CN116813757A - Construction body of nano antibody R14 and application thereof - Google Patents

Construction body of nano antibody R14 and application thereof Download PDF

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CN116813757A
CN116813757A CN202210278872.9A CN202210278872A CN116813757A CN 116813757 A CN116813757 A CN 116813757A CN 202210278872 A CN202210278872 A CN 202210278872A CN 116813757 A CN116813757 A CN 116813757A
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nanobody
ser
gly
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王奇慧
高福
刘红辉
刘博�
仵丽丽
韩鹏程
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Institute of Microbiology of CAS
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Institute of Microbiology of CAS
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Priority to CN202210375857.6A priority Critical patent/CN114805560B/en
Priority to CN202210278872.9A priority patent/CN116813757A/en
Priority to CN202210383740.2A priority patent/CN116813759A/en
Priority to PCT/CN2023/082523 priority patent/WO2023179543A1/en
Priority to PCT/CN2023/082530 priority patent/WO2023179545A1/en
Publication of CN116813757A publication Critical patent/CN116813757A/en
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Abstract

The invention provides a construct (including multivalent nanobody and nanobody fusion protein) based on nanobody R14 which specifically binds SARS-CoV-2RBD, related products and applications thereof; the construct (comprising multivalent nanobody and nanobody fusion protein) of the nanobody R14 based on specific binding SARS-CoV-2RBD can effectively inhibit SARS-CoV-2 infection and variant strain infection thereof, can be atomized and administrated, can directly reach lung, has quick response and long half-life, and provides a more effective treatment strategy for clinically preventing or treating new coronavirus and variant strain infection thereof.

Description

Construction body of nano antibody R14 and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a construct of a nanobody R14 and application thereof, and more particularly relates to a multivalent nanobody of the nanobody R14 based on specific binding SARS-CoV-2RBD, nanobody fusion protein, polynucleotide for encoding the same, nucleic acid construct containing the polynucleotide, expression vector containing the nucleic acid construct, transformed cells containing the polynucleotide, the nucleic acid construct or the expression vector, and pharmaceutical composition containing any one of the products, and application thereof in preparing medicines for preventing or treating novel coronaviruses, and application thereof in preparing reagents or kits for detecting the novel coronaviruses or diagnosing the novel coronavirus infection.
Background
Epidemic caused by the novel coronavirus (SARS-CoV-2) of the Coronaviridae (family Coronaviridae) continues to spread worldwide. In addition, severe acute respiratory syndrome coronavirus (SARS-CoV) of the same genus coronaviridae, middle east respiratory syndrome coronavirus (MERS-CoV) and the like are also main pathogens aiming at human respiratory system, and are mainly transmitted by means of spray, aerosol, contact and the like, so that the viruses causing respiratory diseases seriously jeopardize public health safety.
The current outbreak of new coronapneumonia epidemic situation promotes the development of various vaccines and antiviral drugs, wherein the vaccination can effectively prevent serious infectious diseases, but the applicable subjects of the vaccine are uninfected people, the research and development period is long, and the clinical research process is complex. For patients with definite diagnosis, the treatment can only be carried out through antiviral drugs, wherein one antiviral drug is a therapeutic antibody drug and mainly refers to a neutralizing antibody; the neutralizing antibody medicine is mainly used for preventing specific molecules expressed by pathogenic microorganisms from being combined with cell surface receptors by combining with antigens on the surfaces of the pathogenic microorganisms so as to achieve the effect of neutralization. SARS-CoV-2 virus has glycosylated spike protein (S) on its surface, which interacts with the host cell receptor protein ACE2 and triggers membrane fusion, thus blocking the binding of S protein to ACE2 is an effective way to treat new coronavirus infections.
Conventional monoclonal antibodies are generally administered by intravenous injection, however, the concentration of the drug entering the lungs from the systemic circulation is very low by the administration of the monoclonal antibodies by intravenous injection, which greatly reduces the antiviral effect of the neutralizing antibodies themselves, resulting in an ineffective reduction of the viral load in the lungs. The novel coronavirus initially infects the upper respiratory tract and its first interaction with the immune system occurs primarily at the mucosal surface of the respiratory tract, and in view of this, for the novel coronavirus to infect via the respiratory tract, while focusing on serum antibodies, it is also necessary to think, design and develop appropriate antibody-based drugs from the standpoint of mucosal immunity. For example, aerosolized administration may provide higher local concentrations of antibody drug in the respiratory tract, which may be more effective in blocking viral infection during viral entry.
Nanobodies have been attracting attention as therapeutic drugs, for example, nanobody drug cappucizumab (cablevi TM ) The nanometer antibody is used for treating the acquired thrombotic thrombocytopenic purpura, and is a nanometer antibody medicament which is marketed in the first acquisition lot; for another example, nanobody candidate ALX-0171 is a trivalent form of nanobody for the treatment of pediatric Respiratory Syncytial Virus (RSV) infection, which has been shown to enter phase II clinical (https:// clinicaltrias gov) by aerosolized administration: the nano antibody medicine has safety and feasibility.
Therefore, development of the nano antibody medicine which aims at the novel coronavirus and is suitable for immunization of respiratory mucosa has potential clinical application value and prospect.
Disclosure of Invention
Object of the Invention
The invention aims at providing a construct (comprising multivalent nanobody and nanobody fusion protein) based on nanobody R14 which specifically binds SARS-CoV-2RBD, polynucleotide encoding the same, nucleic acid construct comprising the polynucleotide, expression vector comprising the nucleic acid construct, transformed cell comprising the polynucleotide, nucleic acid construct or expression vector, pharmaceutical composition comprising any one of the above products and application thereof in preparing medicament for preventing or treating novel coronavirus, and application in preparing reagent or kit for detecting novel coronavirus or diagnosing novel coronavirus infection.
The construct (comprising multivalent nanobody and nanobody fusion protein) of the nanobody R14 based on specific binding SARS-CoV-2RBD can effectively inhibit SARS-CoV-2 infection and variant strain infection thereof, can be directly delivered to the lung by atomization, has quick response and long half-life, and provides a more effective treatment strategy for new coronavirus and variant strain infection thereof.
Solution scheme
In order to achieve the above purpose, the present invention provides the following technical solutions:
in a first aspect, the present invention provides a multivalent nanobody comprising two or more VHH chains of nanobody that specifically bind SARS-CoV-2RBD, wherein the VHH chains of nanobody that specifically bind SARS-CoV-2RBD comprise the following CDRs:
CDR1 having an amino acid sequence shown in SEQ ID NO. 1 (i.e., GFTLDYYAIG),
CDR2 having the amino acid sequence shown in SEQ ID NO. 2 (i.e., CISSSDGSTSYADSVKG), and
the amino acid sequence is shown as CDR3 of SEQ ID NO. 3 (i.e., TPATYYSGRYYYQCPAGGMDY).
In a specific embodiment, the VHH chain of the nanobody that specifically binds SARS-CoV-2RBD further comprises 4 framework regions FR1-4, said FR1-4 being staggered sequentially to said CDR1, CDR2 and CDR 3;
preferably, the amino acid sequences of FR1-4 are shown in SEQ ID NO:4 (i.e., QVQLQESGGGLVQPGGSLRLSCAVS), SEQ ID NO:5 (i.e., WFRQAPGKEREGVS), SEQ ID NO:6 (i.e., RFTISRDNAKNTVYLQMNSLKPEDTALYYCAA) and SEQ ID NO:7 (i.e., WGQGTQVTVSS), respectively.
In a preferred embodiment, the VHH chain of the nanobody that specifically binds SARS-CoV-2RBD has an amino acid sequence as set forth in SEQ ID NO. 8, or an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 8; preferably, the amino acid sequence of the VHH chain is as shown in SEQ ID NO 8:
Wherein the underlined parts are the framework regions FR1-4, respectively, and the black-marked parts are CDR1, CDR2 and CDR3, respectively, of the heavy chain variable region.
In a preferred embodiment I, said multivalent nanobody is composed of more than two, preferably three, VHH chains of said nanobody specifically binding SARS-CoV-2RBD, joined by Linker;
wherein the Linker is (GGGGS) n, wherein n=1, 2,3, or 4, preferably n=2 or 3.
As a further preference of embodiment I, the multivalent nanobody is a trivalent nanobody having the amino acid sequence shown in SEQ ID No. 9:
QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSD GSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQC PAGGMDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGL VQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTSYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCPAGGMDYWGQGTQ VTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAVSG FTLDYYAIGWFRQAPGKEREGVSCISSSDGSTSYADSVKGRFTISRDNAKNTVYLQMN SLKPEDTALYYCAATPATYYSGRYYYQCPAGGMDYWGQGTQVTVSS(SEQ ID NO:9)。
in a preferred embodiment II, the multivalent nanobody is an IgM pentamer formed from a fusion protein having a structure from N-terminus to C-terminus as shown in formula (I):
A-L-B (I)
wherein,,
a is a single VHH chain of the nanobody that specifically binds SARS-CoV-2RBD, or a multivalent nanobody as described in preferred embodiment I above;
b is an Fc fragment of human IgM; preferably, the Fc fragment of the humanized IgM has an amino acid sequence as set forth in SEQ ID NO. 10 (i.e., VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTD QVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQD TAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNA TFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSI LTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY), or an amino acid sequence having at least 95%,96%,97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 10;
L is (GGGGS) m, wherein m=0, 1,2,3, or 4.
