SARS-CoV-2 artificially predicted S protein, nucleic acid construct and application

Abstract

The invention belongs to the technical field of biological medicines and genes, and discloses an SARS-CoV-2 artificially predicted S protein, the S protein encodes a protein derived from a coronavirus SARS-CoV-2, and the S protein is an SARS-CoV-2S similar protein. The invention predicts a protein, the coding of the multi-protein is derived from the SARS-CoV-2S protein of the coronavirus, and the multi-protein is obtained by carrying out mutation analysis and integration on the SARS-CoV-2S protein; a vaccine prepared on the basis of the protein sequence or polynucleotide for coding the protein can provide relatively strong immune protection efficacy for various SARS-CoV-2 mutant strains, can induce a remarkably increased immune reaction level when being subjected to sequential immunization with other types of vaccines, and has potential clinical application value and prospect.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedicine and gene technology, and in particular relates to an artificially predicted S protein of SARS-CoV-2, a nucleic acid construct, and their application in vaccines against multiple mutant strains of SARS-CoV-2. Background Art

[0003] Multiple COVID-19 vaccines have been approved for marketing. The main R & D types include viral vector vaccines, nucleic acid vaccines, subunit vaccines, virus-like particle vaccines, inactivated vaccines, and live attenuated vaccines, etc. Among them, the S protein of SARS-CoV-2 is an important protein for the novel coronavirus to recognize and infect target cells, and can be recognized and responded to by the host immune system as an antigen. Therefore, the S protein is the main target for the development of COVID-19 vaccines. Currently, the strains of vaccine antigens are mostly dominant strains with wide spread, which are virus strains existing in nature. However, due to the long vaccine development cycle, the vaccines on the market are against the dominant strains selected during development. For viruses that are prone to mutation, new strains containing multiple mutations will emerge over time, and the previously developed vaccines may have a reduced protection rate against the infection of new strains. Even if the vaccine development is updated to new strains, it is still possible that they cannot cope with the new strains that appear subsequently. To sum up, using virus strains existing in nature to develop vaccines has the hidden danger of being unable to prevent and control new strains.

[0004] In recent years, synthetic biology has developed rapidly. By using artificial intelligence to predict the evolution direction of viruses, and obtaining artificially synthesized sequences integrating potential multiple mutation sites that do not exist in nature as vaccine antigens through synthetic biology, it has the advantage and possibility of preventing and controlling mutant new strains for a long time. The application of artificially predicted antigens in vaccines has not been reported yet. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the existing vaccine antigen selection methods, and provide an artificially predicted S protein of SARS-CoV-2, a nucleic acid construct, and their application in vaccines against multiple mutant strains of SARS-CoV-2.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] An artificially predicted S protein of SARS-CoV-2, wherein the S protein encodes a protein derived from the coronavirus SARS-CoV-2, and the S protein is a SARS-CoV-2 S-like protein.

[0008] Furthermore, the S protein has the amino acid sequence shown in SEQ ID NO.1, which is an artificially predicted amino acid sequence and has immunogenicity;

[0009] Alternatively, the S protein has an amino acid sequence obtained by substituting, deleting, or adding amino acids to the amino acid sequence set forth in SEQ ID NO.1, and having the same or a partial fragment thereof.

[0010] A nucleic acid sequence encoding the S protein as described above, wherein the nucleic acid sequence is DNA and has a nucleic acid sequence of SEQ ID NO.2.

[0011] A nucleic acid construct comprising the S protein as described above or its encoding nucleic acid sequence, wherein the S protein or its encoding nucleic acid sequence in the nucleic acid construct is operably linked to at least one expression regulatory element.

[0012] An expression vector comprising the nucleic acid construct as described above.

[0013] Use of the S protein, nucleic acid sequence, nucleic acid construct, and expression vector as described above in the preparation of a vaccine for preventing and / or treating novel coronavirus.

[0014] Furthermore, the novel coronavirus is a SARS-CoV-2 mutant strain;

[0015] Alternatively, the subject to be administered is a human or non-human mammal;

[0016] Alternatively, the subject to be administered is susceptible to coronavirus or at risk of coronavirus infection;

[0017] Alternatively, the subject to be administered is an adult, child, infant, or elderly person;

[0018] Furthermore, the vaccine is a viral vector vaccine, nucleic acid vaccine, or subunit vaccine;

[0019] Alternatively, the vaccine is in a liquid dosage form, solid dosage form, semi-solid dosage form, or gas dosage form;

[0020] Alternatively, the vaccine is an injection, mucosal immunization preparation, or oral preparation;

[0021] Alternatively, the vaccine is used for single-dose immunization, multiple-dose immunization, or combined use with other COVID-19 vaccines in a subject;

[0022] Alternatively, the vaccine elicits an immune response against coronavirus in the subject to be administered.

