CN114751992A - Universal vaccine for coronavirus and application thereof - Google Patents

Universal vaccine for coronavirus and application thereof Download PDF

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CN114751992A
CN114751992A CN202210574868.7A CN202210574868A CN114751992A CN 114751992 A CN114751992 A CN 114751992A CN 202210574868 A CN202210574868 A CN 202210574868A CN 114751992 A CN114751992 A CN 114751992A
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高福
戴连攀
胥森瑜
刘川玉
徐坤
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Institute of Microbiology of CAS
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Abstract

The invention relates to a general vaccine of coronavirus and application thereof, wherein the general vaccine comprises a coronavirus heterotetramer antigen protein, related products thereof and application thereof in preparing the general vaccine, and an immunogenic composition based on the heterotetramer antigen protein, wherein the coronavirus heterotetramer antigen protein comprises partial amino acid sequences or all amino acid sequences of receptor binding regions of spike proteins of four different coronaviruses which are directly connected in series or connected in series by connecting amino acid sequences. The heterotetrameric antigen protein of the invention can stimulate the generation of a more strong neutralizing antibody level for various coronaviruses (SARS-CoV-2 prototype strain and other coronaviruses Pangolin GX/P4L, SHC014, WIV1, LYRa11, SARS, etc.), i.e. has good immunogenicity and broad spectrum, and the stability and immunogenicity effects of the heterotetrameric antigen proteins P-B-LY-S and P-B-SHC-S relative to P-B-Ra-S and P-B-PgGX-S are more balanced and stable.

Description

Universal vaccine for coronavirus and application thereof
Technical Field
The invention relates to the field of biomedicine, in particular to a coronavirus heterotetramer antigen protein, a preparation method thereof, a polynucleotide encoding the same, a nucleic acid construct containing the polynucleotide, a recombinant vector containing the nucleic acid construct, an expression system cell, application of the protein and the vector to preparation of a universal vaccine, an immunogenic composition serving as the universal vaccine of coronavirus, and application of the protein and the cell to preparation of a medicine for preventing or treating coronavirus.
Background
Coronaviruses belong to the family of the nested virus (Nidovirales) Coronaviridae (Coronaviridae) genus coronaviruses (Coronavirus), and are enveloped positive-strand RNA viruses, with the largest genome among all RNA viruses. Coronaviruses primarily infect mammals and birds. Up to now, 7 kinds of coronaviruses can infect people, of which four kinds of coronaviruses, HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoV-HKU1 can only cause people to have a mild cold, and the patients can recover quickly; there are three more coronaviruses that are lethal, the severe respiratory syndrome coronavirus (SARS-CoV) prevalent in 2002-2003, the middle east respiratory syndrome coronavirus (MERS-CoV) that was outbreaked in 2012 and persisted to date, and the novel coronavirus (SARS-CoV-2) that is still prevalent. In addition, more coronavirus is found in wild animals such as bat, pangolin and the like, such as: RaTG13 isolated from Heps chrysanthemi in 2013, SARS-associated coronavirus RsSHC014 isolated from Heps chrysanthemi in 2013, Shizhen team in 2013, military veterinary institute belonging to Rs3367 branch, Jilin province in WIV1, 2014, in cooperation with endemic prevention and treatment institute in Yunnan province, LYRa11 having 91% homology with SARS-CoV genome was isolated, a virus RmYN02 having 93.3% homology with SARS-CoV-2 genome was isolated from bat sample in Yunnan province in 2019 by New infectious etiology and epidemiology focus laboratory of Shandong medical academy, Pasteur research institute of Cambodia in 2020, RshYN 182 having 92.6% homology with SARS-CoV-2 genome was isolated from Heps, and STT182 and about 90% homology with SARS-CoV-2 sequence GD Pangolin 1/4L GngP and GD 4/L% GD P isolated from squama manitis, however, the difference of strains enables the vaccine to induce the body to generate antibodies to have immune escape. Therefore, it is critical to develop a universal vaccine.
The spike protein (S protein) of coronaviruses is the "key" for the virus to recognize host cell receptors, assisting the virus in invading cells. The S protein contains subunits S1 and S2, and the Receptor Binding Domain (RBD) in the S1 subunit is responsible for binding to the host receptor protein. To date, most antibodies with strong neutralizing activity against coronaviruses are RBD-targeting, and therefore RBD is often selected as the vaccine design target, and we have designed a universal vaccine based on RBD.
Disclosure of Invention
Object of the Invention
The invention aims to provide a coronavirus heterotetramer antigen protein, a preparation method thereof, a polynucleotide for encoding the same, a nucleic acid construct containing the polynucleotide, a recombinant vector containing the nucleic acid construct, an expression system cell, application of the nucleic acid construct, the recombinant vector and the expression system cell in preparation of a universal vaccine, an immunogenic composition serving as the universal vaccine of the coronavirus, and application of the nucleic acid construct, the recombinant vector and the expression system cell in preparation of a medicine for preventing or treating the coronavirus.
The invention adopts the antigen molecules of the coronavirus from different sources to form the heterotetrameric antigen protein, so that the protein not only can be correctly expressed, but also has stable expression quantity, and the expressed antigen can induce an organism to generate more and wider-spectrum neutralizing antibodies aiming at different strains and even various new crown variant strains. Particularly, the heterotetrameric antigen protein P-B-SHC-S, P-B-LY-S not only can effectively induce an organism to generate neutralizing antibodies, but also has stable structure and is not easy to degrade.
Solution scheme
In order to realize the purpose, the invention provides the following technical scheme:
in a first aspect, the present invention provides a coronavirus heterotetrameric antigen protein comprising a partial amino acid sequence or the entire amino acid sequence of the Receptor Binding Domain (RBD) of the spike proteins of four different coronavirus virus strains, directly concatenated or concatenated by a linker sequence;
the four different coronaviruses are selected from four of the following groups: SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain, coronavirus RsSHC014 strain, coronavirus LYRa11 strain, coronavirus RaTG13 strain, coronavirus Pangolin GX/P4L strain, coronavirus Pangolin GD/1 strain, coronavirus WIV1 strain, coronavirus RshSTT182 strain, coronavirus BtKY72 strain, coronavirus YN02 strain, coronavirus SARS-CoV, coronavirus MERS-CoV, coronavirus HCoV-229E, coronavirus HCoV-OC43 strain, coronavirus HCoV-NL63 strain, coronavirus HCoV-HKU1 strain and coronavirus bat-CoV strain.
Further, the four different coronaviruses, optionally, four selected from the group consisting of: SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain, coronavirus RsSHC014 strain, coronavirus LYRa11 strain, coronavirus RaTG13 strain, coronavirus Pangolin GX/P4L strain, coronavirus Pangolin GD/1 strain, coronavirus WIV1 strain, coronavirus RshSTT182 strain, coronavirus BtKY72 strain, coronavirus YN02 strain, and SARS-CoV strain.
Further, the four different coronaviruses are selected from four of the following groups: SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain, coronavirus RsSHC014 strain, coronavirus LYRa11 strain, coronavirus RaTG13 strain, coronavirus Pangolin GX/P4L strain, and coronavirus SARS-CoV strain.
Further, the sequence of the concatenation of the partial amino acid sequences or the complete amino acid sequences of the receptor binding regions of the spike proteins of the four different coronaviruses is: the strains are connected in series in the sequence from high to low in homology.
Further, the heterotetrameric antigenic protein comprises the following four amino acid sequences, either directly or through a linker sequence:
(a) a partial amino acid sequence or the entire amino acid sequence of the receptor-binding region of the spike protein of the SARS-CoV-2 prototype strain, wherein the partial amino acid sequence of the receptor-binding region of the spike protein of the SARS-CoV-2 prototype strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of the entire amino acid sequence thereof;
(b) a partial amino acid sequence or a complete amino acid sequence of a receptor binding region of the spike protein of the SARS-CoV-2 variant strain, wherein the partial amino acid sequence of the receptor binding region of the spike protein of the SARS-CoV-2 variant strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of the complete amino acid sequence thereof;
(c) A partial amino acid sequence or the entire amino acid sequence of the receptor-binding region of the spike protein of strain X coronavirus, wherein the partial amino acid sequence of the receptor-binding region of the spike protein of strain X coronavirus is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of the entire amino acid sequence thereof;
(d) a partial amino acid sequence or the entire amino acid sequence of the receptor-binding region of the spike protein of SARS-CoV, wherein the partial amino acid sequence of the receptor-binding region of the spike protein of the SARS-CoV strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of the entire amino acid sequence thereof;
wherein the coronavirus X strain is at least one selected from coronavirus RsSHC014 strain, coronavirus LYRa11 strain, coronavirus RaTG13 strain and coronavirus Pangolin GX/P4L strain; optionally the coronavirus X strain is selected from coronavirus RsSHC014 strain or coronavirus LYRa11 strain;
wherein the SARS-CoV-2 variant strain is selected from Alpha, Beta, Gamma, Kappa, Lambda, Delta or Omicron strain, optionally the SARS-CoV-2 variant strain is Beta strain.
Further, the series connection is a direct series connection;
as an alternative, the concatenation is by a linker sequence, optionally a linker sequence (GGS) n(ii) a Wherein n is 1,2,3,4 or 5;
further, the four amino acid sequences are connected in series in the sequence of the amino acid sequences (a), (b), (c) and (d).
Further, the heterotetrameric antigenic protein comprises the following four amino acid sequences, either directly or through a linker sequence:
(a) 1 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 1 and having the same or substantially the same immunogenicity as it; or preferably an amino acid sequence having substitution and/or deletion and/or addition of one, two, three or four amino acid residues with the amino acid sequence shown as SEQ ID NO. 1 and having the same or substantially the same immunogenicity as such, which novel amino acid sequence is also considered to be comprised in the scope of the present invention;
(b) an amino acid sequence as set forth in SEQ ID NO. 2, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO. 2 and having the same or substantially the same immunogenicity as it; or preferably an amino acid sequence having substitution and/or deletion and/or addition of one, two, three or four amino acid residues with the amino acid sequence shown as SEQ ID NO. 2 and having the same or substantially the same immunogenicity as such, which novel amino acid sequence is also to be considered as being comprised in the scope of the present invention;
(c) 3, 5, 6 or 7; or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to and having the same or substantially the same immunogenicity as the amino acid sequence set forth as SEQ ID NO 3, 5, 6 or 7; or preferably an amino acid sequence having substitutions and/or deletions and/or additions of one, two, three or four amino acid residues with the amino acid sequence shown as SEQ ID NO. 3, 5, 6 or 7 and having the same or substantially the same immunogenicity as said amino acid sequence, are also to be considered as comprised within the scope of the present invention;
preferably, (c) the amino acid sequence is preferably the amino acid sequence shown as SEQ ID NO 6 or 7; or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth as SEQ ID NO 6 or 7 and having the same or substantially the same immunogenicity as it; or preferably an amino acid sequence having substitutions and/or deletions and/or additions of one, two, three or four amino acid residues with the amino acid sequences shown as SEQ ID NO 6 or 7 and having the same or substantially the same immunogenicity as said amino acid sequences, are also to be considered as comprised within the scope of the present invention;
(d) An amino acid sequence as shown in SEQ ID NO. 4; or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 4 and having the same or substantially the same immunogenicity as it; or preferably an amino acid sequence having substitution and/or deletion and/or addition of one, two, three or four amino acid residues with the amino acid sequence shown in SEQ ID NO. 4 and having the same or substantially the same immunogenicity as such, are also to be considered as being comprised in the scope of the present invention.
Optionally, the series is a direct series;
as an alternative, the concatenation is by a connecting sequence, optionally (GGS)n(ii) a Wherein n is 1,2,3,4 or 5.
In some embodiments, the sequence of amino acid sequences (a), (b), (c), and (d) can be changed or replaced by the RBD amino acid sequence of the corresponding variant strain, and a broad spectrum of immune response can also be obtained. Especially, the amino acid sequences (a) and (b) are both derived from a new coronavirus strain, and the change or the replacement of the connection sequence can also obtain a broad-spectrum immune response.
Further, the heterotetrameric antigenic protein comprises the amino acid sequence shown as SEQ ID NO 8, 9, 10 or 11; or comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to and having the same or substantially the same immunogenicity as the amino acid sequence shown as SEQ ID NO 8, 9, 10 or 11; or comprises an amino acid sequence having substitution and/or deletion and/or addition of one, two, three or four amino acid residues with the amino acid sequence shown as SEQ ID NO 8, 9, 10 or 11 and has the same or substantially the same immunogenicity as it, which novel amino acid sequence is also considered to be comprised in the scope of the present invention;
Further, the heterotetrameric antigenic protein comprises the amino acid sequence shown in SEQ ID NO. 10 or 11; or comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO 10 or 11 and having the same or substantially the same immunogenicity as it; or comprises an amino acid sequence having substitution and/or deletion and/or addition of one, two, three or four amino acid residues with the amino acid sequence shown as SEQ ID NO 10 or 11 and has the same or substantially the same immunogenicity as it, which novel amino acid sequence is also considered to be comprised in the scope of the present invention.
In the above sequence, the amino acid sequence shown in SEQ ID NO:1 is a partial amino acid sequence or a whole amino acid sequence (GenBank: NC-045512.2) of the receptor binding region of the spike protein of the prototype strain of coronavirus SARS-CoV-2, and the amino acid sequence shown in SEQ ID NO:1 may specifically be as follows:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK
in the above sequences, the amino acid sequence shown in SEQ ID NO:2 is a partial amino acid sequence or a whole amino acid sequence (GISAID: EPI _ ISL _825109) of the receptor binding region of the spike protein of the variant strain of coronavirus SARS-CoV-2 (Beta), and the amino acid sequence shown in SEQ ID NO:2 may specifically be as follows:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK
In the above sequences, the amino acid sequence shown in SEQ ID NO:3 is a partial amino acid sequence or a whole amino acid sequence of a receptor binding region of a spike protein of coronavirus RaTG13 (GenBank: MN996532.2), and the amino acid sequence shown in SEQ ID NO:3 may specifically be as follows:
RVQPTDSIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVITGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSKHIDAKEGGNFNYLYRLFRKANLKPFERDISTEIYQAGSKPCNGQTGLNCYYPLYRYGFYPTDGVGHQPYRVVVLSFELLNAPATVCGPKKSTNLVKNK
in the above sequences, the amino acid sequence shown in SEQ ID NO:4 is a partial amino acid sequence or a whole amino acid sequence of a receptor binding region of a spike protein of coronavirus SARS-CoV (GenBank: AAS00003.1), and the amino acid sequence shown in SEQ ID NO:4 may specifically be as follows:
RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQ
in the above sequence, the amino acid sequence shown in SEQ ID NO:5 is a partial amino acid sequence or a whole amino acid sequence of the receptor binding region of the spike protein of coronavirus Pangolin GX/P4L (GenBank: MT040333.1), and the amino acid sequence shown in SEQ ID NO:5 can be specifically as follows:
RVQPTISIVRFPNITNLCPFGEVFNASKFASVYAWNRKRISNCVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVVKGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNSVKQDALTGGNYGYLYRLFRKSKLKPFERDISTEIYQAGSTPCNGQVGLNCYYPLERYGFHPTTGVNYQPFRVVVLSFELLNGPATVCGPKLSTTLVKDK
in the above sequence, the amino acid sequence shown in SEQ ID NO. 6 is a partial amino acid sequence or a whole amino acid sequence of the receptor binding region of the spike protein of coronavirus LYRa11 (GenBank: KF569996.1), and the amino acid sequence shown in SEQ ID NO. 6 may specifically be as follows:
RVSPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSAIKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSSGNFNYKYRSLRHGKLRPFERDISNVPFSPDGKPCTPPAFNCYWPLNDYGFYTTNGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLITNQ
among the above sequences, the amino acid sequence shown in SEQ ID NO:7 is a partial amino acid sequence or a whole amino acid sequence of the receptor binding region of the spike protein of coronavirus RsSHC014 (GenBank: KC881005.1), and the amino acid sequence shown in SEQ ID NO:7 may be specifically as follows:
RVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFLGCVLAWNTNSKDSSTSGNYNYLYRWVRRSKLNPYERDLSNDIYSPGGQSCSAVGPNCYNPLRPYGFFTTAGVGHQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQ
The amino acid sequence of the heterotetrameric antigen protein formed by directly and sequentially connecting SEQ ID NO. 1, 2, 3 and 4 in series is shown as SEQ ID NO. 8, and the nucleotide sequence for coding the heterotetrameric antigen protein is shown as SEQ ID NO. 12.
The amino acid sequence of the heterotetrameric antigen protein formed by directly and sequentially connecting SEQ ID NO. 1, 2, 5 and 4 in series is shown as SEQ ID NO. 9, and the nucleotide sequence for coding the heterotetrameric antigen protein is shown as SEQ ID NO. 13.
