A charge mutant antigen of the receptor binding region of the novel coronavirus and its application

Abstract

The present invention discloses a charge mutant antigen of the receptor binding domain (RBD) of the novel coronavirus, and the mutant is obtained by adding consecutive negatively charged amino acids to the end of the RBD of the novel coronavirus receptor binding domain. Compared with the wild-type RBD antigen, the RBD antigen mutant can significantly increase the neutralizing antibody level of the host against the novel coronavirus after immunization with AL(OH)3 adjuvant. The present invention also provides the application of the RBD antigen mutant in the preparation of drugs or vaccines for the treatment and prevention of the novel coronavirus.

Description

Technical Field

[0001] The present invention relates to a charge mutant antigen of the receptor binding region of the novel coronavirus and its application, belonging to the technical field of polypeptides. Background Art

[0002] The novel coronavirus (SARS-CoV-2) is a newly discovered enveloped positive-sense single-stranded RNA virus, belonging to the Coronaviridae family and the β-coronavirus genus, which can cause a wide range of severe respiratory infectious diseases. Since its report at the end of 2019, the global situation of the novel coronavirus epidemic has been severe, seriously threatening human health and public health security.

[0003] Vaccines are an important means of preventing and controlling newly emerging and sudden infectious diseases. The development of vaccines against the novel coronavirus is urgent. The surface spike protein (Spike, S) is the main target antigen of the novel coronavirus, which has high immunogenicity and can induce the body to produce protective antibodies against the virus and provide protection against virus challenge. The receptor binding domain (RBD) of the S protein, as an independent domain, is responsible for binding to the host receptor angiotensin-converting enzyme 2 (ACE2), and is considered the core region for inducing the body to produce neutralizing antibodies. The recombinant RBD antigen protein can form a correct conformation, induce certain neutralizing antibodies, cellular responses, and immune protection effects in animal models (Nat Rev Immunol 21(2):73-82(2021)).

[0004] Aluminum adjuvant is the most widely used human vaccine adjuvant so far, showing acceptable safety and effectiveness in marketed vaccines. Currently, multiple novel coronavirus recombinant RBD antigen protein vaccines in the clinical stage use aluminum hydroxide (AL(OH)3) as an effective adjuvant component (https: / / www.who.int / emergencies / diseases / novel-coronavirus-2019 / covid-19-vaccines). However, in existing reports (Nature 586:572–577(2020); Cell 182: 722–733(2020)), the combination of the RBD antigen protein and the common AL(OH)3 adjuvant can only stimulate limited antibody and neutralizing antibody responses.

[0005] The purpose of the present invention is to provide a charge mutant antigen of the receptor binding region of the novel coronavirus, so that the antigen combined with the AL(OH)3 adjuvant can stimulate a strong neutralizing antibody response. Summary of the Invention

[0006] For the above purposes, the present invention first provides a novel coronavirus RBD antigen mutant, which is composed of an RBD antigen and 3-24 negatively charged amino acids with an isoelectric point pI less than 7.4 and a hydrophilicity index less than 0 connected to its end.

[0007] The present invention constructs and develops a novel coronavirus charge mutant RBD antigen protein by artificially designing and introducing multiple negatively charged amino acids at the end of the receptor binding domain (RBD) of the novel coronavirus, thereby changing the charge characteristics of the wild-type RBD protein.

[0008] By analyzing the charge properties of the novel coronavirus wild-type RBD (R319-K537) antigen protein, it is found that its isoelectric point pI is 8.95, and it shows a strong positive charge overall under normal physiological conditions (pH about 7.4). The commonly used AL(OH)3 adjuvant in vaccines shows a positive charge on its surface under normal physiological conditions, which is not conducive to the adsorption and slow release of the RBD antigen protein, and may be one of the reasons for the low immune efficacy of the combination of the two. The inventors attempt to artificially design and introduce multiple consecutive negatively charged amino acids at the end of RBD to change the charge characteristics of the wild-type RBD protein, which helps to improve its adsorption and slow release with the AL(OH)3 adjuvant, thereby enhancing the immune efficacy of the vaccine.

