CN114437185A

CN114437185A - Coronavirus trimer subunit vaccine and application thereof

Info

Publication number: CN114437185A
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: 2022-05-06
Anticipated expiration: 2041-12-07
Also published as: CN114437185B

Abstract

The invention discloses a subunit novel coronavirus (SARS-CoV-2), severe acute respiratory syndrome (SARS-CoV) and middle east respiratory syncytial virus (MERS-CoV) vaccine of a trimerization Receptor Binding Domain (RBD). The invention uses the mammal cell to express the SARS-CoV-2S protein trimer forming region and the SARS-CoV-2 RBD region, SARS-CoV RBD region or MERS-CoV RBD region through the mammal cell expression vector, to form the trimer protein SARS-CoV-2 RBDc-trimer, SARS-CoV RBDc-trimer and MERS-CoV RBDc-trimer, and compared with the respective RBD monomer protein, the trimer form protein has better immune effect, and can make the mouse generate higher titer specificity antibody. The trimer form 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 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 single-stranded RNA viruses with envelope, which are spherical or ellipsoidal, and have regularly arranged capsular collagen fiber processes shaped like a crown, hence their name. Coronaviruses belong to the family of coronaviruses of the order Nervirridae, and are divided into two subfamilies, coronaviruses and Circoviruses. Among them, the subfamily coronaviruses have 4 genera of α, β, γ and δ, and the β genus coronaviruses can be divided into 4 independent subgroups A, B, C and D. The viral envelope consists of a bilayer of lipids interspersed with membrane proteins and spike proteins, and some types of coronaviruses may also have hemagglutinin. The virus is internally provided with a nucleoprotein core consisting of RNA and capsid protein and has a spiral structure.

On 12.1.2020, the world health organization formally named "2019 novel coronavirus (2019-nCoV)", and thereafter on 11.12.2.2020, the International Committee on Taxonomy of virues, ICTV, announced the formal classification of 2019 novel coronavirus (2019-nCoV) named Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the World Health Organization (WHO) announced on the global research and innovation forum on the same day as the japanese tile, and the formal name of the disease caused by this virus was "covi-19".

Until 2003, coronavirus, which were known to infect humans and were pathogenic, were generally milder, such as 229E, OC 43. Since the outbreak of SARS-CoV in 2003, several new pathogenic coronaviruses have been discovered in succession in humans. SARS-CoV, MERS-CoV and newly discovered SARS-CoV-2 cause severe respiratory diseases. Numerous virologists and teams have now essentially established SARS-CoV-2 as well as SARS-CoV as belonging to the beta subgroup B. By analyzing 7 conserved non-structural protein sequences and the whole genome sequence, SARS-CoV-2 has genome sequence similarity with SARS-CoV and has extremely high homology with bat-derived coronavirus. The study confirms that SARS-CoV-2 and SARS-CoV invade cells by binding the same receptor, angiotensin converting enzyme 2 (ACE2), ACE2 is present in respiratory epithelium, and the pneumonia symptoms of novel coronavirus infectors and the possibility of spreading are confirmed from molecular level.

To infect a cell, the virus first needs to bind to the host's receptor via the envelope protein. Based on studies of other coronaviruses, particularly SARS-CoV and MERS-CoV, the important envelope protein for receptor binding is 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-terminal (NTD) and C-terminal (CTD) of S1 may be Receptor Binding Domains (RBDs). Through studies on SARS-CoV-2, the team found that CTD is the RBD of this coronavirus, binding to the receptor ACE 2. Therefore, SARS-CoV-2 RBD is used as immunogen to stimulate the organism to produce specific antibody against SARS-CoV-2 RBD. The subunit vaccine of SARS-CoV-2 RBD can be an effective way for inhibiting virus infection. Similarly, subunit vaccines for SARS-CoV RBD and MERS-CoV RBD can also be used to prevent infection by the corresponding viruses.