[0023] Furthermore, the injection is an intramuscular injection; the mucosal immunization preparation is for nasal inhalation or oral inhalation, and is a nasal drop, aerosol, spray, powder for inhalation, powder, liquid preparation, freeze-dried preparation, gel, microsphere, liposome, film, suspension; the oral preparation is a tablet, powder, pill, powder, granule, capsule;

[0024] The immune response includes the production of antibodies that specifically bind to the viral peptides or proteins encoded by the nucleic acid.

[0025] Furthermore, the serum titer of the antibody increases in the administered subject;

[0026] Alternatively, the antibody is a neutralizing antibody against the coronavirus or cells infected with the coronavirus;

[0027] Alternatively, the antibody is a neutralizing antibody against SARS-CoV-2 multi-mutant strain infection or cells infected with the SARS-CoV-2 multi-mutant strain.

[0028] The advantages and positive effects achieved by the present invention are as follows:

[0029] 1. According to the virus evolution law and using methods such as bioinformatics, the present invention predicts a protein. The amino acid sequence is derived from the S protein of the novel coronavirus SARS-CoV-2 and is an artificially synthesized amino acid sequence, which does not exist in nature and has a certain degree of predictability.

[0030] 2. The protein of the present invention contains the mutation sites on the S proteins of multiple SARS-CoV-2 mutant strains and includes the mutation sites that may occur in the future predicted based on bioinformatics and artificial algorithms, which are integrated into one amino acid sequence to obtain a new S protein sequence, having the potential to reflect the antigenicity of multiple mutant strains.

[0031] 3. After immunization, the present invention can stimulate the body to produce antibodies, and the produced antibodies can target the original strain before prediction and the mutant strains after prediction, with higher antibody levels and better quality. The present invention has a certain degree of broad-spectrum immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the S protein containing all amino acid mutations in the present invention, which has eight transmembrane regions;

[0033] Figure 2 It is a schematic diagram of the artificially predicted transmembrane region of the S protein in the present invention;

[0034] Figure 3 It is a structural diagram of the S protein predicted by Alphafold3 in the present invention; wherein, A is a side view of the predicted three-dimensional structure of the S protein trimer, and B is a top view of the predicted three-dimensional structure of the S protein trimer;

[0035] Figure 4 It is a plasmid map of SM2-RBD-pcDNA3.4 in the present invention;

[0036] Figure 5 It is an identification diagram of the expression of the SM2-RBD protein in the present invention;

[0037] Figure 6 This is the antibody level evaluation chart in the present invention; wherein, A is the IgG level against the prototype strain, B is the IgG level against the BA.4 / 5 subtype strain; C is the IgA level against the prototype strain, and D is the IgA level against the BA.4 / 5 subtype. Specific embodiments

[0038] The following further illustrates the present invention in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0039] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For parts not specifically annotated in this article, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literatures, or relevant specifications, manuals, etc. before the application date of the present invention for implementation.

[0040] In a first aspect, the present invention predicts an SARS-CoV-2 S protein, the S protein encodes a protein derived from the coronavirus SARS-CoV-2, and the S protein is an SARS-CoV-2 S-like protein.

[0041] Preferably, the S protein has the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence of the protein has immunogenicity;

[0042] Alternatively, the S protein has an amino acid sequence obtained by substituting, deleting or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.1 and having the same or substantially the same immunogenicity as it.

[0043] The nucleic acid sequence encoding the S protein as described above, the nucleic acid sequence is DNA, and its nucleic acid sequence is SEQ ID NO.2.

[0044] A nucleic acid construct comprising the S protein or its encoding nucleic acid sequence as described above, wherein the S protein or its encoding nucleic acid sequence in the nucleic acid construct is operably linked to at least one expression regulatory element.

[0045] An expression vector comprising the nucleic acid construct as described above.

[0046] Use of the S protein, nucleic acid sequence, nucleic acid construct, and expression vector as described above in the preparation of a vaccine for preventing and / or treating novel coronavirus.

[0047] Preferably, the novel coronavirus is an SARS-CoV-2 mutant strain;

[0048] Alternatively, the administration subject is a human or non-human mammal;

[0049] Alternatively, the subject to be administered is susceptible to coronavirus or at risk of coronavirus infection;

[0050] Alternatively, the subject to be administered is an adult, child, infant, or elderly person;

[0051] Preferably, the vaccine is a viral vector vaccine, nucleic acid vaccine, or subunit vaccine;

[0052] Alternatively, the vaccine is in liquid dosage form, solid dosage form, semi-solid dosage form, or gas dosage form;

[0053] Alternatively, the vaccine is an injection, mucosal immunization preparation, or oral preparation;

[0054] Alternatively, the vaccine is used for single-dose immunization, multiple-dose immunization, or combined use with other COVID-19 vaccines in the subject;

[0055] Alternatively, the vaccine elicits an immune response against coronavirus in the subject to be administered.