[0009] Further analyzing the surface charge distribution of the novel coronavirus RBD (R319-K537) protein, it is found that the C-terminal region has a strong positive charge. Therefore, in a preferred embodiment of the present invention, the negatively charged amino acids are connected to the C-terminal of the RBD antigen.

[0010] In a more preferred embodiment, the negatively charged amino acid - aspartic acid is linked to the C-terminus of the RBD antigen protein. The isoelectric point pI of a protein mainly depends on seven charged amino acids: glutamic acid Glu (isoelectric point pI is 3.22), aspartic acid Asp (isoelectric point pI is 2.77), cysteine Cys (isoelectric point pI is 5.07), tyrosine Tyr (isoelectric point pI is 5.66), histidine His (isoelectric point pI is 7.59), lysine Lys (isoelectric point pI is 9.74), and arginine Arg (isoelectric point pI is 10.76) (Nucleic Acid Res. DOI: 10.1093 / nar / gkab295 (2021)). Under normal physiological conditions (pH is about 7.4), four of these amino acids carry negative charges, namely glutamic acid Glu (isoelectric point pI is 3.22), aspartic acid Asp (isoelectric point pI is 2.77), cysteine Cys (isoelectric point pI is 5.07), and tyrosine Tyr (isoelectric point pI is 5.66). The hydrophilicity of the terminal-linked amino acid is another key factor affecting the expression and immunogenicity of the RBD protein. The hydropathy index of a certain amino acid is a value describing the degree of hydrophilicity or hydrophobicity of its side chain. The "hydropathy index" was proposed by Jack Kyte and Russell Doolittle in 1982. The larger the hydropathy index, the stronger the hydrophobicity of this amino acid. According to the amino acid hydropathy index table, the hydropathy indices of the four negatively charged amino acids are glutamic acid Glu (hydropathy index is -3.5), aspartic acid Asp (hydropathy index is -3.5), cysteine Cys (hydropathy index is 2.5), and tyrosine Tyr (hydropathy index is -1.3). The molecular size of the terminal-linked amino acid has a certain impact on the original spatial structure of the RBD protein. According to the amino acid relative molecular weight table, the relative molecular weights of the four negatively charged amino acids are glutamic acid Glu (relative molecular weight is 147.13), aspartic acid Asp (relative molecular weight is 133.10), cysteine Cys (relative molecular weight is 121.16), and tyrosine Tyr (relative molecular weight is 181.19). By comprehensively considering factors such as the isoelectric point, hydrophilicity, and molecular size of the amino acid, the isoelectric point pI of the linked amino acid should be less than 7.4 and carry a negative charge; the hydropathy index should be less than 0, being a hydrophilic amino acid; in addition, the relative molecular weight should be less than 150, having less impact on the RBD spatial structure. Aspartic acid has an isoelectric point pI of 2.77 and carries a strong negative charge; its hydropathy index is -3.5, showing strong hydrophilicity; its relative molecular weight is 133.10, having less impact on the RBD spatial structure, and it is used as the preferred amino acid to be linked to the C-terminus of the RBD antigen.

[0011] Preferably, the number of aspartic acid is 3 - 24. By analyzing the surface charge distribution of the RBD protein after connecting different numbers of aspartic acid at the RBD end through Discover Studio and ProtParam software, it is found that when 3 - 24 aspartic acid are connected to the C - terminus of RBD, its isoelectric point pI decreases from 8.95 to 8.56 (3 aspartic acid), 7.64 (6 aspartic acid), and 4.30 (24 aspartic acid) respectively. The local charge property directly affects its adsorption and slow - release with the Al(OH)3 adjuvant. When at least 3 aspartic acid are connected, the local charge property at the C - terminus changes from strong positive charge to weak positive charge; when 24 aspartic acid are connected, the local charge property at the C - terminus shifts from strong positive charge to strong negative charge; when more than 24 aspartic acid are connected, the number of added amino acids at the end exceeds 10% of the amino acid sequence length of RBD itself (219 amino acids), and it may have a greater impact on the overall conformation of RBD according to structure prediction. Particularly preferably, when 6 aspartic acid are connected, the local charge property at the C - terminus of RBD can be shifted from positive charge to negative charge, while having a relatively small impact on the overall structure of RBD.

