CN113230395A

CN113230395A - Beta coronavirus antigen, beta coronavirus bivalent vaccine, and preparation method and application thereof

Info

Publication number: CN113230395A
Application number: CN202110605295.5A
Authority: CN
Inventors: 高福; 戴连攀; 郑天依; 韩雨旋; 徐坤
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-05-29
Filing date: 2021-05-31
Publication date: 2021-08-10
Anticipated expiration: 2041-05-31
Also published as: CN113230395B; WO2021239147A1

Abstract

The invention relates to a beta coronavirus antigen, a beta coronavirus bivalent vaccine, and a preparation method and application thereof. The amino acid sequence of the beta coronavirus antigen comprises the following components in sequence from N end to C end: an amino acid sequence arranged in the pattern (A-B) - (A '-B') or (A-B) -C- (A '-B'), wherein: A-B represents a partial amino acid sequence or a complete amino acid sequence of a receptor binding region of a surface spike protein from a beta coronavirus, A '-B' represents a partial amino acid sequence or a complete amino acid sequence of a receptor binding region of a surface spike protein from another beta coronavirus, and C represents a linking amino acid sequence, and the beta coronavirus antigen is a single-chain heterodimer structure. The beta coronavirus antigen is used to obtain a beta coronavirus bivalent vaccine which can stimulate mice to generate strong antibody response.

Description

Beta coronavirus antigen, beta coronavirus bivalent vaccine, and preparation method and application thereof

Cross-referencing

The present application claims priority from an invention patent application entitled "a beta coronavirus antigen, beta coronavirus bivalent vaccine, methods for preparation and use thereof," filed 5/29/2020 and having application number 202010479851.4, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the field of biomedicine, and particularly relates to a beta coronavirus antigen and beta coronavirus bivalent vaccine as well as a preparation method and application thereof.

Background

Coronaviruses belong to the family coronaviridae, which in turn contains 4 genera of coronaviruses. Severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and novel coronavirus (2019-nCoV) (hereinafter abbreviated as nCoV) all belong to the genus beta coronavirus, which contains 4 subgroups (a, B, C, D), SARS-CoV and nCoV belong to subgroup B, and MERS-CoV belongs to subgroup C. They are all positive-strand RNA enveloped viruses capable of infecting humans and animals extensively, and seven kinds of coronaviruses capable of infecting humans have been identified so far, among which SARS-CoV, MERS-CoV and nCoV belonging to the genus β -coronavirus have high lethality and have caused three serious epidemics of diseases in human history. Therefore, the development of corresponding vaccines is of great significance.

The surface spike protein (S protein) is the major neutralizing antigen of coronaviruses. Receptor Binding Domains (RBDs) of the S proteins of MERS-CoV, SARS-CoV, 2019-nCoV are considered to be the most important antigen-target domains for inducing the body to produce neutralizing antibodies. The RBD as a vaccine can focus the neutralizing antibody generated by the stimulation of an organism on the combination of a receptor aiming at the virus, and can improve the immunogenicity and the immune efficiency of the vaccine. MERS-CoV invades cells by binding RBD to a receptor (CD26, also known as DPP4) of the host cell. Furthermore, both SARS-CoV and 2019-nCoV were found to enter the cell by binding of their RBD to the host cell receptor ACE 2.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

Object of the Invention

The invention aims to provide a beta coronavirus antigen, a beta coronavirus bivalent vaccine, and a preparation method and application thereof. Based on the conclusion that the RBD proteins of nCoV, MERS-CoV and SARS-CoV can excite the body to generate neutralizing antibodies, and the dimeric RBD protein can more effectively excite the immune response of the body than the monomeric RBD protein, a beta coronavirus antigen is obtained by connecting MERS-RBD, nCoV-RBD and SARS-RBD in series two by two, and a beta coronavirus bivalent vaccine is obtained by using the beta coronavirus antigen.

Solution scheme

To achieve the object of the present invention, the present embodiment provides a beta coronavirus antigen, wherein an amino acid sequence of the beta coronavirus antigen comprises, in order from N-terminus to C-terminus: an amino acid sequence arranged in the pattern (A-B) - (A '-B') or (A-B) -C- (A '-B'), wherein: A-B represents a partial amino acid sequence or a complete amino acid sequence of a receptor binding region of a surface spike protein from a beta coronavirus, A '-B' represents a partial amino acid sequence or a complete amino acid sequence of a receptor binding region of a surface spike protein from another beta coronavirus, and C represents a linking amino acid sequence, and the beta coronavirus antigen is a single-chain heterodimer structure.

Here, the "species" in the "another" is not a species in "genus species of Comamopsis of the phylum" in the biological taxonomy, but means that A-B and A '-B' are coronaviruses having different names from the genus β -coronavirus, and the expression also corresponds to the "heterologous" mutually. Such as: when A-B is from SARS-CoV, A '-B' cannot be also from SARS-CoV, but A '-B' may be from nCoV, MERS-CoV of the genus β coronavirus.

Here, when the sources of A-B and A '-B' are selected, (A-B) - (A '-B') pattern arrangement or (A-B) -C- (A '-B') pattern arrangement may be, in order from the N-terminus to the C-terminus: part of the amino acid sequence or the entire amino acid sequence of the receptor-binding region of the surface spike protein from nCoV- (C, if necessary) -part of the amino acid sequence or the entire amino acid sequence of the receptor-binding region of the surface spike protein from SARS-CoV-can also be: part of the amino acid sequence or the entire amino acid sequence of the receptor-binding region of the surface spike protein from SARS-CoV- (C, if necessary) -from the surface spike protein from nCoV. And so on.

In one possible implementation, the beta coronavirus is selected from the group consisting of: severe respiratory syndrome coronavirus, middle east respiratory syndrome coronavirus, or a novel coronavirus.

In one possible implementation, the a-B sequence is from a middle east respiratory syndrome coronavirus, the a '-B' sequence is from a severe respiratory syndrome coronavirus;

or, the A-B sequence is from middle east respiratory syndrome coronavirus, and the A '-B' sequence is from novel coronavirus;

alternatively, the A-B sequence is from a novel coronavirus and the A '-B' sequence is from a severe respiratory syndrome coronavirus.

In one possible implementation, the partial amino acid sequence of the receptor binding region of the surface spike protein from the middle east respiratory syndrome coronavirus is SEQ ID No.7 or SEQ ID No. 13. These two sequences are derived from the MERS-CoV S protein (sequences such as GenBank: AFS88936.1) RBD (367- > 606), (367- > 602) sequences, respectively. When the RBD (367-.

In one possible implementation, the partial amino acid sequence of the receptor binding region from the surface spike protein of the novel coronavirus is SEQ ID No.8 or SEQ ID No. 14. These two sequences are derived from the S protein sequence of the WH01 strain of 2019-nCoV (sequence: GenBank: YP-009724390) RBD (319-541) and (319-537), respectively. When the RBD (319) -537) sequence is selected, the protein expression level can be increased by preventing the cysteine at position 538 from disulfide mismatch.