The amino acid sequence of the heterotetrameric antigen protein formed by directly and sequentially connecting SEQ ID NO. 1, 2, 6 and 4 in series is shown as SEQ ID NO. 10, and the nucleotide sequence for coding the heterotetrameric antigen protein is shown as SEQ ID NO. 14.
The amino acid sequence of the heterotetrameric antigen protein formed by directly and sequentially connecting SEQ ID NO. 1, 2, 7 and 4 in series is shown as SEQ ID NO. 11, and the nucleotide sequence for coding the heterotetrameric antigen protein is shown as SEQ ID NO. 15.
Further, the N end and/or the C end of the amino acid sequence of the heterotetrameric antigen protein is also connected with a signal peptide and/or a tag; optionally, the N-terminal of the amino acid sequence of the heterotetrameric antigenic protein is linked to a signal peptide and the C-terminal is linked to a tag; optionally, the tag is selected from at least one of Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, SUMO tag; preferably the tag is a His tag; optionally, the signal peptide is a signal peptide of MERS-CoV spike protein.
In a second aspect, there is provided a method for preparing the coronavirus heterotetramer antigenic protein of the first aspect, comprising the steps of: adding a sequence for coding a signal peptide at the 5 'end of the nucleotide sequence for coding the coronavirus heterotetramer antigen protein in the first aspect, adding a sequence for coding a label at the 3' end of the nucleotide sequence for cloning and expressing, screening a correct recombinant, transfecting cells of an expression system for expression, collecting cell supernatants after expression, and purifying to obtain the coronavirus heterotetramer antigen protein; optionally, the tag is selected from at least one of Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, SUMO tag; preferably the tag is a His tag; optionally, the signal peptide is a signal peptide of MERS-CoV spike protein.
Further, the cell of the expression system comprises a cell that is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell, optionally; the mammalian cells include HEK293T, HEK293F cells, or CHO cells, and the bacterial cells include e.
In a third aspect, there is provided a polynucleotide encoding the coronavirus heterotetramer antigen protein of the first aspect, optionally the polynucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO 12, 13, 14 and/or 15.
In a fourth aspect, there is provided a nucleic acid construct comprising the polynucleotide of the third aspect. Optionally, the vector further comprises a coding sequence, a promoter sequence, a tag sequence and/or a cleavage site sequence of a signal peptide of MERS. For example, the four sequences of the heterotetrameric antigenic protein may be ordered as: kozak sequence-signal peptide-first RBD gene-second RBD gene-third RBD gene-fourth RBD gene-histidine tag-stop codon.
In a fifth aspect, there is provided a recombinant vector comprising the nucleic acid construct of the fourth aspect.
In a sixth aspect, there is provided an expression system cell comprising the recombinant vector of the fifth aspect; preferably, the expression system cell is a mammalian cell; further preferably, the mammalian cell is a HEK293T, HEK293F cell or CHO cell.
In a seventh aspect, there is provided a use of the coronavirus heterotetramer antigenic protein of the first aspect, the polynucleotide of the third aspect, the nucleic acid construct of the fourth aspect, the recombinant vector of the fifth aspect or the expression system cell of the sixth aspect in the preparation of a universal vaccine for coronaviruses.
Preferably, the vaccine is used for immunization alone or in sequential immunization with other types of novel coronavirus vaccines.
In an eighth aspect, there is provided an immunogenic composition comprising a coronavirus heterotetrameric antigen protein of the first aspect, or a heterotetrameric antigen protein prepared by the method of the second aspect, or a polynucleotide of the third aspect, or a nucleic acid construct of the fourth aspect, or a recombinant vector of the fifth aspect, or an expression system cell of the sixth aspect, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and/or diluent.
Further, the immunogenic composition is a coronavirus protein universal vaccine comprising:
(1) the coronavirus heterotetramer antigenic protein of the first aspect; preferably comprising an amino acid sequence as shown in SEQ ID NO 8, 9, 10 and/or 11 or preferably comprising an amino acid sequence with a signal peptide sequence and/or a tag amino acid sequence on the amino acid sequence as shown in SEQ ID NO 8, 9, 10 and/or 11; and
(2) an adjuvant;
optionally, the adjuvant is selected from the group consisting of an aluminum adjuvant, MF59 adjuvant, and MF 59-like adjuvant; alternatively, the MF 59-like adjuvant comprises an Addavax adjuvant;
optionally, the volume ratio of the coronavirus heterotetramer antigenic protein of the first aspect to the adjuvant is 1: 1-2; optionally, the volume ratio is 1: 1.
In some embodiments, the immunogenic composition is a coronavirus protein universal vaccine can be a mixture of a plurality of heterotetrameric antigen proteins.
Further, the immunogenic composition is a coronavirus DNA universal vaccine comprising:
(i) a eukaryotic expression vector; and
(ii) a DNA sequence encoding a coronavirus heterotetramer antigen protein according to the first aspect, constructed into said eukaryotic expression vector; alternatively, the DNA sequence comprises a nucleotide sequence as set forth in SEQ ID NO 12, 13, 14 and/or 15;
preferably, the eukaryotic expression vector is selected from the group consisting of pGX0001, pVAX1, pCAGGS and pcDNA series vectors.
Further, the immunogenic composition is a coronavirus mRNA universal vaccine comprising:
(I) an mRNA sequence encoding the coronavirus heterotetrameric antigen protein of any one of claims 1-5; and (II) lipid nanoparticles.
Further, the immunogenic composition is a coronavirus-viral vector vaccine comprising:
(first) a viral backbone vector; and
(②) a DNA sequence encoding a coronavirus heterotetramer antigen protein as described in the first aspect, constructed into said viral backbone vector; alternatively, the DNA sequence comprises a nucleotide sequence as set forth in SEQ ID NO 12, 13, 14 and/or 15;
Preferably, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors.
Further, the immunogenic composition is in the form of a nasal spray, oral formulation, suppository or parenteral formulation;
preferably, the nasal spray is selected from the group consisting of an aerosol, a spray and a powder spray;
preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets and ointments;
preferably, the parenteral formulation is a transdermal agent, ointment, plaster, topical liquid, injectable or bolus formulation.
In a ninth aspect, there is provided a use of the coronavirus heterotetramer antigenic protein of the first aspect, or the heterotetramer antigenic protein prepared by the method of the second aspect, or the polynucleotide of the third aspect, the nucleic acid construct of the fourth aspect, or the recombinant vector of the fifth aspect, or the expression system cell of the sixth aspect, or the immunogenic composition of the eighth aspect, in the preparation of a medicament for the treatment and/or prevention of a novel coronavirus infection and/or a disease caused by a novel coronavirus;
Alternatively, the coronavirus includes at least one or more of SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain, coronavirus RsSHC014 strain, coronavirus LYRa11 strain, coronavirus RaTG13 strain, coronavirus Pangolin GX/P4L strain, coronavirus Pangolin GD/1 strain, coronavirus WIV1 strain, coronavirus RshSTT182 strain, coronavirus BtKY72 strain, coronavirus RmYN02 strain, coronavirus SARS-CoV, coronavirus MERS-CoV, coronavirus HCoV-229E, coronavirus HCoV-OC43 strain, coronavirus HCoV-63 strain, coronavirus HCoV-HKU1 strain, and coronavirus bat-CoV strain.
In a tenth aspect, there is provided a method of eliciting an immune response against a coronavirus, or treating a coronavirus infection in a subject, by administering to the subject an effective amount of a coronavirus heterotetramer antigenic protein of the first aspect, or a heterotetramer antigenic protein prepared by the method of the second aspect, or a polynucleotide of the third aspect, a nucleic acid construct of the fourth aspect, or a recombinant vector of the fifth aspect, or an expression system cell of the sixth aspect, or an immunogenic composition of the eighth aspect;
alternatively, the coronavirus includes at least one or more of SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain, coronavirus RsSHC014 strain, coronavirus LYRa11 strain, coronavirus RaTG13 strain, coronavirus Pangolin GX/P4L strain, coronavirus Pangolin GD/1 strain, coronavirus WIV1 strain, coronavirus RshSTT182 strain, coronavirus BtKY72 strain, coronavirus RmYN02 strain, coronavirus SARS-CoV, coronavirus MERS-CoV, coronavirus HCoV-229E, coronavirus HCoV-OC43 strain, coronavirus HCoV-63 strain, coronavirus HCoV-HKU1 strain, and coronavirus bat-CoV strain.
Further, the above SARS-CoV-2 variant strain is selected from Alpha, Beta, Gamma, Kappa, Lambda, Delta or Omicron strains, optionally the SARS-CoV-2 variant strain is selected from Beta strains.
Advantageous effects
The heterotetramer can stimulate the generation of various coronavirus strains (SARS-CoV-2 prototype strain, variant strain and other coronavirus Pangolin GX/P4L, SHC014, WIV1, LYRa11, SARS-CoV and the like) with stronger neutralizing antibody level, namely has good immunogenicity and broad spectrum, and the stability and immunogenicity effects of the heterotetramer antigen proteins P-B-LY-S and P-B-SHC-S relative to P-B-Ra-S and P-B-PgGX-S are more balanced and stable.
Drawings
One or more embodiments are illustrated by the figures in the accompanying drawings, which correspond to and 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 preferred or advantageous over other embodiments.
FIG. 1 is a RBD clade of representative strains;
FIG. 2 is a schematic diagram of the construction of four heterotetramers of example 1 of the invention;
FIG. 3 is a molecular sieve elution peak and SDS-PAGE gel plot of four heterotetramers of example 1 of the invention;
FIG. 4 shows the results of an analytical ultracentrifugation of P-P-P-P according to example 2 of the present invention; wherein, the temperature of 4 ℃ and the 1w represent that the mixture is placed at the temperature of 4 ℃ for a week; 4 ℃ and 2w represents 4 ℃ for two weeks; standing at 4 deg.C and 3w at 4 deg.C for three weeks;
FIG. 5 is the result of an analytical ultracentrifugation of P-B-Ra-S according to example 2 of the present invention; wherein, the temperature of 4 ℃ and the 1w represent that the mixture is placed at the temperature of 4 ℃ for a week; 4 ℃ and 2w stands for 4 ℃ for two weeks; standing at 4 deg.C and 3w at 4 deg.C for three weeks;
FIG. 6 shows the results of analytical ultracentrifugation of P-B-PgGX-S according to example 2 of the present invention; wherein, 4 ℃,1w represents that the mixture is placed at 4 ℃ for one week; 4 ℃ and 2w represents 4 ℃ for two weeks; standing at 4 deg.C and 3w at 4 deg.C for three weeks;
FIG. 7 is the results of an analytical ultracentrifugation of P-B-LY-S of example 2 of the present invention; wherein, 4 ℃,1w represents that the mixture is placed at 4 ℃ for one week; 4 ℃ and 2w stands for 4 ℃ for two weeks; standing at 4 deg.C and 3w at 4 deg.C for three weeks;
FIG. 8 shows the results of analytical ultracentrifugation of P-B-SHC-S according to example 2 of the present invention; wherein, 4 ℃,1w represents that the mixture is placed at 4 ℃ for one week; 4 ℃ and 2w stands for 4 ℃ for two weeks; standing at 4 deg.C and 3w at 4 deg.C for three weeks;
FIG. 9 is an experimental grouping of example 3 of the present invention;
FIG. 10 is the immunization and blood collection procedure of example 3 of the present invention;
FIG. 11 is the level of neutralizing antibodies against the SARS-CoV-2 prototype strain pseudovirus in the serum of mice of example 5 of the present invention;
FIG. 12 is the level of neutralizing antibodies against the SARS-CoV-2Beta variant strain pseudovirus in the serum of mice of example 5 of the present invention;
FIG. 13 is the level of neutralizing antibodies against the SARS-CoV-2Delta variant pseudovirus in serum from mice of example 5 of the present invention;
FIG. 14 is the level of neutralizing antibodies against the SARS-CoV-2Omicron (BA.1) variant pseudovirus in the serum of mice of example 5 of the present invention;
FIG. 15 is the level of neutralizing antibodies against the coronavirus Pangolin GD/1 pseudovirus in mouse sera of example 5 of the present invention;
FIG. 16 is the level of neutralizing antibodies against the coronavirus RaTG13 pseudovirus in mouse serum of example 5 of the present invention;
FIG. 17 shows the level of neutralizing antibodies against coronavirus SHC014 pseudovirus in mouse serum according to example 5 of the invention;
FIG. 18 is the level of neutralizing antibodies to coronavirus WIV1 pseudovirus in mouse serum of example 5 of the invention;
FIG. 19 is the level of neutralizing antibodies against the pseudovirus LYRa11 coronavirus in the serum of mice of example 5 of the invention;
FIG. 20 is the level of neutralizing antibodies against coronavirus SARS-CoV pseudovirus in mouse serum of example 5 of the present invention;
FIG. 21 is a statistical difference analysis of the neutralizing antibody levels in the sera of the diabodies of mice immunized with different antigens according to example 5 of the present invention;
FIG. 22 is a statistical difference analysis of neutralizing antibody levels in the sera of the triabodies of mice immunized with different antigens according to example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations such as "comprises" or "comprising", etc., will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some instances, materials, components, methods, procedures, and the like that are well known to those of skill in the art have not been described in detail so as not to obscure the present invention.
The present invention will be described in detail below.
The reagents, enzymes, media, antibiotics, milk and other chemical materials used in the following examples of the present invention are all commercially available products.
Some commonly used biological materials, such as competent cells, vectors, cells to be transformed, murine hybridoma cells, protein a, etc., are also commercially available products.
Some synthetic biomaterials, such as pseudoviral nucleotide sequences, which require artificial synthesis, are committed to the synthesis company.
Example 1: construction and expression purification of heterotetramers
In this example, constructs of four heterotetrameric antigenic proteins were designed, respectively, according to the following specific protocol:
(1) the RBD sequences in the S protein amino acid sequences of a prototype SARS-CoV-2 (the amino acid sequence is shown as SEQ ID NO: 1), a SARS-CoV-2Beta variant (the amino acid sequence is shown as SEQ ID NO: 2), RaTG13 (the amino acid sequence is shown as SEQ ID NO: 3) and SARS-CoV (the amino acid sequence is shown as SEQ ID NO: 4) are connected in series to obtain the amino acid sequence shown as SEQ ID NO:8, the N end of the amino acid sequence shown as SEQ ID NO:8 is added with the signal peptide of MERS-CoV spike protein (MIHSVFLLMFLLTPTES, shown as SEQ ID NO:16) and the C end is added with 6 histidine (HHHHHHHHH) to obtain the construct of the heterotetrameric antigen protein P-B-Ra-S;
(2) The amino acid sequence of RBD in the S protein amino acid sequence of a prototype SARS-CoV-2 (the amino acid sequence is shown as SEQ ID NO: 1), a SARS-CoV-2Beta variant (the amino acid sequence is shown as SEQ ID NO: 2), Pangolin GX/P4L (the amino acid sequence is shown as SEQ ID NO: 5) and SARS-CoV (the amino acid sequence is shown as SEQ ID NO: 4) are connected in series to obtain the amino acid sequence shown as SEQ ID NO:9, the N end of the amino acid sequence shown as SEQ ID NO:9 is added with the signal peptide of MERS-CoV spike protein (MIHSVFLLMFLLTPTES, as SEQ ID NO:16) and the C end is added with 6 histidine (HHHHHHH) to obtain a construction body of the heterologous tetrameric antigen protein P-B-PgGX-S;
(3) the construction of the heterologous tetrameric antigen protein P-B-LY-S is obtained by connecting RBD sequences in the S protein amino acid sequences of a prototype SARS-CoV-2 (the amino acid sequence is shown in SEQ ID NO: 1), a SARS-CoV-2Beta variant (the amino acid sequence is shown in SEQ ID NO: 2), LYRa11 (the amino acid sequence is shown in SEQ ID NO: 6) and SARS-CoV (the amino acid sequence is shown in SEQ ID NO: 4) in series to obtain an amino acid sequence shown in SEQ ID NO:10, adding a MERS-CoV spike protein signal peptide (MIHSVFLLMFLLTPTES, shown in SEQ ID NO:16) at the N end of the amino acid sequence shown in SEQ ID NO:10 and adding 6 histidine (HHHHHHH) at the C end;
(4) The RBD sequences in the S protein amino acid sequences of prototype SARS-CoV-2 (the amino acid sequence is shown in SEQ ID NO: 1), SARS-CoV-2Beta variant (the amino acid sequence is shown in SEQ ID NO: 2), RsSHC014 (the amino acid sequence is shown in SEQ ID NO: 7) and SARS-CoV (the amino acid sequence is shown in SEQ ID NO: 4) are connected in series to obtain the amino acid sequence shown in SEQ ID NO:11, the signal peptide of MERS-CoV spike protein (MIHSVFLLMFLLTPTES, shown in SEQ ID NO:16) is added at the N end of the amino acid sequence shown in SEQ ID NO:11, and 6 histidine (HHHHHHHHH) is added at the C end to obtain the construct of the heterotetrameric antigen protein P-B-SHC-S.