[0012] In a specific embodiment of the present invention, the amino acid sequence of the RBD antigen mutant is as shown in SEQ ID NO: 1.

[0013] By artificially designing to introduce 6 consecutive negatively charged amino acids - aspartic acid (Asp) at its C - terminus, changing the charge characteristic of this region to negative charge, the charge mutant RBD - 6Asp antigen is obtained; after adding a signal peptide and a purification tag, the eukaryotic cell recombinant expression is used to prepare the charge mutant RBD - 6Asp protein, and after purification, it is combined with the Al(OH)3 adjuvant to immunize Balb / C mice.

[0014] The charge mutant RBD - 6Asp protein antigen expressed in the present invention overcomes the disadvantage of insufficient immunogenicity of the wild - type RBD protein. The vaccine prepared by combining with the Al(OH)3 adjuvant can stimulate a stronger immune response and produce higher - titer antibodies that neutralize the invasion of SARS - CoV - 2 virus into target cells. The level of pseudovirus neutralizing antibodies stimulated by the charge mutant RBD - 6Asp reaches 510, which is 13 times that of the wild - type RBD (neutralizing antibody titer is 38); the level of true virus neutralizing antibodies stimulated by the charge mutant RBD - 6Asp reaches 131, which is 8 times that of the wild - type RBD (neutralizing antibody titer is 16), both showing a significant increase (p < 0.05).

[0015] Secondly, the present invention provides the application of the above - mentioned novel coronavirus RBD antigen mutant in the preparation of drugs or vaccines for the treatment and prevention of novel coronavirus.

[0016] The charge mutant RBD-6Asp antigen of the present invention can significantly increase the neutralizing antibody level of the host against the novel coronavirus compared with the wild-type RBD antigen, increasing by 13 times (pseudovirus) and 8 times (true virus) as described above; moreover, the antigen preparation method of the present invention is simple, and AL(OH)3 adjuvant is the most commonly used adjuvant in clinic, having good application prospects.

[0017] Third, the present invention also provides a polynucleotide molecule encoding the above-mentioned RBD antigen mutant.

[0018] In a preferred embodiment of the present invention, the DNA sequence of the polynucleotide molecule is as shown in SEQ ID NO: 2.

[0019] Fourth, the present invention provides the application of the above-mentioned polynucleotide molecule in the preparation of a novel coronavirus DNA vaccine. Based on the above-mentioned polynucleotide molecule as shown in SEQ ID NO: 2 provided by the present invention, the above-mentioned application can be provided by using conventional technical means in the art. For example, using eukaryotic expression vectors such as pcDNA3.1 and pVAX1 as the backbone, designing and adjusting the pre-base sequence of the start codon ATG of the above-mentioned polynucleotide molecule gene to conform to the Kozak rule, constructing a DNA vaccine encoding RBD-6Asp, and after transfecting eukaryotic cells to identify the transient expression of the target protein, immunizing the host by intramuscular injection or other methods to exert an immune protection effect.

[0020] Fifth, the present invention also provides the mRNA transcribed from the above-mentioned polynucleotide molecule. In a preferred embodiment of the present invention, the sequence of the mRNA is as shown in SEQ ID NO: 3.

[0021] Finally, the present invention provides the application of the above-mentioned mRNA in the preparation of a novel coronavirus mRNA vaccine. Based on the above-mentioned mRNA molecule as shown in SEQ ID NO: 3 provided by the present invention, the above-mentioned application can be provided by using conventional technical means in the art. For example, based on the mRNA sequence of the above-mentioned polynucleotide molecule expressing RBD-6Asp, respectively performing selections of 5'UTR or 3'UTR, optimization of the secondary structure of mRNA, optimization of the PolyA tail, optimization of mRNA codons, selection of modified nucleotides, etc., to obtain an optimized sequence with stable expression, and combining with a high-efficiency delivery system such as liposome to deliver the mRNA vaccine into the host to exert an immune protection effect.