In one possible implementation, the partial amino acid sequence of the receptor binding region of the surface spike protein from severe respiratory syndrome coronavirus is SEQ ID No.9 or SEQ ID No. 15. These two sequences are derived from the SARS-CoV S protein (sequence: GenBank: NP-828851) RBD (306-527), (306-523) sequences, respectively. When the RBD (306-523) sequence is selected, the protein expression level can be increased by preventing the cysteine at position 524 from generating disulfide bond mismatch.

In one possible implementation, the amino acid sequence of the beta coronavirus antigen is selected from the group consisting of: SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

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

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

The embodiment of the invention also provides a polynucleotide for coding the beta coronavirus antigen.

The embodiment of the invention also provides a recombinant vector comprising the polynucleotide.

The embodiment of the invention also provides an expression system cell comprising the recombinant vector; preferably, the expression system cell is a mammalian cell, further preferably, the mammalian cell is a 293T cell or a CHO cell.

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

In one possible implementation, the β -coronavirus bivalent vaccine comprises: middle east respiratory syndrome coronavirus-severe respiratory syndrome coronavirus bivalent vaccine, novel coronavirus-severe respiratory syndrome coronavirus bivalent vaccine and middle east respiratory syndrome coronavirus-novel coronavirus bivalent vaccine.

The embodiment of the invention also provides a beta coronavirus bivalent vaccine which comprises the beta coronavirus antigen and an adjuvant.

In one possible implementation, the adjuvant is selected from the group consisting of an aluminum adjuvant, an MF59 adjuvant, and an MF 59-like adjuvant.

In one possible implementation, the volume ratio of the beta coronavirus antigen to the adjuvant is 1: 1-2; alternatively, 1: 1.

the invention also provides a beta coronavirus bivalent DNA vaccine which comprises a DNA sequence for coding the beta coronavirus antigen.

The invention also provides a beta coronavirus bivalent mRNA vaccine which comprises an mRNA sequence coding the beta coronavirus antigen.

The invention also provides a beta coronavirus bigeminy virus vector vaccine, which comprises a polynucleotide for coding the beta coronavirus antigen; optionally, the viral vector is selected from one or more of the following: adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors.

Advantageous effects

(1) The beta coronavirus antigen in the embodiment of the invention is used as an antigen in the beta coronavirus bivalent vaccine, two antibodies can be generated, the times of vaccine injection by the public are reduced, and the compliance is enhanced.

(2) The use of the beta coronavirus antigen in the examples of the present invention as an antigen in a beta coronavirus bivalent vaccine can stimulate mice to produce a significantly stronger antibody response than when two beta coronavirus RBD monomers are used separately as antigens in the vaccine, respectively, e.g., when the beta coronavirus antigen comprises both MERS RBD and nCoV RBD, it can produce a higher level of specific antibodies against MERS RBD and nCoV RBD than when MERS RBD monomers and nCoV RBD monomers are used separately as vaccine antigens, respectively.

Drawings

One or more embodiments are illustrated by the corresponding figures in the drawings, which are not meant to be limiting. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1 is a Western Blot of coronavirus RBD bivalent MERS-SARS, MERS-nCoV, nCoV-SARS according to example 1 of the present invention.

FIG. 2 is a molecular sieve analysis and gel electrophoresis analysis of MERS-nCoV bivalent vaccine antigen in example 2 of the present invention.

FIG. 3 is a molecular sieve analysis and gel electrophoresis analysis of nCoV-SARS bivalent vaccine antigen in example 2 of the present invention.

FIG. 4 shows the molecular sieve analysis and gel electrophoresis analysis of MERS-SARS vaccine antigen in example 2 of the present invention.

FIG. 5 is a schematic view showing the procedure of immunization of a mouse in example 3 of the present invention.

FIG. 6 is the serum nCoV-RBD specific antibody titer induced by day 19 after the bivalent immunization of mice in example 4 of the present invention.

FIG. 7 is the serum MERS-RBD specific antibody titer induced by day 19 after the bivalent immunization of mice in example 4 of the present invention.

FIG. 8 is the serum SARS-RBD specific antibody titer induced by day 19 after the bivalent immunization of mice in example 4 of the present invention.

FIG. 9 is the serum nCoV-RBD specific antibody titer induced by day 33 after the bivalent immunization of mice in example 4 of the present invention.

FIG. 10 is the serum MERS-RBD specific antibody titer induced by day 33 after the bivalent immunization of mice in example 4 of the present invention.

FIG. 11 is the serum SARS-RBD specific antibody titer induced by day 33 after the bivalent immunization of mice in example 4 of the present invention.

FIG. 12 shows the results of measurement of the serum neutralizing antibody titer against the novel coronavirus nCoV after the bivalent immunization of mice in example 5 of the present invention.

FIG. 13 shows the results of serum neutralizing antibody titer against MERS coronavirus after two-vaccine immunization of mice in example 6 of the present invention.

FIG. 14 shows the total viral load (including replication competent live virus, residual killed virus, or viral genome fragments) in mouse lungs 5 days (5dpi) after virus challenge in example 8 of the present invention, as determined by qRT-PCR assay.

FIG. 15 shows the viral load of replication competent viable viruses in the lungs of mice 5 days (5dpi) after virus challenge, measured by qRT-PCR assay in example 8 of the present invention.

FIG. 16 shows the results of measurement of serum neutralizing antibody titer against MERS coronavirus after a second immunization of rhesus monkeys with a bivalent vaccine in example 10 of the present invention.

FIG. 17 shows the result of measurement of the serum neutralizing antibody titer against the neocoronavirus nCoV after the second immunization of rhesus monkeys with two combined vaccines in example 10 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, and the like that are well known to those skilled in the art are not described in detail in order to not unnecessarily obscure the present invention.

Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

In the following examples, the MERS-RBD (367-) (606) sequences were derived from MERS-CoV S proteins (sequences such as GenBank: AFS 88936.1). SARS-RBD (306-523) and (306-527) sequences are derived from SARS-CoV S protein (sequences such as GenBank: NP-828851). nCoV-RBD (319-537), (319-541) sequence is derived from the S protein sequence of WH01 strain of 2019-nCoV (sequence: GenBank: YP-009724390).

Example 1: design and preparation of MERS, SARS, nCoV RBD single-chain hetero-dimer

We have designed single chain RBD heterodimers obtained by concatenating two heterologous RBD subunits in order to induce simultaneous production of neutralizing antibodies against both coronaviruses.