The RBD homology of the selected strains of this example is shown in FIG. 1.
Expression and purification of heterotetrameric antigenic proteins in this example:
the amino acid sequences of the four constructs designed above are optimized by using human codons, and the corresponding DNA coding sequences are respectively shown in SEQ ID NO 17, 18, 19 and 20; a termination codon is added at the 3' end of the DNA coding sequences, four DNA sequences containing the termination codon are synthesized by Nanjing Kisrey Biotechnology Co., Ltd, and are cloned to pCAGGS plasmid through EcoRI and XhoI enzyme cutting sites to respectively obtain expression vectors for expressing the heterologous tetrameric antigen protein P-B-Ra-S, P-B-PgGX-S, P-B-LY-S, P-B-SHC-S.
A schematic representation of the nucleotide sequence of the heterotetramer construct P-B-Ra-S, P-B-PgGX-S, P-B-LY-S, P-B-SHC-S can be found in FIG. 2. Similarly, a schematic representation of the homotetrameric construct is shown in FIG. 2, except that the tetrameric antigenic protein portion is composed of 4 repeated RBD sequences (SEQ ID NO:1) of SARS-CoV-2 (prototype strain) in tandem directly, and the homodimeric protein portion is composed of 2 repeated RBD sequences (SEQ ID NO:1) of SARS-CoV-2 (prototype strain) in tandem directly.
The expression vector of the four heterotetramers is expressed by a mammalian system (such as HEK293F cells), and then purified by Superdex200 Hiload 16/60 column (GE) for molecular sieve chromatography, and the peak pattern is shown in FIG. 3. And (3) performing SDS-PAGE analysis on the protein with the elution volume of about 65mL, wherein protein bands under reducing and non-reducing conditions are about 120kD, which indicates that the constructed expression vector can stably express to obtain the heterotetrameric antigen protein P-B-Ra-S, P-B-PgGX-S, P-B-LY-S, P-B-SHC-S, the expression amount is stable, and 8-12mg of protein can be obtained by purifying every 400mL on average.
Example 2: stability assessment of antigenic proteins
1. The operation steps
(1) Preparing a protein sample: subpackaging four kinds of heterotetramer antigenic proteins frozen at-80 ℃ and four kinds of homotetramer antigenic proteins frozen at-80 ℃ into 0.3 mg/tube, preparing four tubes for each kind of protein, storing at 4 ℃ for 3 weeks, 2 weeks, 1 week and-80 ℃, and simultaneously detecting samples of each kind of protein at 4 ℃ for 3 weeks, 2 weeks and 1 week, and separately detecting samples at-80 ℃.
Adding protein buffer solution 1 × PBS to 400 μ L before detection, centrifuging at 12000rpm/min at 4 deg.C for 30min, and transferring the supernatant to new EP tube;
(2) before the operation of the instrument, radial calibration is carried out at the rotating speed of 3000rpm/min, parameters such as ultraviolet absorption and OD value are checked, and the time is 10 min. Then the mixture is balanced for 1h at 20 ℃ and zero rotating speed.
(3) The experimental method of sedimentation rate is adopted, AN AN60Ti swivel with four holes is used, a sample pool with a center part made of 12mm double-hole aluminum alloy is loaded with 380 mu L of sample and 400 mu L of buffer solution, the absorption wavelength is set to be 280nm, the temperature is 20 ℃, the rpm is 35000/min, data is collected every 3min, 180 spectrograms of sedimentation rate data are collected totally, the obtained data are analyzed by aid of sedfit software, a scatter diagram with a smooth curve is drawn, and the result is shown in figures 4-8.
2. Data analysis
FIG. 4 shows that the homotetrameric antigenic protein P-P-P is hardly degraded after 1 week of four-degree exposure, and the molecular weight is slightly decreased after 2 weeks and 3 weeks of exposure, indicating that degradation is initiated.
The detection results of four kinds of heterotetrameric antigen proteins P-B-Ra-S, P-B-PgGX-S, P-B-LY-S, P-B-SHC-S show that:
FIGS. 7-8 show that the heterotetrameric antigenic protein P-B-LY-S, P-B-SHC-S has little change in molecular weight, no degradation, and good protein stability after being placed at 4 ℃ for 3 weeks.
FIG. 6 shows that the heterotetrameric antigenic protein P-B-PgGX-S decreases in molecular weight after 3 weeks at 4 ℃ indicating evidence of degradation.
FIG. 5 shows that, the heterotetrameric antigenic protein P-B-Ra-S has poor stability, and the molecular weight of the protein is obviously reduced after being placed at 4 ℃ for 2 weeks, which indicates that the protein has obvious degradation.
In conclusion, the heterotetrameric antigenic proteins P-B-LY-S and P-B-SHC-S are the most stable, and P-B-PgGX-S is the less stable, and P-B-Ra-S is the less stable.
Example 3: animal experiments
Addavax is a MF59 adjuvant, and the adjuvant is used as a vaccine component and plays an important guiding role in subsequent clinical tests. We mixed different doses of antigenic proteins with Addavax adjuvant before immunization. The grouping situation is shown in FIG. 9, wherein P-P represents a homodimer obtained by connecting RBDs (R319-K537, RBD amino acid sequence of single new crown prototype strain is shown in SEQ ID NO: 1) of two new crown prototype strains in series, and P-P-P represents a tetramer antigen protein obtained by connecting RBDs (R319-K537, RBD amino acid sequence of single new crown prototype strain is shown in SEQ ID NO: 1) of four new crown prototype strains in series.
The four hetero-tetrameric antigens obtained in example 1 were diluted to two doses of 0.5. mu.g and 2. mu.g in PBS with the homo-dimeric antigenic protein P-P and the homo-tetrameric antigenic protein P-P-P, respectively, and mixed with Addavax for emulsification. 6-8 week old BALB/c mice were then immunized in groups of 6 mice each. The immunization strategy is as shown in fig. 10, i.e. mice were immunized by intramuscular injection at day 0, day 21, and day 103, respectively, with 100 μ L per vaccination. Orbital bleeds were performed on mice 19 days after priming, 14 days after secondary immunization, and 7 days after tertiary immunization, respectively. The mouse serum is obtained by standing and then centrifuging at the rotating speed of 800rpm/min for 15 minutes, and is preserved in a refrigerator at the temperature of minus 80 ℃ after the complement is inactivated at the temperature of 56 ℃ for 30 minutes, and is used for subsequent ELISA and pseudovirus neutralization detection.
Example 4: pseudovirus packaging
The coronavirus pseudovirus used in this example was a pseudovirus displaying coronavirus S protein prepared based on Vesicular Stomatitis Virus (VSV) backbone, and the preparation method was described in the methods section of the published papers of the textgroup (Effects of a Prolongated Booster Interval on neutrallization of Omicron varian, N Engl J Med,2022, PMID: 35081296).
The packaged coronavirus pseudoviruses comprise ten pseudoviruses, namely SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain Beta, SARS-CoV-2 variant strain Delta, SARS-CoV-2 variant strain Omicron (BA.1), coronavirus RaTG13, coronavirus Pangolin GD/1, coronavirus LYRa11, coronavirus WIV1, coronavirus RsSHC014 and coronavirus SARS-CoV, wherein the amino acid sequences of S proteins of the ten pseudoviruses are respectively shown as SEQ ID NO: 21-30, and the nucleotide sequences for coding the S proteins are provided with synthetic services by Jinwei or Jinsry in Suzhou.
SARS-CoV-2 prototype, SARS-CoV-2Beta variant, Delta, and Omicron (BA.1) in the pseudovirus of the packaged coronavirus were detected by Vero, and other strains were detected by HEK293T-ACE2 cell.
Example 5: experiment for neutralizing pseudovirus of immune serum
1. The operation steps
(1) Cell plating: the day before the pseudovirus neutralization experiment, Vero cells or HEK293T-ACE2 cells in logarithmic growth phase are trypsinized, counted, added into a 96-well plate with 100 mu L per well, cultured in a 37 ℃ incubator and 5% CO2The culture was carried out overnight.
(2) Serum dilution: the initial 40-fold dilution followed by the two-fold gradient dilution, 8 gradients for the first serum and 11 gradients for the second serum.
(3) Diluting the virus: each pseudovirus was diluted in DMEM with 10% FBS by appropriate fold and mixed by inversion thoroughly, with serum and virus volumes per well being in the range of 1: 1, mixing evenly, namely 120 mu L/hole.
(4) One positive control (10% FBS in DMEM with pseudovirus in equal volume) was done for each plate and one negative control (10% FBS in DMEM in cells) was done for each batch of neutralization experiments. The serum and pseudovirus mixtures in round bottom 96 well plates were incubated for 1 hour at 37 ℃ in a 5% CO2 incubator.
(5) Plate rotating: cell supernatants from the day prior plates were blotted dry in 96-well flat plates and 100. mu.L of serum and pseudovirus were transferred to the cells in the flat plates. Incubate at 37 ℃ in a 5% CO2 incubator for 15 hours, and read with a confocal laser high content analysis imaging system (Yokogawa, CQ 1).
2. Data analysis
The results of neutralizing antibody levels against different strains of pseudovirus in sera collected 19 days after priming, 14 days after secondary immunization, and 7 days after tertiary immunization in example 3 are shown in fig. 11-20.
The results of the sera from the triones (boost2) of FIGS. 11-16 show that the neutralizing antibody levels against the SARS-CoV-2 prototype strain and strains with RBD homology of 90% or more with the prototype strain, which were elicited by the four heterotetrameric antigenic proteins, have been shown to be even better than the neutralizing antibody levels against the homodimeric antigenic protein P-P and the homodimeric antigenic protein P-P-P-P, and that the neutralizing antibody levels against Omicron (BA.1) of the four heterotetrameric antigenic proteins have been somewhat increased relative to the homotetrameric protein P-P-P-P.
The results of the sera of the diabodies (boost) presented in FIGS. 17-20 show that the four heterotetrameric antigenic proteins induced by the prototype strain of SARS-CoV-2 with RBD homology of 70% -80% (e.g., SHC014, WIV1, LYRa11, SARS-CoV) all gave significantly higher levels of neutralizing antibodies than the homotetrameric antigenic protein and homodimeric antigenic protein, and that the neutralizing effects of the heterotetrameric antigenic proteins P-B-LY-S and P-B-SHC-S on the four strains were very significant compared to the neutralizing effects of the homodimers and homotetramers on the four strains, and also to the neutralizing effects on the four strains relative to the other two heterotetramers P-B-Ra-S and P-B-PgGX-S.
The results of the (boost) sera in FIGS. 17-20 show that the four heterotetrameric antigenic proteins elicited significantly greater levels of neutralization also against strains with 70% -80% RBD homology to the SARS-CoV-2 prototype strain (e.g., SHC014, WIV1, LYRa11, SARS-CoV), especially the heterotetrameric antigenic proteins P-B-LY-S and P-B-SHC-S, compared to the neutralizing antibody levels against these four strains, both homodimers and homotetramers (FIG. 22).
The results in FIGS. 11-22 show that, for the SARS-CoV-2 prototype strain and closely related strains, the heterotetrameric antigenic protein has similar effects to the existing vaccines designed against the prototype strain; in addition, the heterotetrameric antigenic proteins are able to elicit neutralizing antibodies that produce significantly more cross-neutralizing strains more distant from the relatives (SHC014, WIV1, LYRa11, SARS), i.e.the heterotetramers are more broad-spectrum.