As a preferred embodiment of the above fusion protein, it has an amino acid sequence as shown in SEQ ID NO: 11:
QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSD GSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQC PAGGMDYWGQGTQVTVSSVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQI QVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVD HRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWT RQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKY VTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGK PTLYNVSLVMSDTAGTCY(SEQ ID NO:11)。
in a second aspect, the present invention provides a nanobody fusion protein, wherein the structure from the N-terminal to the C-terminal of the nanobody fusion protein is shown in formula (I):
A-L-B (I)
wherein,,
a is a single VHH chain of a nanobody that specifically binds SARS-CoV-2RBD, said VHH chain of a nanobody that specifically binds SARS-CoV-2RBD being as defined in the first aspect above; alternatively, a is a multivalent nanobody according to the preferred embodiment I of the first aspect described above, i.e. a multivalent nanobody consisting of more than two, preferably three, VHH chains of said nanobody specifically binding SARS-CoV-2RBD, connected by Linker, wherein the Linker is (GGGGS) n, wherein n = 1,2,3, or 4, preferably n = 2 or 3;
b is an Fc fragment of human IgM; preferably, the Fc fragment of said humanized IgM has an amino acid sequence as set forth in SEQ ID NO. 10, or an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 10;
L is (GGGGS) m, wherein m=0, 1,2,3, or 4.
As a preferred embodiment of the above fusion protein, it has an amino acid sequence shown as SEQ ID NO. 11.
In a third aspect, the present invention provides a polynucleotide encoding a multivalent nanobody as described in the first aspect above, or encoding a nanobody fusion protein as described in the second aspect above.
In specific embodiments, the polynucleotide is DNA or mRNA;
preferably, the polynucleotide encodes a multivalent nanobody according to preferred embodiment I of the first aspect above, further preferably, the polynucleotide comprises a nucleotide sequence as shown in SEQ ID No. 12 (i.e., CAAGTGCAACTGCAGGAGAGCGGCGGAGGCCTGGTCCAACCTGGCGGCAGCCTG CGGCTGTCTTGTGCTGTGTCTGGATTCACCCTGGATTACTATGCCATCGGCTGGTTT AGACAGGCCCCTGGCAAGGAACGGGAAGGCGTTAGCTGCATCAGCTCTTCCGACG GCTCTACCAGCTACGCTGATTCTGTGAAGGGCCGCTTCACAATCAGCAGAGATAAT GCCAAAAACACGGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCC CTGTACTATTGCGCTGCCACACCCGCCACCTACTACAGCGGCAGATACTACTATCAG TGTCCTGCCGGAGGCATGGATTACTGGGGACAGGGCACCCAGGTGACAGTGAGCA GCGGAGGAGGCGGCAGCGGCGGAGGCGGCAGTGGTGGCGGCGGATCCGGCGGC GGAGGCAGCGGCGGCGGGGGCAGCCAGGTGCAGCTGCAGGAGAGCGGCGGCGG CCTGGTGCAGCCTGGAGGCAGCCTGAGACTGAGCTGTGCCGTGTCCGGTTTCACC CTGGACTACTACGCCATTGGATGGTTCAGACAGGCTCCAGGCAAGGAAAGAGAAG GCGTGTCCTGTATCAGCTCTTCTGATGGATCTACATCTTACGCCGACAGCGTGAAG GGCAGGTTCACCATCTCCAGAGACAATGCCAAGAACACCGTGTACCTGCAGATGA ACAGCCTGAAACCTGAGGATACCGCACTTTATTACTGCGCCGCCACCCCTGCTACA TACTACAGCGGAAGATACTACTACCAGTGCCCCGCCGGCGGCATGGACTACTGGG GCCAGGGCACCCAGGTCACAGTGAGCAGCGGCGGCGGCGGCTCCGGCGGAGGCG GCTCTGGTGGCGGCGGAAGCGGAGGCGGAGGCAGCGGCGGCGGAGGCTCTCAGG TGCAGCTGCAGGAGTCCGGCGGCGGGCTGGTGCAGCCAGGCGGCAGCCTGAGAC TGAGCTGCGCCGTGTCTGGCTTTACACTGGACTACTACGCCATCGGCTGGTTCCGG CAGGCCCCTGGCAAAGAGCGGGAAGGCGTGTCTTGCATCAGCAGCAGCGACGGC AGCACCAGCTACGCCGACAGCGTCAAGGGAAGATTCACCATCTCCCGGGACAACG CCAAGAACACAGTGTACCTGCAAATGAACAGCCTCAAGCCCGAGGACACCGCCCT GTACTACTGCGCCGCTACCCCTGCCACATACTACTCTGGCAGATACTACTACCAGTG CCCTGCCGGCGGCATGGACTACTGGGGCCAGGGCACACAGGTGACCGTGTCCAGC);
Preferably, the polynucleotide encodes a nanobody fusion protein as described in the second aspect above, further preferably, the polynucleotide comprises the nucleotide sequence shown as SEQ ID NO. 13 (i.e., CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTG AGACTCTCCTGTGCAGTCTCTGGATTTACTTTGGATTATTATGCCATAGGCTGGTTC CGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATTAGTAGTAGTGATG GTAGCACATCGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAC GCCAAAAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCC TTTATTACTGTGCAGCAACCCCTGCTACATACTATAGTGGACGTTACTACTACCAAT GTCCCGCGGGGGGCATGGACTACTGGGGCCAGGGGACCCAGGTGACCGTGAGCT CTGTGATCGCCGAGCTGCCCCCCAAGGTGAGCGTGTTCGTGCCCCCTAGAGACGG CTTCTTCGGCAACCCTAGAAAGAGCAAGCTGATCTGCCAAGCCACCGGCTTCTCC CCTAGACAGATCCAAGTGAGCTGGCTGAGAGAGGGCAAGCAAGTGGGCAGCGGC GTCACAACAGACCAAGTGCAAGCCGAGGCCAAGGAGAGCGGCCCCACCACCTAC AAGGTGACAAGCACCCTGACCATCAAGGAGAGCGACTGGCTGGGGCAGAGCATG TTCACCTGCAGAGTGGACCACAGAGGCCTGACCTTTCAGCAGAACGCTAGCAGCA TGTGCGTGCCCGACCAAGACACCGCCATCAGAGTGTTCGCCATCCCCCCTAGCTTC GCTAGCATCTTCCTGACCAAGAGCACCAAGCTGACCTGCCTCGTGACCGATCTGA CCACCTACGACAGCGTGACCATCAGCTGGACAAGACAGAACGGCGAGGCCGTGA AGACCCACACCAACATCAGCGAGAGCCACCCCAACGCCACCTTCAGCGCCGTGG GCGAGGCTAGCATCTGCGAGGACGACTGGAACAGCGGCGAGAGATTCACCTGCA CCGTGACCCACACCGACCTGCCTAGCCCCCTGAAGCAGACCATCAGCAGACCCAA GGGCGTGGCCCTGCACAGACCCGACGTGTACCTGCTGCCCCCCGCTAGAGAGCAG CTGAACCTGAGAGAGAGCGCCACCATCACCTGCCTGGTGACCGGCTTTAGCCCCG CTGACGTGTTCGTGCAGTGGATGCAGAGAGGGCAGCCCCTGAGCCCCGAGAAGTA CGTGACAAGCGCCCCCATGCCCGAGCCCCAAGCCCCCGGCAGATACTTCGCCCAC AGCATCCTGACCGTGAGCGAGGAAGAGTGGAACACCGGCGAGACCTACACCTGC GTGGTGGCCCACGAGGCCCTGCCCAACAGAGTGACCGAGAGAACCGTGGACAAG AGCACCGGCAAGCCCACCCTGTACAACGTGAGCCTGGTGATGAGCGACACCGCCG GCACCTGCTAC).
In a fourth aspect, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the third aspect above, and optionally at least one expression control element operably linked to the polynucleotide. Such as histidine tags, stop codons, etc.
In a fifth aspect, the present invention provides an expression vector comprising a nucleic acid construct as described in the fourth aspect above.
In a sixth aspect, the present invention provides a transformed cell comprising a polynucleotide as described in the third aspect, a nucleic acid construct as described in the fourth aspect or an expression vector as described in the fifth aspect.
In a seventh aspect, the present invention provides a pharmaceutical composition comprising a multivalent nanobody as defined in the first aspect, a nanobody fusion protein as defined in the second aspect, a polynucleotide as defined in the third aspect, a nucleic acid construct as defined in the fourth aspect, an expression vector as defined in the fifth aspect or a transformed cell as defined in the sixth aspect, and a pharmaceutically acceptable carrier and/or excipient.
In particular embodiments, the pharmaceutical composition may be in the form of a nasal spray, oral formulation, suppository or parenteral formulation;
Preferably, the nasal spray is selected from the group consisting of aerosols, sprays and powder sprays;
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, an ointment, a plaster, a topical liquid, an injectable or a bolus formulation.
The amount of the active ingredient to be administered of the pharmaceutical composition of the present invention varies depending on the administration subject, the organ to be administered, the symptoms, the administration method, etc., and can be determined by considering the type of the dosage form, the administration method, the age and weight of the patient, the symptoms of the patient, etc., and the judgment of the doctor.
In an eighth aspect, the present invention provides the use of a multivalent nanobody as defined in the first aspect, a nanobody fusion protein as defined in the second aspect, a polynucleotide as defined in the third aspect, a nucleic acid construct as defined in the fourth aspect, an expression vector as defined in the fifth aspect, a transformed cell as defined in the sixth aspect or a pharmaceutical composition as defined in the seventh aspect for the preparation of a medicament for the prevention and/or treatment of a novel coronavirus infection.
In particular embodiments, the novel coronavirus may be a SARS-CoV-2 original strain and/or a SARS-CoV-2 variant strain;
preferably, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1), delta (B.1.617.2), omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain, more preferably Delta (B.1.617.2) strain, omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain.
In a ninth aspect, the present invention provides the use of a multivalent nanobody as defined in the first aspect, a nanobody fusion protein as defined in the second aspect, a polynucleotide as defined in the third aspect, a nucleic acid construct as defined in the fourth aspect, an expression vector as defined in the fifth aspect or a transformed cell as defined in the sixth aspect, for the preparation of a reagent or kit for detecting a novel coronavirus or for diagnosing a novel coronavirus infection.