We designed the construction of three single-chain heterodimers: (1) the MERS-RBD partial sequence (367-602) is connected with the nCoV-RBD partial sequence (319-537) in series and named as MERS-nCoV (SEQ ID NO. 1); (2) the nCoV-RBD partial sequence (319-537) is connected with the SARS-RBD partial sequence (306-523) in series and is named nCoV-SARS (SEQ ID NO. 2); (3) the MERS-RBD partial sequence (367-602) is connected in series with the SARS-RBD partial sequence (306-523) and named as MERS-SARS (SEQ ID NO. 3). The DNA sequence encoding the following amino acid sequence was inserted between the EcoRI and XhoI cleavage sites of the pCAGGS vector: the C-terminal of the above 3 amino acid sequences is added with 6 histidines, and the N-terminal is connected with a signal peptide (MIHSVFLLMFLLTPTES) (SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and the sequences comprise the sequences coding the histidines and the signal peptide). The promoter contains Kozak sequence gccacc upstream. Plasmids pCAGGS-MERS-nCoV, pCAGGS-nCoV-SARS and pCAGGS-MERS-SARS expressing three heterodimers were obtained by molecular cloning.

293T cells are transfected by the plasmids, after 48 hours, supernatant is taken, the N end of the target protein is provided with a signal peptide, and the expression of the target protein is detected by a Western Blot method (Western Blot). Western Blot results are shown in FIG. 1, and the expression cells can stably express MERS-nCoV, nCoV-SARS and MERS-SARS heterodimer RBD protein.

For comparison with the monomeric RBD antigen, plasmids expressing monomeric MERS-RBD (367-606) (SEQ ID NO.7), nCoV-RBD (319-541) (SEQ ID NO.8) and SARS-RBD (306-527) (SEQ ID NO.9) were constructed, respectively. MERS-RBD (367- & 606), nCoV-RBD (319- & 527) and SARS-RBD (306- & 527) genes are added with a sequence coding a signal peptide at the 5 'end and a sequence coding 6 histidines at the 3' end (SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, the sequences do not comprise sequences coding the histidines and the signal peptide), and are cloned between EcoRI and XhoI enzyme cutting sites of pCAGGS vectors, wherein the upstream of a promoter contains a Kozak sequence gccacc. The plasmids pCAGGS-MERS-RBD, pCAGGS-nCoV-RBD and pCAGGS-SARS-RBD for expressing three monomer RBD protein antigens are obtained by molecular cloning.

Example 2: expression and purification of MERS, SARS, nCoV single-chain heterodimer RBD protein

MERS-nCoV, nCoV-SARS and MERS-SARS single chain heterodimer RBD proteins were expressed using HEK293T cells. Plasmids pCAGGS-MERS-nCoV, pCAGGS-nCoV-SARS and pCAGGS-MERS-SARS were transfected into HEK293T cells, respectively, and after 72 hours, the supernatant was collected, centrifuged to remove the precipitate, and then filtered through a 0.22 μm filter to further remove impurities. The cell supernatant was passed through a nickel affinity column (Histrap, GE H) at 4 deg.Ceathcare) adsorption. Non-specific binding proteins were removed by washing with buffer A (20mM Tris,150mM NaCl, pH 8.0). The protein of interest is then eluted from Histrap with buffer B (20mM Tris,150mM NaCl, pH 8.0,300mM imidazole) and the eluate is concentrated by more than 30 fold using a 10kD concentration tube to buffer A with a final volume of less than 1 ml. Then passes through Superdex^TMThe 200 Increate 10/300GL column (GE Healthcare) was subjected to molecular sieve chromatography to further purify the protein of interest. The molecular sieve chromatography buffer solution is PBS buffer solution (8mM Na)₂HPO4,136mM NaCl,2mM KH₂PO₄2.6mM KCl, pH 7.2). After molecular sieve chromatography, MERS-nCoV (figure 2), nCoV-SARS (figure 3) and MERS-SARS (figure 4) all have an elution peak at about 14.5-15ml, and SDS-PAGE analysis shows that the protein is about 62KD under non-reducing (without DTT) and reducing (with DTT) conditions, and is a dimer.

As can be seen from FIGS. 2, 3 and 4, MERS-nCoV, nCoV-SARS and MERS-SARS can be correctly folded and secreted, and the high purity of the antigen protein can be obtained by purification.

Example 3: MERS-nCoV and nCoV-SARS protein immune mouse experiment

To further examine the immunogenicity of the bivalent vaccine, we immunized BALB/c mice with the purified single-chain heterodimeric protein. The BALB/c mice used were purchased from Witongli, Inc., and were all female, 6-8 weeks old. Groups of mice (5 per group) and vaccine doses are shown in table 1. Mouse immunization experiment setup: a bivalent vaccine group, which immunized mice with MERS-nCoV and nCoV-SARS (obtained in example 2) as immunogens; a single vaccine control group which immunizes mice using RBD monomer proteins of three virus S proteins of MERS-CoV, SARS-CoV and nCoV as immunogens, respectively; negative control group, which treated mice with PBS.

TABLE 1 coronavirus RBD single-chain heterodimer vaccine immunization mouse groups and doses

Diluting the immunogen with PBS to 200. mu.g/ml, and separating the resulting mixtureMF59 adjuvant-AddaVax^TMMixing and emulsifying the immunogen and the immunogen according to the volume ratio of 1:1 to prepare the vaccine. The pooled vaccine was used to immunize 5 mice per group against BALB/c. Experimental protocol in mice as shown in figure 5, all mice received 2 immunizations of vaccine by intramuscular injection on day 0 and day 21, respectively, at a vaccination volume of 100 μ l (200 μ g/ml with 200 μ l antigen +50 μ l adjuvant-500 μ l to 10 μ g). The mice were bled, centrifuged and serum was collected on days 19 and 33, stored in a refrigerator at-80 ℃ and then used for titration of antigen-specific antibody titer and pseudovirus neutralization titer.

Example 4: enzyme linked immunosorbent assay (ELISA) for detecting antigen-specific antibody titer generated by vaccine

(1) The monomeric RBD protein of MERS, SARS or nCoV was diluted to 3. mu.g/ml with an ELISA coating solution (Solebao, C1050), and 100. mu.l was added to each well of a 96-well ELISA plate (Coring, 3590), and left at 4 ℃ for 12 hours.

(2) The coating solution was poured off, PBS was added, and washed once. 5% skim milk prepared with PBS was added to a 96-well plate as a blocking solution in an amount of 100. mu.l per well, blocked, and allowed to stand at room temperature for 1 hour. After blocking, washing with PBS solution.

(3) Mouse serum was diluted during the blocking period. Serum samples were also diluted with blocking solution. Serum samples were serially diluted in 4-fold gradients starting at 20-fold. 152 mul of blocking solution and 8 mul of serum are added into the first well and mixed evenly, and the second dilution is that 120 mul of blocking solution and 40 mul of solution in the first well are mixed evenly and diluted sequentially. After dilution, 100 μ l of blocking solution was added to each well of the ELISA plate, as a negative control, and incubated at 37 degrees for 2 hours, followed by 4 washes with PBST.