In conclusion, for prototype strains and strains with close relativity, the heterotetramer has similar effect with the existing vaccine aiming at SARS-CoV-2 prototype strain, compared with the existing vaccine aiming at the prototype strain, the heterotetramer antigen protein can stimulate to generate stronger neutralizing antibody level for strains with farther relativity, the antibody broad spectrum induced by the heterotetramer antigen protein is stronger, the immunogenicity and the broad spectrum are better than the existing vaccine aiming at the new corona prototype strain, and the effects of the heterotetramer antigen proteins P-B-LY-S and P-B-SHC-S relative to P-B-Ra-S and P-B-PgGX-S are more stable.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
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<221> DOMAIN
<222> (1)..(219)
<400> 3
Arg Val Gln Pro Thr Asp Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr 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 Thr 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 Thr 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 Lys His Ile Asp Ala Lys Glu Gly
115 120 125
Gly Asn Phe Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ala 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 Gln Thr Gly Leu Asn Cys Tyr Tyr Pro Leu Tyr Arg
165 170 175
Tyr Gly Phe Tyr Pro Thr Asp Gly Val Gly His Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys
210 215
<210> 4
<211> 218
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of receptor binding region of spike protein of SARS-CoV coronavirus
<220>
<221> DOMAIN
<222> (1)..(218)
<400> 4
Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val
20 25 30
Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met
100 105 110
Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr
115 120 125
Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg
130 135 140
Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys
145 150 155 160
Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr
165 170 175
Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val
180 185 190
Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro
195 200 205
Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln
210 215
<210> 5
<211> 219
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of receptor-binding region of spike protein of coronavirus Pangolin GX/P4L
<220>
<221> DOMAIN
<222> (1)..(219)
<400> 5
Arg Val Gln Pro Thr Ile Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Ser 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 Ser Thr 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 Val Lys Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Val Lys Gln Asp Ala Leu Thr Gly
115 120 125
Gly Asn Tyr Gly Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Lys 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 Gln Val Gly Leu Asn Cys Tyr Tyr Pro Leu Glu Arg
165 170 175
Tyr Gly Phe His Pro Thr Thr Gly Val Asn Tyr Gln Pro Phe Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu Asn Gly Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Leu Ser Thr Thr Leu Val Lys Asp Lys
210 215
<210> 6
<211> 218
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of receptor binding region of spike protein of coronavirus LYRa11
<220>
<221> DOMAIN
<222> (1)..(218)
<400> 6
Arg Val Ser Pro Ser Lys Glu Val Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Pro Ser Val
20 25 30
Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Ala Ile Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met
100 105 110
Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Ser
115 120 125
Gly Asn Phe Asn Tyr Lys Tyr Arg Ser Leu Arg His Gly Lys Leu Arg
130 135 140
Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys
145 150 155 160
Pro Cys Thr Pro Pro Ala Phe Asn Cys Tyr Trp Pro Leu Asn Asp Tyr
165 170 175
Gly Phe Tyr Thr Thr Asn Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val
180 185 190
Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro
195 200 205
Lys Leu Ser Thr Asp Leu Ile Thr Asn Gln
210 215
<210> 7
<211> 218
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of receptor-binding region of spike protein of coronavirus RsSHC014
<220>
<221> DOMAIN
<222> (1)..(218)
<400> 7
Arg Val Ala Pro Ser Lys Glu Val Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Pro Ser Val
20 25 30
Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Leu
100 105 110
Gly Cys Val Leu Ala Trp Asn Thr Asn Ser Lys Asp Ser Ser Thr Ser
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Trp Val Arg Arg Ser Lys Leu Asn
130 135 140
Pro Tyr Glu Arg Asp Leu Ser Asn Asp Ile Tyr Ser Pro Gly Gly Gln
145 150 155 160
Ser Cys Ser Ala Val Gly Pro Asn Cys Tyr Asn Pro Leu Arg Pro Tyr
165 170 175
Gly Phe Phe Thr Thr Ala Gly Val Gly His Gln Pro Tyr Arg Val Val
180 185 190
Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro
195 200 205
Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln
210 215
<210> 8
<211> 875
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of heterotetrameric antigen protein of P-B-Ra-S
<220>
<221> DOMAIN
<222> (1)..(875)
<400> 8
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val
370 375 380
Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Asp Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Thr Thr Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Thr Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Lys His
545 550 555 560
Ile Asp Ala Lys Glu Gly Gly Asn Phe Asn Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ala Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Gln Thr Gly Leu Asn Cys
595 600 605
Tyr Tyr Pro Leu Tyr Arg Tyr Gly Phe Tyr Pro Thr Asp Gly Val Gly
610 615 620
His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr
660 665 670
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser
675 680 685
Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr
690 695 700
Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly
705 710 715 720
Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala
725 730 735
Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly
740 745 750
Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
755 760 765
Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser
770 775 780
Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu
785 790 795 800
Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly
805 810 815
Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp
820 825 830
Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val
835 840 845
Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly
850 855 860
Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln
865 870 875
<210> 9
<211> 875
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of heterotetrameric antigen protein of P-B-PgGX-S
<220>
<221> DOMAIN
<222> (1)..(875)
<400> 9
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val
370 375 380
Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Ile Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Ser Lys Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Val Lys
545 550 555 560
Gln Asp Ala Leu Thr Gly Gly Asn Tyr Gly Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Lys Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Gln Val Gly Leu Asn Cys
595 600 605
Tyr Tyr Pro Leu Glu Arg Tyr Gly Phe His Pro Thr Thr Gly Val Asn
610 615 620
Tyr Gln Pro Phe Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Gly
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Thr Leu Val Lys Asp
645 650 655
Lys Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr
660 665 670
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser
675 680 685
Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr
690 695 700
Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly
705 710 715 720
Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala
725 730 735
Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly
740 745 750
Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
755 760 765
Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser
770 775 780
Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu
785 790 795 800
Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly
805 810 815
Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp
820 825 830
Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val
835 840 845
Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly
850 855 860
Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln
865 870 875
<210> 10
<211> 874
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of heterotetrameric antigen protein of P-B-LY-S
<220>
<221> DOMAIN
<222> (1)..(874)
<400> 10
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val
370 375 380
Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Ser Pro Ser Lys Glu Val Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Thr Thr Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Ala Ile Lys Leu Asn Asp Leu Cys Phe
500 505 510
Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn
545 550 555 560
Ile Asp Ala Thr Ser Ser Gly Asn Phe Asn Tyr Lys Tyr Arg Ser Leu
565 570 575
Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro
580 585 590
Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Phe Asn Cys Tyr
595 600 605
Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Asn Gly Ile Gly Tyr
610 615 620
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro
625 630 635 640
Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Thr Asn Gln
645 650 655
Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn
660 665 670
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val
675 680 685
Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser
690 695 700
Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val
705 710 715 720
Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp
725 730 735
Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln
740 745 750
Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met
755 760 765
Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr
770 775 780
Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg
785 790 795 800
Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys
805 810 815
Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr
820 825 830
Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val
835 840 845
Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro
850 855 860
Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln
865 870
<210> 11
<211> 874
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of heterotetrameric antigen protein of P-B-SHC-S
<220>
<221> DOMAIN
<222> (1)..(874)
<400> 11
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val
370 375 380
Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Ala Pro Ser Lys Glu Val Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Thr Thr Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Leu Gly Cys Val Leu Ala Trp Asn Thr Asn Ser
545 550 555 560
Lys Asp Ser Ser Thr Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Trp Val
565 570 575
Arg Arg Ser Lys Leu Asn Pro Tyr Glu Arg Asp Leu Ser Asn Asp Ile
580 585 590
Tyr Ser Pro Gly Gly Gln Ser Cys Ser Ala Val Gly Pro Asn Cys Tyr
595 600 605
Asn Pro Leu Arg Pro Tyr Gly Phe Phe Thr Thr Ala Gly Val Gly His
610 615 620
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro
625 630 635 640
Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln
645 650 655
Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn
660 665 670
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val
675 680 685
Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser
690 695 700
Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val
705 710 715 720
Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp
725 730 735
Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln
740 745 750
Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met
755 760 765
Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr
770 775 780
Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg
785 790 795 800
Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys
805 810 815
Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr
820 825 830
Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val
835 840 845
Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro
850 855 860
Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln
865 870
<210> 12
<211> 2625
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence encoding heterotetrameric antigen protein of P-B-Ra-S
<220>
<221> misc_feature
<222> (1)..(2625)
<400> 12
agagtgcagc ctaccgagtc catcgtgcgg ttccccaaca tcaccaatct gtgccctttc 60
ggcgaggtgt tcaacgccac cagattcgcc tctgtgtacg cctggaaccg gaagcggatc 120
agcaattgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag caccttcaag 180
tgctacggcg tgtcccctac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 240
agcttcgtga tcagaggcga cgaagtgcgg cagattgccc ctggacagac aggcaagatc 300
gccgattaca actacaagct gcccgacgac ttcaccggct gtgtgattgc ctggaacagc 360
aacaacctgg acagcaaagt cggcggcaac tacaactacc tgtaccggct gttccggaag 420
tccaacctga agcctttcga gcgggacatc agcaccgaga tctatcaggc cggcagcacc 480
ccttgcaatg gcgtggaagg cttcaactgc tacttcccac tgcagtccta cggcttccag 540
cctacaaacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt cgagctgctg 600
catgctcctg ccacagtgtg cggccctaag aaaagcacca acctggtcaa gaacaagcgg 660
gtgcagccca ctgagagcat tgtgcgcttt ccaaatatca cgaatctctg cccctttggg 720
gaagtcttta atgccacacg cttcgccagc gtctacgctt ggaataggaa gagaatctcc 780
aactgcgtgg cagattactc cgtcctgtac aattccgcca gcttttccac gtttaagtgt 840
tatggcgtca gccccacaaa gctcaatgat ctctgcttta ccaatgtcta tgccgactcc 900
tttgtgattc gcggagatga agtccgccag atcgctccag gacagaccgg aaacattgcc 960
gactataatt acaagctccc ggatgatttc acgggatgcg tgatcgcttg gaactccaac 1020
aatctggatt ccaaagttgg cgggaattac aattatctct atcgcctctt ccgcaagagc 1080
aatctgaagc cctttgagag agacatctct acagaaatct accaggctgg ctctacccca 1140
tgtaacggcg tgaagggctt taattgctat tttcccctgc aatcttacgg gtttcagccc 1200
acctatggcg tgggatatca accctatcgc gtggtggtcc tgtcttttga actgctgcac 1260
gccccagcta ccgtgtgtgg cccaaaaaag tccaccaatc tcgtgaagaa caaaagagtg 1320
cagcccaccg acagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1380
gtgttcaacg ccaccacctt cgctagcgtg tacgcttgga atagaaagag aatcagcaac 1440
tgcgtggccg actacagcgt gctgtacaac agcacaagct tcagcacctt caagtgctat 1500
ggcgtgtccc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1560
gtgatcaccg gcgacgaggt gagacagatc gcccccgggc agaccggcaa gatcgccgac 1620
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaagcac 1680
atcgacgcca aggagggcgg caattttaac tacctgtaca gactgttcag aaaggccaac 1740
ctgaagccct tcgagagaga catcagcacc gagatctacc aagccggcag caagccctgc 1800
aacgggcaga ccggcctgaa ttgctactac cccctgtaca gatacggctt ctaccccacc 1860
gacggcgtgg gccatcagcc ctacagagtg gtcgtgctga gcttcgagct gctgaacgcc 1920
cccgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gcgtgttgtc 1980
ccatccggtg acgttgtccg gtttcctaac atcacaaact tgtgtccctt tggcgaagtc 2040
ttcaatgcta ccaaatttcc cagcgtctac gcgtgggaaa gaaagaaaat atcaaattgt 2100
gttgccgact attccgtcct atataatagc acgttcttct cgacgttcaa gtgttatggt 2160
gtctctgcta cgaaacttaa cgacttatgt ttctcaaacg tgtacgcaga ttctttcgta 2220
gttaaaggtg atgatgtgag gcagattgcg cccggacaaa caggagtaat cgccgattac 2280
aactacaaac tcccggacga ctttatgggg tgtgtgttag cttggaatac gaggaatata 2340
gacgccacga gtaccgggaa ttataattat aagtatcgct atctccgaca tggcaaactc 2400
aggccatttg aacgcgacat tagcaatgtt ccattctctc cggacggcaa accgtgcact 2460
ccaccggctt taaattgtta ttggccgtta aacgactatg gcttttatac aacgacggga 2520
atagggtacc aaccttacag agtagtagta ctaagtttcg agctattaaa tgcgccggcc 2580
accgtatgtg ggcccaagct atcgacggac ctaatcaaga atcag 2625
<210> 13
<211> 2625
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence encoding heterotetrameric antigen protein of P-B-PgGX-S
<220>
<221> misc_feature
<222> (1)..(2625)
<400> 13
agagtgcagc ctaccgagtc catcgtgcgg ttccccaaca tcaccaatct gtgccctttc 60
ggcgaggtgt tcaacgccac cagattcgcc tctgtgtacg cctggaaccg gaagcggatc 120
agcaattgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag caccttcaag 180
tgctacggcg tgtcccctac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 240
agcttcgtga tcagaggcga cgaagtgcgg cagattgccc ctggacagac aggcaagatc 300
gccgattaca actacaagct gcccgacgac ttcaccggct gtgtgattgc ctggaacagc 360
aacaacctgg acagcaaagt cggcggcaac tacaactacc tgtaccggct gttccggaag 420
tccaacctga agcctttcga gcgggacatc agcaccgaga tctatcaggc cggcagcacc 480
ccttgcaatg gcgtggaagg cttcaactgc tacttcccac tgcagtccta cggcttccag 540
cctacaaacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt cgagctgctg 600
catgctcctg ccacagtgtg cggccctaag aaaagcacca acctggtcaa gaacaagcgg 660
gtgcagccca ctgagagcat tgtgcgcttt ccaaatatca cgaatctctg cccctttggg 720
gaagtcttta atgccacacg cttcgccagc gtctacgctt ggaataggaa gagaatctcc 780
aactgcgtgg cagattactc cgtcctgtac aattccgcca gcttttccac gtttaagtgt 840
tatggcgtca gccccacaaa gctcaatgat ctctgcttta ccaatgtcta tgccgactcc 900
tttgtgattc gcggagatga agtccgccag atcgctccag gacagaccgg aaacattgcc 960
gactataatt acaagctccc ggatgatttc acgggatgcg tgatcgcttg gaactccaac 1020
aatctggatt ccaaagttgg cgggaattac aattatctct atcgcctctt ccgcaagagc 1080
aatctgaagc cctttgagag agacatctct acagaaatct accaggctgg ctctacccca 1140
tgtaacggcg tgaagggctt taattgctat tttcccctgc aatcttacgg gtttcagccc 1200
acctatggcg tgggatatca accctatcgc gtggtggtcc tgtcttttga actgctgcac 1260
gccccagcta ccgtgtgtgg cccaaaaaag tccaccaatc tcgtgaagaa caaaagagtg 1320
cagcccacca tcagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1380
gtgttcaacg ctagcaagtt cgctagcgtg tacgcctgga accggaagag aatcagcaac 1440
tgcgtggccg actacagcgt gctgtacaac agcacaagct tcagcacctt caagtgttac 1500
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1560
gtggtgaagg gcgacgaggt gagacagatc gcccccgggc agaccggcgt gatcgccgac 1620
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcgtgaag 1680
caagacgccc tgaccggggg caactacggc tacctgtaca gactgttcag aaagagcaag 1740
ctgaagccct tcgagagaga catcagcacc gagatctacc aagccggcag caccccctgc 1800
aacggccaag tgggcctgaa ctgctactac cccctggaga gatacggctt ccaccccacc 1860
accggcgtga actatcagcc cttcagagtg gtcgtgctga gcttcgagct gctgaacggc 1920
cccgccaccg tgtgcggccc caagctgagc accaccctgg tgaaggacaa gcgtgttgtc 1980
ccatccggtg acgttgtccg gtttcctaac atcacaaact tgtgtccctt tggcgaagtc 2040
ttcaatgcta ccaaatttcc cagcgtctac gcgtgggaaa gaaagaaaat atcaaattgt 2100
gttgccgact attccgtcct atataatagc acgttcttct cgacgttcaa gtgttatggt 2160
gtctctgcta cgaaacttaa cgacttatgt ttctcaaacg tgtacgcaga ttctttcgta 2220
gttaaaggtg atgatgtgag gcagattgcg cccggacaaa caggagtaat cgccgattac 2280
aactacaaac tcccggacga ctttatgggg tgtgtgttag cttggaatac gaggaatata 2340
gacgccacga gtaccgggaa ttataattat aagtatcgct atctccgaca tggcaaactc 2400
aggccatttg aacgcgacat tagcaatgtt ccattctctc cggacggcaa accgtgcact 2460
ccaccggctt taaattgtta ttggccgtta aacgactatg gcttttatac aacgacggga 2520
atagggtacc aaccttacag agtagtagta ctaagtttcg agctattaaa tgcgccggcc 2580
accgtatgtg ggcccaagct atcgacggac ctaatcaaga atcag 2625
<210> 14
<211> 2622
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence encoding heterotetrameric antigen protein of P-B-LY-S
<220>
<221> misc_feature
<222> (1)..(2622)
<400> 14
agagtgcagc ctaccgagtc catcgtgcgg ttccccaaca tcaccaatct gtgccctttc 60
ggcgaggtgt tcaacgccac cagattcgcc tctgtgtacg cctggaaccg gaagcggatc 120
agcaattgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag caccttcaag 180
tgctacggcg tgtcccctac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 240
agcttcgtga tcagaggcga cgaagtgcgg cagattgccc ctggacagac aggcaagatc 300
gccgattaca actacaagct gcccgacgac ttcaccggct gtgtgattgc ctggaacagc 360
aacaacctgg acagcaaagt cggcggcaac tacaactacc tgtaccggct gttccggaag 420
tccaacctga agcctttcga gcgggacatc agcaccgaga tctatcaggc cggcagcacc 480
ccttgcaatg gcgtggaagg cttcaactgc tacttcccac tgcagtccta cggcttccag 540
cctacaaacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt cgagctgctg 600
catgctcctg ccacagtgtg cggccctaag aaaagcacca acctggtcaa gaacaagcgg 660
gtgcagccca ctgagagcat tgtgcgcttt ccaaatatca cgaatctctg cccctttggg 720
gaagtcttta atgccacacg cttcgccagc gtctacgctt ggaataggaa gagaatctcc 780
aactgcgtgg cagattactc cgtcctgtac aattccgcca gcttttccac gtttaagtgt 840
tatggcgtca gccccacaaa gctcaatgat ctctgcttta ccaatgtcta tgccgactcc 900
tttgtgattc gcggagatga agtccgccag atcgctccag gacagaccgg aaacattgcc 960
gactataatt acaagctccc ggatgatttc acgggatgcg tgatcgcttg gaactccaac 1020
aatctggatt ccaaagttgg cgggaattac aattatctct atcgcctctt ccgcaagagc 1080
aatctgaagc cctttgagag agacatctct acagaaatct accaggctgg ctctacccca 1140
tgtaacggcg tgaagggctt taattgctat tttcccctgc aatcttacgg gtttcagccc 1200
acctatggcg tgggatatca accctatcgc gtggtggtcc tgtcttttga actgctgcac 1260
gccccagcta ccgtgtgtgg cccaaaaaag tccaccaatc tcgtgaagaa caaaagagtg 1320
agccctagca aagaggtggt gagattcccc aacatcacca atctgtgccc cttcggcgag 1380
gtgttcaacg ccaccacctt ccctagcgtg tacgcctggg agagaaagag aatcagcaac 1440
tgcgtggccg actacagcgt gctgtacaac agcacaagct tcagcacctt caagtgctac 1500
ggcgtgagcg ccatcaagct gaacgacctg tgcttcagca acgtgtacgc cgacagcttc 1560
gtggtgaagg gcgacgacgt gagacagatc gcccccgggc agaccggcgt gatcgccgac 1620
tacaactaca agctgcccga cgacttcatg ggctgcgtgc tggcctggaa cacaagaaac 1680
atcgacgcca caagcagcgg caacttcaac tacaagtaca gaagcctgag acacggcaag 1740
ctgagaccct tcgagagaga catcagcaac gtgcccttca gccccgacgg caagccctgc 1800
accccccccg ccttcaactg ttactggccc ctgaacgact acggcttcta caccaccaac 1860
ggcattggct accaacccta cagagtggtc gtgctgagct tcgagctgct gaacgctccc 1920
gccaccgtct gcggccccaa gctgagcacc gacctgatca ccaatcagcg tgttgtccca 1980
tccggtgacg ttgtccggtt tcctaacatc acaaacttgt gtccctttgg cgaagtcttc 2040
aatgctacca aatttcccag cgtctacgcg tgggaaagaa agaaaatatc aaattgtgtt 2100
gccgactatt ccgtcctata taatagcacg ttcttctcga cgttcaagtg ttatggtgtc 2160
tctgctacga aacttaacga cttatgtttc tcaaacgtgt acgcagattc tttcgtagtt 2220
aaaggtgatg atgtgaggca gattgcgccc ggacaaacag gagtaatcgc cgattacaac 2280
tacaaactcc cggacgactt tatggggtgt gtgttagctt ggaatacgag gaatatagac 2340
gccacgagta ccgggaatta taattataag tatcgctatc tccgacatgg caaactcagg 2400
ccatttgaac gcgacattag caatgttcca ttctctccgg acggcaaacc gtgcactcca 2460
ccggctttaa attgttattg gccgttaaac gactatggct tttatacaac gacgggaata 2520
gggtaccaac cttacagagt agtagtacta agtttcgagc tattaaatgc gccggccacc 2580
gtatgtgggc ccaagctatc gacggaccta atcaagaatc ag 2622
<210> 15
<211> 2622
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence encoding heterotetrameric antigen protein of P-B-SHC-S
<220>
<221> misc_feature
<222> (1)..(2622)
<400> 15
agagtgcagc ctaccgagtc catcgtgcgg ttccccaaca tcaccaatct gtgccctttc 60
ggcgaggtgt tcaacgccac cagattcgcc tctgtgtacg cctggaaccg gaagcggatc 120
agcaattgcg tggccgacta cagcgtgctg tacaacagcg ccagcttcag caccttcaag 180
tgctacggcg tgtcccctac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 240
agcttcgtga tcagaggcga cgaagtgcgg cagattgccc ctggacagac aggcaagatc 300
gccgattaca actacaagct gcccgacgac ttcaccggct gtgtgattgc ctggaacagc 360
aacaacctgg acagcaaagt cggcggcaac tacaactacc tgtaccggct gttccggaag 420
tccaacctga agcctttcga gcgggacatc agcaccgaga tctatcaggc cggcagcacc 480
ccttgcaatg gcgtggaagg cttcaactgc tacttcccac tgcagtccta cggcttccag 540
cctacaaacg gcgtgggcta ccagccttac agagtggtgg tgctgagctt cgagctgctg 600
catgctcctg ccacagtgtg cggccctaag aaaagcacca acctggtcaa gaacaagcgg 660
gtgcagccca ctgagagcat tgtgcgcttt ccaaatatca cgaatctctg cccctttggg 720
gaagtcttta atgccacacg cttcgccagc gtctacgctt ggaataggaa gagaatctcc 780
aactgcgtgg cagattactc cgtcctgtac aattccgcca gcttttccac gtttaagtgt 840
tatggcgtca gccccacaaa gctcaatgat ctctgcttta ccaatgtcta tgccgactcc 900
tttgtgattc gcggagatga agtccgccag atcgctccag gacagaccgg aaacattgcc 960
gactataatt acaagctccc ggatgatttc acgggatgcg tgatcgcttg gaactccaac 1020
aatctggatt ccaaagttgg cgggaattac aattatctct atcgcctctt ccgcaagagc 1080
aatctgaagc cctttgagag agacatctct acagaaatct accaggctgg ctctacccca 1140
tgtaacggcg tgaagggctt taattgctat tttcccctgc aatcttacgg gtttcagccc 1200
acctatggcg tgggatatca accctatcgc gtggtggtcc tgtcttttga actgctgcac 1260
gccccagcta ccgtgtgtgg cccaaaaaag tccaccaatc tcgtgaagaa caaaagagtg 1320
gcccctagca aagaagtggt gcggttcccc aacatcacca acctgtgccc ttttggggaa 1380
gtgttcaacg ccaccacctt cccctccgtt tacgcctggg aaagaaagag aatctctaac 1440
tgtgtggctg attactctgt gctgtataac agcaccagct tttctacatt caagtgttac 1500
ggcgtgagtg ccacaaagct gaatgatctg tgcttcagca atgtgtatgc cgacagcttc 1560
gtggtgaagg gagatgacgt gcggcagatc gcccctggac agactggcgt gatcgccgat 1620
tacaactaca agctgcctga cgactttctg ggctgtgtgc tggcctggaa caccaacagc 1680
aaggacagca gcacaagcgg aaactacaat tacctgtacc ggtgggtcag aagaagcaag 1740
ctgaacccct acgagcgcga cctgagcaac gacatctaca gccctggcgg ccagtcctgc 1800
agcgccgtgg gccctaactg ctacaaccct ctgagaccct acggcttctt caccaccgcc 1860
ggcgtgggcc accagcctta cagagtggtg gtgctgagct tcgagctgct caacgcccct 1920
gccaccgtgt gcggccccaa gttgtctaca gacctgatca agaaccagcg tgttgtccca 1980
tccggtgacg ttgtccggtt tcctaacatc acaaacttgt gtccctttgg cgaagtcttc 2040
aatgctacca aatttcccag cgtctacgcg tgggaaagaa agaaaatatc aaattgtgtt 2100
gccgactatt ccgtcctata taatagcacg ttcttctcga cgttcaagtg ttatggtgtc 2160
tctgctacga aacttaacga cttatgtttc tcaaacgtgt acgcagattc tttcgtagtt 2220
aaaggtgatg atgtgaggca gattgcgccc ggacaaacag gagtaatcgc cgattacaac 2280
tacaaactcc cggacgactt tatggggtgt gtgttagctt ggaatacgag gaatatagac 2340
gccacgagta ccgggaatta taattataag tatcgctatc tccgacatgg caaactcagg 2400
ccatttgaac gcgacattag caatgttcca ttctctccgg acggcaaacc gtgcactcca 2460
ccggctttaa attgttattg gccgttaaac gactatggct tttatacaac gacgggaata 2520
gggtaccaac cttacagagt agtagtacta agtttcgagc tattaaatgc gccggccacc 2580
gtatgtgggc ccaagctatc gacggaccta atcaagaatc ag 2622
<210> 16
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of signal peptide of MERS-CoV spike protein
<220>
<221> DOMAIN
<222> (1)..(17)
<400> 16
Met Ile His Ser Val Phe Leu Leu Met Phe Leu Leu Thr Pro Thr Glu
1 5 10 15
Ser
<210> 17
<211> 2694
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence of P-B-Ra-S construct
<220>
<221> misc_feature
<222> (1)..(2694)
<400> 17
atgatccaca gcgtgttcct gctgatgttc ctgctcaccc ccacagagag cagagtgcag 60
cctaccgagt ccatcgtgcg gttccccaac atcaccaatc tgtgcccttt cggcgaggtg 120
ttcaacgcca ccagattcgc ctctgtgtac gcctggaacc ggaagcggat cagcaattgc 180
gtggccgact acagcgtgct gtacaacagc gccagcttca gcaccttcaa gtgctacggc 240
gtgtccccta ccaagctgaa cgacctgtgc ttcaccaacg tgtacgccga cagcttcgtg 300
atcagaggcg acgaagtgcg gcagattgcc cctggacaga caggcaagat cgccgattac 360
aactacaagc tgcccgacga cttcaccggc tgtgtgattg cctggaacag caacaacctg 420
gacagcaaag tcggcggcaa ctacaactac ctgtaccggc tgttccggaa gtccaacctg 480
aagcctttcg agcgggacat cagcaccgag atctatcagg ccggcagcac cccttgcaat 540
ggcgtggaag gcttcaactg ctacttccca ctgcagtcct acggcttcca gcctacaaac 600
ggcgtgggct accagcctta cagagtggtg gtgctgagct tcgagctgct gcatgctcct 660
gccacagtgt gcggccctaa gaaaagcacc aacctggtca agaacaagcg ggtgcagccc 720
actgagagca ttgtgcgctt tccaaatatc acgaatctct gcccctttgg ggaagtcttt 780
aatgccacac gcttcgccag cgtctacgct tggaatagga agagaatctc caactgcgtg 840
gcagattact ccgtcctgta caattccgcc agcttttcca cgtttaagtg ttatggcgtc 900
agccccacaa agctcaatga tctctgcttt accaatgtct atgccgactc ctttgtgatt 960
cgcggagatg aagtccgcca gatcgctcca ggacagaccg gaaacattgc cgactataat 1020
tacaagctcc cggatgattt cacgggatgc gtgatcgctt ggaactccaa caatctggat 1080
tccaaagttg gcgggaatta caattatctc tatcgcctct tccgcaagag caatctgaag 1140
ccctttgaga gagacatctc tacagaaatc taccaggctg gctctacccc atgtaacggc 1200
gtgaagggct ttaattgcta ttttcccctg caatcttacg ggtttcagcc cacctatggc 1260
gtgggatatc aaccctatcg cgtggtggtc ctgtcttttg aactgctgca cgccccagct 1320
accgtgtgtg gcccaaaaaa gtccaccaat ctcgtgaaga acaaaagagt gcagcccacc 1380
gacagcatcg tgagattccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 1440
gccaccacct tcgctagcgt gtacgcttgg aatagaaaga gaatcagcaa ctgcgtggcc 1500
gactacagcg tgctgtacaa cagcacaagc ttcagcacct tcaagtgcta tggcgtgtcc 1560
cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcacc 1620
ggcgacgagg tgagacagat cgcccccggg cagaccggca agatcgccga ctacaactac 1680
aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaagca catcgacgcc 1740
aaggagggcg gcaattttaa ctacctgtac agactgttca gaaaggccaa cctgaagccc 1800
ttcgagagag acatcagcac cgagatctac caagccggca gcaagccctg caacgggcag 1860
accggcctga attgctacta ccccctgtac agatacggct tctaccccac cgacggcgtg 1920
ggccatcagc cctacagagt ggtcgtgctg agcttcgagc tgctgaacgc ccccgccacc 1980
gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agcgtgttgt cccatccggt 2040
gacgttgtcc ggtttcctaa catcacaaac ttgtgtccct ttggcgaagt cttcaatgct 2100
accaaatttc ccagcgtcta cgcgtgggaa agaaagaaaa tatcaaattg tgttgccgac 2160
tattccgtcc tatataatag cacgttcttc tcgacgttca agtgttatgg tgtctctgct 2220
acgaaactta acgacttatg tttctcaaac gtgtacgcag attctttcgt agttaaaggt 2280
gatgatgtga ggcagattgc gcccggacaa acaggagtaa tcgccgatta caactacaaa 2340
ctcccggacg actttatggg gtgtgtgtta gcttggaata cgaggaatat agacgccacg 2400
agtaccggga attataatta taagtatcgc tatctccgac atggcaaact caggccattt 2460
gaacgcgaca ttagcaatgt tccattctct ccggacggca aaccgtgcac tccaccggct 2520
ttaaattgtt attggccgtt aaacgactat ggcttttata caacgacggg aatagggtac 2580
caaccttaca gagtagtagt actaagtttc gagctattaa atgcgccggc caccgtatgt 2640
gggcccaagc tatcgacgga cctaatcaag aatcagcacc accaccatca ccac 2694
<210> 18
<211> 2694
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence of P-B-PgGX-S construct
<220>
<221> misc_feature
<222> (1)..(2694)
<400> 18
atgatccaca gcgtgttcct gctgatgttc ctgctcaccc ccacagagag cagagtgcag 60
cctaccgagt ccatcgtgcg gttccccaac atcaccaatc tgtgcccttt cggcgaggtg 120
ttcaacgcca ccagattcgc ctctgtgtac gcctggaacc ggaagcggat cagcaattgc 180
gtggccgact acagcgtgct gtacaacagc gccagcttca gcaccttcaa gtgctacggc 240
gtgtccccta ccaagctgaa cgacctgtgc ttcaccaacg tgtacgccga cagcttcgtg 300
atcagaggcg acgaagtgcg gcagattgcc cctggacaga caggcaagat cgccgattac 360
aactacaagc tgcccgacga cttcaccggc tgtgtgattg cctggaacag caacaacctg 420
gacagcaaag tcggcggcaa ctacaactac ctgtaccggc tgttccggaa gtccaacctg 480
aagcctttcg agcgggacat cagcaccgag atctatcagg ccggcagcac cccttgcaat 540
ggcgtggaag gcttcaactg ctacttccca ctgcagtcct acggcttcca gcctacaaac 600
ggcgtgggct accagcctta cagagtggtg gtgctgagct tcgagctgct gcatgctcct 660
gccacagtgt gcggccctaa gaaaagcacc aacctggtca agaacaagcg ggtgcagccc 720
actgagagca ttgtgcgctt tccaaatatc acgaatctct gcccctttgg ggaagtcttt 780
aatgccacac gcttcgccag cgtctacgct tggaatagga agagaatctc caactgcgtg 840
gcagattact ccgtcctgta caattccgcc agcttttcca cgtttaagtg ttatggcgtc 900
agccccacaa agctcaatga tctctgcttt accaatgtct atgccgactc ctttgtgatt 960
cgcggagatg aagtccgcca gatcgctcca ggacagaccg gaaacattgc cgactataat 1020
tacaagctcc cggatgattt cacgggatgc gtgatcgctt ggaactccaa caatctggat 1080
tccaaagttg gcgggaatta caattatctc tatcgcctct tccgcaagag caatctgaag 1140
ccctttgaga gagacatctc tacagaaatc taccaggctg gctctacccc atgtaacggc 1200
gtgaagggct ttaattgcta ttttcccctg caatcttacg ggtttcagcc cacctatggc 1260
gtgggatatc aaccctatcg cgtggtggtc ctgtcttttg aactgctgca cgccccagct 1320
accgtgtgtg gcccaaaaaa gtccaccaat ctcgtgaaga acaaaagagt gcagcccacc 1380
atcagcatcg tgagattccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 1440
gctagcaagt tcgctagcgt gtacgcctgg aaccggaaga gaatcagcaa ctgcgtggcc 1500
gactacagcg tgctgtacaa cagcacaagc ttcagcacct tcaagtgtta cggcgtgagc 1560
cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtggtgaag 1620
ggcgacgagg tgagacagat cgcccccggg cagaccggcg tgatcgccga ctacaactac 1680
aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcgtgaa gcaagacgcc 1740
ctgaccgggg gcaactacgg ctacctgtac agactgttca gaaagagcaa gctgaagccc 1800
ttcgagagag acatcagcac cgagatctac caagccggca gcaccccctg caacggccaa 1860
gtgggcctga actgctacta ccccctggag agatacggct tccaccccac caccggcgtg 1920
aactatcagc ccttcagagt ggtcgtgctg agcttcgagc tgctgaacgg ccccgccacc 1980
gtgtgcggcc ccaagctgag caccaccctg gtgaaggaca agcgtgttgt cccatccggt 2040
gacgttgtcc ggtttcctaa catcacaaac ttgtgtccct ttggcgaagt cttcaatgct 2100
accaaatttc ccagcgtcta cgcgtgggaa agaaagaaaa tatcaaattg tgttgccgac 2160
tattccgtcc tatataatag cacgttcttc tcgacgttca agtgttatgg tgtctctgct 2220
acgaaactta acgacttatg tttctcaaac gtgtacgcag attctttcgt agttaaaggt 2280
gatgatgtga ggcagattgc gcccggacaa acaggagtaa tcgccgatta caactacaaa 2340
ctcccggacg actttatggg gtgtgtgtta gcttggaata cgaggaatat agacgccacg 2400
agtaccggga attataatta taagtatcgc tatctccgac atggcaaact caggccattt 2460
gaacgcgaca ttagcaatgt tccattctct ccggacggca aaccgtgcac tccaccggct 2520
ttaaattgtt attggccgtt aaacgactat ggcttttata caacgacggg aatagggtac 2580
caaccttaca gagtagtagt actaagtttc gagctattaa atgcgccggc caccgtatgt 2640
gggcccaagc tatcgacgga cctaatcaag aatcagcacc accaccatca ccac 2694
<210> 19
<211> 2691
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence of P-B-LY-S construct
<220>
<221> misc_feature
<222> (1)..(2691)
<400> 19
atgatccaca gcgtgttcct gctgatgttc ctgctcaccc ccacagagag cagagtgcag 60
cctaccgagt ccatcgtgcg gttccccaac atcaccaatc tgtgcccttt cggcgaggtg 120
ttcaacgcca ccagattcgc ctctgtgtac gcctggaacc ggaagcggat cagcaattgc 180
gtggccgact acagcgtgct gtacaacagc gccagcttca gcaccttcaa gtgctacggc 240
gtgtccccta ccaagctgaa cgacctgtgc ttcaccaacg tgtacgccga cagcttcgtg 300
atcagaggcg acgaagtgcg gcagattgcc cctggacaga caggcaagat cgccgattac 360
aactacaagc tgcccgacga cttcaccggc tgtgtgattg cctggaacag caacaacctg 420
gacagcaaag tcggcggcaa ctacaactac ctgtaccggc tgttccggaa gtccaacctg 480
aagcctttcg agcgggacat cagcaccgag atctatcagg ccggcagcac cccttgcaat 540
ggcgtggaag gcttcaactg ctacttccca ctgcagtcct acggcttcca gcctacaaac 600
ggcgtgggct accagcctta cagagtggtg gtgctgagct tcgagctgct gcatgctcct 660
gccacagtgt gcggccctaa gaaaagcacc aacctggtca agaacaagcg ggtgcagccc 720
actgagagca ttgtgcgctt tccaaatatc acgaatctct gcccctttgg ggaagtcttt 780
aatgccacac gcttcgccag cgtctacgct tggaatagga agagaatctc caactgcgtg 840
gcagattact ccgtcctgta caattccgcc agcttttcca cgtttaagtg ttatggcgtc 900
agccccacaa agctcaatga tctctgcttt accaatgtct atgccgactc ctttgtgatt 960
cgcggagatg aagtccgcca gatcgctcca ggacagaccg gaaacattgc cgactataat 1020
tacaagctcc cggatgattt cacgggatgc gtgatcgctt ggaactccaa caatctggat 1080
tccaaagttg gcgggaatta caattatctc tatcgcctct tccgcaagag caatctgaag 1140
ccctttgaga gagacatctc tacagaaatc taccaggctg gctctacccc atgtaacggc 1200
gtgaagggct ttaattgcta ttttcccctg caatcttacg ggtttcagcc cacctatggc 1260
gtgggatatc aaccctatcg cgtggtggtc ctgtcttttg aactgctgca cgccccagct 1320
accgtgtgtg gcccaaaaaa gtccaccaat ctcgtgaaga acaaaagagt gagccctagc 1380
aaagaggtgg tgagattccc caacatcacc aatctgtgcc ccttcggcga ggtgttcaac 1440
gccaccacct tccctagcgt gtacgcctgg gagagaaaga gaatcagcaa ctgcgtggcc 1500
gactacagcg tgctgtacaa cagcacaagc ttcagcacct tcaagtgcta cggcgtgagc 1560
gccatcaagc tgaacgacct gtgcttcagc aacgtgtacg ccgacagctt cgtggtgaag 1620
ggcgacgacg tgagacagat cgcccccggg cagaccggcg tgatcgccga ctacaactac 1680
aagctgcccg acgacttcat gggctgcgtg ctggcctgga acacaagaaa catcgacgcc 1740
acaagcagcg gcaacttcaa ctacaagtac agaagcctga gacacggcaa gctgagaccc 1800
ttcgagagag acatcagcaa cgtgcccttc agccccgacg gcaagccctg cacccccccc 1860
gccttcaact gttactggcc cctgaacgac tacggcttct acaccaccaa cggcattggc 1920
taccaaccct acagagtggt cgtgctgagc ttcgagctgc tgaacgctcc cgccaccgtc 1980
tgcggcccca agctgagcac cgacctgatc accaatcagc gtgttgtccc atccggtgac 2040
gttgtccggt ttcctaacat cacaaacttg tgtccctttg gcgaagtctt caatgctacc 2100
aaatttccca gcgtctacgc gtgggaaaga aagaaaatat caaattgtgt tgccgactat 2160
tccgtcctat ataatagcac gttcttctcg acgttcaagt gttatggtgt ctctgctacg 2220
aaacttaacg acttatgttt ctcaaacgtg tacgcagatt ctttcgtagt taaaggtgat 2280
gatgtgaggc agattgcgcc cggacaaaca ggagtaatcg ccgattacaa ctacaaactc 2340
ccggacgact ttatggggtg tgtgttagct tggaatacga ggaatataga cgccacgagt 2400
accgggaatt ataattataa gtatcgctat ctccgacatg gcaaactcag gccatttgaa 2460
cgcgacatta gcaatgttcc attctctccg gacggcaaac cgtgcactcc accggcttta 2520
aattgttatt ggccgttaaa cgactatggc ttttatacaa cgacgggaat agggtaccaa 2580
ccttacagag tagtagtact aagtttcgag ctattaaatg cgccggccac cgtatgtggg 2640
cccaagctat cgacggacct aatcaagaat cagcaccacc accatcacca c 2691
<210> 20
<211> 2691
<212> DNA
<213> artificial sequence
<220>
<223> nucleotide sequence of P-B-SHC-S construct
<220>
<221> misc_feature
<222> (1)..(2691)
<400> 20
atgatccaca gcgtgttcct gctgatgttc ctgctcaccc ccacagagag cagagtgcag 60
cctaccgagt ccatcgtgcg gttccccaac atcaccaatc tgtgcccttt cggcgaggtg 120
ttcaacgcca ccagattcgc ctctgtgtac gcctggaacc ggaagcggat cagcaattgc 180
gtggccgact acagcgtgct gtacaacagc gccagcttca gcaccttcaa gtgctacggc 240
gtgtccccta ccaagctgaa cgacctgtgc ttcaccaacg tgtacgccga cagcttcgtg 300
atcagaggcg acgaagtgcg gcagattgcc cctggacaga caggcaagat cgccgattac 360
aactacaagc tgcccgacga cttcaccggc tgtgtgattg cctggaacag caacaacctg 420
gacagcaaag tcggcggcaa ctacaactac ctgtaccggc tgttccggaa gtccaacctg 480
aagcctttcg agcgggacat cagcaccgag atctatcagg ccggcagcac cccttgcaat 540
ggcgtggaag gcttcaactg ctacttccca ctgcagtcct acggcttcca gcctacaaac 600
ggcgtgggct accagcctta cagagtggtg gtgctgagct tcgagctgct gcatgctcct 660
gccacagtgt gcggccctaa gaaaagcacc aacctggtca agaacaagcg ggtgcagccc 720
actgagagca ttgtgcgctt tccaaatatc acgaatctct gcccctttgg ggaagtcttt 780
aatgccacac gcttcgccag cgtctacgct tggaatagga agagaatctc caactgcgtg 840
gcagattact ccgtcctgta caattccgcc agcttttcca cgtttaagtg ttatggcgtc 900
agccccacaa agctcaatga tctctgcttt accaatgtct atgccgactc ctttgtgatt 960
cgcggagatg aagtccgcca gatcgctcca ggacagaccg gaaacattgc cgactataat 1020
tacaagctcc cggatgattt cacgggatgc gtgatcgctt ggaactccaa caatctggat 1080
tccaaagttg gcgggaatta caattatctc tatcgcctct tccgcaagag caatctgaag 1140
ccctttgaga gagacatctc tacagaaatc taccaggctg gctctacccc atgtaacggc 1200
gtgaagggct ttaattgcta ttttcccctg caatcttacg ggtttcagcc cacctatggc 1260
gtgggatatc aaccctatcg cgtggtggtc ctgtcttttg aactgctgca cgccccagct 1320
accgtgtgtg gcccaaaaaa gtccaccaat ctcgtgaaga acaaaagagt ggcccctagc 1380
aaagaagtgg tgcggttccc caacatcacc aacctgtgcc cttttgggga agtgttcaac 1440
gccaccacct tcccctccgt ttacgcctgg gaaagaaaga gaatctctaa ctgtgtggct 1500
gattactctg tgctgtataa cagcaccagc ttttctacat tcaagtgtta cggcgtgagt 1560
gccacaaagc tgaatgatct gtgcttcagc aatgtgtatg ccgacagctt cgtggtgaag 1620
ggagatgacg tgcggcagat cgcccctgga cagactggcg tgatcgccga ttacaactac 1680
aagctgcctg acgactttct gggctgtgtg ctggcctgga acaccaacag caaggacagc 1740
agcacaagcg gaaactacaa ttacctgtac cggtgggtca gaagaagcaa gctgaacccc 1800
tacgagcgcg acctgagcaa cgacatctac agccctggcg gccagtcctg cagcgccgtg 1860
ggccctaact gctacaaccc tctgagaccc tacggcttct tcaccaccgc cggcgtgggc 1920
caccagcctt acagagtggt ggtgctgagc ttcgagctgc tcaacgcccc tgccaccgtg 1980
tgcggcccca agttgtctac agacctgatc aagaaccagc gtgttgtccc atccggtgac 2040
gttgtccggt ttcctaacat cacaaacttg tgtccctttg gcgaagtctt caatgctacc 2100
aaatttccca gcgtctacgc gtgggaaaga aagaaaatat caaattgtgt tgccgactat 2160
tccgtcctat ataatagcac gttcttctcg acgttcaagt gttatggtgt ctctgctacg 2220
aaacttaacg acttatgttt ctcaaacgtg tacgcagatt ctttcgtagt taaaggtgat 2280
gatgtgaggc agattgcgcc cggacaaaca ggagtaatcg ccgattacaa ctacaaactc 2340
ccggacgact ttatggggtg tgtgttagct tggaatacga ggaatataga cgccacgagt 2400
accgggaatt ataattataa gtatcgctat ctccgacatg gcaaactcag gccatttgaa 2460
cgcgacatta gcaatgttcc attctctccg gacggcaaac cgtgcactcc accggcttta 2520
aattgttatt ggccgttaaa cgactatggc ttttatacaa cgacgggaat agggtaccaa 2580
ccttacagag tagtagtact aagtttcgag ctattaaatg cgccggccac cgtatgtggg 2640
cccaagctat cgacggacct aatcaagaat cagcaccacc accatcacca c 2691
<210> 21
<211> 1265
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of pseudovirus S protein of coronavirus SARS-CoV-2 prototype strain
<220>
<221> DOMAIN
<222> (1)..(1265)
<400> 21
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn
1010 1015 1020
Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys
1025 1030 1035
Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1040 1045 1050
