column (5mL,GE Healthcare), and the nonspecifically bound proteins were eluted with 20mM Tris, 150mM NaCl, pH8.0, 20mM imidazole, and the target proteins were eluted with 20mM Tris, 150mM NaCl, pH8.0, 100mM imidazole. The protein of interest was collected and concentrated and then subjected to molecular sieve chromatography Superdex 200 Increate10/300 GL or Superdex 200 Hiload 16/60 (GE Healthcare). The peak of the order was determined by SDS-PAGE (reducing). Purified SARS-CoV RBDc-trimer and SARS-CoV RBD-monomer antigen were obtained, and the results are shown in FIGS. 8 and 9. The molecular weight of SARS-CoV RBDc-primer was found to be 120kDa by analytical ultracentrifugation, and it was judged to be a trimer when it was consistent with the theoretical molecular weight of trimer (FIG. 10).

Example 9: mouse immunity and serum ELISA detection

A mouse immunization experiment was performed by using the SARS-CoV RBDc-primer and the SARS-CoV RBD-monomer antigen obtained in example 8 in the same manner as in example 4, and the titer of serum bound to SARS-CoV RBD after immunization of a mouse was detected by the same manner as in example 5.

The results show that the antibody titres binding to the SARS-CoV RBD antigen in the serum of the SARS-CoV RBDc-trimer immunized mice (T2 group 3.1277, T5 group 3.5639) are significantly higher than the SARS-CoV RBD-monomer (M2 group 1.7679, M5 group 1.3215) and PBS control group (1.0000) (FIG. 11), the 2 μg group T2 titres are higher than M2 (P=0.0112), and the 5 μg group T5 titres are also higher than M5 (P < 0.0001).

Example 10: MERS-CoV RBDc-primer, MERS-CoV RBD-monomer protein recombinant expression plasmid construction

The following two MERS-CoV RBD recombinant expression fragments were designed:

MERS-CoV RBDc-primer: 5'-sp+hr1+ linker +hr2+rbd+6×his+ stop codon-3';

MERS-CoV RBD-monomer:5 '-SP+RBD+6XHis+stop codon-3'.

Wherein the method comprises the steps of

SP is MERS-CoV S protein self signal peptide, and the amino acid sequence is SEQ ID NO. 17;

the amino acid sequence of the connecting fragment is SEQ ID NO. 2;

RBD is RBD region E367-N602 of MERS-CoV S protein, and amino acid sequence is SEQ ID NO. 12.

According to the preference of mammalian cell codons, optimizing the nucleic acid sequence of the above amino acid sequence to obtain MERS-CoV RBDc-primer encoding nucleic acid sequence with SP and 6XHis fragments removed optimally as SEQ ID NO. 23, and connecting the MERS-CoV RBD encoding nucleic acid sequence to pCAGGS (purchased from Addgene) vector through two restriction sites of EcoRI and XhoI to form the MERS-CoV RBD recombinant expression plasmid.

Example 11: MERS-CoV RBDc-primer, MERS-CoV RBD-monomer protein test expression and identification

HEK293T cells were cultured in DMEM containing 10% fbs. HEK293T was transfected with the MERS-CoV RBD recombinant expression plasmid containing the antigen-encoding gene obtained in example 11. Cell culture broth was replaced with serum-free DMEM 4-6 hours after transfection, and culture was continued for 3 days, and cell culture supernatants were collected for Western Blot (WB) detection using HRP-labeled Anti-His antibody (purchased from MBL). Judging whether the samples of the MERS-CoV RBDc-trimers can be expressed normally or not and comparing the expression levels of the MERS-CoV RBD-trimers and the MERS-CoV RBD-trimers. We selected the MERS-CoV RBDc-primer construct as the preferred protocol and the MERS-CoV RBD-primer construct as the control construct to proceed with the subsequent experiments.