(4) HRP-conjugated goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:2000 with blocking solution was added and incubated at 37 ℃ for 1.5 hours, followed by PBST washing 5-6 times. Adding 60 μ l TMB color developing solution for color development, adding 60 μ l 2M hydrochloric acid after reacting for a proper time to terminate the reaction, and detecting OD450 reading value on a microplate reader. Antibody titer values were defined as the highest dilution of serum with response values greater than 2.5 times the negative control value. The titer of this sample was defined as half the lowest dilution (limit of detection) at which the response value was still less than 2.5-fold background value, i.e., 1: 10.

And (4) analyzing results:

the results of the immunogenicity measurements of the sera after the primary immunization are shown in FIGS. 6, 7 and 8. The results show that the single chain RBD heterodimers all produced corresponding antibodies after immunization.

The ELISA results of the immune sera against the neocoronavirus nCoV-RBD are shown in FIG. 6. MERS-nCoV bigeminy induced antigen-specific IgG titers of more than about 1:1000, significantly increased over MERS-RBD monomer induced specific antibody levels (× P <0.001), significantly increased over nCoV-RBD monomer induced specific antibody levels (× P <0.0001), and significantly increased over PBS control immunized group induced specific antibody levels (× P < 0.0001). Furthermore, MERS-nCoV bigeminy induced specific antibody levels against nCoV-RBD also significantly increased (P <0.001) over nCoV-SARS production, as shown in fig. 6.

The ELISA results of the immune sera against the new coronavirus MERS-RBD are shown in FIG. 7. MERS-nCoV bigeminy induced antigen specific IgG titers above about 1: 5000. The levels of specific antibodies induced by MERS-RBD monomers were significantly increased (× P <0.0001), compared to nCoV-RBD monomers (× P <0.0001), and compared to PBS control immunized groups (P <0.0001), as shown in fig. 7.

The ELISA results of the immune sera against the novel coronavirus SARS-RBD are shown in FIG. 8. One immunization of nCoV-SARS bigeminy induced antigen specific IgG titer above about 1:500, and compared with nCoV-RBD monomer, SARS-RBD and PBS all induced specific antibody level significantly increased (P < 0.05;. P < 0.01).

Mice were immunized a second time on day 21, sera from the secondary immunization were collected on day 33, and serum antibody titers from mice on day 33 of the ELISA experiment (i.e., post-secondary immunization) were shown in fig. 9, 10, and 11. The results show that the RBD heterodimer induced higher levels of antibodies after immunization of mice, and that there was a significant difference in the level of antibody reactivity of RBD heterodimer with RBD monomer-induced mice.

The results of ELISA against neocoronavirus nCoV-RBD in the sera of the immunized mice are shown in FIG. 9. MERS-nCoV bigeminy induces 1:10⁵The above antigenic peptidesSpecific IgG titer, compared to MERS-RBD monomer induced specific antibody levels significantly increased (. about.P)<0.0001), significantly increased levels of specific antibodies (P) compared to nCoV-RBD monomer induction<0.0001), significantly increased levels of specific antibodies induced by the PBS control immunised group (. about.p)<0.0001). In addition, MERS-nCoV bigeminy induced specific antibody level against nCoV-RBD, also significantly increased over nCoV-SARS production (. about.P.)<0.0001)。

The results of ELISA against the serum of the immunized mice for the new coronavirus MERS-RBD are shown in FIG. 10. MERS-nCoV bigeminy induces approximately 1:10⁶The antigen-specific IgG titer of (1) is obviously improved compared with the specific antibody level (P) induced by MERS-RBD monomer<0.01), significantly higher levels of specific antibodies than induced by nCoV monomers (. about.p)<0.01), induced significant increase in specific antibody levels (. about.p) over PBS control immunized group<0.01)。

The results of ELISA against the novel coronavirus SARS-RBD in the sera of the immunized mice are shown in FIG. 11. nCoV-SARS bigeminy vaccine secondary immunity induced over 1:10⁵The above antigen-specific IgG titers all significantly increased the levels of specific antibodies induced by SARS RBD monomer, nCoV-RBD monomer and PBS group (. about.P.)<0.001；****P<0.0001)。

Example 5 pseudovirus neutralization assay the neutralizing antibody titer against the novel coronavirus produced by the vaccine was tested

The sera obtained in example 3 were diluted in multiples and the serial dilutions were compared with 100TCID₅₀Pseudoviruses were mixed and incubated for 1 hour at 37 ℃. The mixture was added to a 96-well plate that had been confluent with Huh7 cells. After incubation at 37 ℃ for 24 hours, the culture medium was discarded, the cells were washed 2 times with PBS, and cell lysate was added to detect the luciferase activity.

The results of the immunogenicity test of the sera after the secondary immunization are shown in fig. 12, and the results in fig. 12 show: MERS-nCoV bivalent vaccine produced neutralizing antibody after secondary immunization, and the neutralizing titer of mouse serum (NT) is 90 percent₉₀) Can reach more than 1: 1000. Whereas only four of 8 mice immunized with nCoV RBD monomer produced weakly neutralizing antibodies (of which 2 NT were present₉₀NT of 1:20, another 2₉₀1: 40).This result demonstrates that MERS-nCoV bivalent vaccine can induce much higher levels of neutralizing antibodies than nCoV RBD monomer by pseudovirus neutralization experiments<0.01) which is a good candidate vaccine for new coronaviruses.

Example 6 pseudovirus neutralization assay detection of neutralizing antibody titers against MERS tubular virus produced by vaccines

The sera obtained in example 3 were diluted in multiples and the serial dilutions were compared with 100TCID₅₀Pseudoviruses were mixed and incubated for 1 hour at 37 ℃. The mixture was added to a 96-well plate that had been confluent with Huh7 cells. After incubation at 37 ℃ for 48 hours, the culture medium was discarded, the cells were washed 2 times with PBS, and cell lysate was added to detect the luciferase activity.

The results of the immunogenicity assay of sera after the second immunization are shown in fig. 13, which fig. 13 shows: MERS-nCoV bivalent vaccine generates neutralizing antibody, NT after secondary immunization₉₀Can reach more than 1: 1000; comparable to levels of neutralizing antibodies induced by MERS RBD homodimer (n.s.). The result shows that the MERS-nCoV bivalent vaccine can induce mice to generate high-level antibody response and is a good MERS-CoV candidate vaccine proved by a pseudovirus neutralization experiment.