[0056] Preferably, the injection is an intramuscular injection; the mucosal immunization preparation is for nasal inhalation or oral inhalation, and is a nasal drops, aerosol, spray, powder for inhalation, powder, liquid preparation, freeze-dried preparation, gel, microsphere, liposome, film, suspension; the oral preparation is a tablet, powder, pill, powder for oral use, granule, capsule;

[0057] The immune response includes the production of antibodies that specifically bind to the viral peptides or proteins encoded by the nucleic acid.

[0058] Preferably, the serum titer of the antibody increases in the subject to be administered;

[0059] Alternatively, the antibody is a neutralizing antibody against coronavirus or cells infected with coronavirus;

[0060] Alternatively, the antibody is a neutralizing antibody against SARS-CoV-2 multi-mutant strain infection or cells infected with SARS-CoV-2 multi-mutant strain.

[0061] In a second aspect, the present invention provides a nucleic acid construct, which comprises the polynucleotide as described in the first aspect above, and optionally, at least one expression regulatory element operably linked to the polynucleotide.

[0062] The nucleic acid construct encodes an artificially predicted S protein in a specific order, and the elements in the 5' to 3' direction include, but are not limited to, a promoter sequence, a cloning site inserted in effective connection with the promoter, an artificially predicted S protein nucleic acid sequence, a cloning site inserted in effective connection with a terminator, and a terminator.

[0063] In a third aspect, the present invention provides an expression vector comprising the nucleic acid construct as described in the second aspect above.

[0064] The expression vector includes the nucleic acid construct of the second aspect but is not limited to the second aspect. The expression vector can be replicated by a host cell.

[0065] In a fourth aspect, the present invention provides the use of 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 host cell as described in the fourth aspect above in the preparation of a vaccine for preventing and / or treating novel coronavirus;

[0066] In a fifth aspect, the present invention provides a chimeric nucleic acid vaccine or immunogenic composition comprising 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 host cell as described in the fourth aspect above.

[0067] Specifically, the related preparation and detection are as follows:

[0068] An artificially predicted S protein of SARS-CoV-2, and its specific implementation for prediction is as follows:

[0069] 1. Prediction of the S protein sequence

[0070] Through open-source databases such as GenBank and GISAID, obtain the existing natural SARS-CoV-2 S protein sequences. According to the number of infected people and the occurrence time of the strains, input the selected sequences into the prediction model, and output the predicted sequences. Through sequence alignment of MEGA with multiple SARS-CoV-2, an S protein containing artificial prediction is obtained.

[0071] 2. Generation of the artificially predicted S protein

[0072] Analyze the integrated S protein through bioinformatics software, and analyze it in terms of protein physicochemical properties, signal peptides and subcellular localization, prediction of phosphorylation and glycosylation sites, prediction of protein domains, and receptor-binding domains. Specifically, for example, through https: / / services.healthtech.dtu.dk / service.php?TMHMM-2.0, the transmembrane regions are predicted. The S protein containing all amino acid mutations has a total of eight transmembrane regions (such as Figure 1), located at positions 4 - 26, 231 - 253, 224 - 246, 664 - 683, 690 - 709, 853 - 875, 1092 - 1114, 1190 - 1207, 1209 - 1231 respectively, which does not conform to the natural S protein structure. Therefore, the method of artificial analysis to replace some high-frequency mutations and amino acid modifications is adopted to obtain an artificially predicted S protein with only one transmembrane region (such as Figure 2 ). The three-dimensional structure of the S protein predicted by Alphafold3 is as Figure 3 . In some preferred specific embodiments, the amino acid sequence of the artificially predicted SARS-CoV-2 S protein is as shown in SEQ ID NO.1; or an amino acid sequence obtained by substituting, deleting or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.1 and having the same or substantially the same immunogenicity.

[0073] 3. Generation of the coding sequence of the artificially predicted S protein

[0074] According to the artificially predicted S protein sequence and the codon preference of the novel coronavirus, a polynucleotide sequence encoding this protein is generated, and the polynucleotide is a DNA molecule.

[0075] When the artificially predicted S protein is SEQ ID NO.1, the DNA molecule has the DNA sequence as shown in SEQ ID NO.2.

