CN114989308B - Novel coronavirus chimeric nucleic acid vaccine and use thereof - Google Patents

Abstract

The invention relates to a polynucleotide, related products and application thereof in preparing a novel corona vaccine, and a chimeric nucleic acid vaccine or an immunogenic composition based on the polynucleotide; the polynucleotide codes a recombinant chimeric antigen formed by directly connecting an RBD structural domain of S protein of a new coronavirus prototype strain and an RBD structural domain of S protein of a Beta variant strain, or connecting an RBD structural domain of S protein of a Delta variant strain and the RBD structural domain of S protein of the Beta variant strain, or connecting the RBD structural domain of S protein of the Delta variant strain and the RBD structural domain of S protein of an Omicron variant strain in series or through a linker; the chimeric nucleic acid vaccine based on the polynucleotide can provide stronger immune protection efficacy against a plurality of new coronavirus strains, and can induce a remarkably increased immune response level (namely, broad spectrum) against each type of new coronavirus strains when being sequentially immunized with other types of vaccines.

Description

Novel coronavirus chimeric nucleic acid vaccine and its application

technical field

The invention relates to the field of biomedicine, in particular to a novel coronavirus chimeric nucleic acid vaccine and its application.

Background technique

Novel coronavirus pneumonia (also known as COVID-19) is an acute respiratory infectious disease caused by infection with a new type of coronavirus (also known as new coronavirus, SARS-CoV-2). The new coronavirus belongs to the β-coronavirus genus of the Coronaviridae family, has an envelope, and is a single-stranded positive-sense RNA virus. The spike protein (also known as S protein) on the surface of the new coronavirus is responsible for the binding of the virus to the host cell membrane receptor and membrane fusion. There is a receptor binding domain (RBD) on the S protein, which is an important vaccine target and can stimulate The production of neutralizing antibodies has the advantage of immunofocusing.

At present, the new crown pneumonia epidemic is still severe worldwide, and new coronavirus variants continue to emerge and spread, especially the Delta and Omicron variants have swept the world in turn and become the dominant epidemic strains . In particular, there are multiple subtypes of the new coronavirus Omicron variant (for example, BA.1, BA.2, BA.1.1, BA.3 subtypes), which have a transmission speed exceeding Delta and have become the dominant strain in the world ; Among them, the BA.2 subtype strains spread faster than other Omicron subtypes and now account for the largest proportion. There are more than 50 amino acid mutations in the S protein of the Omicron mutant strain, which is much more than that of the previous Delta mutant strain.

Due to the many mutations in the sequence of S protein or RBD in these newly emerged new coronavirus variants, the immune response induced by the existing vaccines designed and developed based on the new coronavirus prototype strain (Prototype) is not effective in the face of mutant strains (such as Omicron). The effectiveness of the mutant strain is greatly reduced, and the phenomenon that the mutant strain breaks through the vaccine and antibody protection is called immune escape. The phenomenon of immune escape is particularly evident in various subtypes of Omicron. In order to solve the immune escape problem of the new coronavirus mutant strain, it is necessary to develop a new type of vaccine to adapt to the emerging mutant strain (such as the Omicron mutant strain and its various subtypes), so that it has a strong protective effect on the current epidemic strain; At the same time, due to the simultaneous prevalence of multiple mutant strains (especially multiple Omicron subtypes), the new vaccine needs to be able to induce a broad-spectrum immune response to protect against multiple new coronavirus strains at the same time as much as possible. Epidemic prevention and control can play a vital role.

The information disclosed in this Background section is only for enhancing the understanding of the general background of the present invention and should not be taken as an acknowledgment or any form of suggestion that the information constitutes the prior art that is already known to those skilled in the art.

Contents of the invention

In order to overcome the problems in the above-mentioned prior art, the present invention provides a polynucleotide, its related products and its use in the preparation of a vaccine for the prevention and/or treatment of new coronavirus, based on the polynucleotide or its related products A chimeric nucleic acid vaccine or an immunogenic composition of a product, and a kit comprising the chimeric nucleic acid vaccine or an immunogenic composition; the polynucleotide code is composed of (1) the S protein RBD domain of the novel coronavirus prototype strain (or a part of it) with the RBD domain of the S protein of the new coronavirus Beta variant (or a part thereof), or (2) the S protein RBD domain of the new coronavirus Delta variant (or a part thereof) with the new coronavirus Beta variant The S protein RBD domain (or a part thereof), or (3) the new coronavirus Delta variant S protein RBD domain (or a part thereof) is directly connected in series with the new coronavirus Omicron variant S protein RBD domain (or a part thereof) Or a recombinant chimeric antigen peptide formed by connecting with a linker; the chimeric nucleic acid vaccine can provide strong immune protection against a variety of new coronavirus strains, and when sequenced with other types of vaccines (such as inactivated vaccines), Consistent immunization can induce a significantly increased level of immune response against various strains of the new coronavirus (ie, broad-spectrum).

Specifically, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a polynucleotide encoding a recombinant chimeric antigen peptide having a structure as shown in formula (I):

(A-B)-C-(A-B')

(I)

In formula (I):

Option 1: A-B represents the amino acid sequence of the S protein RBD domain of the novel coronavirus prototype strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and have identical or substantially identical immunogenic amino acid sequences;

A-B' represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Beta variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and has the same or substantially the same immunogenic amino acid sequence; or

Option 2: A-B represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Delta variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and has the same or substantially the same immunogenic amino acid sequence as it;

A-B' represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Beta variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and has the same or substantially the same immunogenic amino acid sequence; or

Option 3: A-B represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Delta variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and has the same or substantially the same immunogenic amino acid sequence as it;

A-B' represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Omicron variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and has the same or substantially the same immunogenic amino acid sequence as it;

C represents linker (GGS) _n ; where n=0, 1, 2, 3, 4 or 5.

For the above polynucleotide, preferably, a part of the RBD domain of the S protein of the novel coronavirus prototype strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96% of its entire amino acid sequence , 97%, 98% or 99%;

And/or, a part of the RBD domain of the S protein of the novel coronavirus Beta mutant strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%;

And/or, a part of the RBD domain of the S protein of the novel coronavirus Delta variant strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%;

And/or, a part of the RBD domain of the S protein of the novel coronavirus Omicron variant strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%;

And/or n=0,1,2 or 3.

In some preferred specific embodiments, the amino acid sequence of the novel coronavirus prototype strain S protein RBD domain or a part thereof is shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 1 is substituted , an amino acid sequence obtained by deleting or adding one or several amino acids, and having the same or substantially the same immunogenicity as it;

And/or, the amino acid sequence of the S protein RBD domain of the novel coronavirus Beta variant strain or a part thereof is shown in SEQ ID NO: 2, or the amino acid sequence shown in SEQ ID NO: 2 is substituted, deleted or added An amino acid sequence obtained from one or several amino acids and having the same or substantially the same immunogenicity as it;

And/or, the amino acid sequence of the new coronavirus Delta variant S protein RBD domain or a part thereof is shown in SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 3 is substituted, deleted or added An amino acid sequence obtained from one or several amino acids and having the same or substantially the same immunogenicity as it;

And/or, the amino acid sequence of the novel coronavirus Omicron variant S protein RBD domain or a part thereof is shown in SEQ ID NO: 4, or the amino acid sequence shown in SEQ ID NO: 4 is substituted, deleted or added An amino acid sequence obtained from one or several amino acids and having the same or substantially the same immunogenicity as it;

And/or n=0,1 or 2.

In a further preferred embodiment, when the formula (I) is Option 1, the recombinant chimeric antigen peptide having the structure shown in the formula (I) has the amino acid sequence shown in SEQ ID NO:5;

When the formula (I) is Option 2, the recombinant chimeric antigen peptide having the structure shown in the formula (I) has the amino acid sequence shown in SEQ ID NO:6;

When formula (I) is Option 3, the recombinant chimeric antigen peptide having the structure shown in formula (I) has the amino acid sequence shown in SEQ ID NO:7.

In some preferred embodiments, said polynucleotide is a DNA molecule;

Further preferably, when the formula (I) is Option 1, the DNA molecule has a DNA sequence as shown in SEQ ID NO: 8;

Further preferably, when the formula (I) is Option 2, the DNA molecule has a DNA sequence as shown in SEQ ID NO:9;

Further preferably, when the formula (I) is Option 3, the DNA molecule has a DNA sequence as shown in SEQ ID NO:10.

In other preferred embodiments, said polynucleotide is an mRNA molecule;

Further preferably, when the formula (I) is Option 1, the mRNA molecule has an mRNA sequence as shown in SEQ ID NO: 11;

Further preferably, when formula (I) is Option 2, said mRNA molecule has the mRNA sequence as shown in SEQ ID NO:12;

Further preferably, when formula (I) is Option 3, said mRNA molecule has the mRNA sequence as shown in SEQ ID NO:13.

In a second aspect, the present invention provides a nucleic acid construct comprising the polynucleotide described in the first aspect above, and optionally, at least one expression regulatory element operably linked to the polynucleotide.

In a third aspect, the present invention provides an expression vector comprising the nucleic acid construct as described in the second aspect above.

In a fourth aspect, the present invention provides a host cell transformed or transfected with the polynucleotide as described in the first aspect above, the nucleic acid construct as described in the second aspect above or the nucleic acid construct as described in the third aspect above. Expression vector.

In the fifth aspect, the present invention provides the polynucleotide as described in the first aspect above, the nucleic acid construct as described in the second aspect above, the expression vector as described in the third aspect above or the expression vector as described in the fourth aspect above The application of host cells in the preparation of vaccines for the prevention and/or treatment of novel coronaviruses;

Preferably, the vaccine is used for single immunization or sequential immunization with other types of novel coronavirus vaccines; further preferably, the other types of novel coronavirus vaccines include inactivated vaccines.

In the sixth aspect, the present invention provides a chimeric nucleic acid vaccine or immunogenic composition, which comprises the polynucleotide as described in the first aspect above, the nucleic acid construct as described in the second aspect above, and the nucleic acid construct as described in the above-mentioned first aspect. The expression vector of the third aspect or the host cell of the fourth aspect above, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and/or diluent.

In a specific embodiment, the chimeric nucleic acid vaccine or immunogenic composition is a novel coronavirus DNA vaccine, and the DNA vaccine comprises:

(i) eukaryotic expression vectors; and

(ii) a DNA sequence constructed into the eukaryotic expression vector, encoding a recombinant chimeric antigen peptide as defined in the first aspect above and having a structure shown in formula (I), preferably as SEQ ID NO:8 , the DNA sequence shown in 9 or 10;

Preferably, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

In another specific embodiment, the chimeric nucleic acid vaccine or immunogenic composition is a novel coronavirus mRNA vaccine, and the mRNA vaccine comprises:

(1) an mRNA sequence encoding a recombinant chimeric antigen peptide having a structure as shown in formula (I) as defined in the first aspect above, preferably an mRNA sequence as shown in SEQ ID NO: 11, 12 or 13; and

(II) Lipid nanoparticles.

In another specific embodiment, the chimeric nucleic acid vaccine or immunogenic composition is a novel coronavirus-viral vector vaccine, which includes:

(1) a viral backbone vector; and

(2) The DNA sequence of the recombinant chimeric antigen peptide encoding the structure shown in formula (I) as defined in the first aspect above, which is constructed into the viral backbone vector, preferably such as SEQ ID NO: 8, The DNA sequence shown in 9 or 10;

Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vectors, poxvirus vectors, influenza virus vectors, and adeno-associated virus vectors.

In a feasible implementation, the chimeric nucleic acid vaccine or immunogenic composition is in the form of nasal spray, oral formulation, suppository or parenteral formulation;

Preferably, the nasal spray is selected from aerosol, spray and powder;

Preferably, the oral preparation is selected from tablet, powder, pill, powder, granule, fine granule, soft/hard capsule, film-coated agent, pellet, sublingual tablet and ointment;

Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, liquid for external use, injectable or pushable preparation.

In the seventh aspect, the present invention provides a kit, which includes the chimeric nucleic acid vaccine or immunogenic composition as described in the sixth aspect above, and optionally other types of novel coronavirus vaccines, the chimeric The nucleic acid vaccine or immunogenic composition is packaged separately from the other types of novel coronavirus vaccines;

Preferably, said other types of novel coronavirus vaccines are novel coronavirus inactivated vaccines.

Beneficial effect

The inventors of the present invention have designed a polynucleotide, which is composed of (1) the S protein RBD domain of the novel coronavirus prototype strain (or a part thereof) and the novel coronavirus Beta variant strain S protein RBD domain (or a part thereof), or (2) the S protein RBD domain (or a part thereof) of the novel coronavirus Delta variant strain and the S protein RBD domain (or a part thereof) of the novel coronavirus Beta variant strain, or (3) the novel coronavirus A recombinant chimeric antigen peptide formed by direct series connection of the S protein RBD domain (or a part thereof) of the virus Delta variant strain (or a part thereof) and the S protein RBD domain (or a part thereof) of the new coronavirus Omicron variant strain or connected by a linker; based on the polynucleoside The acid chimeric nucleic acid vaccine can provide strong immune protection against a variety of new coronavirus strains, and can induce immunity against various new coronavirus strains (ie, broad-spectrum) when sequentially immunized with other types of vaccines. ), significantly increased immune response levels, are very suitable for the current complex epidemic prevention and control, and have potential clinical application value and prospects.