Example 7 protection experiments against viral challenge in mouse model

32 BALB/c mice were randomly divided into 4 groups of 8 mice each. All BALB/c mice were purchased from Witongli Inc., female, 6-8 weeks old. Mouse immunization experiment setup: a bivalent vaccine group which immunizes mice using MERS-nCoV bivalent vaccine (obtained in example 2) as an immunogen; a single vaccine control group which immunizes mice using RBD monomer protein and RBD homodimer protein of S protein of neocoronavirus, respectively, as immunogens; negative control group, which treated mice with PBS. The vaccine doses for immunization were as in table 1 in example 3. At 39 days after the second immunization (i.e., 74 days after the first immunization), we performed nasal drip infection of mice with Ad5-ACE2 deficient adenovirus to induce lung expression of human ACE2 receptor protein in mice, which were then transferred to the microbiology institute (A) BSL-3 laboratory. On day 79 post immunization, mice were nasally infected with a new coronavirus (hCoV-19/China/CAS-B001/2020(GISAID databases EPI _ ISL _514256-7) strain). The virus infection dose is as follows: virus stock 50. mu.L/mouse. The toxic counteracting approach is nasal drop infection. The specific operation is as follows: diluted tribromoethanol (concentration 20mg/ml) was injected intraperitoneally (i.p.) of mice for anesthesia, 250 μ L per mouse. After the mice were completely anesthetized, 50. mu.L of the virus solution was pipetted using a 200. mu.L pipette to perform nasal drip.

Mice were euthanized 5 days after virus challenge. Then, the lungs were dissected out and placed in 2mL tubes (which had been weighed in advance), and weighed to calculate the net weight of the lungs. The lungs of the mice were ground using a grinder, after which the supernatants were isolated. The resulting supernatant was then carried out of (A) BSL-3 laboratory after virus inactivation. Viral RNA was extracted from the supernatant using a Kit (QIAamp Viral RNA Mini Kit).

Example 8 detection of viral load of lungs of mice 5 days (5dpi) after challenge with New coronavirus by qRT-PCR experiment

In the embodiment, the viral load of the lung of the mouse after virus challenge is detected by using a qRT-PCR method; the probe and the primer can be combined on the N gene of the genome of the novel coronavirus, and the sequences are respectively as follows:

N-F:GACCCCAAAATCAGCGAAAT(SEQ ID NO:16)；

N-R:TCTGGTTACTGCCAGTTGAATCTG(SEQ ID NO:17)；

N-probe:ACCCCGCATTACGTTTGGTGGACC(SEQ ID NO:18)；

the fast king one-step reverse transcription-fluorescence quantitative kit (probe method, product number FP314) of Tiangen Biotechnology company is used, and qRT-PCR experimental operation is carried out according to the kit instruction method; the experimental results showed that the viral load of mice in the MERS-nCoV bivalent vaccine group was about 1000-fold lower than that of the PBS negative control group (as shown in fig. 14). Because a large amount of virus particles are dripped into a mouse in virus challenge, a probe primer targeting the N gene of a virus genome cannot distinguish whether active virus or residual dead virus or virus genome fragments are detected, so another set of probe primer capable of combining with sgRNA of a new coronavirus is used for detection, and the amount of the sgRNA can represent the amount of replication-competent live virus because the sgRNA is generated only in the replication process of active virus cells. Primer probe sequences for detecting sgrnas are as follows:

sgRNA-F:CGATCTCTTGTAGATCTGTTCTC(SEQ ID NO:19)；

sgRNA-R:TGTGTGCGTACTGCTGCAATAT(SEQ ID NO:20)；

sgRNA-probe:ACACTAGCCATCCTTACTGCGCTTCG(SEQ ID NO:21)；

the qRT-PCR assay results are shown in FIG. 15, and the results in FIG. 15 show: in the MERS-nCoV combined vaccine group, no replicative virus can be completely detected, while in the PBS negative control group and the nCoV RBD monomer control group, the replicative virus is still maintained at a higher level, and the result shows that the MERS-nCoV combined vaccine has a good protection effect on mice against new coronavirus infection.

Example 9 protection experiments against viral challenge in the rhesus monkey model

To further examine the protective efficacy of MERS-nCoV bivalent vaccines, we performed protection experiments against virus challenge in the rhesus monkey model. To this end, we immunized rhesus monkeys with purified MERS-nCoV bivalent vaccine protein. Rhesus monkey sources used: institute of medical laboratory animals, 4-6 years old, grade SARS-CoV-2 Free. Rhesus groups (3 per group) and vaccine doses are shown in table 2. Mouse immunization experiment setup: a bivalent vaccine group that immunizes rhesus monkeys using MERS-nCoV as an immunogen: negative control group, rhesus monkey was treated with PBS.

Table 2: experimental groups and dosing of rhesus monkeys

The operation process is as follows: diluting an immunogen MERS-nCoV protein to 100 mu g/ml by PBS, mixing and emulsifying an MF 59-like adjuvant and the immunogen diluent according to the volume ratio of 1:1, and preparing a vaccine; all rhesus monkeys were given 2 vaccinations, each at a volume of 500 μ l, by intramuscular injection, on days 0 and 28, respectively (with 250 μ l immunogen dilution +250 μ l adjuvant mixed, actual dose of immunogen per vaccination 100 μ g/ml 250 μ l to 25 μ g). On day 35, rhesus monkeys were bled, serum was collected by centrifugation, stored in a freezer at-80 ℃ and then used for titration of the pseudovirus neutralizing titer.

Example 10 detection of neutralizing antibody titres generated by rhesus monkeys after vaccine immunization by a pseudovirus neutralization assay

The neutralizing antibody titers against the new coronavirus nCoV and MERS coronavirus in the rhesus monkey sera obtained in example 9 were determined according to the specific procedures described in examples 5 and 6. After the secondary immunization, the detection results of the immunogenicity of the sera against MERS coronavirus and nCoV new coronavirus are respectively shown in FIG. 16 and FIG. 17; the results of FIGS. 16 and 17 show that the MERS-nCoV vaccine immunization group generated neutralizing antibodies against the novel coronavirus and the MERS coronavirus after the secondary immunization, wherein NT against the MERS coronavirus₉₀Up to about 1:10 or more (FIG. 16), NT against neocoronaviruses₉₀Up to about 1:100 or more (fig. 17); the neutralizing antibodies of the PBS control group were negative. The result shows that the MERS-nCoV bivalent vaccine can induce rhesus monkey to generate a neutralizing antibody reaction and is a good bivalent candidate vaccine for resisting the new coronavirus and the MERS coronavirus.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> beta coronavirus antigen, beta coronavirus bivalent vaccine, preparation method and application thereof

<130> 1087-200261F-1

<160> 21

<170> PatentIn version 3.5

<210> 1

<211> 455

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> DOMAIN

<222> (1)..(455)

<223> 367-

319-537 sequence in RBD of the S protein sequence of WH01 strain of nCoV (sequence: GenBank: YP-009724390)

<400> 1

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 Arg Val Gln Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Thr Asn Leu Val Lys Asn Lys

450 455

<210> 2

<211> 437

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> DOMAIN

<222> (1)..(437)

<223> S protein sequence from WH01 strain of 2019-nCoV (sequence as GenBank:

YP-009724390) RBD 319-537 tandem SARS-CoV S protein (sequence as GenBank:

NP-828851) RBD 306-523 sequence

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr

340 345 350

Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp

355 360 365

Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro

370 375 380

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

385 390 395 400

Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu

405 410 415

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

420 425 430

Leu Ile Lys Asn Gln

435

<210> 3

<211> 454

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> DOMAIN

<222> (1)..(454)

<223> MERS-CoV S protein (sequence such as GenBank: AFS88936.1) RBD 367-602 sequence tandem 2019

-nCoV S protein (sequence e.g. GenBank: NP-828851) RBD 306-523 sequence

<400> 3

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 Arg Val Val Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn

355 360 365

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

370 375 380

Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Asp Leu Ile Lys Asn Gln

450

<210> 4

<211> 1434

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> gene

<222> (1)..(1434)

<223> DNA sequence (with DNA sequence coding for histidine and signal peptide) expressing the recombinant protein of SEQ ID NO.1 MERS-nCoV

<400> 4

atgatacact cagtgtttct actgatgttc ttgttaacac ctacagaaag tgaagcaaaa 60

ccttctggct cagttgtgga acaggctgaa ggtgttgaat gtgatttttc acctcttctg 120

tctggcacac ctcctcaggt ttataatttc aagcgtttgg tttttaccaa ttgcaattat 180

aatcttacca aattgctttc acttttttct gtgaatgatt ttacttgtag tcaaatatct 240

ccagcagcaa ttgctagcaa ctgttattct tcactgattt tggattactt ttcataccca 300

cttagtatga aatccgatct cagtgttagt tctgctggtc caatatccca gtttaattat 360

aaacagtcct tttctaatcc cacatgtttg attttagcga ctgttcctca taaccttact 420

actattacta agcctcttaa gtacagctat attaacaagt gctctcgtct tctttctgat 480

gatcgtactg aagtacctca gttagtgaac gctaatcaat actcaccctg tgtatccatt 540

gtcccatcca ctgtgtggga agacggtgat tattatagga aacaactatc tccacttgaa 600

ggtggtggct ggcttgttgc tagtggctca actgttgcca tgactgagca attacagatg 660

ggctttggta ttacagttca atatggtaca gacaccaata gtgtttgccc caagcttgaa 720

tttgctaatg acacaaaaat tgcctctcaa ttaggcaatc gtgttcagcc tactgaatcg 780

atcgtgaggt tcccaaatat taccaatctg tgtccgttcg gagaggtctt caatgcgact 840

cgattcgcgt ctgtttacgc ctggaacagg aaacggatta gcaattgtgt cgctgactat 900

tcggtcttat acaactctgc atcattctca accttcaagt gttatggtgt cagccctaca 960

aagctgaatg acttatgttt caccaatgtt tatgcggaca gtttcgtaat acgaggtgat 1020

gaagtccgcc aaattgcacc cggacaaacc ggcaagatag ccgactataa ttataagctc 1080

cctgatgact ttacgggctg tgtcatagct tggaatagta ataatttgga ctcgaaagtg 1140

ggaggtaatt ataattatct ctatagactg ttccggaaat caaatctcaa gccctttgaa 1200

cgggacataa gtacagaaat ctaccaagct ggttccacgc cgtgtaatgg agtcgagggg 1260

tttaactgtt atttcccgct ccagtcgtat gggttccagc caacgaatgg cgtcggatac 1320

caaccttacc gcgttgtagt attaagcttt gaactgttgc acgcgcccgc gactgtttgt 1380

ggcccgaaga agtcgactaa tctagtaaag aataagcatc atcaccacca ccac 1434

<210> 5

<211> 1380

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> gene

<222> (1)..(1380)

<223> DNA sequence expressing SEQ ID NO.2 nCoV-SARS recombinant protein (DNA sequence with coding histidine and signal peptide)

<400> 5

atgatccact cagtgtttct cttaatgttt ctactaactc ccacggagtc gcgagtgcag 60

cctaccgaaa gcatcgtccg tttcccgaat attactaatc tctgtccatt cggagaagtc 120

ttcaatgcca cccgattcgc ttccgtttac gcgtggaacc gtaaacgaat atctaattgt 180

gttgcggact attccgtgtt gtacaactca gcatcattct ctacttttaa atgctatgga 240

gtgtcgccga ctaaactcaa cgacttgtgt ttcactaatg tttatgctga ctctttcgtt 300

attcgtggag acgaagttcg tcaaatcgca ccagggcaaa ctggcaagat tgcggactat 360

aattataagc tgccagatga ctttaccgga tgtgtaatag cctggaactc aaataatctc 420

gacagtaaag tgggaggcaa ctataattat ctttatcgac tcttcagaaa gtctaacctt 480

aagccatttg aacgtgacat ttctacagaa atttaccaag ccggctctac accttgcaat 540

ggcgtggaag ggtttaactg ttatttccca ttacagtctt atggtttcca gccaactaat 600

ggtgtgggat accaacctta ccgcgtcgtt gtcctgtcgt ttgaattgct tcacgcacca 660

gccaccgttt gtgggccaaa gaagagcact aatctcgtaa agaataaacg tgttgtccca 720

tccggtgacg ttgtccggtt tcctaacatc acaaacttgt gtccctttgg cgaagtcttc 780

aatgctacca aatttcccag cgtctacgcg tgggaaagaa agaaaatatc aaattgtgtt 840

gccgactatt ccgtcctata taatagcacg ttcttctcga cgttcaagtg ttatggtgtc 900

tctgctacga aacttaacga cttatgtttc tcaaacgtgt acgcagattc tttcgtagtt 960

aaaggtgatg atgtgaggca gattgcgccc ggacaaacag gagtaatcgc cgattacaac 1020

tacaaactcc cggacgactt tatggggtgt gtgttagctt ggaatacgag gaatatagac 1080

gccacgagta ccgggaatta taattataag tatcgctatc tccgacatgg caaactcagg 1140

ccatttgaac gcgacattag caatgttcca ttctctccgg acggcaaacc gtgcactcca 1200

ccggctttaa attgttattg gccgttaaac gactatggct tttatacaac gacgggaata 1260

gggtaccaac cttacagagt agtagtacta agtttcgagc tattaaatgc gccggccacc 1320

gtatgtgggc ccaagctatc gacggaccta atcaagaatc agcaccacca ccaccaccac 1380

<210> 6

<211> 1431

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> gene

<222> (1)..(1431)

<223> DNA sequence expressing SEQ ID NO.3 MERS-SARS recombinant protein (DNA sequence with coding histidine and signal peptide)