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1055 1060 1065
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His
1070 1075 1080
Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn
1085 1090 1095
Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln
1100 1105 1110
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1115 1120 1125
Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1130 1135 1140
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1145 1150 1155
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu
1175 1180 1185
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu
1205 1210 1215
Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met
1220 1225 1230
Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys
1235 1240 1245
Ser Cys Gly Ser Cys Cys Lys Asp Tyr Lys Asp Asp Asp Asp Lys
1250 1255 1260
Gly Pro
1265
<210> 22
<211> 1265
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of S protein of coronavirus SARS-CoV-2 variant strain Beta pseudovirus
<220>
<221> DOMAIN
<222> (1)..(1265)
<400> 22
Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val
1 5 10 15
Arg Ala Val Asn Phe Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr
20 25 30
Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser
35 40 45
Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn
50 55 60
Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys
65 70 75 80
Arg Phe Ala Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala
85 90 95
Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr
100 105 110
Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn
115 120 125
Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu
130 135 140
Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe
145 150 155 160
Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln
165 170 175
Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu
180 185 190
Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser
195 200 205
Lys His Thr Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe Ser
210 215 220
Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg
225 230 235 240
Phe Gln Thr Leu His Ile Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Val Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn
1010 1015 1020
Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys
1025 1030 1035
Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1040 1045 1050
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1055 1060 1065
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His
1070 1075 1080
Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn
1085 1090 1095
Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln
1100 1105 1110
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1115 1120 1125
Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1130 1135 1140
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1145 1150 1155
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu
1175 1180 1185
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu
1205 1210 1215
Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met
1220 1225 1230
Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys
1235 1240 1245
Ser Cys Gly Ser Cys Cys Lys Asp Tyr Lys Asp Asp Asp Asp Lys
1250 1255 1260
Gly Pro
1265
<210> 23
<211> 1263
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of S protein of SARS-CoV-2 mutant strain Delta pseudovirus
<220>
<221> DOMAIN
<222> (1)..(1263)
<400> 23
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Arg Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Gly Val Tyr Ser Ser
145 150 155 160
Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp
165 170 175
Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe
180 185 190
Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile
195 200 205
Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Val Leu Glu Pro Leu
210 215 220
Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu
225 230 235 240
Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp
245 250 255
Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr
260 265 270
Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp
275 280 285
Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe
290 295 300
Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro
305 310 315 320
Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe
325 330 335
Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn
340 345 350
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
355 360 365
Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys
370 375 380
Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile
385 390 395 400
Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile
405 410 415
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile
420 425 430
Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn
435 440 445
Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
450 455 460
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly
465 470 475 480
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
485 490 495
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
500 505 510
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
515 520 525
Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu
530 535 540
Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe
545 550 555 560
Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp
565 570 575
Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly
580 585 590
Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val
595 600 605
Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala
610 615 620
Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val
625 630 635 640
Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn
645 650 655
Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr
660 665 670
Gln Thr Gln Thr Asn Ser Arg Arg Arg Ala Arg Ser Val Ala Ser Gln
675 680 685
Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala
690 695 700
Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val
705 710 715 720
Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys
725 730 735
Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu
740 745 750
Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile
755 760 765
Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys
770 775 780
Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe
785 790 795 800
Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile
805 810 815
Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile
820 825 830
Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile
835 840 845
Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr
850 855 860
Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile
865 870 875 880
Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe
885 890 895
Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn
900 905 910
Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala
915 920 925
Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly
930 935 940
Lys Leu Gln Asn Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu
945 950 955 960
Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn
965 970 975
Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp
980 985 990
Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln
995 1000 1005
Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala
1010 1015 1020
Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val
1025 1030 1035
Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser
1040 1045 1050
Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala
1055 1060 1065
Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly
1070 1075 1080
Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr
1085 1090 1095
His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile
1100 1105 1110
Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile
1115 1120 1125
Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu
1130 1135 1140
Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr
1145 1150 1155
Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser
1160 1165 1170
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala
1175 1180 1185
Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys
1190 1195 1200
Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe
1205 1210 1215
Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys
1220 1225 1230
Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys
1235 1240 1245
Gly Ser Cys Cys Lys Asp Tyr Lys Asp Asp Asp Asp Lys Gly Pro
1250 1255 1260
<210> 24
<211> 1262
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of S protein of SARS-CoV-2 mutant strain Omicron pseudovirus
<220>
<221> DOMAIN
<222> (1)..(1262)
<400> 24
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Val Ile Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro
65 70 75 80
Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Ile Glu Lys Ser
85 90 95
Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr
100 105 110
Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val
115 120 125
Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp His Lys Asn Asn
130 135 140
Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn
145 150 155 160
Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly
165 170 175
Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile
180 185 190
Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Ile Val Arg
195 200 205
Glu Pro Glu Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val
210 215 220
Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala
225 230 235 240
Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr
245 250 255
Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe
260 265 270
Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys
275 280 285
Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr
290 295 300
Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr
305 310 315 320
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp
325 330 335
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
340 345 350
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu
355 360 365
Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
370 375 380
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
385 390 395 400
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
405 410 415
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
420 425 430
Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr
435 440 445
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
450 455 460
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val
465 470 475 480
Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro
485 490 495
Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe
500 505 510
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
515 520 525
Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Lys
530 535 540
Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln
545 550 555 560
Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro
565 570 575
Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val
580 585 590
Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu
595 600 605
Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp
610 615 620
Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe
625 630 635 640
Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu Tyr Val Asn Asn Ser
645 650 655
Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln
660 665 670
Thr Gln Thr Lys Ser His Arg Arg Ala Arg Ser Val Ala Ser Gln Ser
675 680 685
Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr
690 695 700
Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr
705 710 715 720
Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr
725 730 735
Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln
740 745 750
Tyr Gly Ser Phe Cys Thr Gln Leu Lys Arg Ala Leu Thr Gly Ile Ala
755 760 765
Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln
770 775 780
Ile Tyr Lys Thr Pro Pro Ile Lys Tyr Phe Gly Gly Phe Asn Phe Ser
785 790 795 800
Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu
805 810 815
Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys
820 825 830
Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys
835 840 845
Ala Gln Lys Phe Lys Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp
850 855 860
Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr
865 870 875 880
Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala
885 890 895
Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val
900 905 910
Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile
915 920 925
Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys
930 935 940
Leu Gln Asp Val Val Asn His Asn Ala Gln Ala Leu Asn Thr Leu Val
945 950 955 960
Lys Gln Leu Ser Ser Lys Phe Gly Ala Ile Ser Ser Val Leu Asn Asp
965 970 975
Ile Phe Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg
980 985 990
Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln
995 1000 1005
Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala
1010 1015 1020
Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp
1025 1030 1035
Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala
1040 1045 1050
Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln
1055 1060 1065
Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys
1070 1075 1080
Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His
1085 1090 1095
Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr
1100 1105 1110
Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly
1115 1120 1125
Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp
1130 1135 1140
Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser
1145 1150 1155
Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val
1160 1165 1170
Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
1175 1180 1185
Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr
1190 1195 1200
Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile
1205 1210 1215
Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys
1220 1225 1230
Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly
1235 1240 1245
Ser Cys Cys Lys Asp Tyr Lys Asp Asp Asp Asp Lys Gly Pro
1250 1255 1260
<210> 25
<211> 1257
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of S protein of RaTG13 pseudovirus coronavirus
<220>
<221> DOMAIN
<222> (1)..(1257)
<400> 25
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Ser
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Leu Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Ile Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Pro Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Asp Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Thr Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Lys His Ile Asp Ala Lys Glu Gly Gly Asn
435 440 445
Phe Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ala Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys
465 470 475 480
Asn Gly Gln Thr Gly Leu Asn Cys Tyr Tyr Pro Leu Tyr Arg Tyr Gly
485 490 495
Phe Tyr Pro Thr Asp Gly Val Gly His Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala Ser Gln Ser Ile
675 680 685
Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser
690 695 700
Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr
705 710 715 720
Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met
725 730 735
Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr
740 745 750
Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val
755 760 765
Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile
770 775 780
Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln
785 790 795 800
Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp
805 810 815
Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln
820 825 830
Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala
835 840 845
Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu
850 855 860
Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser
865 870 875 880
Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met
885 890 895
Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu
900 905 910
Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly
915 920 925
Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu
930 935 940
Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys
945 950 955 960
Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile
965 970 975
Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu
980 985 990
Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu
995 1000 1005
Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr
1010 1015 1020
Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe
1025 1030 1035
Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro
1040 1045 1050
His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu
1055 1060 1065
Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala
1070 1075 1080
His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp
1085 1090 1095
Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr
1100 1105 1110
Asp Asn Thr Phe Val Ser Gly Ser Cys Asp Val Val Ile Gly Ile
1115 1120 1125
Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser
1130 1135 1140
Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro
1145 1150 1155
Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val
1160 1165 1170
Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn
1175 1180 1185
Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu
1190 1195 1200
Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala
1205 1210 1215
Gly Leu Ile Ala Ile Ile Met Val Thr Ile Met Leu Cys Cys Met
1220 1225 1230
Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser
1235 1240 1245
Cys Cys Lys His His His His His His
1250 1255
<210> 26
<211> 1253
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of S protein of Panglin GD/1 pseudovirus of coronavirus
<220>
<221> DOMAIN
<222> (1)..(1253)
<400> 26
Met Leu Phe Phe Phe Phe Leu His Phe Ala Leu Val Asn Ser Gln Cys
1 5 10 15
Val Asn Leu Thr Gly Arg Ala Ala Ile Gln Pro Ser Phe Thr Asn Ser
20 25 30
Ser Gln Arg Gly Val Tyr Tyr Pro Asp Thr Ile Phe Arg Ser Asn Thr
35 40 45
Leu Val Leu Ser Gln Gly Tyr Phe Leu Pro Phe Tyr Ser Asn Val Ser
50 55 60
Trp Tyr Tyr Ala Leu Thr Lys Thr Asn Ser Ala Glu Lys Arg Val Asp
65 70 75 80
Asn Pro Val Leu Asp Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu
85 90 95
Lys Ser Asn Ile Val Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Asn
100 105 110
Thr Ser Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Ile Ile
115 120 125
Lys Val Cys Asn Phe Gln Phe Cys Tyr Asp Pro Tyr Leu Ser Gly Tyr
130 135 140
Tyr His Asn Asn Lys Thr Trp Ser Thr Arg Glu Phe Ala Val Tyr Ser
145 150 155 160
Ser Tyr Ala Asn Cys Thr Phe Glu Tyr Val Ser Lys Ser Phe Met Leu
165 170 175
Asp Ile Ala Gly Lys Ser Gly Leu Phe Asp Thr Leu Arg Glu Phe Val
180 185 190
Phe Arg Asn Val Asp Gly Tyr Phe Lys Ile Tyr Ser Lys Tyr Thr Pro
195 200 205
Val Asn Val Asn Ser Asn Leu Pro Ile Gly Phe Ser Ala Leu Glu Pro
210 215 220
Leu Val Glu Ile Pro Ala Gly Ile Asn Ile Thr Lys Phe Arg Thr Leu
225 230 235 240
Leu Thr Ile His Arg Gly Asp Pro Met Pro Asn Asn Gly Trp Thr Val
245 250 255
Phe Ser Ala Ala Tyr Tyr Val Gly Tyr Leu Ala Pro Arg Thr Phe Met
260 265 270
Leu Asn Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala
275 280 285
Leu Asp Pro Leu Ser Glu Ala Lys Cys Thr Leu Lys Ser Leu Thr Val
290 295 300
Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu
305 310 315 320
Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu
325 330 335
Val Phe Asn Ala Thr Thr Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys
340 345 350
Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr
355 360 365
Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn
370 375 380
Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Val Arg Gly
385 390 395 400
Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Arg Ile Ala Asp