After WB detection of MERS-CoV RBDc-trimers and MERS-CoV RBD-trimers for normal expression (as shown in FIG. 12), we continued the subsequent experiments with MERS-CoV RBDc-trimer construction as the preferred protocol and MERS-CoV RBD-trimers as the control construction.

Example 12: MERS-CoV RBDc-trimer and Monomer protein expression, purification and identification

HEK293T cells were cultured in DMEM containing 10% fbs. HEK293T was transfected with the plasmid containing the antigen-encoding gene obtained in example 11. After 4-6 hours of transfection, the cell culture broth was replaced with serum-free DMEM and the culture was continued for 3 days, after which the supernatant was collected, DMEM was further added and the culture was continued for 4 days, and the supernatant was collected again.

The two supernatants were mixed, centrifuged at 5000rpm for 30 min, filtered through a 0.22 μm filter, bound to a HisTrap excel column (5mL,GE Healthcare), and the nonspecifically bound proteins were eluted with 20mM Tris, 150mM NaCl, pH8.0, 20mM imidazole, and the target proteins were eluted with 20mM Tris, 150mM NaCl, pH8.0, 100mM imidazole. The protein of interest was collected and concentrated and then subjected to molecular sieve chromatography Superdex 200 Increate 10/300GL or Superdex 200 Hiload 16/60 (GE Healthcare). The peak of the order was determined by SDS-PAGE (reducing). Purified MERS-CoV RBDc-trimers and MERS-CoV RBD-monomer antigens were obtained and the results are shown in fig. 13 and 14. The MERS-CoV RBDc-mer was found to have a molecular weight of 133kDa by analytical ultracentrifugation, and was judged to be a trimer when it was consistent with the theoretical molecular weight of the trimer (FIG. 15).

Example 13: mouse immunity and serum ELISA detection

Mouse immunization experiments were performed using MERS-CoV RBDc-primer and MERS-CoV RBD-monomer antigens obtained in example 12 in the same manner as in example 4, and titers of serum binding MERS-CoV RBD after mouse immunization were detected using the same manner as in example 5.

The results showed that the antibody titres binding to MERS-CoV RBD antigen in serum of MERS-CoV RBDc-trimer immunized mice (T2 group 2.7312, T5 group 3.1527) were significantly higher than MERS-CoV RBD-monomer (M2 group 1.4669, M5 group 1.2760) and PBS control group (1.0000) (fig. 16), 2 μg group T2 titres were higher than M2 (p=0.0027), and 5 μg group T5 titres were also higher than M5 (p=0.0059).

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Sequence listing

SEQ ID NO. 1 trimer tag amino acid sequence E918-L966

918 ENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQL 966

SEQ ID NO. 2linker amino acid sequence

LVPRGSGGSGGSGGLEVLFQGP

SEQ ID NO. 3 trimer tag amino acid sequence D1163-L1203

1163 DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 1203 SEQ ID NO:4 SARS-CoV-2 RBD amino acid sequence R319-K537

SEQ ID NO. 5 contains a SARS-CoV-2 RBD-Monomer amino acid sequence of the Signal peptide and the 6XHis tag

SEQ ID NO. 6SARS-CoV-2 RBDc primer amino acid sequence

SEQ ID NO. 7 contains a SARS-CoV-2 RBDc-primer amino acid sequence of the Signal peptide and the 6XHis tag

SEQ ID NO. 8SARS-CoV RBD amino acid sequence R306-Q523

SEQ ID NO. 9 contains a SARS-CoV RBD-Monomer amino acid sequence of the Signal peptide and the 6XHis tag

SEQ ID NO. 10SARS-CoV RBDc-primer amino acid sequence

SEQ ID NO. 11 contains a signal peptide and a SARS-CoV RBDc-primer amino acid sequence of a 6XHis tag

SEQ ID NO. 12MERS-CoV RBD amino acid sequence E367-N602

SEQ ID NO. 13 contains the MERS-CoV RBD-Monomer amino acid sequence of the Signal peptide and the 6XHis tag