Description of drawings

One or more embodiments are exemplified by pictures in the accompanying drawings, and these exemplifications are not intended to limit the embodiments. 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 superior or better than other embodiments.

Fig. 1 is the novel coronavirus prototype strain RBD dimer mRNA vaccine constructed in Example 1 of the present invention (referred to as PP mRNA vaccine, as a control vaccine), the chimeric RBD dimerization formed by the connection of the novel coronavirus prototype strain RBD and the Beta mutant strain RBD. mRNA vaccine (abbreviated as PB mRNA vaccine), chimeric RBD dimer mRNA vaccine (referred to as DB mRNA vaccine) formed by linking RBD of Delta mutant strain and RBD of Beta mutant strain (abbreviated as DB mRNA vaccine), chimeric mRNA vaccine formed by connecting RBD of Delta mutant strain and RBD of Omicron mutant strain Schematic diagram of the structure of the combined RBD dimer mRNA vaccine (referred to as DO mRNA vaccine); the various segments of the mRNA vaccine are marked on the figure, where 5'UTR indicates the 5' untranslated region, and 3'UTR indicates the 3' untranslated region region, SP indicates the signal peptide sequence, Poly(A) indicates the polyadenylation tail, Protype RBD indicates the RBD sequence of the prototype strain, Beta RBD indicates the RBD sequence of the Beta variant strain, Delta RBD indicates the RBD sequence of the Delta variant strain, Omicron (BA.1) RBD indicates the RBD sequence of the Omicron mutant strain, wherein Beta RBD, Delta RBD and Omicron RBD also marked their respective amino acid mutations relative to the prototype strain RBD.

Figure 2 shows the level of humoral immunity induced by the PP, PB, DB, DO mRNA vaccines constructed in Example 1 of the present invention, as detected in Examples 3 and 4, wherein LNP represents the use of lipid nanoparticle immunization Figure 2a is a schematic diagram of the mRNA vaccine immunization mice and sampling procedures; Figure 2b and Figure 2c are the sera collected on the 14th and 28th days after the mice were immunized with the mRNA vaccine respectively against the new coronavirus The RBD antigen binding antibody titers of prototype strain, Delta variant strain, Beta variant strain and Omicron variant strain BA.1, BA.1.1, BA.2, BA.3 subtypes are shown in the bar graphs of Figure 2b and Figure 2c , the numbers above each bar represent the ratio of the antibody titer represented by that bar to the antibody titer represented by the corresponding bar in the LNP group, and, as indicated by the arrows in the figure, the heat on the right side of Figures 2b and 2c The figure is based on these figures; Figure 2d shows that the serum collected on the 28th day after the mice were immunized with the mRNA vaccine neutralized the new coronavirus prototype strain, Delta variant strain, Omicron variant strain BA.1, BA in the pseudovirus neutralization experiment .1.1, BA.2, the NT ₅₀ value of the pseudovirus of BA.3 subtype, the number above each column represents the geometric mean (GMT) of all samples of this experimental group, as shown by the arrow in the figure, the right side Heatmaps were produced based on these figures; all data are presented as GMT ± 95% CI (confidence interval).

Figure 3 shows the level of cellular immunity stimulated by the PP, PB, DB, DO mRNA vaccines constructed in Example 1 of the present invention, as detected in Example 5, LNP represents the negative control group using lipid nanoparticles immunization ; Wherein, Fig. 3a is a schematic diagram of mRNA vaccine immunization mice and sampling procedures; Fig. 3b shows that the splenocytes collected on the 21st day after mRNA vaccine immunization mice by ELISpot test were respectively treated by 4 kinds of peptide libraries (new coronavirus prototype strain, Delta variation strain, Beta mutant strain, and Omicron mutant strain subtype BA.1RBD) stimulated IFNγ+ cell numbers. The ratio of the number of IFNγ+ cells represented by the corresponding columns in the group; all data are presented as Mean±SEM.

Fig. 4 has shown that after adopting inactivated vaccine and the PP, PB, DB, DO mRNA vaccine constructed in the embodiment of the present invention 1 to carry out sequential immunization to mouse, compared with before sequential immunization (that is, without using mRNA vaccine Boosted group), combined with the increase in antibody titer; wherein, Figure 4a is a schematic diagram of sequential immunization and serum sampling procedures for mice, and IV represents inactivated vaccines; ) and the sera collected on day 49 (shown in solid circles) against the new coronavirus prototype strain, Delta variant, Beta variant, and Omicron variant subtypes BA.1, BA.1.1, BA.2, BA.3RBD Antigen-binding antibody titer levels, and the fold increase of the latter (i.e., day 49) relative to the former (i.e., day 35) antibody titers (the "number ×" above each graph indicates the fold increase), where , Fig. 4b(i)~(v) respectively represent two inactivated vaccine + PP mRNA vaccine immunization group, two inactivated vaccine + PB mRNA vaccine immunization group, two inactivated vaccine + DB mRNA vaccine immunization group, two inactivated vaccine immunization group, two inactivated vaccine Live vaccine+DO mRNA vaccine immunization group, three times inactivated vaccine immunization group.

Fig. 5 has shown adopting inactivated vaccine and the PP, PB, DB, DO mRNA vaccine constructed in the embodiment of the present invention 1 to carry out the immune reaction level after mice are sequentially immunized, and its immunization program refers to Fig. 4a; In the graph, "PP" represents the two-time inactivated vaccine + PP mRNA vaccine immunization group, "PB" represents the two-time inactivated vaccine + PB mRNA vaccine immunization group, "DB" represents the two-time inactivated vaccine + DB mRNA vaccine immunization group group, "DO" represents twice inactivated vaccine+DO mRNA vaccine immunization group, "IV" represents three inactivated vaccine immunization group, "LNP" represents twice inactivated vaccine adjuvant (i.e., Al adjuvant) + lipid The nanoparticle (LNP) immunization group was used as a negative control; among them, Figure 5a shows that the serum collected on the 49th day was directed against the new coronavirus prototype strain, Delta mutant strain, Beta mutant strain, Omicron mutant strain subtype BA.1, BA.1.1, The binding antibody titer of BA.2, BA.3 antigen, in the histogram of Fig. 5a, the number of the first line above each column represents the antibody titer represented by this column and inactivated vaccine (IV) group The ratio of the antibody titer represented by the corresponding column in , the number in the second row represents the ratio of the antibody titer represented by the column to the antibody titer represented by the corresponding column in the LNP group, and the arrows in the figure , the heat map on the right side of Figure 5a is based on the second row of numbers; Figure 5b shows that the serum collected on the 49th day neutralized 6 pseudoviruses (prototype strain, Delta variant strain, Omicron variant strain subtype) in the pseudovirus neutralization experiment BA.1, BA.1.1, BA.2, BA.3) NT ₅₀ values, in the bar graph of Figure 5b, the first row of numbers above each bar indicates the NT ₅₀ titer value represented by the bar The ratio of the NT ₅₀ titer value represented by the corresponding column in the inactivated vaccine (IV) group, the second row of numbers above each column represents the geometric mean (GMT) of all samples in the experimental group, and as shown in the figure As indicated by the arrow, the heat map on the right is made based on the numbers in the second row; the data in Figure 5a and Figure 5b are presented as GMT±95%CI (confidence interval); Figure 5c shows the CD8+ and CD4+ cells were respectively stimulated by 4 kinds of peptide libraries (prototype, Delta, Beta, Omicron subtype BA.1 variant RBD constructed peptide library) to produce the proportion of IFNγ+ cells, the data in Figure 5c is displayed as Mean ±SEM; statistical difference was calculated by Mann-Whitney test method (*, p<0.05; **, p<0.01).

Detailed ways

In order to make the purpose, 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. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. the embodiment. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

In addition, in order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It will be understood by those skilled in the art that the present invention may be practiced without certain of the specific details. In some embodiments, materials, components, methods, means, etc. that are well known to those skilled in the art are not described in detail, so as to highlight the gist of the present invention.

Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations thereof such as "includes" or "includes" and the like will be understood to include the stated elements or constituents, and not Other elements or other components are not excluded.

Example 1: New coronavirus prototype strain RBD dimer mRNA vaccine (abbreviated as PP mRNA vaccine, as a control), prototype strain and Beta variant chimeric RBD dimer mRNA vaccine (abbreviated as PB mRNA vaccine), Delta variant and Beta The construction, in vitro preparation and packaging of the chimeric RBD dimer mRNA vaccine (abbreviated as DB mRNA vaccine) of the mutant strain and the chimeric RBD dimer mRNA vaccine (abbreviated as DO mRNA vaccine) of the Delta mutant strain and the Omicron mutant strain

According to the structural representation of PP, PB, DB, DO mRNA vaccine shown in Figure 1, carry out the construction, in vitro preparation and packaging of mRNA vaccine according to the following procedures:

1) In vitro transcription and capping of mRNA vaccines

In this example, the basic plasmid used for in vitro transcription of the mRNA vaccine is pUC57, provided by Nanjing GenScript Biotechnology Co., Ltd.

On the basic plasmid pUC57, the DNA expression elements of mRNA vaccines were introduced by conventional molecular biological means, including: (1) T7 promoter, (2) DNA coding regions of mRNA vaccines (DNA coding sequences of PB, DB, DO mRNA vaccines) Respectively as shown in SEQ ID NO:8,9,10, the DNA coding sequence of the PP mRNA vaccine as a control is shown in SEQ ID NO:14), (3) the 5' end UTR sequence upstream of the coding region (several mRNA The 5' end UTR sequence of the vaccine is the same as shown in SEQ ID NO: 15), (4) signal peptide sequence (i.e. SP, shown in SEQ ID NO: 16), and (5) downstream 3' terminal UTR sequence (several mRNA vaccines have the same 3' terminal UTR sequence, all of which are sequences shown in SEQ ID NO: 17), and polyadenylic acid tail (Poly-A-tail).

First, use the restriction endonuclease BamHI to digest the above-mentioned in vitro transcription plasmid and linearize it; use conventional DNA purification methods to purify to obtain a template for in vitro transcription; then, based on the template, use the T7RNA in vitro transcription kit ( E131-01A, Suzhou Nearshore Protein Technology Co., Ltd.) for in vitro transcription to obtain in vitro transcribed mRNA; finally, using lithium chloride recovery kit (S125, Suzhou Nearshore Protein Technology Co., Ltd.), through lithium chloride precipitation The mRNA was purified by the method to obtain purified in vitro transcribed mRNA.

Then, use the capping enzyme kit Cap1 capping enzyme kit (M082-01B, Suzhou Jinan Protein Technology Co., Ltd.) to cap the 5' end Cap1 of the above-mentioned purified in vitro transcribed mRNA, so that it meets the requirements of eukaryotic expression. The conditions under which the cells are translated; thereafter, the mRNA is purified again using the same lithium chloride precipitation method as above to obtain purified 5'-capped mRNA.

2) Lipid nanoparticle (LNP) packaging mRNA

Mix cationic lipid, phosphatidylcholine, cholesterol and PEG lipid according to the ratio of 50:10:38.5:1.5, and then use the Nanoassemblr Benchtop nano-liposome packaging instrument produced by Precision Nano Systems to combine it with the above 5 ' end capped mRNA for mixing and packaging. After packaging, replace the buffer solution with PBS by centrifugation or dialysis. After the packaging is completed, use the Quan-iT Ribogreen RNAreagent kit from Thermo Fisher to identify the mRNA packaging efficiency, and the packaging efficiency meets the standards for mRNA vaccines.

Example 2: Experimental animal immunization and sample collection

In this embodiment, 6-8 week-old female mice of BALB/c strain (purchased from Weitong Lihua) were used to carry out animal experiments; the experimental components were mRNA vaccine immunization group and negative control group, wherein, mRNA vaccine immunization group Groups include PP mRNA vaccine immunization group, PB mRNA vaccine immunization group, DB mRNA vaccine immunization group and DO mRNA vaccine immunization group, and the negative control group is LNP immunization group. All mice in the mRNA vaccine immunization group were immunized with the same designed mRNA vaccine (i.e., PP, PB, DB or DO mRNA vaccine) on day 0 and day 14, and mice in the negative control group were immunized at the same time, The same amount of empty LNP was injected. The vaccination methods were all intramuscular injections, and the vaccination dose was 5 μg mRNA vaccine or empty LNP per mouse each time. Mouse serum samples were collected on the 14th day and 28th day, respectively, for testing the binding antibody titer and pseudovirus neutralizing antibody titer of the immune serum. In addition, mouse spleen samples were collected on day 21 for testing T cell immunity.

The mice were immunized with the mRNA vaccine and the sampling procedure is shown in Figure 2a.