The new coronavirus (also called new coronavirus for short) is a new virus, and a plurality of uncertain factors exist in vaccine development. At present, inactivated vaccines, nucleic acid vaccines, recombinant protein vaccines, adenoviral vector vaccines and attenuated vaccines of new coronavirus are mainly developed worldwide. And (3) inactivated vaccine: inactivated vaccines are the classical technical route followed by culturing the new coronavirus in vitro and then inactivating it to render it non-toxic. The inactivated vaccine has the advantages of simple and rapid preparation method and higher safety, but the inactivated vaccine also has the disadvantages of large inoculation dose and short immune period, and the most terrible disadvantage is that the antibody dependent enhancement effect (ADE) is sometimes caused to aggravate virus infection. Adenovirus vector vaccine: the adenovirus vector vaccine is prepared by taking the transformed harmless adenovirus as a vector and filling the transformed harmless adenovirus into an S protein gene of a new coronavirus, and has the advantages of safety, high efficiency and less induced adverse reaction, and a human body is stimulated to generate an antibody; the disadvantage is that the recombinant virus vector vaccine takes adenovirus type 5 as a vector, but most people are infected with adenovirus type 5 in the growth process, and antibodies capable of neutralizing the adenovirus vector may exist in vivo, so that 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, and utilizing human body cell to synthesize S protein inside human body to stimulate human body to produce antibody. The nucleic acid vaccine has the advantages that protein or virus does not need to be synthesized during development, the process is simple, and the safety is relatively high; the disadvantage is that there is no precedent for success. Attenuated vaccines: the attenuated influenza virus vaccine which is approved to be on the market is used as a vector, carries S protein of new coronavirus, and stimulates the human body to generate antibodies aiming at the two viruses; since the attenuated influenza virus readily infects the nasal cavity, such vaccines can accomplish vaccination by nasal drip only. Recombinant protein vaccines: the vaccine is also called as gene engineering recombinant subunit vaccine, and is prepared by mass production of S or RBD protein which is most likely to be used as antigen by a new coronavirus through a gene engineering method, and the S or RBD protein is injected into a human body to stimulate the human body to generate antibodies; the recombinant subunit vaccine has the advantages of safety, high efficiency and large-scale production; the successful genetic engineering subunit vaccine is hepatitis B surface antigen vaccine. At present, a plurality of new coronary vaccines enter a clinical stage and achieve certain positive clinical results.

Disclosure of Invention

Aiming at coronavirus, the invention designs a coronavirus subunit vaccine 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 the subunit vaccine; the mammalian cell is used for exogenously expressing SARS-CoV-2S protein trimer formation region to fuse with the RBD region of SARS-CoV-2 protein, the RBD region of SARS-CoV protein or the RBD region of MERS-CoV to form trimer proteins SARS-CoV-2 RBDc-trimer, SARS-CoV RBDc-trimer and MERS-CoV RBDc-trimer which can be normally expressed, and compared with respective RBD monomer protein, the trimer form protein has better immune effect and can make the mouse produce specific antibody aiming at the RBD region with higher titer. The RBD protein in the form of trimer overcomes the defect of insufficient immunogenicity of RBD monomers, and improves the specific antibody aiming at the RBD region generated by a mouse; the design can be used for improving the immunogenicity of the coronavirus RBD, so that the trimer vaccine has excellent immunogenicity, and a brand-new form is provided for the design of the coronavirus vaccine.

Specifically, the present invention provides the following embodiments:

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

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

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

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

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

or the like, or, alternatively,

the direction from the 5 'end to the 3' end of the antigen sequentially comprises:

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

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

or

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

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

preferably the antigen is a trimeric protein.

2. The antigen according to item 1, which comprises an amino acid sequence shown in the following (1), (2), (3) or (4):

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

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

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

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

3. The antigen according to

item

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

4. An isolated polynucleotide encoding the polypeptide of any one of items 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 claim 5, respectively.

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

8. Use of an antigen according to any one of claims 1 to 3 in the manufacture of a medicament, e.g. a vaccine, against a coronavirus.

9. The use of item 8, wherein the antigen is used in combination with an aluminum hydroxide adjuvant.

10. A kit comprising an antigen according to any one of claims 1 to 3, an isolated polynucleotide according to claim 4, or a recombinant vector or expression cassette according to claim 5, a transgenic cell line or recombinant bacterium according to claim 6, preferably the kit further comprises aluminium hydroxide.

In designing the coronavirus trimer vaccine of the present invention, it is particularly important to design the SARS-CoV-2S protein trimer formation region at the 5' end or 3' end of the RBD region of the above three coronaviruses, because the inventors could not express the complete desired protein when trying to design it at the 5' end.