SEQ ID NO. 14MERS-CoV RBDc-primer amino acid sequence

SEQ ID NO. 15 contains the MERS-CoV RBDc-primer amino acid sequence of the Signal peptide and the 6XHis tag

SEQ ID NO. 16 SARS-CoV-2S protein signal peptide

MFVFLVLLPLVSSQ

17 MERS-CoV S protein signal peptide of SEQ ID NO

MIHSVFLLMFLLTPTES

SEQ ID NO. 18 contains a signal peptide and a SARS-CoV-2 RBD-monomer coding sequence of a stop codon and a 6XHis tag

SEQ ID NO. 19 SARS-CoV-2 RBD R319-K537 coding sequence

SEQ ID NO. 20 contains a signal peptide and a SARS-CoV-2 RBDc-primer coding sequence of a stop codon and a 6XHis tag

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SEQ ID NO. 21 SARS-CoV-2 RBDc-primer coding sequence

SEQ ID NO. 22 SARS-CoV RBDc-primer coding sequence

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SEQ ID NO. 23 MERS-CoV RBDc-primer coding sequence

24SARS-CoV S protein signal peptide of SEQ ID NO

1 MFIFLLFLTL TSGS 。

SEQUENCE LISTING

<110> institute of microorganisms at national academy of sciences

<120> coronavirus trimer subunit vaccine and use thereof

<130> IB206208

<150> 202011643340.8

<151> 2020-12-31

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 49

<212> PRT

<213> Artificial Sequence

<220>

<223> trimer tag amino acid sequence E918-L966

<400> 1

Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys

1 5 10 15

Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln

20 25 30

Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln

35 40 45

Leu

<210> 2

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> linker amino acid sequence

<400> 2

Leu Val Pro Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Leu Glu

1 5 10 15

Val Leu Phe Gln Gly Pro

20

<210> 3

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> trimer tag amino acid sequence D1163-L1203

<400> 3

Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn

1 5 10 15

Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn

20 25 30

Glu Ser Leu Ile Asp Leu Gln Glu Leu

35 40

<210> 4

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV-2 RBD amino acid sequence R319-K537

<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

210 215

<210> 5

<211> 239

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV-2 RBD-Monomer amino acid sequence comprising Signal peptide and 6XHis tag

<400> 5

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Arg Val

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys Ser Thr Asn Leu Val Lys Asn Lys His His His His His His

225 230 235

<210> 6

<211> 331

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV-2 RBDc primer amino acid sequence

<400> 6

Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys

1 5 10 15

Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln

20 25 30

Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln

35 40 45

Leu Leu Val Pro Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Leu

50 55 60

Glu Val Leu Phe Gln Gly Pro Asp Val Asp Leu Gly Asp Ile Ser Gly

65 70 75 80

Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys

325 330

<210> 7

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV-2 RBDc-primer amino acid sequence comprising Signal peptide and 6XHis tag

<400> 7

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Glu Asn

1 5 10 15

Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln

20 25 30

Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val

35 40 45

Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Leu

50 55 60

Val Pro Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Leu Glu Val

65 70 75 80

Leu Phe Gln Gly Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

85 90 95

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val

100 105 110

Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Arg Val

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Lys Ser Thr Asn Leu Val Lys Asn Lys His His His His His His

340 345 350

<210> 8

<211> 218

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV RBD amino acid sequence R306-Q523

<400> 8

Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg

130 135 140

Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys

145 150 155 160

Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr

165 170 175

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

180 185 190

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

195 200 205

Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln

210 215

<210> 9

<211> 238

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV RBD-Monomer amino acid sequence comprising Signal peptide and 6XHis tag