Embodiment 3: the inspection of mouse serum antibody titer

With new coronavirus prototype strain (SEQ ID NO:1), Delta variant strain (SEQ ID NO:3), Beta variant strain (SEQ ID NO:2) and Omicron variant strain BA.1 (SEQ ID NO:4), BA. 1.1 (SEQ ID NO: 18), BA.2 (SEQ ID NO: 19), BA.3 (SEQ ID NO: 20) subtypes of RBD antigen protein (0.2 μg/ml) were coated with ELISA plate respectively, and the package The good ELISA plate was blocked in 5% skimmed milk for 1 hour; then, the serum collected from the mice of each experimental group in Example 2 was incubated at 56° C. for 30 minutes to inactivate; the inactivated serum was Samples were serially diluted three-fold starting from 1:200 or 1:1000, and then the dilution was added to each well, and then the ELISA plate was incubated at 37°C for 1 hour; the goat anti-mouse IgG-HRP antibody (purchased from Bo Ao Yijie (EASYBio)) was added to the plate as a secondary antibody and incubated again at 37°C for 1 hour; finally, 3,3',5,5'-tetramethylbenzidine (TMB) substrate was used for visualization After color development, the reaction was terminated with 2M hydrochloric acid, and the absorbance at 450 nm and 630 nm was measured using a microplate reader (PerkinElmer). Absorbance values were calculated by subtracting the absorbance at 630 nm from the absorbance at 450 nm of the same well. The endpoint titer was defined as the dilution factor of the serum at which the serum produced an absorbance (absorbance at 450 nm minus absorbance at 630 nm, as described above) greater than 2.1 times the background value. Antibody titers below the detection limit were defined as one third of the detection limit.

The binding antibody titers of the sera collected from mice in each experimental group on the 14th day and the 28th day of the immunization program against the RBD antigens of the above seven new coronaviruses are shown in Figure 2b and Figure 2c, respectively, and in Figure 2b and Figure 2c In the middle, the left side is the histogram of end-point titer vs vaccine type, and the right side is the corresponding heat map, which is based on the ratio of the end-point antibody titer of each mRNA vaccine to the end-point antibody titer of the LNP group , as described in the figure description.

Figure 2c shows the results on the 28th day when the antibody level was relatively stable, as can be seen from Figure 2c:

(1) PB mRNA vaccine

For the tested novel coronavirus prototype strain and each variant strain, the PB mRNA vaccine can induce a higher level of binding antibody; moreover, the level of binding antibody titer induced by it is comparable to that of the PP mRNA vaccine, or higher than that of the PP mRNA vaccine (For example, 2-3 times higher for BA.1.1 and BA.2 respectively);

(2) DB mRNA vaccine

For the tested novel coronavirus prototype strain and each mutant strain, the DB mRNA vaccine can induce a higher level of binding antibody; moreover, the level of binding antibody titer induced by it against each type of new crown virus strain is much higher than that of PP mRNA vaccine, some as high as 3 times;

(3) DO mRNA vaccine

For the tested novel coronavirus prototype strain and each variant strain, the DO mRNA vaccine can induce higher levels of binding antibodies; in particular, the level of serum antibody titers induced by each subtype of the Omicron variant strain is far Higher than PP mRNA vaccine; for example, for BA.1 subtype, the antibody titer level induced by DO mRNA vaccine is more than 2 times higher than that of PP mRNA vaccine, and for BA.1.1 subtype, the antibody titer induced by DO mRNA vaccine The level of antibody titers induced by BA.2 and BA.3 subtypes was nearly 6 times higher than that of PP mRNA vaccines, and the level of antibody titers induced by BA.3 subtypes was higher than that of PP mRNA vaccines. PP mRNA vaccine is more than 3 times higher; This suggests that DO mRNA vaccine of the present invention can induce significantly higher antibody titer level for each subtype of Omicron variant strain, illustrates that it will have significantly higher antibody titer level for each type of Omicron variant strain. High immune protection efficacy; and, DO mRNA also induced a high level of antibody titer for the new coronavirus prototype strain and other mutant strains, which suggests that it has a good broad-spectrum.

Example 4: Packaging and Serum Neutralization of New Coronavirus Strain Pseudovirus

In this example, the sera of the immunized mice collected in the above-mentioned Example 2 were tested for the pseudotypes of the new coronavirus prototype strain, the Delta variant strain, and the Omicron variant strain BA.1, BA.1.1, BA.2, and BA.3 subtypes. 50% pseudovirus neutralization titer (pVNT ₅₀ ) of virus; Concrete detection method is as follows:

1. Preparation of expression plasmids for the truncated SARS-CoV-2 S protein

The nucleotides at the last 18 amino acids of the S protein encoding the new coronavirus prototype strain, the Delta variant strain and the Omicron variant strain BA.1, BA.1.1, BA.2, and BA.3 subtypes were removed, and the resulting nucleotides Respectively named WT-S-del18, Delta-S-del18, BA.1-S-del18, BA.1.1-S-del18, BA.2-S-del18, BA.3-S-del18, the nucleoside The acid sequences are shown in SEQ ID NO: 21-26, which were synthesized by Suzhou Jinweizhi Company; then, these nucleotide sequences were respectively cloned into the pCAGGS expression vector to obtain the expression plasmids pCAGGS-WT-S-del18, pCAGGS-WT-S-del18, pCAGGS-Delta-S-del18, pCAGGS-BA.1-S-del18, pCAGGS-BA.1.1-S-del18, pCAGGS-BA.2-S-del18, pCAGGS-BA.3-S-del18.

2. Packaging of pseudoviruses of the prototype strain of the new coronavirus and various variant strains

1) Spread HEK293T cells in a 10cm cell culture dish so that the cell density reaches about 80% the next day. The culture medium is DMEM medium containing 10% FBS.

2) Transfect the cells in the culture dish (30 μg/10 cm cell culture dish) with PEI prepared above to express the truncated S protein of each type of the new coronavirus strain. The target plasmid and PEI were mixed at a ratio of 1:3 before transfection, and the culture medium (DMEM medium containing 10% FBS) was changed every 4-6 hours, and cultured at 37°C for 24 hours.

3) Add the pseudovirus packaging skeleton virus G*VSV-delG (Wuhan Privy Brain Science and Technology Co., Ltd.) to the above transfected HEK293T cells, incubate at 37° C. for 2 hours, and change the culture medium (DMEM medium containing 10% FBS), And VSV-G antibody (hybridoma cells expressing this antibody were purchased from ATCC cell bank) was added, and culture was continued for 30 h in the incubator.

4) The supernatant was collected, centrifuged at 3000rpm for 10min, filtered through a 0.45μm sterile filter in an ultra-clean bench to remove cell debris, aliquoted, and stored in a -80°C refrigerator.

Through the above steps, the pseudoviruses of the new coronavirus prototype strain, the Delta variant strain and the Omicron variant strain BA.1, BA.1.1, BA.2, and BA.3 subtypes were respectively obtained.

3. Evaluation of the inhibitory effect of immunized mouse serum on pseudovirus

Incubate the mouse serum of each experimental group collected on the 28th day in Example 2 at 56°C for 30 minutes to inactivate; dilute the inactivated serum samples, and perform a 2-fold serial dilution starting from 1:80 . Then, each pseudovirus was mixed with an equal volume of diluted serum and incubated at 37°C for 1 hour. 100 μl of the virus-serum mixture was added to the pre-plated Vero cells in a 96-well plate. After incubation for 15 hours, use the CQ1 confocal image cytometer to detect the number of transducing units (TU), so as to calculate the effect of the immune mouse serum on the above-mentioned new coronavirus prototype strain, Delta variant strain and Omicron variant strain BA.1, BA.1.1, The neutralizing ability of pseudoviruses of BA.2 and BA.3 subtypes.

The results are shown in Figure 2d; as described in the description of Figure 2d, the left column graph of Figure 2d shows the serum neutralization prototype strain, Delta variant strain and Omicron variant strain BA.1, BA. The pVNT ₅₀ of pseudoviruses of 1.1, BA.2, and BA.3 subtypes (that is, 50% pseudovirus neutralization titer), the heat map on the right shows the ratio of pVNT ₅₀ of each mRNA vaccine to the pVNT ₅₀ of the LNP group .

It can be seen from Figure 2d that:

(1) The serum neutralizing antibody titers induced by PB mRNA vaccine against each subtype of Omicron mutant strains were much higher than that of PP mRNA vaccine; especially, the neutralization antibody titers induced by PB mRNA vaccine against BA.1 subtype The antibody titer level is more than 7 times higher than that of PP mRNA vaccine, the level of neutralizing antibody titer induced by BA.1.1 subtype is more than 6 times higher than that of PP mRNA vaccine, and the level of neutralizing antibody titer induced by BA.2 and BA.3 subtype The neutralizing antibody titer level is about 6 times higher than that of the PP mRNA vaccine; this suggests that the PB mRNA vaccine of the present invention can induce significantly higher neutralizing antibody titer levels for each subtype of the Omicron mutant strain, indicating that it is aimed at Omicron All types of variant strains will have significantly higher immune protection efficacy; in addition, PB mRNA also has a higher level of neutralizing antibody titers for the new coronavirus prototype strain and Delta variant strain, which suggests that it has a good broad-spectrum .

(2) The serum neutralizing antibody titers induced by the DB mRNA vaccine against the prototype strain, Delta variant, and Omicron variant were much higher than that of the PP mRNA vaccine; specifically, the DB mRNA vaccine induced by the prototype strain The level of neutralizing antibody titer is more than 3 times higher than that of PP mRNA vaccine, the level of neutralizing antibody titer induced by the Delta variant strain is more than 5 times higher than that of PP mRNA vaccine, and the level of neutralizing antibody titer induced by the Omicron variant strain BA.1 subtype The neutralizing antibody titer level was nearly 45 times higher than that of the PPmRNA vaccine, and the neutralizing antibody titer level induced by the Omicron variant strain BA.1.1 was nearly 30 times higher than that of the PPmRNA vaccine. The level of neutralizing antibody titer induced is nearly 48 times higher than that of PP mRNA vaccine, and the level of neutralizing antibody titer induced by Omicron variant strain BA.3 subtype is nearly 69 times higher than that of PP mRNA vaccine; this suggests that the present invention DB mRNA vaccine can induce significantly higher levels of neutralizing antibody titers against various strains of the new coronavirus, indicating that it will have significantly higher immune protection efficacy against various strains of the new coronavirus.

(3) The serum neutralizing antibody titers induced by the DO mRNA vaccine against each subtype of the prototype strain, the Delta variant strain, and the Omicron variant strain were much higher than those induced by the PP mRNA vaccine; The level of neutralizing antibody titer was nearly 13 times higher than that of PP mRNA vaccine, the level of neutralizing antibody titer induced by Delta variant was nearly 5 times higher than that of PP mRNA vaccine, and the level of neutralizing antibody titer induced by Omicron variant strain BA.1 The neutralizing antibody titer level was nearly 200 times higher than that of the PPmRNA vaccine, and the neutralizing antibody titer level induced by the Omicron variant strain BA.1.1 was nearly 163 times higher than that of the PPmRNA vaccine. The level of neutralizing antibody titer induced is nearly 230 times higher than that of PPmRNA vaccine, and the level of neutralizing antibody titer induced by Omicron mutant strain BA.3 subtype is nearly 407 times higher than that of PPmRNA vaccine; this suggests that DO mRNA of the present invention The vaccine can induce significantly higher levels of neutralizing antibody titers against various strains of the new coronavirus, indicating that it will have significantly higher immune protection against various strains of the new coronavirus.

Example 5: Evaluation of the level of cellular immunity induced by mRNA vaccines

In this example, the spleen samples of mice in each experimental group collected on day 21 in Example 2 (the schematic diagram of mouse immunization and sampling procedures are shown in Figure 3a) were used to detect the level of cellular immunity induced by the mRNA vaccine. The specific method is as follows:

1) Mouse spleen sample processing

Use a cell homogenizer to prepare mouse spleen cells into single-cell homogenate in 1 ml of serum-free DMEM, filter with a 40 μm cell strainer, and lyse red blood cells with red blood cell lysis buffer (Beijing Suo Laibao Technology Co., Ltd., R1010); then , cells were washed with washing solution (PBS+0.5% FBS), stained with 0.4% trypan blue solution (Gibco, 15250061), and counted using Cell drop FL automatic cell counter.

2) ELISpot test

10 μg/ml anti-mouse IFN-γ antibody (purchased from BD) was incubated overnight at 4°C in a flat-bottomed 96-well plate to coat the flat-bottomed 96-well plate, and blocked at room temperature for 2 hours the next day. Add fresh mouse spleen single-cell suspension (4×10 ⁵ /well) to the 96-well plate coated with the above antibody, and use the RBD of the new coronavirus prototype strain, Delta, Beta, and Omicron variant strain BA.1 subtype respectively The constructed peptide library (each polypeptide 2 μg/ml) was stimulated for 20 hours; the peptide library was designed using the software PeptGen PeptideGenerator on the website https://www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html , the key parameters of the design include: the length of short peptides is 18-20 amino acids, the overlapping amino acid fragments interact with 10 amino acids, etc.; the designed peptide library is synthesized by Zhongke Yaguang Biotechnology Co., Ltd. Positive control wells were stimulated with phytohemagglutinin (PMA) to generate a non-specific cellular immune response, and negative control wells were not stimulated with the peptide library. Then, the cells were discarded, and the 96-well plate was incubated sequentially with biotinylated IFNγ antibody, streptavidin-HRP antibody, and chromogenic substrate. When spots appear on the bottom of the plate, rinse the sample thoroughly with deionized water to stop color development. Finally, Immuno Capture 6.5.0 was used to take pictures and count the number of spots.