[0076] The expression of an artificially predicted S protein fragment of SARS-CoV-2 is as follows in its specific embodiments:

[0077] 1. Construction of the expression vector

[0078] Insert the receptor binding domain RBD sequence in the artificially predicted S protein coding nucleic acid sequence downstream of the CMV promoter sequence of the pcDNA3.4 vector. The plasmid DNA constructed hereby is hereinafter referred to as SM2-RBD-pcDNA3.4. The structural diagram of the plasmid DNA SM2-RBD-pcDNA3.4 constructed hereby is Figure 4 . Insert the RBD sequence in the coding nucleic acid sequence of the prototype strain S protein into the pcDNA3.4 vector in the same method to construct the S-RBD-pcDNA3.4 plasmid.

[0079] 2. Expression of the artificially predicted S protein fragment

[0080] Transfect SM2-RBD-pcDNA3.4 into 293T cells, culture for 72 h, collect and lyse the cells, harvest the cell protein, and detect the expression of the S protein fragment by WB. The primary antibody is rabbit anti-His antibody, and the secondary antibody is goat anti-rabbit IgG antibody. The result after exposure for 5 min is shown in Figure (Figure 5 )。

[0081] From Figure 5 It can be seen that the SM2 protein was successfully expressed and had high specificity. This protein includes mutation sites that may occur in the future predicted by bioinformatics and artificial algorithms, and is an amino acid sequence that does not exist in nature. This result proves that the artificially synthesized sequence that does not exist in nature still has expressibility.

[0082] Immune evaluation of an artificially predicted S protein fragment of SARS-CoV-2 in animals, and the specific implementation plan is as follows:

[0083] 1. Plasmid amplification and extraction

[0084] The constructed SM2-RBD-pcDNA3.4 plasmid was transformed into DH5α engineering strains, inoculated on LB solid medium containing 100 μg / mL ampicillin, and cultured at 37 °C for 16 hours. Single colonies were picked and inoculated into 5 mL of LB liquid medium, cultured at 37 °C with shaking at 200 rpm for 10 hours until the OD600 value reached 0.6. The seed solution was inoculated into 1 L of LB medium at a volume ratio of 1:100, cultured at 37 °C with shaking at 200 rpm for 14 hours until the OD600 value reached 1.8. The cultured bacterial solution was centrifuged at 4 °C and 6000 rpm for 15 minutes to collect the bacterial cell pellet. The supernatant was discarded, and the bacterial cell pellet was resuspended with an appropriate amount of physiological saline (0.9% NaCl by mass) for standby. The resuspended bacterial cell pellet was added with lysis buffer (50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA) at a volume ratio of 1:1, and gently stirred to fully suspend the bacterial cells. Subsequently, lysis solution (0.2 M NaOH, 1% SDS) was added at a volume ratio of 1:1, gently stirred and mixed, and left standing at room temperature for 5 minutes to completely lyse the cells. Neutralization solution (3 M KAc, pH 4.8) was added at a volume ratio of 1:1, gently stirred and mixed, and left standing at room temperature for 10 minutes. The mixture was centrifuged at 12000 rpm for 15 minutes to remove cell debris and other impurities in the precipitate, and the supernatant was collected. An equal volume of isopropanol was added to the supernatant, gently stirred, and left standing at -20 °C for 30 minutes. Subsequently, the mixture was centrifuged at 12000 rpm for 15 minutes to collect the precipitated plasmid DNA. The precipitated plasmid DNA was washed with 70% ethanol, gently dried by shaking off the ethanol, and then dried at room temperature for 10 minutes to remove the residual ethanol. The dried plasmid DNA precipitate was dissolved in an appropriate amount of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for standby.

[0085] 2. Animal immunization

[0086] Using the plasmid extracted above as an antigen, SM2-RBD-pcDNA3.4 was used as the experimental group, S-RBD-pcDNA3.4 was set as the positive control, and the untreated blank group was set as the negative control. SPF mice were immunized at a dose of 50 μg per injection per time, for a total of 3 times, with an interval of 21 days between each immunization, and the immunization method was intramuscular injection.