In particular embodiments, the novel coronavirus may be a SARS-CoV-2 original strain and/or a SARS-CoV-2 variant strain;
preferably, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1), delta (B.1.617.2), omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain, more preferably Delta (B.1.617.2) strain, omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain.
In a tenth aspect, the present invention provides a novel coronavirus detection kit comprising a multivalent nanobody as described in the first aspect above, a nanobody fusion protein as described in the second aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above or a transformed cell as described in the sixth aspect above.
Advantageous effects
The invention aims at the drug development of a nanobody construct of a novel coronavirus, the nanobody R14-based construct of the invention can be combined with SARS-CoV-2RBD with high affinity, and can neutralize pseudoviruses and live viruses of SARS-CoV-2 prototype strains and a series of variant strains with high neutralization activity, which show that: the nanobody R14-based construct is a novel coronavirus (SARS-CoV-2) nanobody capable of binding to SARS-CoV-2RBD with high affinity and having high neutralizing activity.
In particular, the inventor proves through a series of experiments that the trivalent nanometer antibody (TR 14) and IgM pentamer form (MR 14) based on the nanometer antibody R14 have obviously improved neutralization activity and obviously prolonged half-life period relative to the monomer (namely, the nanometer antibody R14), which realizes mucosal immunity, can limit virus propagation and further cross a membrane adhesion barrier, controls virus mucosal propagation, provides potential antibody new drug capable of being subjected to atomization administration for clinical prevention and treatment of novel coronaviruses, and can realize sensitive and reliable detection of the novel coronaviruses.
Drawings
One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used 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 schematic diagram of the structures of nanobody constructs TR14 and MR14 constructed in example 1 of the invention;
FIG. 2 is a graph showing the molecular sieve chromatography and SDS-PAGE identification of the R14 protein described in example 1 of the present invention;
FIG. 3 is a graph showing the results of molecular sieve chromatography and SDS-PAGE identification of the TR14 protein described in example 1 of the present invention;
FIG. 4 is a graph showing the molecular sieve chromatography and SDS-PAGE identification of the MR14 protein described in example 1 of the present invention;
FIG. 5 is a diagram showing the SDS-PAGE identification of SARS-CoV-2RBD-his protein (A), variant strain Omicron (B.1.1.529) subtype BA.1 RBD-his protein (B) and Omicron (B.1.1.529) subtype BA.2 RBD-his protein (C) described in example 2 of the present invention.
FIG. 6 is a graph showing the effect of three antibodies tested in example 5 of the present invention on neutralizing pseudovirus infection of SARS-CoV-2 prototype strain.
FIG. 7 is a graph showing the effect of three antibodies tested in example 5 of the present invention on neutralizing a pseudovirus infection by the SARS-CoV-2 variant Delta (B.1.617.2).
FIG. 8 is a graph showing the effect of three antibodies tested in example 5 of the present invention on neutralizing infection with a SARS-CoV-2 variant strain Omacron (B.1.1.529) subtype BA.1 pseudovirus.
FIG. 9 is a graph showing the effect of three antibodies tested in example 5 of the present invention on neutralizing infection with the SARS-CoV-2 variant strain Omacron (B.1.1.529) subtype BA.2 pseudovirus.
FIG. 10 is the neutralization activity of three antibodies of the invention on pseudoviruses before and after aerosolization as determined in example 7; wherein A is the neutralization activity result of the nano antibody R14 on the SARS-CoV-2 prototype strain pseudovirus before and after atomization, B is the neutralization activity result of the nano antibody construct TR14 on the SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus, and C is the neutralization activity result of the nano antibody construct MR14 on the SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus.
FIG. 11 shows half-lives in blood of three antibodies measured in example 8 of the present invention.
Fig. 12 is an atomizing system used in example 8 of the present invention.
FIG. 13 is the half-life of three antibodies in the lung as determined in example 8 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.
In addition, numerous specific details are set forth in the following description in order to provide a better illustration 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 embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.
The present invention will be described in detail below.
Definition of the definition
"nanobodies", i.e. "heavy chain single domain antibodies", comprise only one heavy chain variable region (VHH), and light chains are naturally deleted compared to other antibodies.
Nanobodies are considered to be very potential antibody-based drugs because of their own biophysical advantages, which can be easily aerosolized and delivered directly to the lungs via an inhaler, thereby treating infections caused by respiratory viruses.
When referring to ligand/receptor, antibody/antigen or other binding pair, "specific" binding refers to determining the presence or absence of binding reaction of a protein, such as a nanobody of the invention, with a SARS-CoV-2 RBD protein in a heterogeneous population of proteins and/or other biological agents. Thus, under the specified conditions, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.
The reagents, enzymes, media, antibiotics, milk and other chemicals used in the following examples of the present invention are all commercially available, e.g., TRIzol is available from Invitrogen, superscript II First-Strand Synthesis System for RT-PCR kit is available from Invitrogen.
Some commonly used biological materials, such as competent cells, vectors, helper phage, cells to be transformed, etc., are also commercially available products, e.g., pCAGGS vectors are available from MiaoLingPlasmid;293F cells, HEK293T cells, and the like were purchased from ATCC; series Sensor Chip SA chip was purchased from GE Healthcare; vero cells were purchased from ATCC CCL81.
Some synthetic biomaterials, such as primers, sequences, etc., that require artificial synthesis, were committed to the synthesis company, e.g., the coding sequence of TR14 in the present invention was synthesized by the company of nanjing gold sri biotechnology limited.
Example 1: construction, expression and purification of antibodies based on the trivalent form of nanobody R14 (TR 14) and IgM pentameric form (MR 14)
The schematic structure of the single chain nanobody and trivalent form and IgM pentameric form in this example is shown in fig. 1.
The basic nano antibody R14 is obtained by simultaneously immunizing alpaca with SARS-CoV-2RBD protein and SARS-CoV-2 NTD protein, constructing an antibody library and screening by utilizing phage display technology in the laboratory; the amino acid sequence of the VHH chain of single-chain nanobody R14 is shown in SEQ ID NO. 8, which is capable of binding SARS-CoV-2RBD with high affinity and specificity (binding constant less than 1E-10M), and is capable of neutralizing SARS-CoV-2 pseudovirus with high neutralization activity in pseudovirus neutralization experiments, all of which indicate: the R14 nanobody is a novel alpaca-derived nanobody of coronavirus (SARS-CoV-2) which can bind with high affinity to SARS-CoV-2RBD and has high neutralizing activity.
The coding sequence of the single-chain nanobody R14 VHH chain (shown as SEQ ID NO: 14) was linked at the 5 'end to a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15), at the 3' end to a coding sequence of 6 histidine tags (hexa-His-tag) and a translation termination codon TGA, which were constructed into a pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, and then the resulting recombinant vector was transfected into 293F cells (purchased from Invitrogen) for expression of the R14-His protein. Cell culture medium containing target protein was subjected to nickel ion affinity chromatography (HisTrap TM excel (GE Healthcare) and gel filtration chromatography (Superdex) TM After 75Increate 10/300GL column (GE Healthcare), the purer target protein can be obtained. The SDS-PAGE of the R14-his protein identified a size of about 15KD, and the results are shown in FIG. 2.
By two stages (GGGGS) 3 The sequence, the coding sequence of the R14 VHH chain of three nanobodies shown as SEQ ID NO. 14 was concatenated end-to-end (synthesized directly by Nanjing Jinsri Biotechnology Co., ltd.) and linked at its 5 'end to the coding sequence of the signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO. 15) and at its 3' end to the 6 histidine tags (hexa-His-tag) and the translation termination codon TGA, which were constructed into pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, and the resulting recombinant vector was transfected into 293F cells (purchased from Invitrogen) for expression of TR14-His protein. Cell culture medium containing target protein was subjected to nickel ion affinity chromatography (HisTrap TM excel (GE Healthcare) and gel filtration chromatography (Superdex) TM After purification of 200Increase 10/300GL column (GE Healthcare), a purer target protein can be obtained. The size of the TR14-his protein was determined to be about 50KD by SDS-PAGE, and the results are shown in FIG. 3.
The coding sequence of the R14 VHH chain of the nanobody (shown as SEQ ID NO: 14) is connected with the coding sequence of the Fc of the human IgM antibody (shown as SEQ ID NO: 16) by homologous recombination, and the 5' end is connected with a signal peptide (ATMHSSALLCCLV)LLTGVRA, SEQ ID NO: 15), 3' end with translation termination codon TGA, was constructed into pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI to obtain pCAGGS-R14-IgM Fc recombinant expression vector. The 3' -end of the coding sequence of the J chain (coding chain) (shown as SEQ ID NO: 17) was ligated with translation termination codon TGA, which was constructed into pCAGGS vector (available from Invitrogen) through restriction enzyme sites EcoRI and XhoI, to obtain pCAGGS-J chain recombinant expression vector. The two recombinant expression vectors pCAGGS-R14-IgM Fc and pCAGGS-J chain were co-transfected into 293F cells (purchased from Invitrogen) to express R14-IgM Fc fusion protein and J chain, and then self-assembled to form MR14 protein in IgM form. The obtained MR14 protein passes through HiTrap TM IgM Purification HP (GE Healthcare) and Superose TM 6 incrustase 10/300GL (GE Healthcare) was purified and identified by SDS-PAGE. The SDS-PAGE of the MR14 protein identified a size of about 70KD, and the results are shown in FIG. 4.
Example 2: expression and purification of SARS-CoV-2 and its variant strain RBD
The coding sequence of 6 histidine tags (hexa-His-tag) and a translation termination codon TGA are connected to the 3' end of the coding sequence of RBD protein (the amino acid sequence of which is shown as SEQ ID NO: 18) of SARS-CoV-2 original strain, and the coding sequence is constructed into pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, and then the obtained recombinant vector is transfected into 293F cells (purchased from Invitrogen) for expression of SARS-CoV-2RBD-His protein.