The results are shown in Figure 3b. It can be seen from Figure 3b that the number of IFN-γ+ cells produced by spleen cells of mice immunized with PB, DB, and DO mRNA vaccines was comparable to that of PP after being stimulated with the above four new coronavirus RBD peptide libraries The mice immunized with mRNA vaccines were comparable, and they were all much higher than the LNP control group, which indicated that PB, DB, and DO mRNA vaccines could effectively stimulate cellular immune responses, and the level of cellular immunity they stimulated was comparable to that of PP mRNA vaccines.

Embodiment 6: the sequential immunization of mRNA vaccine and inactivated vaccine

In this example, 6-8 week-old female mice of BALB/c strain (purchased from Weitong Lihua) were used for animal experiments, and the inactivated vaccine used was from Sinopharm Zhongsheng BBBIP-CorV.

The experimental groups were: three inactivated vaccine immunization groups (ie, "IV" group), two inactivated vaccine + PP mRNA vaccine immunization groups (abbreviated as "PP" group), two inactivated vaccine + PB mRNA vaccine immunization groups ( "PB" group for short), twice inactivated vaccine+DB mRNA vaccine immunization group (abbreviated as "DB" group), twice inactivated vaccine+DO mRNA vaccine immunization group (abbreviated as "DO" group) and inactivated vaccine adjuvant+ LNP immune group (referred to as "LNP" group, as negative control group).

Group "IV": All mice were inoculated with one dose of inactivated vaccine on day 0, day 21 and day 35;

"PB" group, "DB" group, and "DO" group: All mice were inoculated with a dose of inactivated vaccine on day 0 and day 21, and then received a dose of each mRNA vaccine on day 35;

"LNP" group: All mice were inoculated with inactivated vaccine adjuvant—Al adjuvant on day 0, day 21, and empty LNP on day 35.

The inoculation methods of the above-mentioned vaccines are all intramuscular injections, wherein the inoculation dose of the inactivated vaccine is 2.6 U per mouse (0.4 doses for human dose), and the inoculation dose of each mRNA vaccine or empty LNP is 2.6 U per mouse. 5 μg each time for mice.

Mouse serum samples were collected on the 35th day and 49th day, respectively, for testing the binding antibody titer and pseudovirus neutralizing antibody titer of the immune serum. In addition, mouse spleen samples were collected on day 49 to examine T cell immunity.

A schematic diagram of the sequential immunization and serum sampling procedures for mice is shown in Figure 4a.

Example 7: Sequential immunization of mouse serum to detect the level of binding antibody titer to the RBD antigen of each strain of the new coronavirus

In this example, using the method described in Example 3, it was tested that the sera of mice in each immune group collected in Example 6 were effective against the new coronavirus prototype strain, Delta mutant strain, Beta mutant strain, and Omicron mutant strain BA.1 , BA.1.1, BA.2, BA.3 subtypes of RBD antigen-binding antibody titers.

The result is shown in Fig. 4b. Among them, Fig. 4b(i)～(v) respectively shows the group of the third booster immunization with PP, PB, DB, DO and inactivated vaccine, the first four groups are sequential immunization, and the last group is the control; As described in the description of the figure, it shows that the sera collected on the 35th day (shown in open circles) and 49th day (shown in solid circles) of each immunization program against the new coronavirus prototype strain, Delta variant strain, and Beta variant strain , Omicron variant subtypes BA.1, BA.1.1, BA.2, BA.3 RBD antigen binding antibody titer levels, and the latter (ie, day 49) relative to the former (ie, day 35) antibodies The multiple of the titer increase (the "number ×" above each graph indicates the multiple of increase), the multiple of the increase of the serum antibody titer reflects the level of antibody titer after sequential immunization with mRNA vaccine compared with that before sequential immunization It can be seen from these results that after sequential immunization with each mRNA vaccine, its serum antibody titer level is significantly improved compared with that before sequential immunization, indicating that the mRNA vaccine of the present application can be used for sequential immunization to strengthen the immune response level.

In addition, compared with the PP mRNA vaccine sequential immunization group:

1) PB mRNA vaccine sequential immunization group

For the above seven new coronavirus strains, the serum antibody titers on the 49th day were much higher than those of the PP mRNA vaccine sequential immunization group, up to 10 times higher than the serum antibody titers on the 35th day many;

2) DB mRNA vaccine sequential immunization group

For the new coronavirus prototype strain, Delta variant strain, Beta variant strain and Omicron subtype BA.2 variant strain, the serum antibody titers on the 49th day were much higher than those on the 35th day. PP mRNA vaccine sequential immunization group, up to nearly 5 times;

3) DO mRNA vaccine sequential immunization group

For the above seven new coronavirus strains, the serum antibody titers on the 49th day were much higher than those in the PP mRNA vaccine sequential immunization group, up to 5 times higher than the serum antibody titers on the 35th day .

In addition, the sera collected on the 49th day of each immunization group had binding antibodies against the new coronavirus prototype strain, Delta variant strain, Beta variant strain, and Omicron variant subtype BA.1, BA.1.1, BA.2, and BA.3 antigens The titer results are shown in Figure 5a.

Figure 5a shows that compared with the PP mRNA vaccine sequential immunization group:

1) PB mRNA vaccine sequential immunization group

For the above-mentioned seven new coronavirus strains, the level of binding antibody titers induced by them is much higher than that of PP mRNA vaccine, up to 3 times;

2) DB mRNA vaccine sequential immunization group

For the new coronavirus prototype strain, Delta variant strain, Beta variant strain and Omicron subtype BA.2 variant strain, the level of binding antibody titers induced by them is much higher than that of PP mRNA vaccine;

3) DO mRNA vaccine sequential immunization group

For the above seven new coronavirus strains, the titers of binding antibodies induced by them are much higher than that of PP mRNA vaccine.

Example 8: Evaluation of the inhibitory effect of sequentially immunized mouse serum on pseudoviruses of various strains of the new coronavirus

In this example, using the method described in Example 4, it was detected that the sera of mice in each immune group collected on the 49th day in Example 6 were effective against the new coronavirus prototype strain, Delta variant strain, Beta variant strain, and Omicron variant. Neutralizing antibody titers of pseudoviruses of strain BA.1, BA.1.1, BA.2, BA.3 subtype.

The results are shown in Figure 5b. Figure 5b shows: compared with the PP mRNA vaccine sequential immunization group:

1) PB mRNA vaccine sequential immunization group

For the pseudovirus of the new coronavirus prototype strain, Delta, Omicron BA.1.1, the level of neutralizing antibody titer induced by it is higher than that of PP mRNA vaccine, or equivalent to that of PP mRNA vaccine;

However, for the pseudoviruses of the new coronavirus Omicron BA.1, BA.2, and BA.3, the level of neutralizing antibody titers induced by them is more than two times higher than that of the PP mRNA vaccine, up to about 3 times;

2) DB mRNA vaccine sequential immunization group

Except for the new coronavirus Omicron BA.1.1, the neutralizing antibody titers induced by pseudoviruses against all other types of strains were significantly higher than that of PP mRNA vaccine;

3) DO mRNA vaccine sequential immunization group

For the pseudoviruses of each subtype strain of the new coronavirus Omicron variant, the level of neutralizing antibody titer induced by it is higher than that of the PP mRNA vaccine, or equivalent to it.

Example 9: Evaluation of the level of cellular immunity induced by sequential immunization

In this example, the method described in Example 5 and the spleen samples of mice in each immunized group collected on day 49 in Example 6 were used to detect the level of cellular immunity induced by sequential immunization.

The results are shown in Figure 5c. It can be seen from Figure 5c that after being stimulated with the above four new coronavirus RBD peptide libraries, the IFN-γ+CD4+, IFN The number of -γ+CD8+ cells is equivalent to that of mice sequentially immunized with PP mRNA vaccine, and they are much higher than that of LNP control group, which shows that sequential immunization with PB, DB, and DO mRNA vaccines can effectively stimulate cellular immunity Response, and the level of cellular immunity it stimulated was equivalent to that of the PPmRNA vaccine sequential immunization group.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still be Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Institute of Microbiology, Chinese Academy of Sciences

<120> Novel coronavirus chimeric nucleic acid vaccine and its application

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<170> PatentIn version 3.5

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

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

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

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

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

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

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

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

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Cys Pro Phe Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr

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

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

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

65 70 75 80

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

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

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

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

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Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr

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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

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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

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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

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

210 215 220

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

225 230 235 240

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

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

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

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

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

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

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

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

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

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

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Asn Lys

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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

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

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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 Arg 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 Lys

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 Val Gln Pro Thr Glu Ser Ile Val

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

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Asn Lys

<210> 7

<211> 434

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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 Arg 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 Lys

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 Val Gln Pro Thr Glu Ser Ile Val

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn

370 375 380

Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val

385 390 395 400

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

405 410 415

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

420 425 430

Asn Lys

<210> 8

<211> 1302

<212>DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> DNA coding sequence of PB mRNA vaccine

<222> (1)..(1302)

<400> 8

cgagtgcagc ctaccgaaag catcgtccgt ttcccgaata ttactaatct ctgtccattc 60

ggagaagtct tcaatgccac ccgattcgct tccgtttacg cgtggaaccg taaacgaata 120

tctaattgtg ttgcggacta ttccgtgttg tacaactcag catcattctc tacttttaaa 180

tgctatggag tgtcgccgac taaactcaac gacttgtgtt tcactaatgt ttatgctgac 240

tctttcgtta ttcgtggaga cgaagttcgt caaatcgcac cagggcaaac tggcaagatt 300

gcggactata attataagct gccagatgac tttaccggat gtgtaatagc ctggaactca 360

aataatctcg acagtaaagt gggaggcaac tataattatc tttatcgact cttcagaaag 420

tctaacctta agccatttga acgtgacatt tctacagaaa tttaccaagc cggctctaca 480

ccttgcaatg gcgtggaagg gtttaactgt tatttcccat tacagtctta tggtttccag 540

ccaactaatg gtgtgggata ccaaccttac cgcgtcgttg tcctgtcgtt tgaattgctt 600

cacgcaccag ccaccgtttg tgggccaaag aagagcacta atctcgtagt tcagcctact 660

gaatcgatcg tgaggttccc aaatattacc aatctgtgtc cgttcggaga ggtcttcaat 720

gcgactcgat tcgcgtctgt ttacgcctgg aacaggaaac ggattagcaa ttgtgtcgct 780

gactattcgg tcttatacaa ctctgcatca ttctcaacct tcaagtgtta tggtgtcagc 840

cctacaaagc tgaatgactt atgtttcacc aatgtttatg cggacagttt cgtaatacga 900

ggtgatgaag tccgccaaat tgcacccgga caaaccggca acatagccga ctataattat 960

aagctccctg atgactttac gggctgtgtc atagcttgga atagtaataa tttggactcg 1020

aaagtggggag gtaattataa ttatctctat agactgttcc ggaaatcaaa tctcaagccc 1080

tttgaacggg acataagtac agaaatctac caagctggtt ccacgccgtg taatggagtc 1140

aaggggttta actgttattt cccgctccag tcgtatgggt tccagccaac gtatggcgtc 1200

ggataccaac cttaccgcgt tgtagtatta agctttgaac tgttgcacgc gcccgcgact 1260

gtttgtggcc cgaagaagtc gactaatcta gtaaagaata ag 1302

<210> 9

<211> 1302

<212>DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> DNA coding sequence of DB mRNA vaccine

<222> (1)..(1302)