Drawings

FIG. 1: WB identification of SARS-CoV-2 RBDc-trimer, SARS-CoV-2 RBD-monomer;

FIG. 2: SARS-CoV-2 RBDc-trimer molecular sieve chromatography and SDS-PAGE identification;

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

FIG. 4: determination of SARS-CoV-2 RBDc-trimer by analytical ultracentrifugation;

FIG. 5: measuring the serum antibody titer of the mouse after SARS-CoV-2 RBDc-dimer and SARS-CoV-2 RBD-monomer immunization;

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

FIG. 7: WB identification of SARS-CoV RBDc-dimer, SARS-CoV RBD-monomer;

FIG. 8: SARS-CoV RBDc-trimer molecular sieve chromatography and SDS-PAGE identification;

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

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

FIG. 11: measuring the serum antibody titer of the mouse after SARS-CoV RBDc-dimer and SARS-CoV RBD-monomer immunization;

FIG. 12: WB identification of MERS-CoV RBDc-trimer and MERS-CoV RBD-monomer;

FIG. 13: MERS-CoV RBDc-trimer molecular sieve chromatography and SDS-PAGE identification;

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

FIG. 15: performing analytical ultracentrifugation determination on MERS-CoV RBDc-trimer;

FIG. 16: and (3) determining the serum antibody titer of the mice after the immunization of MERS-CoV RBDc-trimer and MERS-CoV RBD-monomer.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

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

The following two SARS-CoV-2 RBD recombinant expression fragments were designed:

SARS-CoV-2 RBDc-trimer: 5'-SP + HR1+ linker + HR2+ RBD + 6XHis + stop codon-3';

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

Wherein

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

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

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

HR2 is a SARS-CoV-2S protein trimer formation region D1163-L1203, and has an amino acid sequence of SEQ ID NO. 3;

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

The nucleic acid sequences coding the amino acid sequences are optimized according to the preference of mammalian cell codons to respectively obtain optimized coding nucleic acid sequences SEQ ID NO:20 and SEQ ID NO:18, wherein the optimized coding nucleic acid sequences of the fragments except SP and 6XHis are SEQ ID NO:21 and SEQ ID NO:19, and the optimized coding nucleic acid sequences are connected to a pCAGGS (purchased from Addgene) vector through two enzyme cleavage sites of EcoRI and XhoI to form SARS-CoV-2 RBD recombinant expression plasmids.

Example 2: test expression and identification of SARS-CoV-2 RBDc-dimer and SARS-CoV-2 RBD-monomer protein

HEK293T cells were cultured in DMEM containing 10% FBS. HEK293T (in duplicate) was transfected with the SARS-CoV-2 RBD recombinant expression plasmid containing the antigen encoding gene obtained in example 1. Cell culture media were changed to serum-free DMEM 4-6 hours after transfection and incubation 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-monomer 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-monomer (as shown in figure 1), so we select SARS-CoV-2 RBDc-trimer construction as the preferred scheme, and SARS-CoV-2 RBD-monomer as the control construction to continue the subsequent experiments.

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. And (3) after 4-6 hours of transfection, changing the cell culture solution into serum-free DMEM, continuing to culture for 3 days, collecting the supernatant, supplementing DMEM, continuing to culture for 4 days, and collecting the supernatant again.

The supernatants collected twice were mixed, centrifuged at 5000rpm for 30 minutes, filtered through a 0.22 μm filter, bound to a HisTrap excel column (5mL, GE Healthcare), and the non-specifically 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. After the target protein is collected and concentrated, the protein is subjected to molecular sieve chromatography Superdex 200 Increate 10/300GL or Superdex 200 Hiload 16/60(GE Healthcare). The objective peak was determined by SDS-PAGE (reduction), and the results are shown in FIGS. 2 and 3. Obtaining the purified SARS-CoV-2 RBDc-dimer and SARS-CoV-2 RBD-monomer antigen. After the analysis of ultracentrifugation, the molecular weight of SARS-CoV-2 RBDc-trimer was measured to be 151kDa, which was consistent with the theoretical molecular weight of trimer, and it was determined to be trimer, as shown in FIG. 4.

Example 4: immunization experiment of mice

The SARS-CoV-2 RBDc-trimer antigen obtained in example 3 was diluted in physiological saline according to the method described in Table 1 below, and was emulsified in groups with an equal volume of adjuvant, followed by group immunization of 6-week-old Balb/c mice (purchased from Witongliwa). The immunization strategy was by means of intramuscular injection into the thigh, each mouse being given 1 vaccine immunization on day 0 and 14, respectively, at a vaccination volume of 100. mu.l each time. Mice were tail bled on day 28. The serum of the mouse is separated out after standing for a period of time, the serum is obtained by centrifuging for 10 minutes at 3000rpm, and the serum is frozen in a refrigerator at the temperature of minus 80 ℃ after being inactivated at the temperature of 56 ℃ for 30 minutes and is used for ELISA binding detection.