[0087] 3. Evaluation of the effect of immunizing animals

[0088] Fourteen days after the last immunization, whole blood of the mice was collected, serum was obtained by centrifugation, and immunological evaluation was performed by ELISA. The results were as Figure 6 . As shown in Figures A and B, both S and SM2 could produce IgG and IgA antibodies against the prototype strain and BA.4 / 5 subtype. Among them, the OD450 value of the IgG or IgA antibody level produced by the S-RBD group against the prototype strain was higher, and the data was statistically significantly different (p < 0.05). The antibody level produced by the SM2-RBD group was statistically significantly different from that of the blank group (p < 0.01), indicating that SM2, as an artificial predicted protein, has the immunogenicity of the S protein and can produce antibodies against the original prototype strain; Figures C and D show that the IgG and IgA antibodies produced by S and SM2 against the Omicron subtype BA.4 / 5 were higher than the antibody level produced by S, and there were statistically significant differences (p < 0.05 or p < 0.01), indicating that SM2 can stimulate the body to produce high-level antibodies against mutant strains. The above results show that SM2, as an artificial predicted protein, can produce higher-level and higher-quality antibodies against the original prototype strain and the Omicron subtype BA.4 / 5 mutant strain compared with the prototype S protein existing in nature, indicating that this protein has certain broad-spectrum immunogenicity.

[0089] In a specific embodiment, the nucleic acid vaccine is a novel coronavirus DNA vaccine, and the DNA vaccine includes:

[0090] (1) A eukaryotic expression vector; and

[0091] (2) A DNA sequence encoding an artificial predicted S protein as defined in the first aspect above, which is constructed into the eukaryotic expression vector;

[0092] In another specific embodiment, the nucleic acid vaccine is a novel coronavirus mRNA vaccine, and the mRNA vaccine includes:

[0093] (1) An mRNA sequence encoding an artificial predicted S protein as defined in the first aspect above;

[0094] (2) Lipid nanoparticles.

[0095] In another specific embodiment, the nucleic acid vaccine is a novel coronavirus - viral vector vaccine, which comprises:

[0096] (1) A viral backbone vector; and

[0097] Construct a DNA sequence encoding an artificially predicted S protein as defined in the first aspect above;

[0098] (2) Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, adeno - associated virus vector.

[0099] In a feasible implementation, the vaccine is an injection, a mucosal immunization preparation or an oral preparation;

[0100] Preferably, the injection is an intramuscular injection;

[0101] Preferably, the mucosal immunization preparation is for nasal inhalation or oral inhalation, and is a nasal drop, an aerosol, a spray, a powder inhaler, a powder, a liquid preparation, a freeze - dried preparation, a gel, a microsphere, a liposome, a film, a suspension;

[0102] Preferably, the oral preparation is a tablet, a powder, a pill, a powder for oral use, a granule, a capsule.

[0103] In a sixth aspect, the present invention provides an immunization strategy, and the vaccine is used for single - dose immunization, multiple - dose immunization or combined use with other COVID - 19 vaccines in a subject.

[0104] In a specific embodiment, the vaccine elicits an immune response against the coronavirus in the administered subject.

[0105] In a specific embodiment, the immune response elicited by the vaccine in the administered subject includes the production of antibodies that specifically bind to the viral peptides or proteins encoded by the nucleic acid.

[0106] In a specific embodiment, the immune response elicited by the vaccine in the administered subject is associated with an increase in the serum titer of the antibody in the administered subject.

[0107] In a specific embodiment, the immune response elicited by the vaccine in the administered subject is such that the antibody is a neutralizing antibody against the coronavirus or cells infected with the coronavirus.

[0108] In a specific embodiment, the immune response elicited by the vaccine in the administered subject is such that the antibody is a neutralizing antibody against SARS - CoV - 2 multi - mutant strain infection or cells infected with the SARS - CoV - 2 multi - mutant strain.

[0109] The relevant sequences used in the present invention are as follows:

[0110] SEQ ID NO.1 Sequence - SM2 - pro Sequence:

[0111] MLGFFFLFSLVFSHCFNFITTTQSHINSFIRGFYYPDKVFRSSVLYSIRDFFLPFFSNLTRYVTE

[0112] VINKFANTFIPFNDGFYFSFIVEFNTTKGWVFGTTLDLKTQSLFIVNNATNAFVKVCEFQFFN

[0113] FDQETTNILMKGIIVKNCTFEYVSRSFFINFVAKHSDSKSFKEFLFKNVGGYFKIYSKHMLTL

[0114] GREPEGFPHSFLSLQPLVHFPIGVNITKFQIYSFILGDFFSSLTTVSVAYYVGYLQPRTFLLKYN

[0115] QNGIIIDAIDCALDPLSEIKCTLKSLILQKGIYKTSNFRIQPIESIIRFSNITNLCPLDKVFNSTKF

[0116] SSVYSWKRRKISNCFADYSFIYNFAPFFAFKCYGLSSIKLNDLCFTSVYADSFVIRGNEVSQIS

[0117] PGRTGNISDYNYKLPVDFIGCVVAWNSKKLDSNVSGNNDYRFRFFRKFSLKSFERDISNQIY

[0118] QVSNKSCNGFAGVNCYLPLRPYSFRRTYGVGHQPHRVVVLFFQLLHSPPTVCGPKKSTSLV

[0119] KNKCVSFNFNGLKGIGILIDSKKNFLPFQQLGRDIDDIINSVRDPQTLDIFDITPCSFGGVSVIT

[0120] PGTNISNQVAVLYHGINCIEVSFVIRSDKLTSNWRVYSIGFSVLQTHSGCLIGVQYVNSSYDC

[0121] DIPIGAGVCVTYHIHIKFHRWVRSLVSQSIIVYTMSLGVKNLVVYSNNSVAIPINFIVSVITEIL

[0122] PVFIIKTAGDCIIYICGDSIECSNLLLQYGSFCTQLKHALTVISIEQDKNIKQVFAQVKHIYKIPP

[0123] NRYFDGFNFSQILPDSSKPIKRSFVEDLFFNKVILTDVGFTKRYGYCLSDVSVRDLLCSQNFK

[0124] GIIVLPPLLTDEMVSQYTSALLSGTITSGWTLGVGVALQIPFSMQMAYRFNGVGVTQNVLYE

[0125] NQKFISNQFNIAIGKIQHSFFFSSPALRKLQNVVNHNALALNMLVKQLSSKFGAISSVLSEIFA

[0126] RLDKVEAEVQIDRLITGRLQSLQIYVIQQLIRAAEIRSSANLASIKMSECVLGQSKRVDFCGK

[0127] GYHIISFPQSAPYGVVFFHVTYFPSLETSFITSSAVCYEGKSHFPHEGVFVSNDTHWFLTQRN

[0128] FYQPKTVTIHNTFLSVNCDVIIGVINNTVYHPFQLQLHLFKDELYKYFNNHTSSYADLVGVS

[0129] VINVSFLNVQKEIYHLYDVANNLNQSLIYFHEFGNYDHYIKWPWYIWLCFTTGFIAILMLSI

[0130] MIFFIISCCIFLKGFFSFVFFCKFDDYYYELLFRGVKLHDT

[0131] SEQ ID NO.2 - Sequence SM2 - dna sequence:

[0132] ATGCTTGGTTTTTTTTTTTTATTTTCACTAGTCTTTAGTCACTGTTTTAATTTTATAACCAC

[0133] AACTCAATCACACATTAATTCTTTCATACGTGGTTTTTATTACCCTGACAAAGTTTTCAGA

[0134] TCCTCAGTTTTATATTCAATTCGGGACTTTTTCTTACCTTTCTTTTCCAATCTTACTAGGTA

[0135] TGTTACAGAGGTTATTAACAAGTTTGCTAACACTTTCATACCATTTAATGATGGTTTTTATT

[0136] TTTCTTTCATTGTGGAGTTTAACACAACAAAAGGCTGGGTTTTTGGTACTACTTTAGATT

[0137] TGAAGACCCAGTCCCTATTTATTGTTAATAACGCTACTAATGCTTTTGTTAAAGTCTGTGA

[0138] ATTTCAATTTTTTAATTTTGATCAGGAAACCACCAACATTTTGATGAAAGGGATTATTGTG

[0139] AAGAATTGCACTTTTGAATATGTCTCTCGGTCTTTTTTTATTAACTTTGTAGCAAAACATA

[0140] GTGATTCCAAAAGTTTTAAGGAATTTCTGTTTAAGAATGTTGGTGGTTATTTTAAAATATA

[0141] TTCTAAGCACATGCTTACTTTAGGGCGTGAGCCAGAAGGTTTCCCTCATAGTTTTTTGTC

[0142] TTTACAACCATTGGTACATTTTCCAATAGGTGTTAACATCACTAAGTTTCAAATTTATTCTT

[0143] TTATTCTTGGTGATTTTTTTTCAAGTTTGACAACTGTTTCTGTAGCTTATTATGTGGGTTAT

[0144] CTTCAACCTAGGACTTTTCTATTAAAATATAATCAAAATGGAATCATTATAGATGCTATAGA

[0145] CTGTGCACTTGACCCTCTCTCAGAAATAAAGTGTACGTTGAAATCCCTCATTCTACAAAA

[0146] AGGAATCTATAAAACTTCTAACTTTAGAATCCAACCAATAGAATCTATTATTAGATTTTCT

[0147] AATATTACAAACTTGTGCCCTCTTGATAAAGTTTTTAACTCCACCAAATTTTCATCTGTTT

[0148] ATTCTTGGAAGAGGAGGAAAATCAGCAACTGTTTTGCTGATTATTCTTTCATATATAATTT

[0149] CGCACCATTTTTCGCTTTTAAGTGTTATGGACTGTCTTCTATTAAATTAAATGATCTCTGCT

[0150] TTACTAGTGTCTATGCAGATTCATTTGTAATTAGAGGTAATGAAGTCAGTCAAATCTCTCC

[0151] AGGGCGAACTGGAAACATTTCTGATTATAATTATAAATTACCAGTTGATTTTATAGGCTGC

[0152] GTTGTAGCTTGGAATTCTAAGAAGCTTGATTCTAACGTTAGTGGTAATAATGATTACCGGT

[0153] TTAGATTTTTTAGGAAGTTTAGTCTCAAATCTTTTGAGAGAGATATTTCAAATCAAATCTA

[0154] TCAGGTCAGTAACAAATCTTGTAATGGTTTTGCAGGTGTTAATTGTTACCTTCCTTTACGA

[0155] CCATATAGTTTCCGACGCACTTATGGTGTTGGTCACCAACCACACAGAGTAGTAGTACTT

[0156] TTTTTTCAACTTCTACATTCACCACCAACTGTTTGTGGACCTAAAAAGTCTACTAGTTTG

[0157] GTTAAAAACAAATGTGTCAGTTTCAACTTCAATGGTTTAAAAGGCATAGGTATTCTTATT

[0158] GATTCTAAGAAAAACTTTCTGCCTTTCCAACAACTTGGCAGAGACATTGATGACATTATT

[0159] AATTCTGTCCGTGATCCACAGACACTTGATATTTTTGACATTACACCATGTTCTTTTGGTG

[0160] GTGTCAGTGTTATAACACCAGGAACAAATATTTCTAACCAGGTTGCTGTTCTTTATCATGG

[0161] TATTAACTGCATAGAAGTCTCTTTTGTTATTCGTTCAGATAAACTTACTTCTAATTGGCGT

[0162] GTTTATTCTATAGGTTTTAGTGTTCTTCAAACACATTCAGGCTGTTTAATAGGGGTTCAAT

[0163] ATGTCAACAGCTCATATGATTGTGACATACCCATTGGTGCAGGTGTATGCGTTACTTATCA

[0164] TATTCATATTAAGTTTCATCGGTGGGTACGTAGTCTAGTTAGTCAATCCATCATTGTCTACA

[0165] CTATGTCACTTGGTGTAAAAAATTTAGTTGTTTACTCTAATAACTCTGTTGCCATACCCAT

[0166] AAATTTTATTGTTAGTGTTATCACAGAAATTCTACCAGTGTTTATTATCAAGACAGCAGGA

[0167] GATTGTATAATTTACATTTGTGGTGATTCAATTGAATGCAGCAATCTTTTGTTGCAATATGG

[0168] CAGTTTTTGTACACAATTAAAGCATGCTTTAACTGTAATATCTATTGAACAAGACAAAAA

[0169] CATCAAACAAGTTTTTGCACAAGTCAAACATATTTACAAAATACCACCAAATAGATATTT

[0170] TGATGGTTTTAATTTTTCACAAATATTACCAGATTCATCAAAACCAATCAAGAGGTCATTT

[0171] GTTGAAGATCTATTTTTCAACAAAGTGATACTTACAGATGTTGGCTTCACCAAACGATAT

[0172] GGTTATTGCCTTAGTGATGTTTCTGTTAGAGACCTCCTTTGTTCACAAAACTTTAAGGGC

[0173] ATTATTGTTTTGCCACCTTTGCTCACAGATGAAATGGTTTCTCAATACACTTCTGCACTGT

[0174] TATCGGGTACAATCACTTCTGGTTGGACCCTTGGTGTAGGTGTTGCATTACAAATACCATT

[0175] TTCTATGCAAATGGCTTATAGGTTTAATGGTGTTGGAGTTACACAGAATGTTCTCTATGAG

[0176] AACCAAAAATTTATTTCCAACCAATTTAATATTGCTATTGGCAAAATTCAACACTCATTTT

[0177] TTTTCTCATCACCTGCACTTCGAAAACTTCAAAATGTGGTCAACCATAATGCACTAGCTT

[0178] TAAACATGCTTGTTAAACAACTTAGCTCCAAGTTTGGTGCAATTTCAAGTGTTTTAAGTG

[0179] AAATCTTTGCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACA

[0180] GGCAGACTTCAAAGTTTGCAGATATATGTGATTCAACAATTAATTAGAGCTGCAGAAATC

[0181] AGATCTTCTGCTAATCTTGCTTCTATTAAAATGTCAGAGTGTGTACTTGGACAATCAAAA

[0182] AGAGTTGATTTTTGTGGAAAGGGCTATCATATTATTTCCTTCCCTCAGTCAGCACCTTATG

[0183] GTGTAGTCTTCTTTCATGTGACTTATTTCCCTTCACTAGAAACGAGCTTCATAACTTCTTC

[0184] TGCCGTTTGTTATGAAGGAAAATCACACTTTCCTCATGAAGGTGTCTTTGTTTCAAATGA

[0185] CACACACTGGTTTCTAACACAAAGGAATTTTTATCAACCAAAAACCGTTACTATACACAA

[0186] CACATTTCTGTCTGTTAACTGTGATGTTATAATAGGAGTTATCAACAACACAGTTTATCAT

[0187] CCTTTTCAACTTCAATTACACTTATTCAAGGATGAGTTATATAAATATTTTAACAATCATAC