[0022] In the present invention, multiple negatively charged amino acids - aspartic acid (Asp) are artificially designed and introduced at the end of the receptor-binding domain RBD (R319-K537) of the novel coronavirus, thereby changing the charge characteristics of the wild-type RBD protein. A charged mutant RBD antigen protein is constructed and developed. At the same time, a tPA signal peptide and a His purification tag are added, and the recombinant protein is expressed through a plasmid transfection eukaryotic cell expression system. The obtained charged mutant RBD-6Asp antigen can overcome the disadvantage of insufficient immunogenicity when the wild-type RBD is combined with the adjuvant AL(OH)3, effectively stimulate a stronger immune response, and significantly improve the neutralizing antibody level of the host against SARS-CoV-2. Through the immunization experiment of Balb / c mice, comparing the immunization efficacy of the charged mutant RBD-6Asp antigen and the wild-type RBD antigen, it has been confirmed that after immunization with the adjuvant AL(OH)3, both the SARS-CoV-2 pseudovirus neutralizing antibody and the true virus neutralizing antibody stimulated by the RBD-6Asp antigen are significantly higher than those of the wild-type RBD antigen. The antigen preparation method of the present invention is simple and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . Schematic diagram of the design of the charged mutant antigen RBD-6Asp of the receptor-binding domain of the novel coronavirus;

[0024] Figure 2 . SDS-PAGE diagrams of the wild-type RBD and the charged mutant RBD-6Asp antigen proteins;

[0025] Figure 3 . Schematic diagram of the immunization strategy for Balb / c mice. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are merely exemplary and do not constitute any limitation to the protection scope defined by the claims of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0027] Example 1 Expression and purification of the charged mutant RBD of the receptor-binding domain of the novel coronavirus

[0028] The C-terminus of the amino acid sequence of the receptor-binding domain (RBD) of the novel coronavirus (the sequence is as in Genebank, YP_009724390.1, R319-K537) was added with 6 consecutive negatively charged amino acids - aspartic acid (Asp), and a secretory signal peptide tPA and a His purification tag were added to its N-terminus to obtain the RBD-6Asp amino acid sequence (as shown in SEQ ID NO: 1), and the details of the sequence construction are shown in Figure 1. After the sequence was artificially optimized for mammalian cell codons, the RBD-6Asp nucleotide sequence (as shown in SEQ ID NO: 2) was obtained. The nucleotide sequence before the start codon ATG was designed and adjusted to conform to the Kozak rule, and the translation termination codon TGA was added at the 3' end. The gene was ligated to the pcDNA3.1 eukaryotic expression vector through the EcoRI (GAATTC) and HindIII (AAGCTT) restriction enzyme sites.

[0029] The wild-type RBD and RBD-6Asp antigen proteins were expressed using the Expi293F mammalian cell expression system. First, the Expi293F suspension cells were cultured and expanded, and the cell density was adjusted to 3×10 6 cells / mL. Subsequently, 30 μg of the wild-type RBD and RBD-6Asp expression plasmids were prepared and transfected into 30 mL of cells using the Expi293 transfection kit. Then the transfected cells were placed on a shaker for culture, and the culture conditions were 120 rpm, 37 °C, relative humidity ≥ 80%, and carbon dioxide concentration of 8%. 72 h after transfection, the cell culture medium was taken and centrifuged at 3000 g for 15 min. The supernatant was filtered through a 0.45 μm needle filter to remove cell debris. The filtered supernatant was used for subsequent protein purification.