405 410 415
Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp
420 425 430
Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu
435 440 445
Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile
450 455 460
Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu
465 470 475 480
Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe His Pro Thr
485 490 495
Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu
500 505 510
Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro Lys Gln Ser Thr Asn
515 520 525
Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly
530 535 540
Thr Gly Val Leu Thr Glu Ser Ser Lys Lys Phe Leu Pro Phe Gln Gln
545 550 555 560
Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln
565 570 575
Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser
580 585 590
Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr
595 600 605
Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln
610 615 620
Leu Thr Pro Thr Trp Ser Val Tyr Ser Thr Gly Ser Asn Val Phe Gln
625 630 635 640
Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr
645 650 655
Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr
660 665 670
Gln Thr Asn Ser Arg Ser Val Ser Ser Gln Ala Ile Ile Ala Tyr Thr
675 680 685
Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ala Asn Asn Ser Ile
690 695 700
Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro
705 710 715 720
Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly
725 730 735
Asp Ser Ile Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys
740 745 750
Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys
755 760 765
Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro
770 775 780
Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp
785 790 795 800
Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn
805 810 815
Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys
820 825 830
Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn
835 840 845
Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln
850 855 860
Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe
865 870 875 880
Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr
885 890 895
Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln
900 905 910
Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp
915 920 925
Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val
930 935 940
Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser
945 950 955 960
Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu
965 970 975
Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg
980 985 990
Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala
995 1000 1005
Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu
1010 1015 1020
Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly
1025 1030 1035
Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val
1040 1045 1050
Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Lys Asn Phe Thr
1055 1060 1065
Thr Thr Pro Ala Ile Cys His Glu Gly Lys Ala His Phe Pro Arg
1070 1075 1080
Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln
1085 1090 1095
Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe
1100 1105 1110
Val Ser Gly Ser Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr
1115 1120 1125
Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu
1130 1135 1140
Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu
1145 1150 1155
Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys
1160 1165 1170
Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser
1175 1180 1185
Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys
1190 1195 1200
Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala
1205 1210 1215
Ile Ile Met Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys
1220 1225 1230
Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys His
1235 1240 1245
His His His His His
1250
<210> 27
<211> 1247
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of S protein of coronavirus LYRa11 pseudovirus
<220>
<221> DOMAIN
<222> (1)..(1247)
<400> 27
Met Phe Leu Thr Cys Phe Ile Leu Ser Phe Ser Leu Phe Cys Val Ser
1 5 10 15
Gly Asp Ser Ile Asp Thr Cys Glu Thr Phe Asp Asp Val Ser Pro Pro
20 25 30
Gln Gln Asn Leu Val Ser Ser Ser Lys Arg Gly Val Tyr Tyr Pro Asp
35 40 45
Asp Ile Tyr Arg Ser Asp Val His His Leu Val Gln Asp Leu Phe Leu
50 55 60
Pro Phe Asn Ser Asn Val Val Gly Leu Met Ser Phe Asn Tyr Arg Phe
65 70 75 80
Asp Asn Pro Ile Ile Pro Phe Lys Asp Gly Val Tyr Phe Ala Ala Thr
85 90 95
Glu Lys Ser Asn Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn
100 105 110
Asn Lys Ser Gln Ser Val Ile Ile Met Asn Asn Ser Thr Asn Val Val
115 120 125
Ile Arg Ala Cys Asn Phe Gln Leu Cys Asp Asn Pro Phe Phe Ala Val
130 135 140
Ile Arg Pro Thr Ser Gln Gln Ile Glu Thr Ile Leu Phe Glu Asn Ala
145 150 155 160
Phe Asn Cys Thr Phe Glu Tyr Val Ser Asp Ser Phe Leu Met Asp Val
165 170 175
Gly Glu Lys Pro Gly Asn Phe Lys His Leu Arg Glu Phe Ile Phe Lys
180 185 190
Asn Lys Asp Gly Phe Leu Asn Ile Tyr Ser Gly Phe Gln Asn Ile Asp
195 200 205
Val Ala Asn Gly Leu Pro Ser Gly Phe Ser Leu Leu Lys Pro Leu Leu
210 215 220
Lys Leu Pro Leu Gly Leu Asn Ile Thr Asn Phe Arg Val Leu Leu Thr
225 230 235 240
Ala Phe Ile Pro Asn Ile Gly Thr Trp Gly Thr Ser Pro Val Ala Tyr
245 250 255
Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Tyr
260 265 270
Asn Gly Thr Ile Val Asp Ala Val Asp Cys Ser Gln His Pro Leu Ala
275 280 285
Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr
290 295 300
Gln Thr Ser Asn Phe Arg Val Ser Pro Ser Lys Glu Val Val Arg Phe
305 310 315 320
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
325 330 335
Thr Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys
340 345 350
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe
355 360 365
Lys Cys Tyr Gly Val Ser Ala Ile Lys Leu Asn Asp Leu Cys Phe Ser
370 375 380
Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln
385 390 395 400
Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu
405 410 415
Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile
420 425 430
Asp Ala Thr Ser Ser Gly Asn Phe Asn Tyr Lys Tyr Arg Ser Leu Arg
435 440 445
His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe
450 455 460
Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Phe Asn Cys Tyr Trp
465 470 475 480
Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Asn Gly Ile Gly Tyr Gln
485 490 495
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala
500 505 510
Thr Val Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Thr Asn Gln Cys
515 520 525
Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro
530 535 540
Ser Leu Lys Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Phe Ser
545 550 555 560
Asp Phe Thr Asp Ser Val Arg Asp Pro Lys Thr Leu Glu Val Leu Asp
565 570 575
Ile Ser Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr
580 585 590
Asn Ile Ser Ser Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr
595 600 605
Asp Val Pro Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg
610 615 620
Ile Tyr Ser Ala Gly Val Asn Val Phe Gln Thr Gln Ala Gly Cys Leu
625 630 635 640
Ile Gly Ala Glu His Val Asn Ala Ser Tyr Glu Cys Asp Ile Pro Ile
645 650 655
Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Ala Ser Leu Leu Arg Asn
660 665 670
Thr Asp Gln Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Glu
675 680 685
Asn Ser Ile Ala Tyr Ala Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe
690 695 700
Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr
705 710 715 720
Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Gln Glu Cys Ser
725 730 735
Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala
740 745 750
Leu Ser Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe
755 760 765
Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Ala Ile Lys Asp Phe Gly
770 775 780
Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Thr Lys
785 790 795 800
Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp
805 810 815
Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Ser Ala
820 825 830
Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro
835 840 845
Pro Leu Leu Thr Asp Glu Met Ile Ala Ala Tyr Thr Ala Ala Leu Val
850 855 860
Ser Gly Thr Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu
865 870 875 880
Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly
885 890 895
Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln
900 905 910
Phe Asn Lys Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser
915 920 925
Thr Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala
930 935 940
Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser
945 950 955 960
Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu
965 970 975
Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr
980 985 990
Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala
995 1000 1005
Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser
1010 1015 1020
Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe
1025 1030 1035
Pro Gln Ala Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr
1040 1045 1050
Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys
1055 1060 1065
His Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Ser
1070 1075 1080
Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro
1085 1090 1095
Gln Leu Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp
1100 1105 1110
Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln
1115 1120 1125
Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys
1130 1135 1140
Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Arg Ile
1145 1150 1155
Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn
1160 1165 1170
Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu
1175 1180 1185
Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp
1190 1195 1200
Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Ala Thr Ile
1205 1210 1215
Leu Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala
1220 1225 1230
Cys Ser Cys Gly Ser Cys Cys Lys His His His His His His
1235 1240 1245
<210> 28
<211> 1246
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of S protein of coronavirus WIV1 pseudovirus
<220>
<221> DOMAIN
<222> (1)..(1246)
<400> 28
Met Lys Leu Leu Val Leu Val Phe Ala Thr Leu Val Ser Ser Tyr Thr
1 5 10 15
Ile Glu Lys Cys Leu Asp Phe Asp Asp Arg Thr Pro Pro Ala Asn Thr
20 25 30
Gln Phe Leu Ser Ser His Arg Gly Val Tyr Tyr Pro Asp Asp Ile Phe
35 40 45
Arg Ser Asn Val Leu His Leu Val Gln Asp His Phe Leu Pro Phe Asp
50 55 60
Ser Asn Val Thr Arg Phe Ile Thr Phe Gly Leu Asn Phe Asp Asn Pro
65 70 75 80
Ile Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser
85 90 95
Asn Val Ile Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser
100 105 110
Gln Ser Val Ile Ile Met Asn Asn Ser Thr Asn Leu Val Ile Arg Ala
115 120 125
Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Val Val Leu Lys Ser
130 135 140
Asn Asn Thr Gln Ile Pro Ser Tyr Ile Phe Asn Asn Ala Phe Asn Cys
145 150 155 160
Thr Phe Glu Tyr Val Ser Lys Asp Phe Asn Leu Asp Leu Gly Glu Lys
165 170 175
Pro Gly Asn Phe Lys Asp Leu Arg Glu Phe Val Phe Arg Asn Lys Asp
180 185 190
Gly Phe Leu His Val Tyr Ser Gly Tyr Gln Pro Ile Ser Ala Ala Ser
195 200 205
Gly Leu Pro Thr Gly Phe Asn Ala Leu Lys Pro Ile Phe Lys Leu Pro
210 215 220
Leu Gly Ile Asn Ile Thr Asn Phe Arg Thr Leu Leu Thr Ala Phe Pro
225 230 235 240
Pro Arg Pro Asp Tyr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly
245 250 255
Tyr Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys
275 280 285
Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Ala Pro Ser Lys Glu Val Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Pro
325 330 335
Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr
420 425 430
Gln Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Ser Leu Arg His Gly Lys
435 440 445
Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp
450 455 460
Gly Lys Pro Cys Thr Pro Pro Ala Phe Asn Cys Tyr Trp Pro Leu Asn
465 470 475 480
Asp Tyr Gly Phe Tyr Ile Thr Asn Gly Ile Gly Tyr Gln Pro Tyr Arg
485 490 495
Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys
500 505 510
Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe
515 520 525
Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys
530 535 540
Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr
545 550 555 560
Asp Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro
565 570 575
Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser
580 585 590
Ser Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Pro
595 600 605
Val Ala Ile His Ala Asp Gln Leu Thr Pro Ser Trp Arg Val His Ser
610 615 620
Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala
625 630 635 640
Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly
645 650 655
Ile Cys Ala Ser Tyr His Thr Val Ser Ser Leu Arg Ser Thr Ser Gln
660 665 670
Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile
675 680 685
Ala Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser
690 695 700
Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp
705 710 715 720
Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu
725 730 735
Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly
740 745 750
Ile Ala Val Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val
755 760 765
Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys Asp Phe Gly Gly Phe Asn
770 775 780
Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe
785 790 795 800
Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe
805 810 815
Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu
820 825 830
Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu
835 840 845
Thr Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr
850 855 860
Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro
865 870 875 880
Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln
885 890 895
Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys
900 905 910
Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu
915 920 925
Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr
930 935 940
Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu
945 950 955 960
Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile
965 970 975
Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr
980 985 990
Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala
995 1000 1005
Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val
1010 1015 1020
Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala
1025 1030 1035
Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser
1040 1045 1050
Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly
1055 1060 1065
Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr
1070 1075 1080
Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile
1085 1090 1095
Thr Thr Asp Asn Thr Phe Val Ser Gly Ser Cys Asp Val Val Ile
1100 1105 1110
Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu
1115 1120 1125
Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr
1130 1135 1140
Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser
1145 1150 1155
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala
1160 1165 1170
Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys
1175 1180 1185
Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe
1190 1195 1200
Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys
1205 1210 1215
Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys
1220 1225 1230
Gly Ser Cys Cys Lys Asp Tyr Lys Asp Asp Asp Asp Lys
1235 1240 1245
<210> 29
<211> 1246
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of S protein of coronavirus SHC014 pseudovirus
<220>
<221> DOMAIN
<222> (1)..(1246)
<400> 29
Met Lys Leu Leu Val Leu Val Phe Ala Thr Leu Val Ser Ser Tyr Thr
1 5 10 15
Ile Glu Lys Cys Leu Asp Phe Asp Asp Arg Thr Pro Pro Ala Asn Thr
20 25 30
Gln Phe Leu Ser Ser His Arg Gly Val Tyr Tyr Pro Asp Asp Ile Phe
35 40 45
Arg Ser Asn Val Leu His Leu Val Gln Asp His Phe Leu Pro Phe Asp
50 55 60
Ser Asn Val Thr Arg Phe Ile Thr Phe Gly Leu Asn Phe Asp Asn Pro
65 70 75 80
Ile Ile Pro Phe Arg Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser
85 90 95
Asn Val Ile Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser
100 105 110
Gln Ser Val Ile Ile Met Asn Asn Ser Thr Asn Leu Val Ile Arg Ala
115 120 125
Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Val Val Leu Lys Ser
130 135 140
Asn Asn Thr Gln Ile Pro Ser Tyr Ile Phe Asn Asn Ala Phe Asn Cys
145 150 155 160
Thr Phe Glu Tyr Val Ser Lys Asp Phe Asn Leu Asp Leu Gly Glu Lys
165 170 175
Pro Gly Asn Phe Lys Asp Leu Arg Glu Phe Val Phe Arg Asn Lys Asp
180 185 190
Gly Phe Leu His Val Tyr Ser Gly Tyr Gln Pro Ile Ser Ala Ala Ser
195 200 205
Gly Leu Pro Thr Gly Phe Asn Ala Leu Lys Pro Ile Phe Lys Leu Pro
210 215 220
Leu Gly Ile Asn Ile Thr Asn Phe Arg Thr Leu Leu Thr Ala Phe Pro
225 230 235 240
Pro Arg Pro Asp Tyr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly
245 250 255
Tyr Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys
275 280 285
Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Ala Pro Ser Lys Glu Val Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Thr Phe Pro
325 330 335
Ser Val Tyr Ala Trp Glu Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Leu Gly Cys Val Leu Ala Trp Asn Thr Asn Ser Lys Asp Ser Ser
420 425 430
Thr Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Trp Val Arg Arg Ser Lys
435 440 445
Leu Asn Pro Tyr Glu Arg Asp Leu Ser Asn Asp Ile Tyr Ser Pro Gly
450 455 460
Gly Gln Ser Cys Ser Ala Val Gly Pro Asn Cys Tyr Asn Pro Leu Arg
465 470 475 480
Pro Tyr Gly Phe Phe Thr Thr Ala Gly Val Gly His Gln Pro Tyr Arg
485 490 495
Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys
500 505 510
Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe
515 520 525
Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys
530 535 540
Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr
545 550 555 560
Asp Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro
565 570 575
Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser
580 585 590
Ser Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Pro
595 600 605
Val Ala Ile His Ala Asp Gln Leu Thr Pro Ser Trp Arg Val Tyr Ser
610 615 620
Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala
625 630 635 640
Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly
645 650 655
Ile Cys Ala Ser Tyr His Thr Val Ser Ser Leu Arg Ser Thr Ser Gln
660 665 670
Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile
675 680 685
Ala Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser
690 695 700
Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp
705 710 715 720
Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu
725 730 735
Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly
740 745 750
Ile Ala Val Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val
755 760 765
Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys Asp Phe Gly Gly Phe Asn
770 775 780
Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe
785 790 795 800
Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe
805 810 815
Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu
820 825 830
Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu
835 840 845
Thr Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr
850 855 860
Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro
865 870 875 880
Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln
885 890 895
Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys
900 905 910
Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu
915 920 925
Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr
930 935 940
Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu
945 950 955 960
Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile
965 970 975
Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr
980 985 990
Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala
995 1000 1005
Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val
1010 1015 1020
Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala
1025 1030 1035
Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser
1040 1045 1050
Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly
1055 1060 1065
Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr
1070 1075 1080
Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile
1085 1090 1095
Thr Thr Asp Asn Thr Phe Val Ser Gly Ser Cys Asp Val Val Ile
1100 1105 1110
Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu
1115 1120 1125
Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr
1130 1135 1140
Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser
1145 1150 1155
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala
1160 1165 1170
Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys
1175 1180 1185
Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe
1190 1195 1200
Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys
1205 1210 1215
Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys
1220 1225 1230
Gly Ser Cys Cys Lys Asp Tyr Lys Asp Asp Asp Asp Lys
1235 1240 1245
<210> 30
<211> 1216
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of S protein of coronavirus SARS-COV pseudovirus
<220>
<221> DOMAIN
<222> (1)..(1216)
<400> 30
Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu
1 5 10 15
Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln
20 25 30
His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg
35 40 45
Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser
50 55 60
Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Asp Asn Pro Val
65 70 75 80
Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn
85 90 95
Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln
100 105 110
Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys
115 120 125
Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met
130 135 140
Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr
145 150 155 160
Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser
165 170 175
Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly
180 185 190
Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp
195 200 205
Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu
210 215 220
Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro
225 230 235 240
Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr
245 250 255
Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile
260 265 270
Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys
275 280 285
Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn
290 295 300
Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr
305 310 315 320
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser
325 330 335
Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr
340 345 350
Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly
355 360 365
Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala
370 375 380
Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly
385 390 395 400
Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
405 410 415
Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser
420 425 430
Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu
435 440 445
Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly
450 455 460
Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp
465 470 475 480
Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val
485 490 495
Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly
500 505 510
Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn
515 520 525
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg
530 535 540
Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp
545 550 555 560
Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys
565 570 575
Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser
580 585 590
Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr
595 600 605
Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr
610 615 620
Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu
625 630 635 640
His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile
645 650 655
Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys
660 665 670
Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala
675 680 685
Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile
690 695 700
Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys
705 710 715 720
Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu
725 730 735
Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile
740 745 750
Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys
755 760 765
Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe
770 775 780
Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile
785 790 795 800
Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met
805 810 815
Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile
820 825 830
Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr
835 840 845
Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala
850 855 860
Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe
865 870 875 880
Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn
885 890 895
Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala
900 905 910
Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly
915 920 925
Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu
930 935 940
Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn
945 950 955 960
Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp
965 970 975
Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln
980 985 990
Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala
995 1000 1005
Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp
1010 1015 1020
Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala
1025 1030 1035
Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln
1040 1045 1050
Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys
1055 1060 1065
Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser
1070 1075 1080
Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr
1085 1090 1095
Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly
1100 1105 1110
Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp
1115 1120 1125
Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser
1130 1135 1140
Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val
1145 1150 1155
Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
1160 1165 1170
Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr
1175 1180 1185
Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile
1190 1195 1200
Ala Gly Leu Ile Ala Asp Tyr Lys Asp Asp Asp Asp Lys
1205 1210 1215

Claims (19)

1. A coronavirus heterotetrameric antigen protein comprising a partial amino acid sequence or a complete amino acid sequence of the receptor binding region of the spike proteins of four different coronavirus virus strains, directly concatenated or concatenated by a linker sequence;
the four different coronaviruses are selected from four of the following groups: SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain, coronavirus RsSHC014, coronavirus LYRa11, coronavirus RaTG13, coronavirus Pangolin GX/P4L, coronavirus Pangolin GD/1, coronavirus WIV1, coronavirus RshSTT182, coronavirus BtKY72, coronavirus RmYN02, coronavirus SARS-CoV, coronavirus MERS-CoV, coronavirus HCoV-229E, coronavirus HCoV-OC43, coronavirus HCoV-NL63, coronavirus HCoV-HKU1, and coronavirus bat-CoV.
2. The coronavirus heterotetrameric antigen protein of claim 1, optionally comprising four of the following groups: SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain, coronavirus RsSHC014, coronavirus LYRa11, coronavirus RaTG13, coronavirus Pangolin GX/P4L, coronavirus Pangolin GD/1, coronavirus WIV1, coronavirus RshSTT182, coronavirus BtKY72, coronavirus RmYN02, and coronavirus SARS-CoV;
optionally, the four different coronaviruses are selected from four of the following groups: SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain, coronavirus RsSHC014, coronavirus LYRa11, coronavirus RaTG13, coronavirus Pangolin GX/P4L, and coronavirus SARS-CoV;
alternatively, the sequence of the concatenation of the partial amino acid sequences or the complete amino acid sequences of the receptor binding regions of the spike proteins of the four different coronaviruses is: the strains are connected in series in the sequence from high to low in homology.
3. The coronavirus heterotetrameric antigen protein of claim 1, characterized in that it comprises the following four amino acid sequences, either directly in series or in series through a linker sequence:
(a) A partial amino acid sequence or the entire amino acid sequence of the receptor-binding region of the spike protein of the SARS-CoV-2 prototype strain, wherein the partial amino acid sequence of the receptor-binding region of the spike protein of the SARS-CoV-2 prototype strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of the entire amino acid sequence thereof;
(b) a partial amino acid sequence or a complete amino acid sequence of the receptor binding region of the spike protein of the SARS-CoV-2 variant strain, wherein the partial amino acid sequence of the receptor binding region of the spike protein of the SARS-CoV-2 variant strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of the complete amino acid sequence thereof;
(c) a partial amino acid sequence or the entire amino acid sequence of the receptor-binding region of the spike protein of coronavirus X, wherein the partial amino acid sequence of the receptor-binding region of the spike protein of coronavirus X is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of the entire amino acid sequence thereof;
(d) a partial amino acid sequence or the entire amino acid sequence of the receptor-binding region of the spike protein of SARS-CoV, wherein the partial amino acid sequence of the receptor-binding region of the spike protein of SARS-CoV is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of the entire amino acid sequence thereof;
Wherein, the coronavirus X is selected from at least one of coronavirus RsSHC014, coronavirus LYRa11, coronavirus RaTG13 and coronavirus Pangolin GX/P4L; optionally, the coronavirus X is selected from coronavirus RsSHC014 or coronavirus LYRa 11;
wherein the SARS-CoV-2 variant strain is selected from Alpha, Beta, Gamma, Kappa, Lambda, Delta or Omicron strain, optionally the SARS-CoV-2 variant strain is Beta strain;
optionally, the series is a direct series;
optionally, the tandem is tandem via a linker sequence, optionally the linker sequence is (GGS)n(ii) a Wherein n is 1,2,3,4 or 5;
alternatively, the four amino acid sequences are connected in series in the sequence of the amino acid sequences (a), (b), (c) and (d).
4. The coronavirus heterotetrameric antigen protein of claim 1, characterized in that it comprises the following four amino acid sequences, either directly or through a linker sequence in series:
(a) 1 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No. 1 and having the same or substantially the same immunogenicity as it;
(b) An amino acid sequence as set forth in SEQ ID NO. 2, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO. 2 and having the same or substantially the same immunogenicity as it;
(c) 3, 5, 6 or 7; or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to and having the same or substantially the same immunogenicity as the amino acid sequence set forth as SEQ ID NO 3, 5, 6 or 7; preferably, (c) an amino acid sequence as shown in SEQ ID NO 6 or 7; or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to and having the same or substantially the same immunogenicity as the amino acid sequence set forth as SEQ ID NO 6 or 7;
(d) an amino acid sequence as shown in SEQ ID NO. 4; or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to and having the same or substantially the same immunogenicity as the amino acid sequence set forth as SEQ ID NO. 4;
optionally, the series is a direct series;
Optionally, the concatenation is by a connecting sequence, optionally the connecting sequence is (GGS)n(ii) a Wherein n is 1,2,3,4 or 5.
5. The coronavirus heterotetrameric antigen protein of claim 4, characterized in that it comprises an amino acid sequence as shown in SEQ ID NO 8, 9, 10 or 11; or comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to and having the same or substantially the same immunogenicity as the amino acid sequence shown as SEQ ID NO 8, 9, 10 or 11;
alternatively, the heterotetrameric antigenic protein comprises the amino acid sequence shown in SEQ ID NO 10 or 11; or comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO 10 or 11 and having the same or substantially the same immunogenicity as it;
and/or the N end and/or the C end of the amino acid sequence of the heterotetrameric antigen protein are/is also connected with a signal peptide and/or a tag; optionally, the N-terminal of the amino acid sequence of the heterotetrameric antigenic protein is linked to a signal peptide and the C-terminal is linked to a tag; optionally, the tag is selected from at least one of Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, SUMO tag; preferably the tag is a His tag; optionally, the signal peptide is a signal peptide of MERS-CoV spike protein.
6. A method for preparing the coronavirus heterotetramer antigenic protein of any one of claims 1-5, characterized in that: the method comprises the following steps: adding a sequence encoding a signal peptide to the 5 'end and a sequence encoding a tag to the 3' end of the nucleotide sequence encoding the coronavirus heterotetramer antigen protein of any one of claims 1-5, cloning and expressing, screening correct recombinants, transfecting cells of an expression system for expression, collecting cell supernatants after expression, and purifying to obtain the coronavirus heterotetramer antigen protein; optionally, the tag is selected from at least one of Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, SUMO tag; preferably the tag is a His tag; optionally, the signal peptide is a signal peptide of MERS-CoV spike protein.
7. The method of claim 6, wherein: the cell of the expression system comprises a mammalian cell, an insect cell, a yeast cell, or a bacterial cell, optionally; the mammalian cells include HEK293T, HEK293F cells or CHO cells, and the bacterial cells include E.coli cells.
8. A polynucleotide encoding the coronavirus heterotetramer antigen protein of any one of claims 1-5, optionally the polynucleotide sequence comprises the nucleotide sequence shown in SEQ ID NOs 12, 13, 14 and/or 15.
9. A nucleic acid construct comprising the polynucleotide of claim 8.
10. A recombinant vector comprising the nucleic acid construct of claim 9.
11. An expression system cell comprising the recombinant vector of claim 10; preferably, the expression system cell is a mammalian cell; further preferably, the mammalian cell is a HEK293T, HEK293F cell or CHO cell.
12. Use of the coronavirus heterotetrameric antigen protein of any one of claims 1 to 5, the polynucleotide of claim 8, the nucleic acid construct of claim 9, the recombinant vector of claim 10 or the expression system cell of claim 11 for the preparation of a universal vaccine for coronaviruses;
preferably, the vaccine is used for immunization alone or in sequential immunization with other types of novel coronavirus vaccines.
13. An immunogenic composition comprising the coronavirus heterotetrameric antigen protein of any one of claims 1-5, or the heterotetrameric antigen protein prepared by the method of claim 6 or 7, or the polynucleotide of claim 8, or the nucleic acid construct of claim 9, or the recombinant vector of claim 10, or the expression system cell of claim 11, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and/or diluent.
14. The immunogenic composition according to claim 13, which is a coronavirus protein universal vaccine comprising:
(1) the coronavirus heterotetrameric antigen protein of any one of claims 1-5; preferably comprising an amino acid sequence as shown in SEQ ID NO 8, 9, 10 and/or 11 or preferably comprising an amino acid sequence with a signal peptide sequence and/or a tag amino acid sequence over the amino acid sequence as shown in SEQ ID NO 8, 9, 10 and/or 11; and
(2) an adjuvant;
optionally, the adjuvant is selected from the group consisting of an aluminum adjuvant, an MF59 adjuvant, and an MF 59-like adjuvant; alternatively, the MF 59-like adjuvant comprises an Addavax adjuvant;
alternatively, the coronavirus heterotetrameric antigen protein of any one of claims 1-5 and adjuvant in a volume ratio of 1: 1-2; optionally, the volume ratio is 1: 1.
15. the immunogenic composition according to claim 13, characterized in that it is a coronavirus DNA universal vaccine comprising:
(i) a eukaryotic expression vector; and
(ii) a DNA sequence encoding the coronavirus heterotetramer antigen protein of any one of claims 1-5 constructed into said eukaryotic expression vector, optionally said DNA sequence comprising a nucleotide sequence as set forth in SEQ ID NOs 12, 13, 14 and/or 15;
Preferably, the eukaryotic expression vector is selected from the group consisting of pGX0001, pVAX1, pCAGGS and pcDNA series vectors.
16. The immunogenic composition according to claim 13, characterized in that it is a coronavirus mRNA universal vaccine comprising:
(I) an mRNA sequence encoding the coronavirus heterotetrameric antigen protein of any one of claims 1-5; and
(II) lipid nanoparticles.
17. The immunogenic composition according to claim 13, characterized in that it is a coronavirus-viral vector vaccine comprising:
(first) a viral backbone vector; and
(② a DNA sequence encoding a coronavirus heterotetramer antigen protein according to any one of claims 1-5, optionally comprising a nucleotide sequence as shown in SEQ ID NO 12, 13, 14 and/or 15, constructed into said viral backbone vector;
preferably, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors.
18. The immunogenic composition of any one of claims 13 to 17, wherein the immunogenic composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation;
Preferably, the nasal spray is selected from the group consisting of an aerosol, a spray and a powder spray;
preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets and ointments;
preferably, the parenteral formulation is a transdermal agent, ointment, plaster, topical liquid, injectable or bolus formulation.
19. Use of a coronavirus heterotetrameric antigen protein of any one of claims 1-5, or a heterotetrameric antigen protein prepared by the method of claim 6 or 7, or a polynucleotide of claim 8, or a nucleic acid construct of claim 9, or a recombinant vector of claim 10, or an expression system cell of claim 11, or an immunogenic composition of any one of claims 13-18 for the preparation of a medicament for the treatment and/or prevention of a novel coronavirus infection and/or a disease caused by a novel coronavirus;
alternatively, the coronavirus includes at least one or more of SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain, coronavirus RsSHC014 strain, coronavirus LYRa11 strain, coronavirus RaTG13 strain, coronavirus Pangolin GX/P4L strain, coronavirus Pangolin GD/1 strain, coronavirus WIV1 strain, coronavirus RshSTT182 strain, coronavirus BtKY72 strain, coronavirus RYN 02 strain, coronavirus SARS-CoV, coronavirus MERS-CoV, coronavirus HCoV-229E, coronavirus HCoV-OC43, coronavirus HCoV-63, coronavirus HCoV-HKU1 strain, and coronavirus bat-CoV.
CN202210574868.7A 2022-05-25 2022-05-25 Universal vaccine for coronavirus and application thereof Pending CN114751992A (en)

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