Table 1: animal immunization group status

Example 5: mouse immune post-serum ELISA detection experiment

The serum of the mouse prepared in example 4 of the previous step was subjected to ELISA to determine the level of RBD-specific antibodies. 200ng of the purified SARS-CoV-2 RBD-monomer protein obtained in example 3 was added to each well of the ELISA plate, and each well was coated with ELISA coating solution (50mM Na)₂CO₃、NaHCO₃Buffer, ph9.6) was coated overnight at 4 ℃; after discarding the coating solution, 150. mu.l of 5% skim milk powder was added to each well and sealed at room temperature for 1 hour. After the blocking is finished, washing the ELISA plate for 2 times by PBS containing 0.05% Tween 20, adding 100 mu L of serum after antigen immunization or serum after PBS immunization in each hole, and incubating for 1 hour at room temperature; after discarding the supernatant, washing the ELISA plate 5 times with PBS containing 0.05% Tween 20; 100 mul of goat anti-mouse IgG antibody (secondary antibody, purchased from Zhongshan Jinqiao) diluted with horseradish peroxidase at a ratio of 1:3000 was added to each well, and incubated at room temperature for 1 hour; after discarding the secondary antibody, the ELISA plate was washed 5 times with PBS containing 0.05

% Tween

20, 50. mu.L of ELISA developing solution was added to each well to develop for 15 minutes, and 50. mu.L of 2M H was added to each well₂SO₄The reaction is stopped, and the enzyme-linked immunosorbent assay (OD) is read by an enzyme-linked immunosorbent assay (ELISA) instrument₄₅₀A numerical value; the antibody Titer (Titer) was calculated by taking lg value as the lowest dilution factor higher than the value of negative control x 2.1 times of the group to which no serum was added, and calculating the antibody Titer of each group. Results displayThe antibody titer (4.4654 of T2 group and 4.7914 of T5 group) of SARS-CoV-2 RBD immune group mouse serum combined with SARS-CoV-2 RBD antigen is obviously higher than that of SARS-CoV-2 RBD-monomer (1.6193 of M2 group and 3.9383 of M5 group) and PBS control group (1.0000) (figure 5), and the T2 titer of 2 mug group is higher than that of M2 (P2)<0.0001), the T5 titer was also higher in the 5 μ g group than M5(P ═ 0.0059).

SARS-CoV and MERS-CoV and SARS-CoV-2 belong to the same beta coronavirus, and the structures of RBD of the three (PDB:3BGF, 6WAR, 6LZG) were compared by using PDB data, and they were found to have high similarity (FIG. 6), so that SARS-CoV RBD and MERS-CoV RBD can be prepared into a trimeric protein vaccine by the method of examples 1 to 5 as well as SARS-CoV-2 RBD. The above point is demonstrated experimentally below.

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

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

SARS-CoV RBDc-trimer: 5'-SP + HR1+ linker + HR2+ RBD + 6XHis + stop codon-3';

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

Wherein

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

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

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

According to the preference of mammalian cell codon, optimizing the nucleic acid sequence for coding the amino acid sequence to obtain the SARS-CoV RBDc-trimer with optimized SP and 6XHis fragments removed and the coding nucleic acid sequence is SEQ ID NO. 22, and then connecting the SARS-CoV RBDc-trimer with pCAGGS (purchased from Addgene) vector through two enzyme cutting sites of EcoRI and XhoI to form SARS-CoV-2 RBD recombinant expression plasmid.

Example 7: test expression and identification of SARS-CoV RBDc-dimer and SARS-CoV RBD-monomer protein

HEK293T cells were cultured in DMEM containing 10% FBS. The SARS-CoV RBD recombinant expression plasmid containing the antigen-encoding gene obtained in example 6 was used to transfect HEK 293T. Cell culture media were changed to serum-free DMEM 4-6 hours after transfection and incubation 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 SARS-CoV RBDc-dimer sample and SARS-CoV RBD-monomer can be normally expressed or not and comparing the expression levels of the two samples. We chose SARS-CoV RBDc-trimer construction as the preferred protocol and SARS-CoV RBD-monomer as the control construction to proceed with the subsequent experiments.