<400> 9

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Arg Val

1 5 10 15

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

20 25 30

Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr Ala

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn

130 135 140

Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg Pro Phe

145 150 155 160

Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys Pro Cys

165 170 175

Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly Phe

180 185 190

Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val Val Leu

195 200 205

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

210 215 220

Ser Thr Asp Leu Ile Lys Asn Gln His His His His His His

225 230 235

<210> 10

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV RBDc-primer amino acid sequence

<400> 10

Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys

1 5 10 15

Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln

20 25 30

Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln

35 40 45

Leu Leu Val Pro Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Leu

50 55 60

Glu Val Leu Phe Gln Gly Pro Asp Val Asp Leu Gly Asp Ile Ser Gly

65 70 75 80

Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn

85 90 95

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

100 105 110

Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg

245 250 255

Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys

260 265 270

Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr

275 280 285

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

290 295 300

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

305 310 315 320

Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln

325 330

<210> 11

<211> 350

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV RBDc-trimer amino acid sequence comprising Signal peptide and 6XHis tag

<400> 11

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Glu Asn

1 5 10 15

Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln

20 25 30

Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val

35 40 45

Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Leu

50 55 60

Val Pro Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Leu Glu Val

65 70 75 80

Leu Phe Gln Gly Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

85 90 95

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val

100 105 110

Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Arg Val

115 120 125

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

130 135 140

Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr Ala

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn

245 250 255

Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg Pro Phe

260 265 270

Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys Pro Cys

275 280 285

Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly Phe

290 295 300

Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val Val Leu

305 310 315 320

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

325 330 335

Ser Thr Asp Leu Ile Lys Asn Gln His His His His His His

340 345 350

<210> 12

<211> 236

<212> PRT

<213> Artificial Sequence

<220>

<223> MERS-CoV RBD amino acid sequence E367-N602

<400> 12

Glu Ala Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu

1 5 10 15

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

20 25 30

Phe Lys Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu

35 40 45

Leu Ser Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro

50 55 60

Ala Ala Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe

65 70 75 80

Ser Tyr Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly

85 90 95

Pro Ile Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys

100 105 110

Leu Ile Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro

115 120 125

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

130 135 140

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

145 150 155 160

Val Ser Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg

165 170 175

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

180 185 190

Ser Thr Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr

195 200 205

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

210 215 220

Ala Asn Asp Thr Lys Ile Ala Ser Gln Leu Gly Asn

225 230 235

<210> 13

<211> 259

<212> PRT

<213> Artificial Sequence

<220>

<223> MERS-CoV RBD-Monomer amino acid sequence comprising Signal peptide and 6XHis tag