The coding sequence of RBD protein of SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 (its amino acid sequence is shown as SEQ ID NO: 19) and the coding sequence of RBD protein of Omicron (B.1.1.529) subtype BA.2 (its amino acid sequence is shown as SEQ ID NO: 20) are respectively connected with the 3' -end of coding sequence of 6 histidine tags (hexa-His-tag) and translation termination codon TGA, and expressed by bac-to-bac baculovirus expression system (Invitrogen). The pFastbac1 plasmid containing the gene of interest was transformed into DH10Bac competent cells to produce recombinant bacmids. Transfection of recombinant bacmid into Sf9 cells for viral expansion and in Protein expression was performed in Hi5 cells. After 48h expression, hi5 cell supernatants were collected using a HisTrap TM excel (GE Healthcare) the soluble proteins were purified by nickel affinity chromatography.
Subjecting cell culture solution containing target protein to nickel ion affinity chromatography column HisTrap TM excel (GE Healthcare) and gel filtration chromatography Superdex TM After purification of 200Increase 10/300GL column (GE Healthcare), a purer target protein can be obtained. SDS-PAGE of SARS-CoV-2RBD-his protein, variant strain Omicron (B.1.1.529) subtype BA.1 RBD-his protein and Omicron (B.1.1.529) subtype BA.2 RBD-his protein identified about 30KD, as shown in FIGS. 5A-C, respectively.
Example 3: surface Plasmon Resonance (SPR) technology for detecting binding capability of antibodies to RBD protein of SARS-CoV-2 original strain and variant strain thereof
Surface plasmon resonance analysis was performed using Biacore 8K (Biacore inc.). The method comprises the following specific steps:
the single-chain nanobody R14 prepared in the above example and its constructs TR14 and MR14 were biotinylated, respectively, and then immobilized on Series Sensor Chip SA chip (Cytiva Life Sciences); with PBST buffer (2.7 mM KCl,137mM NaCl,4.3mM Na) 2 HPO 4 ，1.4mM KH 2 PO 4 0.05% tween) the RBD proteins of the SARS-CoV-2 original strain and its variant strain prepared in the above examples were diluted by multiple ratios, and loaded one by one onto the chip from low concentration to high concentration. The binding kinetics constants were calculated using BIAevaluation software K (Biacore, inc.) software. Equilibrium dissociation constant (K) between each antibody and each RBD D ) As shown in table 1, the results of table 1 indicate: both the single-chain nanobody R14 and the constructs TR14 and MR14 can be combined with RBD proteins of SARS-CoV-2 prototype strain and variant strains Omicron subtype BA.1 and Omicron subtype BA.2 with higher affinity.
TABLE 1 Single chain nanobody R14 and results of affinity between MR14 and its constructs TR14 and MR14 and RBD of SARS-CoV-2 original strain and variant strain
Example 4: packaging of SARS-CoV-2 original strain and variant strain pseudovirus
1) The genes encoding the 18 th amino acid after the S protein of SARS-CoV-2 original strain (WT) and variant strain Delta (B.1.617.2), omicron (B.1.1.529) subtype BA.1 and Omicron (B.1.1.529) subtype BA.2 are respectively removed, and the rest sequences of S protein are synthesized (the synthesis service is provided by Suzhou Jin Weizhi), so that the nucleotide sequences of SARS-CoV-2-WT-S-del18, B.1.617.2-S-del18, B.1.1.529-BA.1-S-del18 and B.1.1.529-BA.2-S-del18 genes are respectively shown as SEQ ID NO. 21-24.
2) Cloning the protein gene obtained in 1) onto pCAGGS vector to obtain expression plasmids pCAGGS-SARS-CoV-2-WT-S-del 18, pCAGGS-B.1.617.2-S-del18, pCAGGS-B.1.529-BA.1-S-del 18 and pCAGGS-B.1.1.529-BA.2-S-del18.
The packaging steps of the SARS-CoV-2 original strain and the variant strain pseudovirus are as follows:
a. cell preparation: HEK293T cells (purchased from ATCC CRL-3216) were plated in 10cm cell culture dishes to give a confluency of cells of about 80% the next day. The culture medium was DMEM medium containing 10% fbs.
b. Transfection: taking the expression plasmids of the S proteins in the step 2), transfecting 30 mug of plasmids/10 cm cell culture dishes by PEI, evenly mixing the target plasmids and PEI according to a ratio of 1:3, then transfecting, changing culture solution (DMEM culture medium containing 10% FBS) for 4-6 hours, and culturing at 37 ℃ for 24 hours.
c. And (3) adding poison: pseudovirus packaging backbone virus G VSV-delG (purchased from wu han kokumi brain science technologies limited) was added to HEK293T cells transfected as described above, incubated at 37 ℃ for 2h, medium was changed (DMEM medium containing 10% fbs), and VSV-G antibody (hybridoma cells expressing the antibody were purchased from ATCC cell bank) was added and culture was continued in an incubator for 30h.
d. And (3) toxin collection: the supernatant was collected and centrifuged at 3000rpm for 10min, filtered through a 0.45 μm sterile filter in an ultra-clean bench to remove cell debris, sub-packaged and frozen in a refrigerator at-80 ℃.
Pseudoviruses of the SARS-CoV-2 prototype strain (SARS-CoV-2 WT), variant strain Delta (B.1.617.2), omicron (B.1.1.529) subtype BA.1 and Omicron (B.1.1.529) subtype BA.2 were obtained, respectively.
Example 5: detection of antibody neutralization pseudovirus infection
The purified single-chain nanobody R14 and its constructs TR14 and MR14 (prepared in example 1) were diluted 5-fold to the 9 th gradient (2.56 pg/mL), respectively, and the dilutions were combined with 1.6X10 4 TCID 50 The pseudoviruses of the series of SARS-CoV-2 original and variant strains obtained in example 4 were mixed, respectively, and incubated at 37℃for 1 hour, and then added to a 96-well plate pre-inoculated with Vero cells (purchased from ATCC CCL 81). After 18-20 hours incubation, detection was by CQ1 Confocal Quantitative Image Cytometer (Yokogawa). The neutralizing capacity of the antibodies against the pseudoviruses of the series of SARS-CoV-2 prototype strains and variant strains Delta, BA.1 and BA.2 was calculated based on the number of cells with GFP fluorescence, the results are shown in FIGS. 6 to 9, and the statistics of the results are shown in Table 2; the results show that the pseudo-virus neutralization effect of both the constructs TR14 and MR14 is improved relative to the single chain nanobody R14.
TABLE 2 neutralization Capacity of Single-chain nanobody R14 and its constructs TR14 and MR14 on pseudoviruses of SARS-CoV-2 original strain and variant strain
Note that: IC (integrated circuit) 50 (μg/mL) is the half inhibitory concentration of the antibody.
Example 6: detection of antibody-neutralized live virus infection
In this example, the neutralizing effect of each antibody on live virus of the new coronavirus was determined by a live virus neutralization test based on cytopathic effect (CPE). The method comprises the following specific steps:
the purified single-chain nanobody R14 and TR14 and MR14 (prepared in example 1) were diluted 2-fold to 11 th gradient, 4 replicate wells per gradient, 50. Mu.L per well, respectively, and the dilutions were combined with an equal volume of 100 TCID 50 SARS-CoV-2 origin of (C)Strain or variant strain Delta, omicron subtype ba.1 was incubated at 37 ℃. 1. After hours, the mixture was added to the suspended Vero cells and incubation was continued for 3 days at 37 ℃. And observing and recording the pathological condition of the cells. Calculation of IC for nanobody and its constructs using GraphPad Prism 7.0 50 . Experiments were all performed in the biosafety three-level laboratory (BSL 3) of the chinese disease prevention control center.
The neutralization effect of the single-chain nano antibody R14 and TR14 and MR14 on the live viruses of the original strain and the variant strain of the novel coronavirus is shown in Table 3, and the results in Table 3 show that: TR14 and MR14 have good inhibition effect on live viruses of original strains and variant strains of the novel coronaviruses.
TABLE 3 neutralizing Capacity of Single-chain nanobody R14 and its constructs TR14 and MR14 against live virus of SARS-CoV-2 original strain and variant strain
And (3) injection: IC (integrated circuit) 50 (μg/mL) is the half inhibitory concentration of the antibody. # indicates that 100% inhibition was still achieved for live virus at the lowest concentration of 0.001. Mu.g/mL.
Example 7: detection of stability of antibodies before and after nebulization
Single-chain nanobody R14 and its constructs TR14 and MR14 were aerosolized using an Aerogen Solo (Aerogen Inc., chicago, USA) nebulizer, respectively, and the aerosolized antibodies were then collected using a full glass SKC (weight Fourier, pa., USA) containing 20mL PBS and subjected to a pseudovirus neutralization assay as described in example 5. The results are shown in FIG. 10, wherein A is the result of neutralizing activity of nanobody R14 against SARS-CoV-2 prototype strain pseudovirus before and after atomization, B is the result of neutralizing activity of nanobody construct TR14 against SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus, and C is the result of neutralizing activity of nanobody construct MR14 against SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus. The results in fig. 10 show that: the nanobody constructs TR14 and MR14 of the invention remain stable for the neutralizing activity of pseudoviruses of the novel coronavirus prototype strain or variant strain thereof before and after aerosolization, suggesting that they are both suitable for administration by the aerosolization route.