[0030] Protein purification was performed using a His-trap affinity chromatography column from GE. The chromatography column was installed on an AKTA protein purifier, and the chromatography column was equilibrated with the equilibration buffer. The supernatant containing the protein was loaded onto the chromatography column. It was equilibrated with a buffer containing 50 mM imidazole with a volume of 5 times the column volume, and then eluted with an elution buffer containing 500 mM imidazole. The eluates containing the wild-type RBD and RBD-6Asp proteins were exchanged using ultrafiltration. After obtaining the recombinant proteins, the protein concentration was measured using the BCA method and a UV spectrophotometer, and the purified proteins were identified using reducing and non-reducing SDS-PAGE experiments. The purity of both the wild-type RBD protein and the RBD-6Asp protein was greater than 95%. Under reducing conditions, the molecular weights were both between 25 kDa and 35 kDa, which was in line with the theoretical expectation (see Figure 2 ).

[0031] Example 2 Pseudovirus neutralization experiment of immune mouse serum

[0032] 1. Mouse immunization

[0033] Female BALB / c mice aged 6 - 8 weeks, 8 mice in each group, were selected. The wild-type RBD and RBD-6Asp proteins obtained in Example 1 were diluted to 100 μg / mL with PB buffer and mixed with an equal volume of AL(OH)3 adjuvant (Alhydrogel, 1000 μg / mL), and then adsorbed at room temperature for 1 h. Then, the 6 - 8-week-old female Balb / c mice were grouped for immunization, and the grouping is shown in Table 1. The immunization strategy is as Figure 3 shown, that is, by intramuscular injection, each mouse received three vaccinations at day 0, the second week (14 days), and the fourth week (28 days), with each vaccination volume of 100 μL (5 μg antigen, 50 μg aluminum adjuvant). On the 35th day, blood was collected from the tail vein of the mice. The blood was allowed to stand at room temperature for 4 h, and after the blood was layered, it was centrifuged at 8000 g for 15 min to obtain mouse serum for subsequent SARS-CoV-2 pseudovirus neutralizing antibody detection.

[0034] Table 1. Grouping of mouse immunization

[0035] Antigen or control Antigen content Adjuvant Number of animals PB 0 <![CDATA[50μg AL(OH)3]]> 8 RBD 5 μg <![CDATA[50μg AL(OH)3]]> 8 RBD-6Asp 5 μg <![CDATA[50μg AL(OH)3]]> 8

[0036] 2. Pseudovirus packaging

[0037] According to the nucleotide coding sequence of the Spike protein of SARS-CoV-2 published by NCBI (NC_045512.2), it was synthesized and inserted into the pCAGGS expression vector. The expression vector of the Spike protein of SARS-CoV-2 and the pNL4.3-Luc-R-E backbone plasmid (Nat Commun 11, 4081(2020)) were co-transfected into 293T cells. After 6 hours of transfection, the supernatant was replaced with MEM medium containing 10% FBS. After 48 hours, the supernatant was collected, filtered through a 0.45 μm sterile filter, and the packaged pseudovirus was obtained. After quantification, it was aliquoted and stored at -80 °C for use.

[0038] 3. Pseudovirus neutralization assay

[0039] The serum of the mice 35 days after immunization was inactivated at 56 °C for 30 min. The serially diluted serum was added to a 96-well cell culture plate and serially diluted. 50 μL of pseudovirus solution was added to each well and incubated at 37 °C for 1 h. 100 μl of ACE2-293T cells were added to each well of the 96-well plate, so that the number of cells in each well was 2.5×10 4Cells were counted and seeded at a density of 1×10⁴ cells / well in a 96-well plate, with a total of 96 wells. The 96-well plate was gently shaken back and forth and from side to side to evenly disperse the cells in the wells. The 96-well plate was then placed in a cell culture incubator and cultured at 37 °C with 5% CO₂ for 48 hours. A 5× lysis buffer was used, and the mixture was shaken at 500 rpm for 15 min. 20 μl of the lysate in the 96-well plate was aspirated and added to the corresponding 96-well chemiluminescence detection plate, and the luminescence value was read using a chemiluminescence detector. The neutralization inhibition rate was calculated as follows: Inhibition rate = [1 - (mean luminescence intensity of the sample group - mean CC value of the blank control) / (mean VC value of the luminescence intensity of the negative group - CC value of the blank control)] × 100%. Based on the results of the neutralization inhibition rate, the pseudovirus half-maximal neutralization titer (NT50) of the mouse serum samples was calculated.