<400> 9

cgagtgcagc ctaccgaaag catcgtccgt ttcccgaata ttactaatct ctgtccattc 60

ggagaagtct tcaatgccac ccgattcgct tccgtttacg cgtggaaccg taaacgaata 120

tctaattgtg ttgcggacta ttccgtgttg tacaactcag catcattctc tacttttaaa 180

tgctatggag tgtcgccgac taaactcaac gacttgtgtt tcactaatgt ttatgctgac 240

tctttcgtta ttcgtggaga cgaagttcgt caaatcgcac cagggcaaac tggcaagatt 300

gcggactata attataagct gccagatgac tttaccggat gtgtaatagc ctggaactca 360

aataatctcg acagtaaagt gggaggcaac tataattatc gttatcgact cttcagaaag 420

tctaacctta agccatttga acgtgacatt tctacagaaa tttaccaagc cggctctaag 480

ccttgcaatg gcgtggaagg gtttaactgt tatttcccat tacagtctta tggtttccag 540

ccaactaatg gtgtgggata ccaaccttac cgcgtcgttg tcctgtcgtt tgaattgctt 600

cacgcaccag ccaccgtttg tgggccaaag aagagcacta atctcgtagt tcagcctact 660

gaatcgatcg tgaggttccc aaatattacc aatctgtgtc cgttcggaga ggtcttcaat 720

gcgactcgat tcgcgtctgt ttacgcctgg aacaggaaac ggattagcaa ttgtgtcgct 780

gactattcgg tcttatacaa ctctgcatca ttctcaacct tcaagtgtta tggtgtcagc 840

cctacaaagc tgaatgactt atgtttcacc aatgtttatg cggacagttt cgtaatacga 900

ggtgatgaag tccgccaaat tgcacccgga caaaccggca acatagccga ctataattat 960

aagctccctg atgactttac gggctgtgtc atagcttgga atagtaataa tttggactcg 1020

aaagtggggag gtaattataa ttatctctat agactgttcc ggaaatcaaa tctcaagccc 1080

tttgaacggg acataagtac agaaatctac caagctggtt ccacgccgtg taatggagtc 1140

aaggggttta actgttattt cccgctccag tcgtatgggt tccagccaac gtatggcgtc 1200

ggataccaac cttaccgcgt tgtagtatta agctttgaac tgttgcacgc gcccgcgact 1260

gtttgtggcc cgaagaagtc gactaatcta gtaaagaata ag 1302

<210> 10

<211> 1302

<212>DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> DNA coding sequence of DO mRNA vaccine

<222> (1)..(1302)

<400> 10

cgagtgcagc ctaccgaaag catcgtccgt ttcccgaata ttactaatct ctgtccattc 60

ggagaagtct tcaatgccac ccgattcgct tccgtttacg cgtggaaccg taaacgaata 120

tctaattgtg ttgcggacta ttccgtgttg tacaactcag catcattctc tacttttaaa 180

tgctatggag tgtcgccgac taaactcaac gacttgtgtt tcactaatgt ttatgctgac 240

tctttcgtta ttcgtggaga cgaagttcgt caaatcgcac cagggcaaac tggcaagatt 300

gcggactata attataagct gccagatgac tttaccggat gtgtaatagc ctggaactca 360

aataatctcg acagtaaagt gggaggcaac tataattatc gttatcgact cttcagaaag 420

tctaacctta agccatttga acgtgacatt tctacagaaa tttaccaagc cggctctaag 480

ccttgcaatg gcgtggaagg gtttaactgt tatttcccat tacagtctta tggtttccag 540

ccaactaatg gtgtgggata ccaaccttac cgcgtcgttg tcctgtcgtt tgaattgctt 600

cacgcaccag ccaccgtttg tgggccaaag aagagcacta atctcgtagt tcagcctact 660

gaatcgatcg tgaggttccc aaatattacc aatctgtgtc cgttcgacga ggtcttcaat 720

gcgactcgat tcgcgtctgt ttacgcctgg aacaggaaac ggattagcaa ttgtgtcgct 780

gactattcgg tcttatacaa cttggcacca ttcttcacct tcaagtgtta tggtgtcagc 840

cctacaaagc tgaatgactt atgtttcacc aatgtttatg cggacagttt cgtaatacga 900

ggtgatgaag tccgccaaat tgcacccgga caaaccggca acatagccga ctataattat 960

aagctccctg atgactttac gggctgtgtc atagcttgga atagtaataa gttggactcg 1020

aaagtgtcag gtaattataa ttatctctat agactgttcc ggaaatcaaa tctcaagccc 1080

tttgaacggg acataagtac agaaatctac caagctggta acaagccgtg taatggagtc 1140

gcagggttta actgttattt cccgctccgg tcgtattcct tccggccaac gtatggcgtc 1200

ggacaccaac cttaccgcgt tgtagtatta agctttgaac tgttgcacgc gcccgcgact 1260

gtttgtggcc cgaagaagtc gactaatcta gtaaagaata ag 1302

<210> 11

<211> 1302

<212> RNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> mRNA coding sequence of PB mRNA vaccine

<222> (1)..(1302)

<400> 11

cgagugcagc cuaccgaaag caucguccgu uucccgaaua uuacuaaucu cuguccauuc 60

ggagaagucu ucaaugccac ccgauucgcu uccguuuacg cguggaaccg uaaacgaaua 120

ucuaauugug uugcggacua uuccguguug uacaacucag caucauucuc uacuuuuaaa 180

ugcuauggag ugucgccgac uaaacucaac gacuuguguu ucacuaaugu uuaugcugac 240

ucuuucguua uucguggaga cgaaguucgu caaaucgcac cagggcaaac uggcaagauu 300

gcggacuaua auuauaagcu gccagaugac uuuaccggau guguaauagc cuggaacuca 360

aauaaucucg acaguaaagu gggaggcaac uauaauuauc uuuaucgacu cuucagaaag 420

ucuaaccuua agccauuuga acgugacauu ucuacagaaa uuuaccaagc cggcucuaca 480

ccuugcaaug gcguggaagg guuuaacugu uauuucccau uacagucuua ugguuuccag 540

ccaacuaaug guguggaua ccaaccuuac cgcgucguug uccugucguu ugaauugcuu 600

cacgcaccag ccaccguuug ugggccaaag aagagcacua aucucguagu ucagccuacu 660

gaaucgaucg ugagguuccc aaauauuacc aaucuguc cguucggaga ggucuucaau 720

gcgacucgau ucgcgucugu uuacgccugg aacaggaaac ggauuagcaa uugugucgcu 780

gacuauucgg ucuuauacaa cucugcauca uucucaaccu ucaaguguua uggugucagc 840

ccuacaaagc ugaaugacuu auguuucacc aauguuuaug cggacaguuu cguaauacga 900

ggugaugaag uccgccaaau ugcacccgga caaaccggca acauagccga cuauaauuau 960

aagcucccug augacuuuac gggcuguc auagcuugga auaguaauaa uuuggacucg 1020

aaaguggggag guaauuauaa uuaucucuau agacuguucc ggaaaucaaa ucucaagccc 1080

uuugaacggg acauaaguac agaaaucuac caagcuggu ccacgccgug uaauggaguc 1140

aagggguuua acuguuauuu cccgcuccag ucguaugggu uccagccaac guauggcguc 1200

ggauaccaac cuuaccgcgu uguaguauua agcuuugaac uguugcacgc gcccgcgacu 1260

guuuguggcc cgaagaaguc gacuaaucua guaaagaaua ag 1302

<210> 12

<211> 1302

<212> RNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> mRNA coding sequence of DB mRNA vaccine

<222> (1)..(1302)

<400> 12

cgagugcagc cuaccgaaag caucguccgu uucccgaaua uuacuaaucu cuguccauuc 60

ggagaagucu ucaaugccac ccgauucgcu uccguuuacg cguggaaccg uaaacgaaua 120

ucuaauugug uugcggacua uuccguguug uacaacucag caucauucuc uacuuuuaaa 180

ugcuauggag ugucgccgac uaaacucaac gacuuguguu ucacuaaugu uuaugcugac 240

ucuuucguua uucguggaga cgaaguucgu caaaucgcac cagggcaaac uggcaagauu 300

gcggacuaua auuauaagcu gccagaugac uuuaccggau guguaauagc cuggaacuca 360

aauaaucucg acaguaaagu gggaggcaac uauaauuauc guuaucgacu cuucagaaag 420

ucuaaccuua agccauuuga acgugacauu ucuacagaaa uuuaccaagc cggcucuaag 480

ccuugcaaug gcguggaagg guuuaacugu uauuucccau uacagucuua ugguuuccag 540

ccaacuaaug guguggaua ccaaccuuac cgcgucguug uccugucguu ugaauugcuu 600

cacgcaccag ccaccguuug ugggccaaag aagagcacua aucucguagu ucagccuacu 660

gaaucgaucg ugagguuccc aaauauuacc aaucuguc cguucggaga ggucuucaau 720

gcgacucgau ucgcgucugu uuacgccugg aacaggaaac ggauuagcaa uugugucgcu 780

gacuauucgg ucuuauacaa cucugcauca uucucaaccu ucaaguguua uggugucagc 840

ccuacaaagc ugaaugacuu auguuucacc aauguuuaug cggacaguuu cguaauacga 900

ggugaugaag uccgccaaau ugcacccgga caaaccggca acauagccga cuauaauuau 960

aagcucccug augacuuuac gggcuguc auagcuugga auaguaauaa uuuggacucg 1020

aaaguggggag guaauuauaa uuaucucuau agacuguucc ggaaaucaaa ucucaagccc 1080

uuugaacggg acauaaguac agaaaucuac caagcuggu ccacgccgug uaauggaguc 1140

aagggguuua acuguuauuu cccgcuccag ucguaugggu uccagccaac guauggcguc 1200

ggauaccaac cuuaccgcgu uguaguauua agcuuugaac uguugcacgc gcccgcgacu 1260

guuuguggcc cgaagaaguc gacuaaucua guaaagaaua ag 1302

<210> 13

<211> 1302

<212> RNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> mRNA coding sequence of DO mRNA vaccine

<222> (1)..(1302)

<400> 13

cgagugcagc cuaccgaaag caucguccgu uucccgaaua uuacuaaucu cuguccauuc 60

ggagaagucu ucaaugccac ccgauucgcu uccguuuacg cguggaaccg uaaacgaaua 120

ucuaauugug uugcggacua uuccguguug uacaacucag caucauucuc uacuuuuaaa 180

ugcuauggag ugucgccgac uaaacucaac gacuuguguu ucacuaaugu uuaugcugac 240

ucuuucguua uucguggaga cgaaguucgu caaaucgcac cagggcaaac uggcaagauu 300

gcggacuaua auuauaagcu gccagaugac uuuaccggau guguaauagc cuggaacuca 360

aauaaucucg acaguaaagu gggaggcaac uauaauuauc guuaucgacu cuucagaaag 420

ucuaaccuua agccauuuga acgugacauu ucuacagaaa uuuaccaagc cggcucuaag 480

ccuugcaaug gcguggaagg guuuaacugu uauuucccau uacagucuua ugguuuccag 540

ccaacuaaug guguggaua ccaaccuuac cgcgucguug uccugucguu ugaauugcuu 600

cacgcaccag ccaccguuug ugggccaaag aagagcacua aucucguagu ucagccuacu 660

gaaucgaucg ugagguuccc aaauauuacc aaucuguc cguucgacga ggucuucaau 720

gcgacucgau ucgcgucugu uuacgccugg aacaggaaac ggauuagcaa uugugucgcu 780

gacuauucgg ucuuauacaa cuuggcacca uucuucaccu ucaaguguua uggugucagc 840

ccuacaaagc ugaaugacuu auguuucacc aauguuuaug cggacaguuu cguaauacga 900

ggugaugaag uccgccaaau ugcacccgga caaaccggca acauagccga cuauaauuau 960

aagcucccug augacuuac gggcuguc auagcuugga auaguaauaa guuggacucg 1020

aaagugucag guaauuauaa uuaucucuau agacuguucc ggaaaucaaa ucucaagccc 1080

uuugaacggg acauaaguac agaaaucuac caagcugga acaagccgug uaauggaguc 1140

gcaggguuua accuguuauuu cccgcuccgg ucguauuccu uccggccaac guauggcguc 1200

ggacaccaac cuuaccgcgu uguaguauua agcuuugaac uguugcacgc gcccgcgacu 1260

guuuguggcc cgaagaaguc gacuaaucua guaaagaaua ag 1302

<210> 14

<211> 1302

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> DNA coding sequence of PP mRNA vaccine

<222> (1)..(1302)

<400> 14

cgagtgcagc ctaccgaaag catcgtccgt ttcccgaata ttactaatct ctgtccattc 60

ggagaagtct tcaatgccac ccgattcgct tccgtttacg cgtggaaccg taaacgaata 120

tctaattgtg ttgcggacta ttccgtgttg tacaactcag catcattctc tacttttaaa 180

tgctatggag tgtcgccgac taaactcaac gacttgtgtt tcactaatgt ttatgctgac 240

tctttcgtta ttcgtggaga cgaagttcgt caaatcgcac cagggcaaac tggcaagatt 300

gcggactata attataagct gccagatgac tttaccggat gtgtaatagc ctggaactca 360

aataatctcg acagtaaagt gggaggcaac tataattatc tttatcgact cttcagaaag 420

tctaacctta agccatttga acgtgacatt tctacagaaa tttaccaagc cggctctaca 480

ccttgcaatg gcgtggaagg gtttaactgt tatttcccat tacagtctta tggtttccag 540

ccaactaatg gtgtgggata ccaaccttac cgcgtcgttg tcctgtcgtt tgaattgctt 600

cacgcaccag ccaccgtttg tgggccaaag aagagcacta atctcgtagt tcagcctact 660

gaatcgatcg tgaggttccc aaatattacc aatctgtgtc cgttcggaga ggtcttcaat 720

gcgactcgat tcgcgtctgt ttacgcctgg aacaggaaac ggattagcaa ttgtgtcgct 780

gactattcgg tcttatacaa ctctgcatca ttctcaacct tcaagtgtta tggtgtcagc 840

cctacaaagc tgaatgactt atgtttcacc aatgtttatg cggacagttt cgtaatacga 900

ggtgatgaag tccgccaaat tgcacccgga caaaccggca agatagccga ctataattat 960

aagctccctg atgactttac gggctgtgtc atagcttgga atagtaataa tttggactcg 1020

aaagtggggag gtaattataa ttatctctat agactgttcc ggaaatcaaa tctcaagccc 1080

tttgaacggg acataagtac agaaatctac caagctggtt ccacgccgtg taatggagtc 1140

gaggggttta actgttattt cccgctccag tcgtatgggt tccagccaac gaatggcgtc 1200

ggataccaac cttaccgcgt tgtagtatta agctttgaac tgttgcacgc gcccgcgact 1260

gtttgtggcc cgaagaagtc gactaatcta gtaaagaata ag 1302

<210> 15

<211> 47

<212>DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> 5' UTR

<222> (1)..(47)