Example 8: determination of antibody half-life
By means of intraperitoneal injection, the half-life of nanobody R14 and its constructs TR14 and MR14 in blood was studied, and the procedure was as follows: female SPF grade BALB/c mice (purchased from Venetuno Liwa (Vital River)) from 6-8 weeks were anesthetized with isoflurane and 200 μl 2mg/mL (20 mg/kg body weight) of R14, TR14 and MR14, respectively, were slowly injected by intraperitoneal injection. Blood samples were collected by orbital blood sampling at 1 hour, 6 hours, 10 hours, 24 hours, 48 hours, 72 hours, and 96 hours, respectively, after injection. After centrifugation, the supernatant was taken and the concentration of antibodies in serum was determined by ELISA assay. Half-lives (t) of nanobody R14 and its constructs TR14 and MR14 were calculated using software PKSolver 1/2 ). The results of the half-lives of R14, TR14 and MR14 in blood are shown in fig. 11, and the results of fig. 11 show: constructs TR14 and MR14 exhibit significantly prolonged half-lives relative to the mono-valent antibody R14, which is advantageous for enhancing the antiviral effect of the antibody in vivo.
Next, again by means of nebulized administration, the residence time of nanobody R14 and its constructs TR14 and MR14 in the lung was investigated. Three antibodies R14, TR14 and MR14 were each aerosolized at 20mg/ml using an aerosolization system (as shown in FIG. 12), and after sustained exposure of the mice in the aerosolization chamber for 10 minutes, the mice were anesthetized with tribromoethanol by intraperitoneal injection, bronchoalveolar lavage fluid was collected at 0, 1, 6 and 24 hours, respectively, the antibody concentration in the lavage fluid was determined by ELISA, and the half-life of the nanobody in the lung was calculated using PKSolver (t 1/2 ). The results of the pulmonary half-life of R14, TR14 and MR14 are shown in fig. 13, and the results of fig. 13 show: constructs TR14 and MR14 exhibit significantly prolonged half-lives relative to monovalent antibody R14, which is advantageous for enhancing the antiviral effect of antibodies in vivo following administration via nebulized route.
The above results indicate that the nanobody R14-based constructs TR14 and MR14 of the invention have the potential to be developed into highly neutralizing active antibody drugs, in particular aerosolized drugs, for the treatment of novel coronaviruses and their variant strains of infection.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the 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 scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced equivalently; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
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Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ala Thr Pro Ala Thr Tyr Tyr Ser Gly Arg Tyr Tyr Tyr Gln Cys
100 105 110
Pro Ala Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln
145 150 155 160
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
165 170 175
Cys Ala Val Ser Gly Phe Thr Leu Asp Tyr Tyr Ala Ile Gly Trp Phe
180 185 190
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser Cys Ile Ser Ser
195 200 205
Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
210 215 220
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
225 230 235 240
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala Thr Pro Ala
245 250 255
Thr Tyr Tyr Ser Gly Arg Tyr Tyr Tyr Gln Cys Pro Ala Gly Gly Met
260 265 270
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly
275 280 285
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
290 295 300
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
305 310 315 320
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly
325 330 335
Phe Thr Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly
340 345 350
Lys Glu Arg Glu Gly Val Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr
355 360 365
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
370 375 380
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
385 390 395 400
Thr Ala Leu Tyr Tyr Cys Ala Ala Thr Pro Ala Thr Tyr Tyr Ser Gly
405 410 415
Arg Tyr Tyr Tyr Gln Cys Pro Ala Gly Gly Met Asp Tyr Trp Gly Gln
420 425 430
Gly Thr Gln Val Thr Val Ser Ser
435 440
<210> 10
<211> 349
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Polypeptide
<220>
<221> MISC_FEATURE
<223> Fc fragment of human IgM
<400> 10
Val Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg
1 5 10 15
Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala
20 25 30
Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly
35 40 45
Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala
50 55 60
Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile
65 70 75 80
Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp
85 90 95
His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro
100 105 110
Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala
115 120 125
Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp
130 135 140
Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly
145 150 155 160
Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala
165 170 175
Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn
180 185 190
Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser
195 200 205
Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val Ala Leu His Arg
210 215 220
Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg
225 230 235 240
Glu Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp
245 250 255
Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys
260 265 270
Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr
275 280 285
Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp Asn Thr Gly
290 295 300
Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val
305 310 315 320
Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn
325 330 335
Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr
340 345
<210> 11
<211> 479
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Polypeptide
<220>
<221> MISC_FEATURE
<223> amino acid sequence of nanobody fusion protein
<400> 11
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ala Thr Pro Ala Thr Tyr Tyr Ser Gly Arg Tyr Tyr Tyr Gln Cys
100 105 110
Pro Ala Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
Ser Ser Val Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro
130 135 140
Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys
145 150 155 160
Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg
165 170 175
Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala
180 185 190
Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu
195 200 205
Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg
210 215 220
Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys
225 230 235 240
Val Pro Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser
245 250 255
Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val
260 265 270
Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln
275 280 285
Asn Gly Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro
290 295 300
Asn Ala Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp
305 310 315 320
Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu
325 330 335
Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val Ala Leu
340 345 350
His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn
355 360 365
Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser Pro
370 375 380
Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro
385 390 395 400
Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly
405 410 415
Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp Asn
420 425 430
Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn
435 440 445
Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu
450 455 460
Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr
465 470 475
<210> 12
<211> 1320
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> coding sequence of trivalent nanobody
<400> 12
caagtgcaac tgcaggagag cggcggaggc ctggtccaac ctggcggcag cctgcggctg 60
tcttgtgctg tgtctggatt caccctggat tactatgcca tcggctggtt tagacaggcc 120
cctggcaagg aacgggaagg cgttagctgc atcagctctt ccgacggctc taccagctac 180
gctgattctg tgaagggccg cttcacaatc agcagagata atgccaaaaa cacggtgtac 240
ctgcagatga acagcctgaa gcccgaggac accgccctgt actattgcgc tgccacaccc 300
gccacctact acagcggcag atactactat cagtgtcctg ccggaggcat ggattactgg 360
ggacagggca cccaggtgac agtgagcagc ggaggaggcg gcagcggcgg aggcggcagt 420
ggtggcggcg gatccggcgg cggaggcagc ggcggcgggg gcagccaggt gcagctgcag 480
gagagcggcg gcggcctggt gcagcctgga ggcagcctga gactgagctg tgccgtgtcc 540
ggtttcaccc tggactacta cgccattgga tggttcagac aggctccagg caaggaaaga 600
gaaggcgtgt cctgtatcag ctcttctgat ggatctacat cttacgccga cagcgtgaag 660
ggcaggttca ccatctccag agacaatgcc aagaacaccg tgtacctgca gatgaacagc 720
ctgaaacctg aggataccgc actttattac tgcgccgcca cccctgctac atactacagc 780
ggaagatact actaccagtg ccccgccggc ggcatggact actggggcca gggcacccag 840
gtcacagtga gcagcggcgg cggcggctcc ggcggaggcg gctctggtgg cggcggaagc 900
ggaggcggag gcagcggcgg cggaggctct caggtgcagc tgcaggagtc cggcggcggg 960
ctggtgcagc caggcggcag cctgagactg agctgcgccg tgtctggctt tacactggac 1020
tactacgcca tcggctggtt ccggcaggcc cctggcaaag agcgggaagg cgtgtcttgc 1080
atcagcagca gcgacggcag caccagctac gccgacagcg tcaagggaag attcaccatc 1140
tcccgggaca acgccaagaa cacagtgtac ctgcaaatga acagcctcaa gcccgaggac 1200
accgccctgt actactgcgc cgctacccct gccacatact actctggcag atactactac 1260
cagtgccctg ccggcggcat ggactactgg ggccagggca cacaggtgac cgtgtccagc 1320
<210> 13
<211> 1437
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> coding sequence of nanobody fusion protein
<400> 13
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag tctctggatt tactttggat tattatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaggg ggtctcatgt attagtagta gtgatggtag cacatcgtat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaaaaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acagcccttt attactgtgc agcaacccct 300
gctacatact atagtggacg ttactactac caatgtcccg cggggggcat ggactactgg 360
ggccagggga cccaggtgac cgtgagctct gtgatcgccg agctgccccc caaggtgagc 420