WB detection shows that SARS-CoV RBDc-trimer and SARS-CoV RBD-monomer can be expressed normally (as shown in FIG. 7), we continued the subsequent experiments with the construction of SARS-CoV RBDc-trimer as the preferred protocol and SARS-CoV RBD-monomer as the control.

Example 8: expression, purification and identification of SARS-CoV RBDc-dimer and monomer protein

HEK293T cells were cultured in DMEM containing 10% FBS. HEK293T was transfected with the plasmid containing the antigen encoding gene obtained in example 6. And (3) after 4-6 hours of transfection, replacing the cell culture solution with serum-free DMEM, continuing to culture for 3 days, collecting the supernatant, supplementing the DMEM again, continuing to culture for 4 days, and collecting the supernatant again.

The supernatants collected twice were mixed, centrifuged at 5000rpm for 30 minutes, filtered through a 0.22 μm filter, bound to a HisTrap excel column (5mL, GE Healthcare), and the non-specifically 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. After the target protein is collected and concentrated, the protein is subjected to molecular sieve chromatography Superdex 200 Increate 10/300GL or Superdex 200 Hiload 16/60(GE Healthcare). The peak of interest was determined by SDS-PAGE (reduction). The results of obtaining purified SARS-CoV RBDc-dimer and SARS-CoV RBD-monomer antigen are shown in FIGS. 8 and 9. After analytical ultracentrifugation, the molecular weight of SARS-CoV RBDc-trimer is measured to be 120kDa, and if the molecular weight is consistent with the theoretical molecular weight of trimer, it can be determined as trimer (FIG. 10).

Example 9: mouse immunization and serum ELISA detection

A mouse immunization experiment was carried out using the SARS-CoV RBDc-dimer and SARS-CoV RBD-monomer antigen obtained in example 8 in the same manner as in example 4, and the titer of serum-binding SARS-CoV RBD after the immunization of the mouse was measured in the same manner as in example 5.

The results showed that the antibody titer against SARS-CoV RBDc-trimer antigen in serum of mice in SARS-CoV RBDc-trimer immunization group (3.1277 in T2 and 3.5639 in T5) was significantly higher than that of SARS-CoV RBD-monomer (1.7679 in M2 and 1.3215 in M5) and PBS control group (1.0000) (fig. 11), the T2 titer in 2 μ g group was higher than that of M2(P ═ 0.0112), and the T5 titer in 5 μ g group was also higher than that of M5(P < 0.0001).

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

Designing the following two MERS-CoV RBD recombinant expression fragments:

MERS-CoV RBDc-trimer: 5'-SP + HR1+ linker + HR2+ RBD + 6XHis + stop codon-3';

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

Wherein

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;

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

The nucleic acid sequence coding the amino acid sequence is optimized according to the preference of mammalian cell codons, the encoding nucleic acid sequence of MERS-CoV RBDc-trimer with the SP and 6XHis fragments removed in an optimized mode is SEQ ID NO:23, and the encoding nucleic acid sequence is connected to pCAGGS (purchased from Addgene) vector through EcoRI and XhoI enzyme cleavage sites to form MERS-CoV RBD recombinant expression plasmid.

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

HEK293T cells were cultured in DMEM containing 10% FBS. The MERS-CoV RBD recombinant expression plasmid containing the antigen-encoding gene obtained in example 11 was used to transfect HEK 293T. Cell culture media were changed to serum-free DMEM 4-6 hours after transfection and incubation 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). And (4) judging whether the MERS-CoV RBDc-trimer sample and the MERS-CoV RBD-monomer can be normally expressed or not and comparing the expression levels of the MERS-CoV RBDc-trimer sample and the MERS-CoV RBD-monomer. We chose MERS-CoV RBDc-trimer construction as the preferred protocol and MERS-CoV RBD-monomer as the control construction to proceed with subsequent experiments.

The MERS-CoV RBDc-trimer and the MERS-CoV RBD-monomer can be normally expressed by WB detection (as shown in figure 12), and the MERS-CoV RBDc-trimer is constructed as a preferred scheme and the MERS-CoV RBD-monomer is constructed as a control to continue subsequent experiments.

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. And (3) after 4-6 hours of transfection, replacing the cell culture solution with serum-free DMEM, continuing to culture for 3 days, collecting the supernatant, supplementing the DMEM again, continuing to culture for 4 days, and collecting the supernatant again.