[0188] ATCATCATATGCTGATTTAGTTGGCGTCTCTGTCATTAATGTTTCATTTCTAAACGTTCAAA

[0189] AAGAAATTTACCACCTCTATGATGTTGCCAACAATTTAAATCAATCTCTCATCTATTTCCA

[0190] TGAATTTGGAAACTATGATCATTATATAAAATGGCCATGGTACATTTGGCTATGTTTTACA

[0191] ACTGGCTTTATTGCCATACTAATGCTGTCAATTATGATTTTCTTTATTATCAGTTGCTGTATT

[0192] TTTCTCAAGGGCTTTTTTTCTTTTGTATTCTTCTGCAAATTTGATGATTACTACTATGAGCT

[0193] ACTGTTCAGAGGAGTCAAATTACATGACACATAA

[0194] Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will understand that: various substitutions, changes, and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. An artificially predicted S protein of SARS-CoV-2, characterized in that: The S protein encodes a protein derived from the coronavirus SARS-CoV-2, and the S protein is a SARS-CoV-2S similar protein.

2. The S protein according to claim 1, characterized in that: The S protein has an amino acid sequence shown in SEQ ID NO.1, which is an artificially predicted amino acid sequence and has immunogenicity; Alternatively, the S protein has an amino acid sequence obtained by replacing, deleting or adding amino acids to the amino acid sequence described in SEQ ID NO.1, or has an amino acid sequence that is identical to or contains a partial fragment thereof.

3. The nucleic acid sequence encoding the S protein according to claim 1 or 2, characterized in that: The nucleic acid sequence is DNA, and its nucleic acid sequence is SEQ ID NO.

2.

4. A nucleic acid construct comprising the S protein according to any one of claims 1 to 3 or a nucleic acid sequence encoding the S protein, characterized in that: In the nucleic acid construct, the S protein or the nucleic acid sequence encoding it is operably linked to at least one expression regulatory element.

5. An expression vector comprising the nucleic acid construct according to claim 4.

6. Use of the S protein, nucleic acid sequence, nucleic acid construct, and expression vector as described in any one of claims 1 to 5 in the preparation of a vaccine for preventing and / or treating a new coronavirus.

7. The use according to claim 6, characterized in that: The novel coronavirus is a mutant strain of SARS-CoV-2; Alternatively, the administration subject is a human or non-human mammal; Alternatively, the subject is susceptible to coronavirus or is at risk of coronavirus infection; Alternatively, the administration subjects are adults, children, infants and the elderly.

8. The use according to claim 6, characterized in that: The vaccine is a viral vector vaccine, a nucleic acid vaccine or a subunit vaccine; Alternatively, the vaccine is in liquid, solid, semisolid or gaseous dosage form; Alternatively, the vaccine is an injectable, mucosal immunization preparation or an oral preparation; Alternatively, the vaccine is used for single or multiple administrations to a subject for immunization or is used in combination with other COVID-19 vaccines; Alternatively, the vaccine induces an immune response against the coronavirus in the subject of administration.

9. The use according to claim 8, characterized in that: The injection is an intramuscular injection; the mucosal immunization preparation is inhaled through the nose or mouth, and is a nasal drop, aerosol, spray, powder spray, powder, liquid preparation, freeze-dried preparation, gel, microsphere, liposome, film, suspension; the oral preparation is a tablet, powder, pill, powder, granule, capsule; The immune response includes the production of antibodies that specifically bind to the viral peptide or protein encoded by the nucleic acid.

10. The use according to claim 9, characterized in that: The serum titer of the antibody increases in the administered subject; Alternatively, the antibody is a neutralizing antibody against coronavirus or cells infected by coronavirus; Alternatively, the antibody is a neutralizing antibody against cells infected with or infected by multiple mutant strains of SARS-CoV-2.

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