[0040] The results are shown in Table 2. After immunizing mice with wild-type RBD antigen combined with AL(OH)₃ for 35 days, the level of pseudovirus-neutralizing antibodies elicited was relatively low. Among 8 mice, only 4 had a pseudovirus-neutralizing antibody titer above 30, with an average value of 38 (95% CI: 9 - 68). In contrast, compared with the wild-type RBD antigen, after immunizing mice with the charge mutant RBD-6Asp antigen combined with AL(OH)₃ for 35 days, the pseudovirus-neutralizing antibody titer was significantly increased. All 8 mice produced high-titer neutralizing antibodies, with an average value of 510 (95% CI: 27 - 993), which was 13 times that of the wild-type RBD. The pseudovirus-neutralizing antibody titers of the two groups of antigens were statistically analyzed by t-test, and the difference was significant (p < 0.05), indicating that the charge mutant RBD-6Asp as an antigen can significantly increase the level of neutralizing antibodies in the host against the SARS-CoV-2 pseudovirus.

[0041] Table 2. Detection of the level of pseudovirus-neutralizing antibodies elicited by RBD-6Asp antigen and wild-type RBD in mice

[0042]

[0043] Example 3 Neutralization experiment of live virus with immune mouse serum

[0044] Serum from mice 35 days after immunization was used for the neutralization experiment with the live SARS-CoV-2 virus (SARS-CoV-2 / human / CHN / Beijing_IME-BJ01 / 2020 (Genbank No. MT291831), which has been publicly reported in Nat Commun 11, 4081 (2020)). The heat-inactivated mouse serum was serially diluted and incubated with 100 TCID 50 of the SARS-CoV-2 IME-BJ01 strain at 37 °C for 2 hours. The serum-virus complex was added to a 96-well plate pre-coated with Vero E6 cells and incubated Hours. The cells were stained with 0.05% crystal violet for 30 minutes. After adding the decolorizing solution, the OD was measured at 570 nm / 630 nm. According to the neutralization inhibition rate results, the true virus half-maximal neutralization titer (NT50) of the mouse serum samples was calculated.

[0045] As shown in Table 3, after immunizing mice with wild-type RBD antigen combined with AL(OH)3 for 35 days, the level of true virus neutralizing antibodies elicited was low, and the average neutralizing antibody titer of 8 mice was only 16 (95% CI was 8 - 24); while for the charge mutant RBD-6Asp antigen compared with the wild-type RBD antigen, after immunizing mice with it combined with AL(OH)3 for 35 days, the true virus neutralizing antibody titer elicited was significantly increased. The average neutralizing antibody titer of 8 mice was 131 (95% CI was 50 - 213), which was 8 times that of the wild-type RBD. The true virus neutralizing antibody titers of the two groups of antigens were statistically analyzed by t-test, and the difference was significant (p < 0.05), indicating that the charge mutant RBD-6Asp as an antigen could significantly increase the level of neutralizing antibodies of the host against the true virus of the novel coronavirus and had a very significant protective effect against the true virus of SARS-CoV-2.