gtgttcgtgc cccctagaga cggcttcttc ggcaacccta gaaagagcaa gctgatctgc 480
caagccaccg gcttctcccc tagacagatc caagtgagct ggctgagaga gggcaagcaa 540
gtgggcagcg gcgtcacaac agaccaagtg caagccgagg ccaaggagag cggccccacc 600
acctacaagg tgacaagcac cctgaccatc aaggagagcg actggctggg gcagagcatg 660
ttcacctgca gagtggacca cagaggcctg acctttcagc agaacgctag cagcatgtgc 720
gtgcccgacc aagacaccgc catcagagtg ttcgccatcc cccctagctt cgctagcatc 780
ttcctgacca agagcaccaa gctgacctgc ctcgtgaccg atctgaccac ctacgacagc 840
gtgaccatca gctggacaag acagaacggc gaggccgtga agacccacac caacatcagc 900
gagagccacc ccaacgccac cttcagcgcc gtgggcgagg ctagcatctg cgaggacgac 960
tggaacagcg gcgagagatt cacctgcacc gtgacccaca ccgacctgcc tagccccctg 1020
aagcagacca tcagcagacc caagggcgtg gccctgcaca gacccgacgt gtacctgctg 1080
ccccccgcta gagagcagct gaacctgaga gagagcgcca ccatcacctg cctggtgacc 1140
ggctttagcc ccgctgacgt gttcgtgcag tggatgcaga gagggcagcc cctgagcccc 1200
gagaagtacg tgacaagcgc ccccatgccc gagccccaag cccccggcag atacttcgcc 1260
cacagcatcc tgaccgtgag cgaggaagag tggaacaccg gcgagaccta cacctgcgtg 1320
gtggcccacg aggccctgcc caacagagtg accgagagaa ccgtggacaa gagcaccggc 1380
aagcccaccc tgtacaacgt gagcctggtg atgagcgaca ccgccggcac ctgctac 1437
<210> 14
<211> 390
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> coding sequence of nanobody R14 VHH chain
<400> 14
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag tctctggatt tactttggat tattatgcca taggctggtt ccgccaggcc 120
ccagggaagg agcgtgaggg ggtctcatgt attagtagta gtgatggtag cacatcgtat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaaaaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acagcccttt attactgtgc agcaacccct 300
gctacatact atagtggacg ttactactac caatgtcccg cggggggcat ggactactgg 360
ggcaaaggga cccaggtgac cgtgagctct 390
<210> 15
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Polypeptide
<220>
<221> MISC_FEATURE
<223> Signal peptide sequence
<400> 15
Ala Thr Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr
1 5 10 15
Gly Val Arg Ala
20
<210> 16
<211> 1047
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> coding sequence of Fc of human IgM antibody
<400> 16
gtgatcgccg agctgccccc caaggtgagc gtgttcgtgc cccctagaga cggcttcttc 60
ggcaacccta gaaagagcaa gctgatctgc caagccaccg gcttctcccc tagacagatc 120
caagtgagct ggctgagaga gggcaagcaa gtgggcagcg gcgtcacaac agaccaagtg 180
caagccgagg ccaaggagag cggccccacc acctacaagg tgacaagcac cctgaccatc 240
aaggagagcg actggctggg gcagagcatg ttcacctgca gagtggacca cagaggcctg 300
acctttcagc agaacgctag cagcatgtgc gtgcccgacc aagacaccgc catcagagtg 360
ttcgccatcc cccctagctt cgctagcatc ttcctgacca agagcaccaa gctgacctgc 420
ctcgtgaccg atctgaccac ctacgacagc gtgaccatca gctggacaag acagaacggc 480
gaggccgtga agacccacac caacatcagc gagagccacc ccaacgccac cttcagcgcc 540
gtgggcgagg ctagcatctg cgaggacgac tggaacagcg gcgagagatt cacctgcacc 600
gtgacccaca ccgacctgcc tagccccctg aagcagacca tcagcagacc caagggcgtg 660
gccctgcaca gacccgacgt gtacctgctg ccccccgcta gagagcagct gaacctgaga 720
gagagcgcca ccatcacctg cctggtgacc ggctttagcc ccgctgacgt gttcgtgcag 780
tggatgcaga gagggcagcc cctgagcccc gagaagtacg tgacaagcgc ccccatgccc 840
gagccccaag cccccggcag atacttcgcc cacagcatcc tgaccgtgag cgaggaagag 900
tggaacaccg gcgagaccta cacctgcgtg gtggcccacg aggccctgcc caacagagtg 960
accgagagaa ccgtggacaa gagcaccggc aagcccaccc tgtacaacgt gagcctggtg 1020
atgagcgaca ccgccggcac ctgctac 1047
<210> 17
<211> 477
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> coding sequence of J chain
<400> 17
atgaagaacc acctgctgtt ctggggcgtg ctggccgtgt tcatcaaggc cgtgcacgtg 60
aaggcccaag aggacgagag aatcgtgctg gtggacaaca agtgcaagtg cgctagaatc 120
acaagcagaa tcatcagaag cagcgaggac cccaacgagg acatcgtgga gagaaacatc 180
agaatcatcg tgcccctgaa caacagagag aacatcagcg accccacaag ccccctgaga 240
acaagattcg tgtaccacct gagcgacctg tgcaagaagt gcgaccccac cgaggtggag 300
ctggacaatc agatcgtgac cgccacacag agcaacatct gcgacgagga cagcgccacc 360
gagacctgct acacctacga cagaaacaag tgctacaccg ccgtggtgcc cctggtgtac 420
ggcggcgaga ccaagatggt ggagaccgcc ctgacccccg acgcctgcta ccccgac 477
<210> 18
<211> 238
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Polypeptide
<220>
<221> MISC_FEATURE
<223> amino acid sequence of RBD protein of SARS-CoV-2 original Strain
<400> 18
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg
1 5 10 15
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
20 25 30
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
35 40 45
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
50 55 60
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
65 70 75 80
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
85 90 95
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
100 105 110
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
115 120 125
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
130 135 140
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
145 150 155 160
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
165 170 175
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
180 185 190
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
195 200 205
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
210 215 220
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
225 230 235
<210> 19
<211> 261
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Polypeptide
<220>
<221> MISC_FEATURE
<223> amino acid sequence of RBD protein of variant strain Omicron (B.1.1.529) subtype BA.1
<400> 19
Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr
1 5 10 15
Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala
20 25 30
Ala His Ser Ala Phe Ala Arg Val Gln Pro Thr Glu Ser Ile Val Arg
35 40 45
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala
50 55 60
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
65 70 75 80
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr
85 90 95
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
100 105 110
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
115 120 125
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
130 135 140
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys
145 150 155 160
Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
165 170 175
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
180 185 190
Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys
195 200 205
Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly
210 215 220
His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
225 230 235 240
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
245 250 255
Lys Cys Val Asn Phe
260
<210> 20
<211> 261
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Polypeptide
<220>
<221> MISC_FEATURE
<223> amino acid sequence of RBD protein of variant strain Omicron (B.1.1.529) subtype BA.2
<400> 20
Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr
1 5 10 15
Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala
20 25 30
Ala His Ser Ala Phe Ala Arg Val Gln Pro Thr Glu Ser Ile Val Arg
35 40 45
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala
50 55 60
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
65 70 75 80
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Phe Ala Pro Phe Phe Ala
85 90 95
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
100 105 110
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asn Glu Val Ser
115 120 125
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
130 135 140
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys
145 150 155 160
Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
165 170 175
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
180 185 190
Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys
195 200 205
Tyr Phe Pro Leu Arg Ser Tyr Gly Phe Arg Pro Thr Tyr Gly Val Gly
210 215 220
His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
225 230 235 240
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
245 250 255
Lys Cys Val Asn Phe
260
<210> 21
<211> 3789
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> nucleotide sequence of SARS-CoV-2-WT-S-del18 Gene
<400> 21
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc aatgcgtgaa cctgaccaca 60
agaacacagc tgccccccgc ctacaccaac agcttcacaa gaggcgtgta ctaccccgac 120
aaggtgttca gaagcagcgt cctccacagc acccaagacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catcagcggc accaacggca ccaagagatt cgacaacccc 240
gtgctgccct tcaacgacgg cgtgtacttc gctagcaccg agaagagcaa catcatcaga 300
ggctggatct tcggcaccac cctggacagc aaaacacaga gcctgctgat cgtgaacaac 360
gccacaaacg tggtgatcaa ggtgtgcgag tttcagttct gcaacgaccc cttcctgggc 420
gtgtaccaca agaacaacaa gagctggatg gagagcgagt tccgggtgta cagcagcgcc 480
aacaactgca ccttcgagta cgtgagccaa cccttcctga tggacctgga gggcaagcaa 540
ggcaatttta agaacctgag agagttcgtg ttcaagaaca tcgacggcta cttcaagatc 600
tacagcaagc acacccccat caacctggtg agagacctgc cccaaggctt cagcgccctg 660
gagcccctgg tggacctgcc catcggcatc aacatcacaa gatttcagac cctgctggcc 720
ctgcacagaa gctatctgac ccccggcgac agcagcagcg gctggaccgc cggcgccgcc 780
gcttactacg tgggctacct gcagcctaga accttcctgc tgaagtacaa cgagaacggc 840
acaatcaccg acgccgtcga ctgcgccctg gaccccctga gcgagaccaa gtgcaccctg 900
aagagcttca ccgtggagaa gggcatctat cagacaagca acttcagagt gcagcccacc 960
gagagcatcg tgagattccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 1020
gccacaagat tcgctagcgt gtacgcctgg aacagaaaga gaatcagcaa ctgcgtggcc 1080
gactacagcg tgctgtacaa cagcgctagc ttcagcacct tcaagtgcta cggcgtcagc 1140
cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcaga 1200
ggcgacgagg tgagacagat cgcccccggg cagaccggca agatcgccga ctacaactac 1260
aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggactcc 1320
aaggtgggcg gcaactacaa ctacctgtac agactgttca gaaagagcaa cctgaagccc 1380
ttcgagagag acatcagcac cgagatctac caagccggca gcaccccctg caacggcgtg 1440
gagggcttca actgctactt ccccctgcag agctacggct ttcagcccac ctacggcgtg 1500
ggctatcagc cctacagagt ggtcgtgctg agcttcgagc tgctgcacgc ccccgccacc 1560
gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttcaacttc 1620
aacggcctca ccgggaccgg cgtgctgacc gagagcaaca agaagttcct gcctttccaa 1680
cagttcggca gagacatcga cgacaccacc gacgccgtca gagaccctca gaccctggag 1740
atcctggaca tcacaccctg cagcttcggc ggcgtgagcg tgatcacccc cggcaccaac 1800