<400> 6

atgatacact cagtgtttct actgatgttc ttgttaacac ctacagaaag tgaagcaaaa 60

ccttctggct cagttgtgga acaggctgaa ggtgttgaat gtgatttttc acctcttctg 120

tctggcacac ctcctcaggt ttataatttc aagcgtttgg tttttaccaa ttgcaattat 180

aatcttacca aattgctttc acttttttct gtgaatgatt ttacttgtag tcaaatatct 240

ccagcagcaa ttgctagcaa ctgttattct tcactgattt tggattactt ttcataccca 300

cttagtatga aatccgatct cagtgttagt tctgctggtc caatatccca gtttaattat 360

aaacagtcct tttctaatcc cacatgtttg attttagcga ctgttcctca taaccttact 420

actattacta agcctcttaa gtacagctat attaacaagt gctctcgtct tctttctgat 480

gatcgtactg aagtacctca gttagtgaac gctaatcaat actcaccctg tgtatccatt 540

gtcccatcca ctgtgtggga agacggtgat tattatagga aacaactatc tccacttgaa 600

ggtggtggct ggcttgttgc tagtggctca actgttgcca tgactgagca attacagatg 660

ggctttggta ttacagttca atatggtaca gacaccaata gtgtttgccc caagcttgaa 720

tttgctaatg acacaaaaat tgcctctcaa ttaggcaatc gtgttgtccc atccggtgac 780

gttgtccggt ttcctaacat cacaaacttg tgtccctttg gcgaagtctt caatgctacc 840

aaatttccca gcgtctacgc gtgggaaaga aagaaaatat caaattgtgt tgccgactat 900

tccgtcctat ataatagcac gttcttctcg acgttcaagt gttatggtgt ctctgctacg 960

aaacttaacg acttatgttt ctcaaacgtg tacgcagatt ctttcgtagt taaaggtgat 1020

gatgtgaggc agattgcgcc cggacaaaca ggagtaatcg ccgattacaa ctacaaactc 1080

ccggacgact ttatggggtg tgtgttagct tggaatacga ggaatataga cgccacgagt 1140

accgggaatt ataattataa gtatcgctat ctccgacatg gcaaactcag gccatttgaa 1200

cgcgacatta gcaatgttcc attctctccg gacggcaaac cgtgcactcc accggcttta 1260

aattgttatt ggccgttaaa cgactatggc ttttatacaa cgacgggaat agggtaccaa 1320

ccttacagag tagtagtact aagtttcgag ctattaaatg cgccggccac cgtatgtggg 1380

cccaagctat cgacggacct aatcaagaat cagcaccacc accaccacca c 1431

<210> 7

<211> 240

<212> PRT

<213> MERS-CoV

<220>

<221> DOMAIN

<222> (1)..(240)

<223> MERS-CoV S protein (sequence such as GenBank: AFS88936.1) RBD 367-606 sequence

<400> 7

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 Cys Val Glu Tyr

225 230 235 240

<210> 8

<211> 223

<212> PRT

<213> nCoV

<220>

<221> DOMAIN

<222> (1)..(223)

<223> S protein sequence from WH01 strain of 2019-nCoV (sequence as GenBank:

YP-009724390) RBD 319-541 sequences

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210> 9

<211> 222

<212> PRT

<213> SARS-CoV

<220>

<221> DOMAIN

<222> (1)..(222)

<223> RBD 306-527 sequence from SARS-CoV S protein (sequence: GenBank: NP-828851)

<400> 9

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 Cys Val Asn Phe

210 215 220

<210> 10

<211> 720

<212> DNA

<213> MERS-CoV

<220>

<221> gene

<222> (1)..(720)

<223> DNA sequence encoding SEQ ID NO.7 MERS-CoV S protein RBD 367-606 sequence

<400> 10

gaagcaaaac cttctggctc agttgtggaa caggctgaag gtgttgaatg tgatttttca 60

cctcttctgt ctggcacacc tcctcaggtt tataatttca agcgtttggt ttttaccaat 120

tgcaattata atcttaccaa attgctttca cttttttctg tgaatgattt tacttgtagt 180

caaatatctc cagcagcaat tgctagcaac tgttattctt cactgatttt ggattacttt 240

tcatacccac ttagtatgaa atccgatctc agtgttagtt ctgctggtcc aatatcccag 300

tttaattata aacagtcctt ttctaatccc acatgtttga ttttagcgac tgttcctcat 360

aaccttacta ctattactaa gcctcttaag tacagctata ttaacaagtg ctctcgtctt 420

ctttctgatg atcgtactga agtacctcag ttagtgaacg ctaatcaata ctcaccctgt 480

gtatccattg tcccatccac tgtgtgggaa gacggtgatt attataggaa acaactatct 540

ccacttgaag gtggtggctg gcttgttgct agtggctcaa ctgttgccat gactgagcaa 600

ttacagatgg gctttggtat tacagttcaa tatggtacag acaccaatag tgtttgcccc 660

aagcttgaat ttgctaatga cacaaaaatt gcctctcaat taggcaattg cgtggaatac 720

<210> 11

<211> 669

<212> DNA

<213> nCoV

<220>

<221> gene

<222> (1)..(669)

<223> DNA sequence encoding the sequence of the S protein RBD 319-541 of the WH01 strain of SEQ ID NO. 82019-nCoV

<400> 11

agagtgcaac ctacagaatc aatcgtgaga tttcctaaca tcacaaacct ttgccctttc 60

ggcgaggtgt ttaacgcaac aagatttgca tcagtgtacg catggaacag aaagcgtata 120

tcaaactgcg tggcagatta ctcagtgctt tacaactcag catcattcag tacgtttaaa 180

tgctacggag tgtcacctac aaagctaaat gatctttgct ttacaaacgt gtacgcagat 240

tcatttgtga tcagaggaga tgaagtgaga caaatcgcac ctggacaaac aggaaagatt 300

gccgattaca actacaaact tcctgatgat ttcaccggct gcgtgatcgc atggaactca 360

aacaaccttg attcaaaggt aggtggtaat tataattatt tgtataggct ctttcgtaag 420

agcaacttaa agccatttga gcgagatatc tcaacagaaa tctaccaagc aggatcaaca 480

ccttgcaacg gagtggaagg atttaactgc tactttcctc ttcaatcata cggatttcaa 540

cctacaaacg gagtgggata ccaaccttac agagtggtgg tgctttcatt tgaacttctt 600

cacgcacctg caacagtgtg cggacctaag aagagcacga accttgtgaa gaataagtgc 660

gtgaacttt 669

<210> 12

<211> 666

<212> DNA

<213> SARS-CoV

<220>

<221> gene

<222> (1)..(666)