<400> 15

gggaaataag agagaaaaga agagtaagaa gaaatataag agccacc 47

<210> 16

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> DNA coding sequence for signal peptide

<222> (1)..(45)

<400> 16

atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgc 45

<210> 17

<211> 110

<212>DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> 3' end UTR

<222> (1)..(110)

<400> 17

tgataatagg ctggagcctc ggtggccatg cttcttgccc cttgggcctc cccccagccc 60

ctcctcccct tcctgcaccc gtacccccgt ggtctttgaa taaagtctga 110

<210> 18

<211> 223

<212> PRT

<213> SARS-CoV-2 Omicron variant strain BA.1.1 subtype

<220>

<221> RBD antigen protein of SARS-CoV-2 Omicron variant strain BA.1.1 subtype

<222> (1)..(223)

<400> 18

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

1 5 10 15

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

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 Asn Lys

145 150 155 160

Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser

165 170 175

Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly His 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> 19

<211> 223

<212> PRT

<213> SARS-CoV-2 Omicron variant BA.2 subtype

<220>

<221> RBD antigen protein of SARS-CoV-2 Omicron variant strain BA.2 subtype

<222> (1)..(223)

<400> 19

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

1 5 10 15

Leu Cys Pro Phe Asp 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 Phe Ala Pro Phe Phe Ala 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 Asn Glu Val Ser Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Asn 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 Lys 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 Asn Lys

145 150 155 160

Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser

165 170 175

Tyr Gly Phe Arg Pro Thr Tyr Gly Val Gly His 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> 20

<211> 223

<212> PRT

<213> SARS-CoV-2 Omicron variant BA.3 subtype

<220>

<221> RBD antigen protein of SARS-CoV-2 Omicron variant strain BA.3 subtype

<222> (1)..(223)

<400> 20

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

1 5 10 15

Leu Cys Pro Phe Asp 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 Phe Ala Pro Phe Phe 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 Asn Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Asn 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 Lys Leu Asp Ser Lys Val Ser

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 Asn Lys

145 150 155 160

Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser

165 170 175

Tyr Gly Phe Arg Pro Thr Tyr Gly Val Gly His 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> 21

<211> 3765

<212>DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> Nucleotide sequence of WT-S-del18

<222> (1)..(3765)

<400> 21

atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60

cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120

aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180

aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac 240

aacccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300

atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360

aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420

ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agtctgagtt cagagtgtac 480

tctagcgcca acaactgcac cttcgagtac gtgtcccagc ctttcctcat ggacctggag 540

ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat tgacggctac 600

ttcaagattt actctaagca caccccaatt aacctcgtga gggacctccc tcagggcttc 660

tccgccttag aaccactggt ggacctccct attggcatta acatcacg cttccagaca 720

ctgctcgccc tccaccggtc ttacctgacc ccaggcgact ctagctctgg ctggacagcc 780

ggcgccgccg cctactacgt gggctacctg cagcctagga ccttcctcct gaagtacaac 840

gagaacggca caattaccga cgccgtggac tgcgccctgg accactgtc cgagacaaag 900

tgcacactga agtccttcac agtggagaag ggcatttacc agacatctaa cttccgggtg 960

cagcctacag agtctattgt gcggttccca aacatcacaa acctgtgccc tttcggcgag 1020

gtgttcaacg ccaccccggtt cgcctctgtg tacgcctgga accggaagcg gatctctaac 1080

tgcgtggccg actactccgt gctgtacaac tccgcctctt tctctacatt caagtgctac 1140

ggcgtgtccc ctacaaagct gaacgacctg tgcttcacca acgtgtacgc cgactctttc 1200

gtgattatagg gcgacgaggt gaggcagatt gcccccggcc agacaggcaa gatcgccgac 1260

tacaactaca agctgcccga cgacttcaca ggctgcgtga tcgcctggaa ctctaacaac 1320

ctggactcta aggtgggcgg caactacaac tacctgtaca gactgttccg gaagtctaac 1380

ctgaagccat tcgagaggga cattagcacc gagattacc aggccggctc taccccatgc 1440

aacggcgtgg agggcttcaa ctgctacttc ccactgcagt cctacggctt ccagcctaca 1500

aacggcgtgg gctaccagcc ttaccgggtg gtggtgctgt ctttcgagct gctccacgcc 1560

cccgccacag tgtgcggccc aaagaagagc acaaacctcg tgaagaacaa gtgcgtgaac 1620

ttcaacttca acggcctcac aggcacaggc gtgctcaccg agtctaacaa gaagttcctc 1680

cctttccagc agttcggccg cgacattgcc gacaccaccg acgccgtgcg ggaccctcag 1740

acactggaaa ttctcgacat caccccttgc agcttcggcg gcgtgtccgt gatcacccca 1800

ggcacaaaca catctaacca ggtggccgtg ctgtaccagg acgtgaactg caccgaggtg 1860

ccagtggcca tccacgccga ccagctcacc ccaacatgga gggtgtacag cacaggctct 1920

aacgtgttcc agacccgggc cggctgcctc attggcgccg agcacgtgaa caactcttac 1980

gagtgcgaca tccctattgg cgccggcatt tgcgcctctt accagaccca gacaaactct 2040

ccacggagag cccggtctgt ggcctctcag agcattattg cttacaccat gtctctgggc 2100

gccgagaact ctgtggccta ctctaacaac tctattgcca tccctacaaa cttcacaatt 2160

tctgtgacca ccgagattct cccagtgtct atgaccaaga catctgtgga ctgcaccatg 2220

tacatttgcg gcgactccac cgagtgctct aacctcctgc tccagtacgg ctctttctgc 2280

accccagctca accgcgccct gacaggcatc gccgtggagc aggacaagaa cacccaggag 2340

gtgttcgccc aggtgaagca gatttacaag acccccccaa ttaaggactt cggcggcttc 2400

aacttctctc agattctccc cgacccatcc aagcctagca agcggtcctt cattgaggac 2460

ctcctgttca acaaggtgac actggccgac gccggcttca ttaagcagta cggcgactgc 2520

ctgggcgaca ttgccgcccg ggacctgatt tgcgcccaga agttcaacgg cctcacagtg 2580

ctccccccac tgctcaccga cgagatgatt gcccagtaca catctgccct cctggccggc 2640

acaattacat ctggctggac cttcggcgcc ggcgccgccc tgcagatccc tttcgccatg 2700

cagatggcct accgcttcaa cggcatcggc gtgacacaga acgtgctgta cgagaaccag 2760

aagctgatcg ccaaccagtt caacagcgcc attggcaaga ttcaggactc tctgagcagc 2820

acagccagcg ccctgggcaa gctgcaggac gtggtgaacc agaacgccca ggccctgaac 2880

acactggtga agcagctgtc ttctaacttc ggcgccattt ctagcgtgct gaacgacatt 2940

ctgtcgcggc tggacaaggt ggaggccgag gtgcagattg acaggctcat cacaggcaga 3000

ctgcagtctc tgcagacata cgtgacccag cagctgatta gagccgccga gattagagcc 3060

tccgccaacc tggccgccac caagatgagc gagtgcgtgc tcggccagtc taagcgggtg 3120

gacttctgcg gcaagggcta ccacctcatg tctttccctc agtccgcccc tcacggcgtg 3180

gtgttcctcc acgtgacata cgtgcccgcc caggagaaga acttcaccac agcccccgcc 3240

atttgccacg acggcaaggc ccacttccct aggggagggcg tgttcgtgtc taacggcacc 3300

cactggttcg tgacccagcg gaacttctac gagcctcaga ttattaccac agacaacaca 3360

ttcgtgagcg gcaactgcga cgtggtgatt ggcattgtga acaacacagt gtacgaccca 3420

ctgcagcctg agttggactc tttcaaggag gaactcgaca agtacttcaa gaaccacaca 3480

tctcctgacg tggacctggg cgacattagc ggcattaacg cctctgtggt gaacattcag 3540

aaggagattg acagactgaa cgaggtggcc aagaacctga acgagtctct cattgacctg 3600

caggagctgg gcaagtacga gcagtacatt aagtggcctt ggtacatttg gctgggcttc 3660

attgccggcc tgatcgccat tgtgatggtg accatcatgc tgtgctgcat gacatcttgc 3720

tgcagctgcc tgaagggctg ctgctcttgc ggctcttgct gcaag 3765

<210> 22

<211> 3759

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> Nucleotide sequence of Delta-S-del18

<222> (1)..(3759)

<400> 22

atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgagaaca 60

cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120

aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180

aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac 240

aacccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300

atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360

aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420

ctggacgtgt actaccacaa gaacaacaag tcttggatgg agtctggcgt gtactctagc 480

gccaacaact gcaccttcga gtacgtgtcc cagcctttcc tcatggacct ggagggcaag 540

cagggcaact tcaagaacct gagagagttc gtgttcaaga aattgacgg ctacttcaag 600

atttactcta agcacacccc aattaacctc gtgagggacc tccctcaggg cttctccgtg 660

ttagaaccac tggtggacct ccctattggc attaacatca cacgcttcca gacactgctc 720

gccctccacc ggtcttacct gaccccaggc gactctagct ctggctggac agccggcgcc 780

gccgcctact acgtgggcta cctgcagcct aggaccttcc tcctgaagta caacgagaac 840

ggcacaatta ccgacgccgt ggactgcgcc ctggacccac tgtccgagac aaagtgcaca 900

ctgaagtcct tcacagtgga gaagggcatt taccagacat ctaacttccg ggtgcagcct 960

acagagtcta ttgtgcggtt cccaaacatc acaaacctgt gccctttcgg cgaggtgttc 1020

aacgccacccc ggttcgcctc tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 1080