[0046] Table 3. Detection of the level of true virus neutralizing antibodies elicited by RBD-6Asp antigen and wild-type RBD in mice

[0047]

[0048] The above embodiments are the preferred embodiments of the present invention, but are not intended to limit the present invention. Any other changes and modifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention. Sequence Listing <110> Academy of Military Medical Sciences, Academy of Military Sciences of the Chinese People's Liberation Army <120> A charge mutant antigen of the receptor-binding domain of the novel coronavirus and its application <150> CN202110508745.9 <151> 2021-05-11 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 254 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 1 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Asn Ser His His His His His His Arg Val Gln 20 25 30 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 35 40 45 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 50 55 60 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 65 70 75 80 Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr 85 90 95 Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val 100 105 110 Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys 115 120 125 Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val 130 135 140 Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr 145 150 155 160 Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu 165 170 175 Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn 180 185 190 Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 195 200 205 Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu 210 215 220 Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 225 230 235 240 Ser Thr Asn Leu Val Lys Asn Lys Asp Asp Asp Asp Asp Asp 245 250 <210> 2 <211> 777 <212> DNA <213> Artificial Sequence <400> 2 atggacgcca tgaagagggg cctgtgttgt gtgctgctgc tgtgcggcgc cgtgtttgtg 60 tccaattccc accaccacca ccatcacagg gtgcagccca ccgagtccat cgtgagattc 120 cctaacatca caaacctgtg ccccttcggc gaggtgttca acgccaccag gttcgccagc 180 gtgtacgcct ggaatagaaa gagaatctcc aattgtgtgg ccgactacag cgtgctgtac 240 aacagcgcca gctttagcac cttcaagtgc tacggcgtgt cccctaccaa gctgaatgat 300 ctgtgcttta ccaatgtgta cgccgactcc tttgtgatca gaggcgatga ggtgagacag 360 atcgcccctg gccagacagg caagatcgcc gactacaact acaagctgcc tgatgacttt 420 acaggctgcg tgatcgcctg gaacagcaac aatctggatt ccaaggtggg cggcaattac 480 aactacctgt acagactgtt tagaaagtcc aacctgaagc cctttgagag agatatctcc 540 acagagatct accaggccgg cagcacacct tgcaatggcg tggagggctt caattgctac 600 ttccctctgc agtcctacgg ctttcagccc accaacggcg tgggctacca gccctacaga 660 gtggtggtgc tgtccttcga gctgctgcac gcccccgcca ccgtgtgtgg accaaagaag 720 tccaccaacc tggtgaagaa caagtgcgtg aattttgatg acgacgatga tgattga 777 <210> 3 <211> 777 <212> RNA <213> Artificial Sequence <400> 3 auggacgcca ugaagagggg ccuguguugu gugcugcugc ugugcggcgc cguguuugug 60 uccaauuccc accaccacca ccaucacagg gugcagccca ccgaguccau cgugagauuc 120 ccuaacauca caaaccugug ccccuucggc gagguguuca acgccaccag guucgccagc 180 guguacgccu ggaauagaaa gagaaucucc aauugugugg ccgacuacag cgugcuguac 240 aacagcgcca gcuuuagcac cuucaagugc uacggcgugu ccccuaccaa gcugaaugau 300 cugugcuuua ccaaugugua cgccgacucc uuugugauca gaggcgauga ggugagacag 360 aucgccccug gccagacagg caagaucgcc gacuacaacu acaagcugcc ugaugacuuu 420 acaggcugcg ugaucgccug gaacagcaac aaucuggauu ccaagguggg cggcaauuac 480 aacuaccugu acagacuguu uagaaagucc aaccugaagc ccuuugagag agauaucucc 540 acagagaucu accaggccgg cagcacaccu ugcaauggcg uggagggcuu caauugcuac 600 uucccucugc aguccuacgg cuuucagccc accaacggcg ugggcuacca gcccuacaga 660 gugguggugc uguccuucga gcugcugcac gcccccgcca ccgugugugg accaaagaag 720 uccaccaacc uggugaagaa caagugcgug aauuuugaug acgacgauga ugauuga 777

Claims

1. A novel coronavirus RBD antigen mutant, characterized in that, The amino acid sequence of the RBD antigen mutant is shown as SEQ ID NO:

1.

2. Use of the novel coronavirus RBD antigen mutant according to claim 1 in the preparation of a medicament or vaccine for the treatment and prevention of the novel coronavirus.

3. A polynucleotide molecule encoding the novel coronavirus RBD antigen mutant according to claim 1.

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