<223> DNA sequence encoding SEQ ID NO.9 SARS-CoV S protein RBD 306-527 sequence

<400> 12

cgtgttgtcc catccggtga cgttgtccgg tttcctaaca tcacaaactt gtgtcccttt 60

ggcgaagtct tcaatgctac caaatttccc agcgtctacg cgtgggaaag aaagaaaata 120

tcaaattgtg ttgccgacta ttccgtccta tataatagca cgttcttctc gacgttcaag 180

tgttatggtg tctctgctac gaaacttaac gacttatgtt tctcaaacgt gtacgcagat 240

tctttcgtag ttaaaggtga tgatgtgagg cagattgcgc ccggacaaac aggagtaatc 300

gccgattaca actacaaact cccggacgac tttatggggt gtgtgttagc ttggaatacg 360

aggaatatag acgccacgag taccgggaat tataattata agtatcgcta tctccgacat 420

ggcaaactca ggccatttga acgcgacatt agcaatgttc cattctctcc ggacggcaaa 480

ccgtgcactc caccggcttt aaattgttat tggccgttaa acgactatgg cttttataca 540

acgacgggaa tagggtacca accttacaga gtagtagtac taagtttcga gctattaaat 600

gcgccggcca ccgtatgtgg gcccaagcta tcgacggacc taatcaagaa tcagtgtgtt 660

aatttc 666

<210> 13

<211> 236

<212> PRT

<213> MERS-CoV

<220>

<221> DOMAIN

<222> (1)..(236)

<223> sequence derived from MERS-CoV S protein (sequence such as GenBank: AFS88936.1) RBD 367-

<400> 13

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> 14

<211> 219

<212> PRT

<213> nCoV

<220>

<221> DOMAIN

<222> (1)..(219)

<223> S protein sequence from WH01 strain of 2019-nCoV (sequence as GenBank:

YP _009724390) RBD 319-537 sequences

<400> 14

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> 15

<211> 218

<212> PRT

<213> SARS-CoV

<220>

<221> DOMAIN

<222> (1)..(218)

<223> sequence of RBD 306-523 from SARS-CoV S protein (sequence: GenBank: NP-828851)

<400> 15

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> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<223> Forward primer N-F for detecting N gene of novel coronavirus genome

<400> 16

gaccccaaaa tcagcgaaat 20

<210> 17

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<223> reverse primer N-R for detecting N gene of novel coronavirus genome

<400> 17

tctggttact gccagttgaa tctg 24

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<223> Probe N-Probe for detecting N Gene of novel coronavirus genome

<400> 18

accccgcatt acgtttggtg gacc 24

<210> 19

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<223> forward primer sgRNA-F for detecting sgRNA of novel coronavirus

<400> 19

cgatctcttg tagatctgtt ctc 23

<210> 20

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<223> reverse primer sgRNA-R for detecting sgRNA of novel coronavirus

<400> 20

tgtgtgcgta ctgctgcaat at 22

<210> 21

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<223> Probe sgRNA-probe for detecting sgRNA of novel coronavirus

<400> 21

acactagcca tccttactgc gcttcg 26

Claims

1. a betacoronavirus antigen, is characterized in that: the aminoacid sequence of described betacoronavirus antigen comprises according to the sequence from N end to C end: the aminoacid sequence arranged according to (A-B)-(A'-B') pattern or Amino acid sequences arranged in (A-B)-C-(A'-B') pattern, wherein: A-B represents the partial amino acid sequence or the entire amino acid sequence of the receptor binding region of the surface spike protein from betacoronavirus, A'-B ' represents the partial amino acid sequence or the entire amino acid sequence of the receptor binding region of the surface spike protein from another betacoronavirus, C represents the connecting amino acid sequence, and the betacoronavirus antigen is a single-chain heterodimer structure.

2. betacoronavirus antigen according to claim 1, is characterized in that: betacoronavirus is selected from: severe respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus or novel coronavirus.

3. betacoronavirus antigen according to claim 1, is characterized in that: A-B sequence is from Middle East respiratory syndrome coronavirus, and A'-B' sequence is from severe respiratory syndrome coronavirus;

Or, the A-B sequence is from Middle East respiratory syndrome coronavirus, and the A'-B' sequence is from the new coronavirus;

Or, the A-B sequence is from the new coronavirus, and the A'-B' sequence is from the severe respiratory syndrome coronavirus.

4. betacoronavirus antigen according to claim 3 is characterized in that: the partial amino acid sequence of the receptor binding region from the surface spike protein of Middle East Respiratory Syndrome Coronavirus is SEQ ID NO.7 and SEQ ID NO. 13;

And/or, the partial amino acid sequence of the receptor binding region of the surface spike protein from the novel coronavirus is SEQ ID NO.8 or SEQ ID NO.14;

And/or, the partial amino acid sequence of the receptor binding region of the surface spike protein from severe respiratory syndrome coronavirus is SEQ ID NO.9 or SEQ ID NO.15.

5. betacoronavirus antigen according to claim 1, is characterized in that: the amino acid sequence of betacoronavirus antigen is selected from: SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

6. a method for preparing the described betacoronavirus antigen of one of claims 1-5, is characterized in that: comprise the following steps: in the nucleotide of the described betacoronavirus antigen described in one of coding claims 1-5 The sequence encoding the signal peptide is added to the 5' end of the sequence, and the sequence encoding histidine is added to the 3' end to clone and express, screen the correct recombinant, and then transfect the cells of the expression system for expression, and collect the cell supernatant after expression. , purified to obtain betacoronavirus antigen.

7. The method according to claim 6, wherein: the cells of the expression system include mammalian cells, insect cells, yeast cells or bacterial cells, optionally; the mammalian cells include 293T cells or CHO cells cells, including Escherichia coli cells.

8. A polynucleotide encoding the betacoronavirus antigen of one of claims 1-5.

9. A recombinant vector comprising the polynucleotide of claim 8.

10. An expression system cell comprising the recombinant vector of claim 9; preferably, the expression system cell is a mammalian cell; further preferably, the mammalian cell is a 293T cell or a CHO cell.

11. the described βcoronavirus antigen of one of claim 1-5, the described polynucleotide of claim 8, the described recombinant vector of claim 9 or the described expression system cell of claim 10 are preparing β The application of coronavirus dual vaccine.

12. A betacoronavirus dual vaccine comprising the betacoronavirus antigen and adjuvant described in one of claims 1-5.

13. betacoronavirus dual vaccine according to claim 12, is characterized in that: described betacoronavirus dual vaccine comprises: Middle East respiratory syndrome coronavirus-severe respiratory syndrome coronavirus dual vaccine, novel coronavirus - Severe respiratory syndrome coronavirus dual vaccine, Middle East respiratory syndrome coronavirus-novel coronavirus dual vaccine.

14. The betacoronavirus dual vaccine according to claim 12 or 13, wherein the adjuvant is selected from aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.

15. The betacoronavirus dual vaccine according to any one of claims 12-14, wherein the volume ratio of the betacoronavirus antigen described in one of claims 1-5 and the adjuvant is 1:1-2 ; optionally, 1:1.

16. A betacoronavirus duplex DNA vaccine comprising a DNA sequence encoding the betacoronavirus antigen described in one of claims 1-5.

17. A betacoronavirus duplex mRNA vaccine comprising the mRNA sequence encoding the betacoronavirus antigen described in one of claims 1-5.

18. a betacoronavirus dual viral vector vaccine, it comprises the polynucleotide of the described betacoronavirus antigen of one of coding claims 1-5; Alternatively, viral vector is selected from following one or more : Adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.