<400> 13

Met Ile His Ser Val Phe Leu Leu Met Phe Leu Leu Thr Pro Thr Glu

1 5 10 15

Ser Glu Ala Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val

20 25 30

Glu Cys Asp Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr

35 40 45

Asn Phe Lys Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys

50 55 60

Leu Leu Ser Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser

65 70 75 80

Pro Ala Ala Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr

85 90 95

Phe Ser Tyr Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala

100 105 110

Gly Pro Ile Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr

115 120 125

Cys Leu Ile Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys

130 135 140

Pro Leu Lys Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp

145 150 155 160

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

165 170 175

Cys Val Ser Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr

180 185 190

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

195 200 205

Gly Ser Thr Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile

210 215 220

Thr Val Gln Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu

225 230 235 240

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

245 250 255

His His His

<210> 14

<211> 348

<212> PRT

<213> Artificial Sequence

<220>

<223> MERS-CoV RBDc-primer amino acid sequence

<400> 14

Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys

1 5 10 15

Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln

20 25 30

Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln

35 40 45

Leu Leu Val Pro Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Leu

50 55 60

Glu Val Leu Phe Gln Gly Pro Asp Val Asp Leu Gly Asp Ile Ser Gly

65 70 75 80

Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn

85 90 95

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

100 105 110

Glu Ala Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu

115 120 125

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

130 135 140

Phe Lys Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu

145 150 155 160

Leu Ser Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro

165 170 175

Ala Ala Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe

180 185 190

Ser Tyr Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly

195 200 205

Pro Ile Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys

210 215 220

Leu Ile Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro

225 230 235 240

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

245 250 255

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

260 265 270

Val Ser Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg

275 280 285

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

290 295 300

Ser Thr Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr

305 310 315 320

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

325 330 335

Ala Asn Asp Thr Lys Ile Ala Ser Gln Leu Gly Asn

340 345

<210> 15

<211> 371

<212> PRT

<213> Artificial Sequence

<220>

<223> MERS-CoV RBDc-primer amino acid sequence comprising Signal peptide and 6XHis tag

<400> 15

Met Ile His Ser Val Phe Leu Leu Met Phe Leu Leu Thr Pro Thr Glu

1 5 10 15

Ser Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly

20 25 30

Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu

35 40 45

Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys

50 55 60

Gln Leu Leu Val Pro Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

65 70 75 80

Leu Glu Val Leu Phe Gln Gly Pro Asp Val Asp Leu Gly Asp Ile Ser

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Glu Cys Asp Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr

145 150 155 160

Asn Phe Lys Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys

165 170 175

Leu Leu Ser Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser

180 185 190

Pro Ala Ala Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr

195 200 205

Phe Ser Tyr Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala

210 215 220

Gly Pro Ile Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr

225 230 235 240

Cys Leu Ile Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys

245 250 255

Pro Leu Lys Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp

260 265 270

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

275 280 285

Cys Val Ser Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr

290 295 300

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

305 310 315 320

Gly Ser Thr Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile

325 330 335

Thr Val Gln Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu

340 345 350

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

355 360 365

His His His

370

<210> 16

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV-2S protein Signal peptide

<400> 16

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln

1 5 10

<210> 17

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> MERS-CoV S protein signal peptide

<400> 17

Met Ile His Ser Val Phe Leu Leu Met Phe Leu Leu Thr Pro Thr Glu

1 5 10 15

Ser

<210> 18

<211> 720

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-CoV-2 RBD-monomer coding sequence comprising Signal peptide and 6XHis tag and stop codon

<400> 18

atgtttgttt ttcttgtttt attgccacta gtctctagtc agcgggtgca gcctacagag 60

tctattgtgc ggttcccaaa catcacaaac ctgtgccctt tcggcgaggt gttcaacgcc 120

acccggttcg cctctgtgta cgcctggaac cggaagcgga tctctaactg cgtggccgac 180

tactccgtgc tgtacaactc cgcctctttc tctacattca agtgctacgg cgtgtcccct 240

acaaagctga acgacctgtg cttcaccaac gtgtacgccg actctttcgt gattagaggc 300

gacgaggtga ggcagattgc ccccggccag acaggcaaga tcgccgacta caactacaag 360

ctgcccgacg acttcacagg ctgcgtgatc gcctggaact ctaacaacct ggactctaag 420

gtgggcggca actacaacta cctgtacaga ctgttccgga agtctaacct gaagccattc 480

gagagggaca ttagcaccga gatttaccag gccggctcta ccccatgcaa cggcgtggag 540

ggcttcaact gctacttccc actgcagtcc tacggcttcc agcctacaaa cggcgtgggc 600

taccagcctt accgggtggt ggtgctgtct ttcgagctgc tccacgcccc cgccacagtg 660

tgcggcccaa agaagagcac aaacctcgtg aagaacaagc accatcatca ccaccactga 720

<210> 19

<211> 657

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-CoV-2 RBD R319-K537 coding sequence