acaagcaacc aagtggccgt gctgtaccaa ggcgtgaact gcaccgaggt gcccgtggcc 1860
atccacgccg atcagctgac ccccacctgg agagtgtaca gcaccggcag caacgtgttt 1920
cagacaagag ccggctgcct gatcggcgcc gagcacgtga acaacagcta cgagtgcgac 1980
atccccatcg gcgccggcat ctgcgctagc tatcagacac agaccaacag ccacagaaga 2040
gctagaagcg tggctagcca aagcatcatc gcctacacca tgagcctggg cgccgagaac 2100
agcgtggcct acagcaacaa cagcatcgcc atccccacca acttcaccat cagcgtgacc 2160
accgaaatcc tgcctgtgag catgaccaag acaagcgtgg actgcaccat gtacatctgc 2220
ggcgacagca ccgagtgcag caacctgctc ctgcagtacg gcagcttctg cattcagctg 2280
aacagagccc tgaccggcat cgccgtggag caagacaaga acacccaaga ggtgttcgcc 2340
caagtgaagc agatctacaa gacccccccc atcaaggact tcggcggctt caacttcagc 2400
caaatcctgc ctgaccctag caagcctagc aagagaagct tcatcgagga cctgctgttc 2460
aacaaggtga ccctggccga cgccggcttc atcaagcagt acggcgactg cctgggcgac 2520
atcgccgcta gagacctgat ctgcgctcag aagttcaacg gcctgaccgt gctgcccccc 2580
ctgctgaccg acgagatgat cgctcagtac acaagcgccc tgctcgctgg caccatcaca 2640
agcgggtgga ccttcggcgc cggggccgcc ctgcagatcc ccttcgccat gcagatggcc 2700
tacagattca acggcatcgg cgtgacacag aacgtgctgt acgagaatca gaagctgatc 2760
gccaatcagt tcaacagcgc catcggcaag atccaagaca gcctgagcag caccgctagc 2820
gccctgggca agctgcaaga cgtggtgaat cagaacgccc aagccctgaa caccctggtg 2880
aagcagctga gcagcaactt cggcgccatc agcagcgtgc tgaacgacat cctggctaga 2940
ctggacaagg tggaggccga ggtgcagatc gatagactga tcaccggcag actgcagagc 3000
ctgcagacct acgtgacaca gcagctgatc agagccgccg agatcagagc tagcgccaac 3060
ctggccgcca ccaagatgag cgagtgcgtg ctggggcaga gcaagagagt ggacttctgc 3120
ggcaagggct accacctgat gagcttccct cagagcgccc cccacggcgt ggtgttcctg 3180
cacgtgacct acgtgcccgc ccaagagaag aacttcacca ccgcccccgc catctgccac 3240
gacggcaagg cccacttccc tagagagggc gtgttcgtga gcaacggcac ccactggttc 3300
gtgacacaga gaaacttcta cgagcctcag atcatcacca cccacaacac cttcgtgagc 3360
ggcaactgcg acgtggtgat cggcatcgtg aacaacaccg tgtacgaccc tctgcagccc 3420
gagctggaca gcttcaagga ggagctggac aagtacttca agaaccacac aagccccgac 3480
gtggacctgg gcgacatcag cgggatcaac gctagcgtgg tgaacattca gaaggaaatc 3540
gacagactga atgaggtggc caagaacctg aacgagagcc tgatcgacct gcaagagctg 3600
ggcaagtacg agcagtacat caagtggccc tggtacatct ggctgggctt catcgccggc 3660
ctgatcgcca tcgtgatggt gaccatcatg ctgtgctgca tgacaagctg ctgctcctgt 3720
ctgaaggggt gctgcagctg cggcagctgc tgcaaggact acaaggacga tgacgacaag 3780
ggcccctga 3789
<210> 22
<211> 3792
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> nucleotide sequence of B.1.617.2-S-del18 Gene
<400> 22
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgagaaca 60
cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120
aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180
aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac 240
aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300
atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360
aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420
ctggacgtgt actaccacaa gaacaacaag tcttggatgg agtctggcgt gtactctagc 480
gccaacaact gcaccttcga gtacgtgtcc cagcctttcc tcatggacct ggagggcaag 540
cagggcaact tcaagaacct gagagagttc gtgttcaaga acattgacgg ctacttcaag 600
atttactcta agcacacccc aattaacctc gtgagggacc tccctcaggg cttctccgtg 660
ttagaaccac tggtggacct ccctattggc attaacatca cacgcttcca gacactgctc 720
gccctccacc ggtcttacct gaccccaggc gactctagct ctggctggac agccggcgcc 780
gccgcctact acgtgggcta cctgcagcct aggaccttcc tcctgaagta caacgagaac 840
ggcacaatta ccgacgccgt ggactgcgcc ctggacccac tgtccgagac aaagtgcaca 900
ctgaagtcct tcacagtgga gaagggcatt taccagacat ctaacttccg ggtgcagcct 960
acagagtcta ttgtgcggtt cccaaacatc acaaacctgt gccctttcgg cgaggtgttc 1020
aacgccaccc ggttcgcctc tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 1080
gccgactact ccgtgctgta caactccgcc tctttctcta cattcaagtg ctacggcgtg 1140
tcccctacaa agctgaacga cctgtgcttc accaacgtgt acgccgactc tttcgtgatt 1200
agaggcgacg aggtgaggca gattgccccc ggccagacag gcaagatcgc cgactacaac 1260
tacaagctgc ccgacgactt cacaggctgc gtgatcgcct ggaactctaa caacctggac 1320
tctaaggtgg gcggcaacta caactacaga tacagactgt tccggaagtc taacctgaag 1380
ccattcgaga gggacattag caccgagatt taccaggccg gctctaagcc atgcaacggc 1440
gtggagggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacaaacggc 1500
gtgggctacc agccttaccg ggtggtggtg ctgtctttcg agctgctcca cgcccccgcc 1560
acagtgtgcg gcccaaagaa gagcacaaac ctcgtgaaga acaagtgcgt gaacttcaac 1620
ttcaacggcc tcacaggcac aggcgtgctc accgagtcta acaagaagtt cctccctttc 1680
cagcagttcg gccgcgacat tgccgacacc accgacgccg tgcgggaccc tcagacactg 1740
gaaattctcg acatcacccc ttgcagcttc ggcggcgtgt ccgtgatcac cccaggcaca 1800
aacacatcta accaggtggc cgtgctgtac cagggcgtga actgcaccga ggtgccagtg 1860
gccatccacg ccgaccagct caccccaaca tggagggtgt acagcacagg ctctaacgtg 1920
ttccagaccc gggccggctg cctcattggc gccgagcacg tgaacaactc ttacgagtgc 1980
gacatcccta ttggcgccgg catttgcgcc tcttaccaga cccagacaaa ctctagacgg 2040
agagcccggt ctgtggcctc tcagagcatt attgcctaca ccatgtctct gggcgccgag 2100
aactctgtgg cctactctaa caactctatt gccatcccta caaacttcac aatttctgtg 2160
accaccgaga ttctcccagt gtctatgacc aagacatctg tggactgcac catgtacatt 2220
tgcggcgact ccaccgagtg ctctaacctc ctgctccagt acggctcttt ctgcacccag 2280
ctcaaccgcg ccctgacagg catcgccgtg gagcaggaca agaacaccca ggaggtgttc 2340
gcccaggtga agcagattta caagaccccc ccaattaagg acttcggcgg cttcaacttc 2400
tctcagattc tccccgaccc atccaagcct agcaagcggt ccttcattga ggacctcctg 2460
ttcaacaagg tgacactggc cgacgccggc ttcattaagc agtacggcga ctgcctgggc 2520
gacattgccg cccgggacct gatttgcgcc cagaagttca acggcctcac agtgctcccc 2580
ccactgctca ccgacgagat gattgcccag tacacatctg ccctcctggc cggcacaatt 2640
acatctggct ggaccttcgg cgccggcgcc gccctgcaga tccctttcgc catgcagatg 2700
gcctaccgct tcaacggcat cggcgtgaca cagaacgtgc tgtacgagaa ccagaagctg 2760
atcgccaacc agttcaacag cgccattggc aagattcagg actctctgag cagcacagcc 2820
agcgccctgg gcaagctgca gaacgtggtg aaccagaacg cccaggccct gaacacactg 2880
gtgaagcagc tgtcttctaa cttcggcgcc atttctagcg tgctgaacga cattctgtcg 2940
cggctggaca aggtggaggc cgaggtgcag attgacaggc tcatcacagg cagactgcag 3000
tctctgcaga catacgtgac ccagcagctg attagagccg ccgagattag agcctccgcc 3060
aacctggccg ccaccaagat gagcgagtgc gtgctcggcc agtctaagcg ggtggacttc 3120
tgcggcaagg gctaccacct catgtctttc cctcagtccg cccctcacgg cgtggtgttc 3180
ctccacgtga catacgtgcc cgcccaggag aagaacttca ccacagcccc cgccatttgc 3240
cacgacggca aggcccactt ccctagggag ggcgtgttcg tgtctaacgg cacccactgg 3300
ttcgtgaccc agcggaactt ctacgagcct cagattatta ccacagacaa cacattcgtg 3360
agcggcaact gcgacgtggt gattggcatt gtgaacaaca cagtgtacga cccactgcag 3420
cctgagttgg actctttcaa ggaggaactc gacaagtact tcaagaacca cacatctcct 3480
gacgtggacc tgggcgacat tagcggcatt aacgcctctg tggtgaacat tcagaaggag 3540
attgacagac tgaacgaggt ggccaagaac ctgaacgagt ctctcattga cctgcaggag 3600
ctgggcaagt acgagcagta cattaagtgg ccttggtaca tttggctggg cttcattgcc 3660
ggcctgatcg ccattgtgat ggtgaccatc atgctgtgct gcatgacatc ttgctgcagc 3720
tgcctgaagg gctgctgctc ttgcggctct tgctgcaagg actacaagga cgacgatgac 3780
aagggacctt aa 3792
<210> 23
<211> 3795
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> nucleotide sequence of B.1.1.529-BA.1-S-del18 Gene
<400> 23
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60
cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120
aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180
aacgtgacat ggttccacgt gatctctggc acaaacggca caaagcggtt cgacaacccc 240
gtgctccctt tcaacgacgg cgtgtacttc gccagcattg agaagtctaa cattatccgg 300
ggctggattt tcggcaccac actcgactct aagacacagt ccctcctgat tgtgaacaac 360
gccacaaacg tggtgattaa ggtgtgcgag ttccagttct gcaacgaccc tttcctggac 420
cacaagaaca acaagtcttg gatggagtct gagttcagag tgtactctag cgccaacaac 480
tgcaccttcg agtacgtgtc ccagcctttc ctcatggacc tggagggcaa gcagggcaac 540
ttcaagaacc tgagagagtt cgtgttcaag aacattgacg gctacttcaa gatttactct 600
aagcacaccc caattattgt gagggaacca gaagacctcc ctcagggctt ctccgcctta 660
gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720
ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780
gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840
acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900
aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960
gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020
gccacccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080
gactactccg tgctgtacaa cctggcccct ttcttcacat tcaagtgcta cggcgtgtcc 1140
cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200
ggcgacgagg tgaggcagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260
aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320
aaggtgtctg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380
ttcgagaggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440
gccggcttca actgctactt cccactgcgc tcctactcct tccggcctac atacggcgtg 1500
ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560
gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620
aacggcctca agggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680
cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740
attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800
acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860
atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920
caaacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980
atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040
gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100
tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160
accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220
ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280
aagcgcgccc tgacaggcat cgccgtggag caggacaaga acacccagga ggtgttcgcc 2340
caggtgaagc agatttacaa gaccccccca attaagtact tcggcggctt caacttctct 2400
cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460
aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520
attgccgccc gggacctgat ttgcgcccag aagttcaagg gcctcacagt gctcccccca 2580
ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640
tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700
taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760
gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820
gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880
aagcagctgt cttctaagtt cggcgccatt tctagcgtgc tgaacgacat tttctcgcgg 2940