gccgactact ccgtgctgta caactccgcc tctttctcta cattcaagtg ctacggcgtg 1140

tcccctacaa agctgaacga cctgtgcttc accaacgtgt acgccgactc tttcgtgatt 1200

agaggcgacg aggtgaggca gattgccccc ggccagacag gcaagatcgc cgactacaac 1260

tacaagctgc ccgacgactt cacaggctgc gtgatcgcct ggaactctaa caacctggac 1320

tctaaggtgg gcggcaacta caactacaga tacagactgt tccggaagtc taacctgaag 1380

ccattcgaga gggacattag caccgagatt taccaggccg gctctaagcc atgcaacggc 1440

gtggagggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacaaacggc 1500

gtgggctacc agccttaccg ggtggtggtg ctgtctttcg agctgctcca cgcccccgcc 1560

acagtgtgcg gcccaaagaa gagcacaaac ctcgtgaaga acaagtgcgt gaacttcaac 1620

ttcaacggcc tcacaggcac aggcgtgctc accgagtcta acaagaagtt cctccctttc 1680

cagcagttcg gccgcgacat tgccgacacc accgacgccg tgcgggaccc tcagacactg 1740

gaaattctcg acatcacccc ttgcagcttc ggcggcgtgt ccgtgatcac cccaggcaca 1800

aacacatcta accaggtggc cgtgctgtac cagggcgtga actgcaccga ggtgccagtg 1860

gccatccacg ccgaccagct caccccaaca tggagggtgt acagcacagg ctctaacgtg 1920

ttccagaccc gggccggctg cctcattggc gccgagcacg tgaacaactc ttacgagtgc 1980

gacatcccta ttggcgccgg catttgcgcc tcttaccaga cccagacaaa ctctagacgg 2040

agagcccggt ctgtggcctc tcagagcatt attgcctaca ccatgtctct gggcgccgag 2100

aactctgtgg cctactctaa caactctatt gccatcccta caaacttcac aatttctgtg 2160

accaccgaga ttctcccagt gtctatgacc aagacatctg tggactgcac catgtacatt 2220

tgcggcgact ccaccgagtg ctctaacctc ctgctccagt acggctcttt ctgcacccag 2280

ctcaaccgcg ccctgacagg catcgccgtg gagcaggaca agaacaccca ggaggtgttc 2340

gcccaggtga agcagattta caagacccccc ccaattaagg acttcggcgg cttcaacttc 2400

tctcagattc tccccgaccc atccaagcct agcaagcggt ccttcattga ggacctcctg 2460

ttcaacaagg tgacactggc cgacgccggc ttcattaagc agtacggcga ctgcctgggc 2520

gacattgccg cccgggacct gatttgcgcc cagaagttca acggcctcac agtgctcccc 2580

ccactgctca ccgacgagat gattgcccag tacacatctg ccctcctggc cggcacaatt 2640

acatctggct ggaccttcgg cgccggcgcc gccctgcaga tccctttcgc catgcagatg 2700

gcctaccgct tcaacggcat cggcgtgaca cagaacgtgc tgtacgagaa ccagaagctg 2760

atcgccaacc agttcaacag cgccattggc aagattcagg actctctgag cagcacagcc 2820

agcgccctgg gcaagctgca gaacgtggtg aaccagaacg cccaggccct gaacacactg 2880

gtgaagcagc tgtcttctaa cttcggcgcc atttctagcg tgctgaacga cattctgtcg 2940

cggctggaca aggtggaggc cgaggtgcag attgacaggc tcatcacagg cagactgcag 3000

tctctgcaga catacgtgac ccagcagctg attagagccg ccgagattag agcctccgcc 3060

aacctggccg ccaccaagat gagcgagtgc gtgctcggcc agtctaagcg ggtggacttc 3120

tgcggcaagg gctaccacct catgtctttc cctcagtccg cccctcacgg cgtggtgttc 3180

ctccacgtga catacgtgcc cgcccaggag aagaacttca ccacagcccc cgccatttgc 3240

cacgacggca aggcccactt ccctagggag ggcgtgttcg tgtctaacgg cacccactgg 3300

ttcgtgaccc agcggaactt ctacgagcct cagattatta ccacagacaa cacattcgtg 3360

agcggcaact gcgacgtggt gattggcatt gtgaacaaca cagtgtacga cccactgcag 3420

cctgagttgg actctttcaa ggaggaactc gacaagtact tcaagaacca cacatctcct 3480

gacgtggacc tgggcgacat tagcggcatt aacgcctctg tggtgaacat tcagaaggag 3540

attgacagac tgaacgaggt ggccaagaac ctgaacgagt ctctcattga cctgcaggag 3600

ctgggcaagt acgagcagta cattaagtgg ccttggtaca tttggctggg cttcattgcc 3660

ggcctgatcg ccattgtgat ggtgaccatc atgctgtgct gcatgacatc ttgctgcagc 3720

tgcctgaagg gctgctgctc ttgcggctct tgctgcaag 3759

<210> 23

<211> 3756

<212>DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> Nucleotide sequence of BA.1-S-del18

<222> (1)..(3756)

<400> 23

atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60

cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120

aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180

aacgtgacat ggttccacgt gatctctggc acaaacggca caaagcggtt cgacaaccccc 240

gtgctccctt tcaacgacgg cgtgtacttc gccagcattg agaagtctaa cattatccgg 300

ggctggattt tcggcaccac actcgactct aagacacagt ccctcctgat tgtgaacaac 360

gccacaaacg tggtgattaa ggtgtgcgag ttccagttct gcaacgaccc tttcctggac 420

cacaagaaca acaagtcttg gatggagtct gagttcagag tgtactctag cgccaacaac 480

tgcaccttcg agtacgtgtc ccagcctttc ctcatggacc tggagggcaa gcagggcaac 540

ttcaagaacc tgagagagtt cgtgttcaag aacattgacg gctacttcaa gatttactct 600

aagcacaccc caattattgt gagggaacca gaagacctcc ctcagggctt ctccgcctta 660

gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720

ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780

gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840

acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900

aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960

gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020

gccaccccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080

gactactccg tgctgtacaa cctggcccct ttcttcacat tcaagtgcta cggcgtgtcc 1140

cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200

ggcgacgagg tgaggcagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260

aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320

aaggtgtctg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380

ttcgagagggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440

gccggcttca actgctactt cccactgcgc tcctactcct tccggcctac atacggcgtg 1500

ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560

gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620

aacggcctca agggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680

cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740

attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800

acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860

atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920

caaacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980

atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040

gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100

tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160

accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220

ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280

aagcgcgccc tgacaggcat cgccgtggag caggacaaga aacccagga ggtgttcgcc 2340

caggtgaagc agattacaa gaccccccca attaagtact tcggcggctt caacttctct 2400

cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460

aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520

attgccgccc gggacctgat ttgcgcccag aagttcaagg gcctcacagt gctcccccca 2580

ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640

tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700

taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760

gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820

gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880

aagcagctgt cttctaagtt cggcgccatt tctagcgtgc tgaacgacat tttctcgcgg 2940

ctggacaagg tggaggccga ggtgcagatt gacaggctca tcacaggcag actgcagtct 3000

ctgcagacat acgtgaccca gcagctgatt agagccgccg agattagagc ctccgccaac 3060

ctggccgcca ccaagatgag cgagtgcgtg ctcggccagt ctaagcgggt ggacttctgc 3120

ggcaagggct accacctcat gtctttccct cagtccgccc ctcacggcgt ggtgttcctc 3180

cacgtgacat acgtgcccgc ccaggagaag aacttcacca cagcccccgc catttgccac 3240

gacggcaagg cccacttccc tagggagggc gtgttcgtgt ctaacggcac ccactggttc 3300

gtgacccagc ggaacttcta cgagcctcag atttattacca cagacaacac attcgtgagc 3360

ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420

gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480

gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540

gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600

ggcaagtacg agcagtacat taagtggcct tggtacattt ggctgggctt cattgccggc 3660

ctgatcgcca ttgtgatggt gaccatcatg ctgtgctgca tgacatcttg ctgcagctgc 3720

ctgaagggct gctgctcttg cggctcttgc tgcaag 3756

<210> 24

<211> 3756

<212>DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> Nucleotide sequence of BA.1.1-S-del18

<222> (1)..(3756)

<400> 24

atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60

cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120

aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180

aacgtgacat ggttccacgt gatctctggc acaaacggca caaagcggtt cgacaaccccc 240

gtgctccctt tcaacgacgg cgtgtacttc gccagcattg agaagtctaa cattatccgg 300

ggctggattt tcggcaccac actcgactct aagacacagt ccctcctgat tgtgaacaac 360

gccacaaacg tggtgattaa ggtgtgcgag ttccagttct gcaacgaccc tttcctggac 420

cacaagaaca acaagtcttg gatggagtct gagttcagag tgtactctag cgccaacaac 480

tgcaccttcg agtacgtgtc ccagcctttc ctcatggacc tggagggcaa gcagggcaac 540

ttcaagaacc tgagagagtt cgtgttcaag aacattgacg gctacttcaa gatttactct 600

aagcacaccc caattattgt ggaaccagaa agggacctcc ctcagggctt ctccgcctta 660

gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720

ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780

gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840

acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900

aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960

gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020

gccaccaagt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080

gactactccg tgctgtacaa cctggcccct ttcttcacat tcaagtgcta cggcgtgtcc 1140

cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200

ggcgacgagg tgaggcagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260

aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320

aaggtgtctg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380

ttcgagagggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440

gccggcttca actgctactt cccactgcgc tcctactcct tccggcctac atacggcgtg 1500

ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560

gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620

aacggcctca agggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680

cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740

attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800

acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860

atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920

caaacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980

atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040

gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100

tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160

accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220

ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280

aagcgcgccc tgacaggcat cgccgtggag caggacaaga aacccagga ggtgttcgcc 2340

caggtgaagc agattacaa gaccccccca attaagtact tcggcggctt caacttctct 2400

cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460

aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520

attgccgccc gggacctgat ttgcgcccag aagttcaagg gcctcacagt gctcccccca 2580

ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640

tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700

taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760

gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820

gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880

aagcagctgt cttctaagtt cggcgccatt tctagcgtgc tgaacgacat tttctcgcgg 2940

ctggacaagg tggaggccga ggtgcagatt gacaggctca tcacaggcag actgcagtct 3000

ctgcagacat acgtgaccca gcagctgatt agagccgccg agattagagc ctccgccaac 3060

ctggccgcca ccaagatgag cgagtgcgtg ctcggccagt ctaagcgggt ggacttctgc 3120

ggcaagggct accacctcat gtctttccct cagtccgccc ctcacggcgt ggtgttcctc 3180

cacgtgacat acgtgcccgc ccaggagaag aacttcacca cagcccccgc catttgccac 3240

gacggcaagg cccacttccc tagggagggc gtgttcgtgt ctaacggcac ccactggttc 3300

gtgacccagc ggaacttcta cgagcctcag atttattacca cagacaacac attcgtgagc 3360

ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420

gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480

gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540

gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600

ggcaagtacg agcagtacat taagtggcct tggtacattt ggctgggctt cattgccggc 3660

ctgatcgcca ttgtgatggt gaccatcatg ctgtgctgca tgacatcttg ctgcagctgc 3720

ctgaagggct gctgctcttg cggctcttgc tgcaag 3756

<210> 25

<211> 3756

<212>DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> Nucleotide sequence of BA.2-S-del18

<222> (1)..(3756)

<400> 25

atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgatcaca 60

cggacccaga gctacacaaa ctctttcacc cggggcgtgt actacccga caaggtgttc 120

cggtctagcg tgctccactc tacacaggac ctgttcctcc ctttcttcag caacgtgaca 180

tggttccacg ccatccacgt gtctggcaca aacggcacaa agcggttcga caaccccgtg 240

ctccctttca acgacggcgt gtacttcgcc agcaccgaga agtctaacat tatccggggc 300

tggattttcg gcaccaacact cgactctaag acacagtccc tcctgattgt gaacaacgcc 360

acaaacgtgg tgattaaggt gtgcgagttc cagttctgca acgacccttt cctggacgtg 420

tactaccaca agaacaacaa gtcttggatg gagtctgagt tcagagtgta ctctagcgcc 480

aacaactgca ccttcgagta cgtgtcccag cctttcctca tggacctgga gggcaagcag 540

ggcaacttca agaacctgag agagttcgtg ttcaagaaca ttgacggcta cttcaagatt 600

tactctaagc acaccccaat taacctcggc agggacctcc ctcagggctt ctccgcctta 660

gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720

ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780

gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840

acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900

aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960

gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020

gccaccccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080

gactactccg tgctgtacaa cttcgccccc ttcttcgcct tcaagtgcta cggcgtgtcc 1140

cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200

ggcaacgagg tgagccagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260

aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320

aaggtgggcg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380

ttcgagagggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440

gccggcttca actgctactt cccactgcgg tcctacggct tccggcctac atacggcgtg 1500

ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560

gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620

aacggcctca caggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680

cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740

attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800

acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860

atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920

cagacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980

atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040

gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100

tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160

accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220

ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280

aagcgcgccc tgacaggcat cgccgtggag caggacaaga aacccagga ggtgttcgcc 2340

caggtgaagc agattacaa gaccccccca attaagtact tcggcggctt caacttctct 2400

cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460

aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520

attgccgccc gggacctgat ttgcgcccag aagttcaacg gcctcacagt gctcccccca 2580

ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640

tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700

taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760

gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820

gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880

aagcagctgt cttctaagtt cggcgccatt agcagcgtgc tgaacgacat tctgtcgcgg 2940

ctggacaagg tggaggccga ggtgcagatt gacaggctca tcacaggcag actgcagtct 3000

ctgcagacat acgtgaccca gcagctgatt agagccgccg agattagagc ctccgccaac 3060

ctggccgcca ccaagatgag cgagtgcgtg ctcggccagt ctaagcgggt ggacttctgc 3120

ggcaagggct accacctcat gtctttccct cagtccgccc ctcacggcgt ggtgttcctc 3180

cacgtgacat acgtgcccgc ccaggagaag aacttcacca cagcccccgc catttgccac 3240

gacggcaagg cccacttccc tagggagggc gtgttcgtgt ctaacggcac ccactggttc 3300

gtgacccagc ggaacttcta cgagcctcag atttattacca cagacaacac attcgtgagc 3360

ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420

gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480

gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540

gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600

ggcaagtacg agcagtacat taagtggcct tggtacattt ggctgggctt cattgccggc 3660

ctgatcgcca ttgtgatggt gaccatcatg ctgtgctgca tgacatcttg ctgcagctgc 3720

ctgaagggct gctgctcttg cggctcttgc tgcaag 3756

<210> 26

<211> 3747

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic polynucleotides

<220>

<221> Nucleotide sequence of BA.3-S-del18

<222> (1)..(3747)