<400> 19

cgggtgcagc ctacagagtc tattgtgcgg ttcccaaaca tcacaaacct gtgccctttc 60

ggcgaggtgt tcaacgccac ccggttcgcc tctgtgtacg cctggaaccg gaagcggatc 120

tctaactgcg tggccgacta ctccgtgctg tacaactccg cctctttctc tacattcaag 180

tgctacggcg tgtcccctac aaagctgaac gacctgtgct tcaccaacgt gtacgccgac 240

tctttcgtga ttagaggcga cgaggtgagg cagattgccc ccggccagac aggcaagatc 300

gccgactaca actacaagct gcccgacgac ttcacaggct gcgtgatcgc ctggaactct 360

aacaacctgg actctaaggt gggcggcaac tacaactacc tgtacagact gttccggaag 420

tctaacctga agccattcga gagggacatt agcaccgaga tttaccaggc cggctctacc 480

ccatgcaacg gcgtggaggg cttcaactgc tacttcccac tgcagtccta cggcttccag 540

cctacaaacg gcgtgggcta ccagccttac cgggtggtgg tgctgtcttt cgagctgctc 600

cacgcccccg ccacagtgtg cggcccaaag aagagcacaa acctcgtgaa gaacaag 657

<210> 20

<211> 1056

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-CoV-2 RBDc-primer coding sequence comprising Signal peptide and 6XHis tag and stop codon

<400> 20

atgtttgttt ttcttgtttt attgccacta gtctctagtc aggagaacca gaagctgatc 60

gccaaccagt tcaatagcgc catcggcaag atccaggact ccctgagctc caccgcatct 120

gccctgggca agctgcagga tgtggtgaac cagaatgccc aggccctgaa tacactggtg 180

aagcagctgc tggtgccaag gggatctgga ggaagcggag gctccggagg actggaggtg 240

ctgtttcagg gacctgacgt ggatctgggc gacatctctg gcatcaacgc cagcgtggtg 300

aatatccaga aggagatcga cagactgaac gaggtggcca agaacctgaa tgagtccctg 360

atcgatctgc aggagctgcg ggtgcagcct acagagtcta ttgtgcggtt cccaaacatc 420

acaaacctgt gccctttcgg cgaggtgttc aacgccaccc ggttcgcctc tgtgtacgcc 480

tggaaccgga agcggatctc taactgcgtg gccgactact ccgtgctgta caactccgcc 540

tctttctcta cattcaagtg ctacggcgtg tcccctacaa agctgaacga cctgtgcttc 600

accaacgtgt acgccgactc tttcgtgatt agaggcgacg aggtgaggca gattgccccc 660

ggccagacag gcaagatcgc cgactacaac tacaagctgc ccgacgactt cacaggctgc 720

gtgatcgcct ggaactctaa caacctggac tctaaggtgg gcggcaacta caactacctg 780

tacagactgt tccggaagtc taacctgaag ccattcgaga gggacattag caccgagatt 840

taccaggccg gctctacccc atgcaacggc gtggagggct tcaactgcta cttcccactg 900

cagtcctacg gcttccagcc tacaaacggc gtgggctacc agccttaccg ggtggtggtg 960

ctgtctttcg agctgctcca cgcccccgcc acagtgtgcg gcccaaagaa gagcacaaac 1020

ctcgtgaaga acaagcacca tcatcaccac cactga 1056

<210> 21

<211> 993

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-CoV-2 RBDc-primer coding sequence