ctggacaagg tggaggccga ggtgcagatt gacaggctca tcacaggcag actgcagtct 3000
ctgcagacat acgtgaccca gcagctgatt agagccgccg agattagagc ctccgccaac 3060
ctggccgcca ccaagatgag cgagtgcgtg ctcggccagt ctaagcgggt ggacttctgc 3120
ggcaagggct accacctcat gtctttccct cagtccgccc ctcacggcgt ggtgttcctc 3180
cacgtgacat acgtgcccgc ccaggagaag aacttcacca cagcccccgc catttgccac 3240
gacggcaagg cccacttccc tagggagggc gtgttcgtgt ctaacggcac ccactggttc 3300
gtgacccagc ggaacttcta cgagcctcag attattacca cagacaacac attcgtgagc 3360
ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420
gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480
gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540
gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600
ggcaagtacg agcagtacat taagtggcct tggtacattt ggctgggctt cattgccggc 3660
ctgatcgcca ttgtgatggt gaccatcatg ctgtgctgca tgacatcttg ctgcagctgc 3720
ctgaagggct gctgctcttg cggctcttgc tgcaaggact acaaggacga cgatgacaag 3780
ggaccttaac tcgag 3795
<210> 24
<211> 2913
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> nucleotide sequence of B.1.1.529-BA.2-S-del18 Gene
<400> 24
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgatcaca 60
cggacccaga gctacacaaa ctctttcacc cggggcgtgt actaccccga caaggtgttc 120
cggtctagcg tgctccactc tacacaggac ctgttcctcc ctttcttcag caacgtgaca 180
tggttccacg ccatccacgt gtctggcaca aacggcacaa agcggttcga caaccccgtg 240
ctccctttca acgacggcgt gtacttcgcc agcaccgaga agtctaacat tatccggggc 300
tggattttcg gcaccacact cgactctaag acacagtccc tcctgattgt gaacaacgcc 360
acaaacgtgg tgattaaggt gtgcgagttc cagttctgca acgacccttt cctggacgtg 420
tactaccaca agaacaacaa gtcttggatg gagtctgagt tcagagtgta ctctagcgcc 480
aacaactgca ccttcgagta cgtgtcccag cctttcctca tggacctgga gggcaagcag 540
ggcaacttca agaacctgag agagttcgtg ttcaagaaca ttgacggcta cttcaagatt 600
tactctaagc acaccccaat taacctcggc agggacctcc ctcagggctt ctccgcctta 660
gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720
ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780
gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840
acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900
aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960
gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020
gccacccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080
gactactccg tgctgtacaa cttcgccccc ttcttcgcct tcaagtgcta cggcgtgtcc 1140
cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200
ggcaacgagg tgagccagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260
aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320
aaggtgggcg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380
ttcgagaggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440
gccggcttca actgctactt cccactgcgg tcctacggct tccggcctac atacggcgtg 1500
ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560
gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620
aacggcctca caggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680
cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740
attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800
acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860
atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920
cagacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980
atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040
gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100
tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160
accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220
ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280
aagcgcgccc tgacaggcat cgccgtggag caggacaaga acacccagga ggtgttcgcc 2340
caggtgaagc agatttacaa gaccccccca attaagtact tcggcggctt caacttctct 2400
cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460
aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520
attgccgccc gggacctgat ttgcgcccag aagttcaacg gcctcacagt gctcccccca 2580
ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640
tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700
taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760
gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820
gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880
aagcagctgt cttctaagtt cggcgccatt agc 2913

Claims (17)

1. A multivalent nanobody comprising two or more VHH chains of nanobody that specifically bind SARS-CoV-2RBD, wherein the VHH chains of nanobody that specifically bind SARS-CoV-2RBD comprise the following CDRs: CDR1 with the amino acid sequence shown as SEQ ID NO. 1, CDR2 with the amino acid sequence shown as SEQ ID NO. 2, and CDR3 with the amino acid sequence shown as SEQ ID NO. 3.
2. The multivalent nanobody of claim 1, wherein the VHH chain of the nanobody that specifically binds SARS-CoV-2RBD further comprises 4 framework regions FR1-4, the FR1-4 being staggered in sequence with the CDR1, CDR2 and CDR 3;
preferably, the amino acid sequences of the FR1-4 are shown as SEQ ID NOs 4, 5, 6 and 7, respectively.
3. The multivalent nanobody of claim 1, wherein the VHH chain of the nanobody that specifically binds SARS-CoV-2RBD has an amino acid sequence as shown in SEQ ID No. 8, or an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No. 8.
4. A multivalent nanobody according to any of claims 1-3, which consists of more than two, preferably three, VHH chains of said nanobody specifically binding SARS-CoV-2RBD, connected by Linker;
Wherein the Linker is (GGGGS) n, wherein n=1, 2,3, or 4, preferably n=2 or 3;
preferably, the multivalent nanobody is a trivalent nanobody, and has an amino acid sequence shown as SEQ ID NO. 9.
5. A multivalent nanobody according to any of claims 1-3, wherein the multivalent nanobody is an IgM pentamer formed from a fusion protein having the structure from N-terminus to C-terminus as shown in formula (I):
A-L-B (I)
wherein,,
a is a single VHH chain of the nanobody that specifically binds SARS-CoV-2RBD, or a multivalent nanobody according to claim 4;
b is an Fc fragment of human IgM; preferably, the Fc fragment of said humanized IgM has an amino acid sequence as set forth in SEQ ID NO. 10, or an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 10;
l is (GGGGS) m, wherein m = 0,1,2,3, or 4;
preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO. 11.
6. A nanobody fusion protein, which is characterized in that the structure from N end to C end of the nanobody fusion protein is shown as a formula (I):
A-L-B (I)
Wherein,,
a is a single VHH chain of a nanobody that specifically binds SARS-CoV-2RBD, said VHH chain of a nanobody that specifically binds SARS-CoV-2RBD being as defined in any one of claims 1-3, or a is a multivalent nanobody according to claim 4;
b is an Fc fragment of human IgM; preferably, the Fc fragment of said humanized IgM has an amino acid sequence as set forth in SEQ ID NO. 9, or an amino acid sequence having at least 95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 9;
l is (GGGGS) m, wherein m = 0,1,2,3, or 4;
preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO. 11.
7. A polynucleotide encoding the multivalent nanobody of any one of claims 1 to 5, or encoding the nanobody fusion protein of claim 6.
8. The polynucleotide of claim 7, wherein the polynucleotide is DNA or mRNA;
preferably, the polynucleotide encodes a multivalent nanobody as claimed in claim 4, further preferably, the polynucleotide comprises a nucleotide sequence as shown in SEQ ID No. 12;
preferably, the polynucleotide encodes a nanobody fusion protein according to claim 6, further preferably, the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO. 13.
9. A nucleic acid construct comprising the polynucleotide of claim 7 or 8, and optionally, at least one expression regulatory element operably linked to the polynucleotide.
10. An expression vector comprising the nucleic acid construct of claim 9.
11. A transformed cell comprising the polynucleotide of claim 7 or 8, the nucleic acid construct of claim 9, or the expression vector of claim 10.
12. A pharmaceutical composition comprising the multivalent nanobody of any of claims 1 to 5, nanobody fusion protein of claim 6, polynucleotide of claim 7 or 8, nucleic acid construct of claim 9, expression vector of claim 10 or transformed cell of claim 11, and pharmaceutically acceptable carrier and/or excipient.
13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation;
preferably, the nasal spray is selected from the group consisting of aerosols, sprays and powder sprays;
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, an ointment, a plaster, a topical liquid, an injectable or a bolus formulation.
14. Use of a multivalent nanobody according to any one of claims 1 to 5, a nanobody fusion protein according to claim 6, a polynucleotide according to claim 7 or 8, a nucleic acid construct according to claim 9, an expression vector according to claim 10, a transformed cell according to claim 11 or a pharmaceutical composition according to claim 12 or 13 for the preparation of a medicament for the prevention and/or treatment of novel coronavirus infections.
15. Use of a multivalent nanobody according to any one of claims 1 to 5, a nanobody fusion protein according to claim 6, a polynucleotide according to claim 7 or 8, a nucleic acid construct according to claim 9, an expression vector according to claim 10 or a transformed cell according to claim 11 for the preparation of a reagent or kit for detecting a novel coronavirus or for diagnosing a novel coronavirus infection.
16. The use according to claim 14 or 15, wherein the novel coronavirus is a SARS-CoV-2 original strain and/or a SARS-CoV-2 variant strain;
Preferably, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1), delta (B.1.617.2), omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain, more preferably Delta (B.1.617.2) strain, omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain.
17. A novel coronavirus detection kit comprising the multivalent nanobody of any one of claims 1 to 5, nanobody fusion protein of claim 6, polynucleotide of claim 7 or 8, nucleic acid construct of claim 9, expression vector of claim 10 or transformed cell of claim 11.
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