<400> 26

atgttcgtgt tcctggtgct cctgcccctc gtgagctctc agtgcgtgaa cctgaccacc 60

cggacacagc tgccacctgc ctacaccaac tctttcacaa gaggcgtgta ctaccccgac 120

aaggtgttcc ggagcagcgt gctgcacagc acacaggatc tgttcctgcc cttcttcagc 180

aacgtgacct ggttccacgt gatcagcggc accaacggaa caaaaagatt tgacaacccc 240

gtgctgcctt ttaacgatgg cgtctacttc gcctccaccg agaagagcaa catcatccgc 300

ggctggatct tcggtacaac cctggattcc aagaccaga gcctgctgat cgtgaacaat 360

gccacaaacg tggtgatcaa ggtgtgtgag ttccagttct gtaacgaccc ttttctggga 420

cacaagaata acaagagctg gatggaaagc gagttccgag tgtactccag cgccaacaac 480

tgcactttcg agtacgtgag ccagcctttc ctgatggacc tggaaggcaa gcagggaaat 540

ttcaagaacc tgcgggagtt cgtgtttaag aacattgatg gctactttaa gatctacagc 600

aagcacaccc caatcatcgt gcgggacctg cctcaaggct tcagcgccct cgaacctctg 660

gtggacctgc ccatcggaat caacatcaca cggtttcaga ccctgctggc cctgcatagg 720

agctacctga cacctggcga cagcagctcc ggctggacag ccggagctgc cgcttattac 780

gttggctacc tgcagcctcg tacattcctg cttaagtata atgagaatgg cacaatcacc 840

gacgccgtgg actgcgccct ggaccccctg tctgagacaa aatgcaccct gaagtctttc 900

accgtggaaa agggcatcta ccagacctct aacttccgcg tccagcctac cgagtccatc 960

gtccggttcc ctaacataac caacctgtgc cctttcgacg aggtgtttaa cgccaccaga 1020

ttcgcttctg tgtacgcctg gaacagaaag agaatcagca attgtgtggc tgactacagc 1080

gtgctctaca actttgcccc ttttttcaca ttcaagtgct acggagtgag ccctacaaag 1140

ctgaacgacc tgtgcttcac caacgtgtac gccgacagct ttgttatccg gggcaatgag 1200

gtgagacaga tcgcccctgg acagaccgga aacatcgccg attacaacta caaactgcca 1260

gatgacttca ccggctgcgt gatcgcctgg aactccaaca agctggactc taaggtgagc 1320

ggcaattaca actacctgta cagactgttt cggaagagca acctgaagcc tttcgagaga 1380

gatataagca ccgagatcta ccaggctggc aataaacctt gcaacggcgt tgccggcttc 1440

aactgctact tccctctgag aagctacggc tttaggccca cctacggcgt gggccaccag 1500

ccctaccggg tggtggtgct gagcttcgag ctgctgcacg cccccgcaac cgtgtgcggc 1560

cctaagaaat ctacaaatct cgtgaaaaat aagtgcgtca acttcaactt caatggcctg 1620

accggcacgg gtgtactgac cgagtctaac aagaaattcc tgcccttcca acagttcggc 1680

agagacatcg ccgacaccac cgatgccgtg cgggacccac aaacccttga gatcctggat 1740

atcacacctt gtagttttgg cggcgtgtct gtcatcaccc ctggcaccaa cacctctaac 1800

caagtggccg tcctctacca gggcgttaat tgcaccgagg tccctgtggc aatccacgcc 1860

gaccagctga cccccacatg gagagtgtac agcacaggca gcaacgtgtt ccaaacaaga 1920

gccggctgcc tgatcggcgc tgaatacgtg aataacagct acgagtgcga catccccatc 1980

ggggctggga tctgcgccag ctaccagacc cagaccaaaa gccacagaag agcccggagc 2040

gttgccagcc agtcaatcat cgcctacacc atgagcctcg gcgctgagaa cagcgtggcc 2100

tattccaaca atagtatcgc catccctacc aatttcacca tctcggtgac caccgaaatc 2160

ctgcctgtga gtatgaccaa aacatcagtg gactgcacca tgtacatctg cggcgatagc 2220

accgagtgca gcaacctgct gctgcagtac gggagcttct gcacccaact gaagcgcgct 2280

ctgaccggca tcgctgtgga acaggataag aacacacagg aggtgttcgc ccaggtgaag 2340

cagatctaca agacgcctcc tatcaagtac ttcggcggct tcaacttttc tcagatcctg 2400

cctgacccct caaagcccag caagcggtcc ttcatcgagg acctgctctt taacaaggtg 2460

acgctggccg acgctggctt catcaaacag tatggggatt gcctgggcga catcgccgcc 2520

agagacctga tttgtgccca gaagttcaac ggcctgaccg tcctccctcc tctgctgaca 2580

gacgaaatga tcgcccagta cacaagcgct ctgctggccg gcacaatcac tagcggctgg 2640

accttcggcg ccggagccgc tctgcaaatc cctttcgcca tgcagatggc ctacagattc 2700

aacggcattg gtgttaccca gaacgtgctg tatgagaacc agaagctgat cgccaaccag 2760

tttaatagcg ccatcggaaa gatccaagac tctctgagca gcaccgccag cgccttagga 2820

aagctgcagg acgtggtgaa ccacaacgcc caggccctga atacactggt gaagcagctg 2880

agctccaagt tcggcgccat ctcatctgtc cttaacgaca ttctgagtag actggacaag 2940

gtggaagccg aagtgcagat cgacagactg atcaccggca gactgcaaag cctgcagaca 3000

tatgtgaccc agcagctgat cagagcggcc gagatcagag ccagcgctaa tctggctgcc 3060

acaaagatgt ccgaatgcgt gctcggccag tccaagagag tggatttctg cggcaaaggc 3120

taccacctga tgagcttccc ccagagcgcc cctcacggcg tggtgtttct gcatgtgacc 3180

tacgtgcctg ctcaggaaaa gaacttcaca acagctcctg ccatctgtca cgacggcaag 3240

gcccacttcc ccagagaggg cgtattcgtg tctaacggca cccactggtt cgtgacccag 3300

agaaacttct acgagcctca gatcatcaca accgacaaca ccttcgtgag cggcaactgt 3360

gatgtggtga tcggcatcgt gaacaacacc gtttacgacc ccttacagcc tgagctggat 3420

tctttcaagg aagaactgga taaatacttc aagaatcaca caagtcccga cgtggaccta 3480

ggggacatct ctggcataaa cgcctccgtc gtgaacatcc agaaagaaat cgatagactg 3540

aacgaagtgg ccaagaacct gaacgagagc ctgatcgacc tgcaggagct gggcaaatac 3600

gagcagtaca tcaagtggcc ttggtacatc tggctgggct tcatcgccgg actgatcgcc 3660

atcgtgatgg tgaccatcat gctgtgttgc atgaccagct gctgcagctg cctgaaggga 3720

tgttgctctt gtggctcatg ctgtaaa 3747

Claims (26)

1. A polynucleotide encoding a recombinant chimeric antigenic peptide of structure as shown in formula (I): (A-B)-C-(A-B') (I) In formula (I): Option 1: A-B represents the amino acid sequence of the S protein RBD domain segment of the novel coronavirus prototype strain as shown in SEQ ID NO: 1; A-B' represents the amino acid sequence of the S protein RBD domain segment of the novel coronavirus Beta mutant strain as shown in SEQ ID NO: 2; or Option 2: A-B represents the amino acid sequence of the S protein RBD domain segment of the new coronavirus Delta variant strain as shown in SEQ ID NO:3; A-B' represents the amino acid sequence of the S protein RBD domain segment of the novel coronavirus Beta mutant strain as shown in SEQ ID NO: 2; or Option 3: A-B represents the amino acid sequence of the S protein RBD domain segment of the new coronavirus Delta variant strain as shown in SEQ ID NO:3; A-B' represents the amino acid sequence of the S protein RBD domain segment of the novel coronavirus Omicron mutant strain as shown in SEQ ID NO:4; C represents linker (GGS) _n ; where n=0. 2. The polynucleotide according to claim 1, wherein when formula (I) is option 1, the amino acid sequence of the recombinant chimeric antigen peptide of structure shown in formula (I) is as SEQ ID NO:5 shown; Or, when formula (I) is option 2, the amino acid sequence of the recombinant chimeric antigen peptide having the structure shown in formula (I) is as shown in SEQ ID NO:6; Alternatively, when formula (I) is option 3, the amino acid sequence of the recombinant chimeric antigen peptide having the structure shown in formula (I) is shown in SEQ ID NO:7. 3. The polynucleotide according to any one of claims 1-2, wherein the polynucleotide is a DNA molecule; When formula (I) is option 1, the DNA sequence of described DNA molecule is as shown in SEQ ID NO:8; When formula (I) is option 2, the DNA sequence of described DNA molecule is as shown in SEQ ID NO:9; When formula (I) is option 3, the DNA sequence of described DNA molecule is as shown in SEQ ID NO:10. 4. The polynucleotide according to any one of claims 1-2, wherein the polynucleotide is an mRNA molecule; When formula (I) is option 1, the mRNA sequence of described mRNA molecule is as shown in SEQ ID NO:11; When formula (I) is option 2, the mRNA sequence of described mRNA molecule is as shown in SEQ ID NO:12; When formula (I) is option 3, the mRNA sequence of described mRNA molecule is as shown in SEQ ID NO:13. 5. A nucleic acid construct comprising the polynucleotide of any one of claims 1-4, and optionally at least one expression regulatory element operably linked to the polynucleotide. 6. An expression vector comprising the nucleic acid construct as claimed in claim 5. 7. A host cell transformed or transfected with the polynucleotide according to any one of claims 1-4, the nucleic acid construct according to claim 5 or the expression vector according to claim 6. 8. The polynucleotide as claimed in any one of claims 1-4, the nucleic acid construct as claimed in claim 5, the expression vector as claimed in claim 6 or the host cell as claimed in claim 7 are prepared Application in vaccines for the prevention of novel coronaviruses. 9. A chimeric nucleic acid vaccine or immunogenic composition, comprising the polynucleotide as claimed in any one of claims 1-4, the nucleic acid construct as claimed in claim 5, the nucleic acid construct as claimed in claim 6 The expression vector of or the host cell as claimed in claim 7, and a physiologically acceptable vehicle, adjuvant and/or carrier. 10. The chimeric nucleic acid vaccine or immunogenic composition according to claim 9, wherein the carrier is a diluent. 11. chimeric nucleic acid vaccine or immunogenic composition according to claim 9, it is novel coronavirus DNA vaccine, and described DNA vaccine comprises: (i) eukaryotic expression vectors; and (ii) the DNA sequence of the recombinant chimeric antigen peptide encoding the structure shown in formula (I) constructed into the eukaryotic expression vector. 12. chimeric nucleic acid vaccine or immunogenic composition according to claim 11, is characterized in that, the dna sequence of the recombinant chimeric antigen peptide of described structure as shown in formula (I) is as SEQ ID NO: The DNA sequence shown in 8, 9 or 10. 13. The chimeric nucleic acid vaccine or immunogenic composition according to claim 11, wherein the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors. 14. chimeric nucleic acid vaccine or immunogenic composition according to claim 9, it is novel coronavirus mRNA vaccine, and described mRNA vaccine comprises: (I) the mRNA sequence of the recombinant chimeric antigen peptide encoding the structure shown in formula (I); and (II) Lipid nanoparticles. 15. chimeric nucleic acid vaccine or immunogenic composition according to claim 14, is characterized in that, the mRNA sequence of the recombinant chimeric antigen peptide of described coding structure as shown in formula (I) is such as SEQ ID NO: The mRNA sequence shown in 11, 12 or 13. 16. The chimeric nucleic acid vaccine or immunogenic composition according to claim 9, which is a novel coronavirus-viral vector vaccine, comprising: (1) a viral backbone vector; and (2) The DNA sequence of the recombinant chimeric antigen peptide encoding the structure shown in formula (I) constructed into the viral backbone vector. 17. chimeric nucleic acid vaccine or immunogenic composition according to claim 16, is characterized in that, the dna sequence of the recombinant chimeric antigen peptide of described coding structure as shown in formula (I) is such as SEQ ID NO: The DNA sequence shown in 8, 9 or 10. 18. The chimeric nucleic acid vaccine or immunogenic composition according to claim 16, wherein the viral backbone carrier is selected from one or more of the following viral vectors: adenovirus vectors, poxvirus vectors, Influenza virus vector, adeno-associated virus vector. 19. The chimeric nucleic acid vaccine or immunogenic composition according to any one of claims 9-18, wherein the vaccine or immunogenic composition is nasal spray, oral preparation, suppository or gastrointestinal The form of external preparations. 20. The chimeric nucleic acid vaccine or immunogenic composition according to claim 19, wherein the nasal spray is selected from aerosol, spray and powder spray. 21. chimeric nucleic acid vaccine or immunogenic composition according to claim 19, is characterized in that, described oral preparation is selected from tablet, powder, pill, granule, soft/hard capsule, film coating potions and ointments. 22. The chimeric nucleic acid vaccine or immunogenic composition according to claim 21, wherein the tablet is a sublingual tablet; And/or, the granules are fine granules; And/or, the powder is a powder; And/or, the pill is a pellet. 23. The chimeric nucleic acid vaccine or immunogenic composition according to claim 19, wherein the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid or an injectable preparation. 24. The chimeric nucleic acid vaccine or immunogenic composition according to claim 23, wherein the injectable formulation is a bolus injectable formulation. 25. A kit comprising the chimeric nucleic acid vaccine or immunogenic composition according to any one of claims 9-18, and optionally other types of novel coronavirus vaccines, said chimeric nucleic acid vaccine Or the immunogenic composition is packaged separately with the other types of novel coronavirus vaccines. 26. The kit according to claim 25, wherein said other types of novel coronavirus vaccines are novel coronavirus inactivated vaccines.

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