<400> 21

gagaaccaga agctgatcgc caaccagttc aatagcgcca tcggcaagat ccaggactcc 60

ctgagctcca ccgcatctgc cctgggcaag ctgcaggatg tggtgaacca gaatgcccag 120

gccctgaata cactggtgaa gcagctgctg gtgccaaggg gatctggagg aagcggaggc 180

tccggaggac tggaggtgct gtttcaggga cctgacgtgg atctgggcga catctctggc 240

atcaacgcca gcgtggtgaa tatccagaag gagatcgaca gactgaacga ggtggccaag 300

aacctgaatg agtccctgat cgatctgcag gagctgcggg tgcagcctac agagtctatt 360

gtgcggttcc caaacatcac aaacctgtgc cctttcggcg aggtgttcaa cgccacccgg 420

ttcgcctctg tgtacgcctg gaaccggaag cggatctcta actgcgtggc cgactactcc 480

gtgctgtaca actccgcctc tttctctaca ttcaagtgct acggcgtgtc ccctacaaag 540

ctgaacgacc tgtgcttcac caacgtgtac gccgactctt tcgtgattag aggcgacgag 600

gtgaggcaga ttgcccccgg ccagacaggc aagatcgccg actacaacta caagctgccc 660

gacgacttca caggctgcgt gatcgcctgg aactctaaca acctggactc taaggtgggc 720

ggcaactaca actacctgta cagactgttc cggaagtcta acctgaagcc attcgagagg 780

gacattagca ccgagattta ccaggccggc tctaccccat gcaacggcgt ggagggcttc 840

aactgctact tcccactgca gtcctacggc ttccagccta caaacggcgt gggctaccag 900

ccttaccggg tggtggtgct gtctttcgag ctgctccacg cccccgccac agtgtgcggc 960

ccaaagaaga gcacaaacct cgtgaagaac aag 993

<210> 22

<211> 990

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-CoV RBDc-primer coding sequence