The supernatants collected twice were mixed, centrifuged at 5000rpm for 30 minutes, filtered through a 0.22 μm filter, bound to a HisTrap excel column (5mL, GE Healthcare), and the non-specifically 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. After the target protein is collected and concentrated, the protein is subjected to molecular sieve chromatography Superdex 200 Increate 10/300GL or Superdex 200 Hiload 16/60(GE Healthcare). The peak of interest was determined by SDS-PAGE (reduction). Purified MERS-CoV RBDc-trimer and MERS-CoV RBD-monomer antigens were obtained, and the results are shown in FIGS. 13 and 14. After analytical ultracentrifugation, the molecular weight of MERS-CoV RBDc-trimer is 133kDa, and if the molecular weight is consistent with the theoretical molecular weight of trimer, the trimer can be judged (FIG. 15).

Example 13: mouse immunization and serum ELISA detection

The MERS-CoV RBDc-dimer and MERS-CoV RBD-monomer antigens obtained in example 12 were subjected to a mouse immunization experiment in the same manner as in example 4, and the titer of serum-bound MERS-CoV RBD in the mouse after immunization was measured in the same manner as in example 5.

The results showed that the titer of antibodies binding to MERS-CoV RBD antigen in sera of mice in MERS-CoV RBDc-trimer immunization group (2.7312 in T2, group 3.1527 in T5) was significantly higher than MERS-CoV RBD-monomer (1.4669 in M2, group 1.2760 in M5) and PBS control group (1.0000) (fig. 16), the titer of T2 in 2 μ g group was higher than M2(P ═ 0.0027), and the titer of T5 in 5 μ g group was also higher than M5(P ═ 0.0059).

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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

918 ENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQL 966

2linker amino acid sequence of SEQ ID NO

LVPRGSGGSGGSGGLEVLFQGP

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

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

SEQ ID NO 5 SARS-CoV-2 RBD-monomer amino acid sequence comprising signal peptide and 6XHis tag

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

SEQ ID NO 7 SARS-CoV-2 RBDc-trimer amino acid sequence comprising signal peptide and 6XHis tag

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

SEQ ID NO 9 SARS-CoV RBD-monomer amino acid sequence comprising signal peptide and 6XHis tag

SEQ ID NO 10SARS-CoV RBDc-trimer amino acid sequence

SEQ ID NO. 11 SARS-CoV RBDc-trimer amino acid sequence comprising signal peptide and 6XHis tag

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

SEQ ID NO 13 MERS-CoV RBD-monomer amino acid sequence comprising a signal peptide and a 6XHis tag

14MERS-CoV RBDc-trimer amino acid sequence of SEQ ID NO

SEQ ID NO 15 MERS-CoV RBDc-trimer amino acid sequence comprising a signal peptide and a 6XHis tag

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

MFVFLVLLPLVSSQ

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

MIHSVFLLMFLLTPTES

SEQ ID NO 18 SARS-CoV-2 RBD-monomer coding sequence comprising signal peptide, 6XHis tag and stop codon

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

SEQ ID NO. 20 SARS-CoV-2 RBDc-trimer coding sequence containing signal peptide, 6XHis tag and stop codon

SEQ ID NO 21 SARS-CoV-2 RBDc-trimer coding sequence

SEQ ID NO 22 SARS-CoV RBDc-trimer coding sequence

23 MERS-CoV RBDc-trimer coding sequence of SEQ ID NO

SEQ ID NO 24SARS-CoV S protein signal peptide

1 MFIFLLFLTL TSGS 。

SEQUENCE LISTING

<110> institute of microbiology of Chinese academy of sciences

<120> coronavirus trimer subunit vaccine and application 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-tagged 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 trimer 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-trimer 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-trimer 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-trimer 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-trimer 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, 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-trimer coding sequence comprising signal peptide, 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-trimer 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-trimer 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-trimer 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

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

or the like, or, alternatively,

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

or

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

preferably the antigen is a trimeric protein.

2. The antigen of claim 1, comprising an amino acid sequence as set forth in (1), (2), (3), or (4) below:

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

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

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

3. The antigen of claim 1 or 2, wherein the antigen further comprises a secretory signal peptide (e.g., 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) added at the 5 'end, a purification TAG (e.g., a 6XHis TAG) added at the 3' end, and a stop codon (e.g., TAA, TAG, or TGA).

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 claim 4.

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 to 3 in the manufacture of a medicament, such as a vaccine, against a coronavirus.

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

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