<400> 22

gagaaccaga agctgatcgc caaccagttc aatagcgcca tcggcaagat ccaggactcc 60

ctgagctcca ccgcatctgc cctgggcaag ctgcaggatg tggtgaacca gaatgcccag 120

gccctgaata cactggtgaa gcagctgctg gtgccaaggg gatctggagg aagcggaggc 180

tccggaggac tggaggtgct gtttcaggga cctgacgtgg atctgggcga catctctggc 240

atcaacgcca gcgtggtgaa tatccagaag gagatcgaca gactgaacga ggtggccaag 300

aacctgaatg agtccctgat cgatctgcag gagctgaggg tggtgcccag cggcgacgtg 360

gtgaggttcc ccaacatcac caacctgtgc cccttcggcg aggtgttcaa cgccaccaag 420

ttccccagcg tatacgcctg ggagaggaag aagatcagca actgcgtggc cgactacagc 480

gtgctgtaca acagcacctt cttcagcacc ttcaagtgct acggcgtgag cgccaccaag 540

ctgaacgacc tgtgcttcag caacgtgtac gccgatagct tcgtggtgaa gggcgacgac 600

gtgaggcaga tcgctcctgg acagaccggc gtgatcgccg actacaacta caagctgccc 660

gacgacttca tgggctgcgt gctggcctgg aacaccagga acatcgacgc caccagcacc 720

ggcaactaca actacaagta caggtacctg aggcacggca agctgaggcc cttcgagagg 780

gacatcagca acgtgccatt cagccctgac ggcaagccct gcacaccacc tgccctgaac 840

tgctactggc cactgaacga ctacggcttc tacaccacca ccggcatcgg ctaccagccc 900

tacagggtgg tggtgctgag cttcgagctg ctgaacgctc ctgccaccgt gtgcggccct 960

aagctgagca ccgacctgat caagaaccag 990

<210> 23

<211> 1044

<212> DNA

<213> Artificial Sequence

<220>

<223> MERS-CoV RBDc-primer coding sequence

<400> 23

gagaaccaga agctgatcgc caaccagttc aatagcgcca tcggcaagat ccaggactcc 60

ctgagctcca ccgcatctgc cctgggcaag ctgcaggatg tggtgaacca gaatgcccag 120

gccctgaata cactggtgaa gcagctgctg gtgccaaggg gatctggagg aagcggaggc 180

tccggaggac tggaggtgct gtttcaggga cctgacgtgg atctgggcga catctctggc 240

atcaacgcca gcgtggtgaa tatccagaag gagatcgaca gactgaacga ggtggccaag 300

aacctgaatg agtccctgat cgatctgcag gagctggaag caaaaccttc tggctcagtt 360

gtggaacagg ctgaaggtgt tgaatgtgat ttttcacctc ttctgtctgg cacacctcct 420

caggtttata atttcaagcg tttggttttt accaattgca attataatct taccaaattg 480

ctttcacttt tttctgtgaa tgattttact tgtagtcaaa tatctccagc agcaattgct 540

agcaactgtt attcttcact gattttggat tacttttcat acccacttag tatgaaatcc 600

gatctcagtg ttagttctgc tggtccaata tcccagttta attataaaca gtccttttct 660

aatcccacat gtttgatttt agcgactgtt cctcataacc ttactactat tactaagcct 720

cttaagtaca gctatattaa caagtgctct cgtcttcttt ctgatgatcg tactgaagta 780

cctcagttag tgaacgctaa tcaatactca ccctgtgtat ccattgtccc atccactgtg 840

tgggaagacg gtgattatta taggaaacaa ctatctccac ttgaaggtgg tggctggctt 900

gttgctagtg gctcaactgt tgccatgact gagcaattac agatgggctt tggtattaca 960

gttcaatatg gtacagacac caatagtgtt tgccccaagc ttgaatttgc taatgacaca 1020

aaaattgcct ctcaattagg caat 1044

<210> 24

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> SARS-CoV S protein Signal peptide

<400> 24

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser

1 5 10

Claims

1. A coronavirus antigen, wherein the antigen consists of, in order from the 5 'end to the 3' end:

a connecting fragment containing 7-25 amino acids, the amino acid sequence of which is SEQ ID NO. 2,

SARS-CoV-2S protein trimer forming region D1163-L1203 with amino acid sequence of SEQ ID NO. 3,

the RBD region R319-K537 of SARS-CoV-2S protein has an amino acid sequence of SEQ ID NO. 4;

or alternatively, the first and second heat exchangers may be,

the antigen consists of the following components in sequence from the 5 'end to the 3' end:

or (b)

the antigen is a trimeric protein.

2. The antigen according to claim 1, wherein the amino acid sequence is the amino acid sequence shown in (1), (2) or (3):

(1) An amino acid sequence shown as SEQ ID NO. 6;

(2) An amino acid sequence as shown in SEQ ID NO. 10;

(3) The amino acid sequence shown in SEQ ID NO. 14.

3. The antigen of claim 1 or 2, wherein the antigen further comprises a secretion signal peptide added at the 5 'end and a purification tag added at the 3' end.

4. The antigen according to claim 3, wherein the secretion signal peptide is a SARS-CoV-2S protein self-signal peptide having an amino acid sequence of SEQ ID NO. 16, a MERS-CoV S protein self-signal peptide having an amino acid sequence of SEQ ID NO. 17, or a SARS-CoV S protein self-signal peptide having an amino acid sequence of SEQ ID NO. 24; the purification tag is a 6×his tag.

5. An isolated polynucleotide encoding the antigen of any one of claims 1-4, said isolated polynucleotide comprising a sequence as set forth in SEQ ID No. 21, SEQ ID No. 22 or SEQ ID No. 23.

6. A recombinant vector or expression cassette comprising the isolated polynucleotide of claim 5.

7. A transgenic cell line or recombinant bacterium comprising the recombinant vector or expression cassette, respectively, of claim 6.

8. The transgenic cell line or recombinant bacterium of claim 7, wherein the transgenic cell line is a mammalian cell.

9. Use of an antigen according to any one of claims 1-4 for the preparation of an anti-coronavirus medicament.

10. The use of claim 9, wherein the medicament is a vaccine.

11. The use of claim 10, wherein the antigen is combined with an aluminium hydroxide adjuvant.

12. A kit comprising an antigen according to any one of claims 1 to 4, an isolated polynucleotide according to claim 5, or a recombinant vector or expression cassette according to claim 6, a transgenic cell line or recombinant bacterium according to claim 7.

13. The kit of claim 12, further comprising aluminum hydroxide.