Constructs of nanobody r14 and uses thereof

Abstract

The application provides a construct based on a nanobody R14 specifically binding to SARS-CoV-2 RBD (including a multivalent nanobody and a nanobody fusion protein), a related product thereof and application thereof; the construct based on the nanobody R14 specifically binding to SARS-CoV-2 RBD (including the multivalent nanobody and the nanobody fusion protein) can effectively inhibit SARS-CoV-2 infection and infection of a variant strain thereof, can be administered in a nebulization mode, can directly reach the lung, has a relatively fast effect and has a long half-life, and provides a more effective treatment strategy for preventing or treating a new coronavirus and a variant strain thereof.

Description

[0001] This application is a divisional application of the invention patent application with the application date of March 21, 2022, the application number of 202210278872.9, and the invention name of "Constructs of Nanobody R14 and Applications Thereof". TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, in particular to constructs of nanobody R14 and applications thereof, more particularly to multivalent nanobodies, nanobody fusion proteins based on nanobody R14 that specifically binds to SARS-CoV-2 RBD, polynucleotides encoding the same, nucleic acid constructs comprising the polynucleotides, expression vectors comprising the nucleic acid constructs, transformed cells comprising the polynucleotides, nucleic acid constructs or expression vectors, and pharmaceutical compositions comprising any of the foregoing, and the use thereof in the preparation of a medicament for preventing or treating novel coronavirus, and the use thereof in the preparation of a reagent or kit for detecting novel coronavirus or diagnosing novel coronavirus infection. BACKGROUND

[0003] Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), etc. of the same family of Coronaviridae are also major pathogens of the respiratory system of humans, mainly transmitted through droplets, aerosols and contact, etc. They have strong infectivity and are easy to cause public panic, so such viruses that cause respiratory diseases seriously endanger public health and safety, especially the frequent occurrence of respiratory infectious diseases in recent years and the continuous mutation of viruses, which pose a great threat to the health and safety of the people, the development of the national economy and social stability.

[0004] The current outbreak of COVID-19 has promoted the development of various vaccines and antiviral drugs. Vaccination can effectively prevent the occurrence of serious infectious diseases, but the vaccine is suitable for uninfected people, and the development cycle is long, and the clinical research process is complex. For confirmed patients, only antiviral drugs can be used for treatment, one of which is therapeutic antibody drugs, mainly neutralizing antibodies; neutralizing antibody drugs mainly bind to the surface antigens of pathogenic microorganisms, preventing specific molecules expressed by pathogenic microorganisms from binding to cell surface receptors, achieving the effect of "neutralization". The SARS-CoV-2 virus surface has a glycosylated spike protein (S), which can interact with the host cell receptor protein ACE2 and trigger membrane fusion, so blocking the binding of the S protein to ACE2 is an effective way to treat COVID-19 infection.

[0005] Conventional monoclonal antibodies are generally administered by intravenous injection, however, the drug concentration of monoclonal antibodies administered by intravenous injection from the systemic circulation into the lungs is very low, which greatly reduces the antiviral effect of the neutralizing antibodies themselves, resulting in the inability to effectively reduce the viral load in the lungs. The novel coronavirus initially infects the upper respiratory tract, and the first interaction with the immune system mainly occurs on the respiratory mucosal surface. In view of this, for the novel coronavirus infected through the respiratory tract, it is necessary to consider, design and develop suitable antibody drugs from the perspective of mucosal immunity in addition to focusing on serum antibodies. For example, aerosol administration can achieve higher local concentration of antibody drugs in the respiratory tract, which can more effectively block viral infection when the virus invades.

[0006] Nanobodies have been paid much attention as therapeutic drugs, for example, Caplacizumab (Cabivi TM ) developed by Ablynx company is used for treating acquired thrombotic thrombocytopenic purpura, and is the first nanobody drug approved for listing; for another example, nanobody candidate drug ALX-0171 is a trivalent form of nanobody for treating pediatric respiratory syncytial virus (RSV) infection, which is administered by aerosol and has entered the clinical phase II (https: / / clinicaltrials.gov), which indicates that nanobody drugs have safety and feasibility.

[0007] Therefore, it has potential clinical application value and prospect to develop nanobody drugs for the novel coronavirus suitable for respiratory mucosal immunity. SUMMARY

[0008] Invention objectives

[0009] The present application aims to provide a construct based on the nanobody R14 specifically binding to SARS-CoV-2 RBD (including multivalent nanobody and nanobody fusion protein), a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a transformed cell comprising the polynucleotide, the nucleic acid construct or the expression vector, and a pharmaceutical composition comprising any of the above products, and the use of the same in the preparation of a medicament for preventing or treating the novel coronavirus, and the use of the same in the preparation of a reagent or kit for detecting the novel coronavirus or diagnosing the infection of the novel coronavirus.

[0010] The construct based on the nanobody R14 specifically binding to the RBD of SARS-CoV-2 of the present application (including the multivalent nanobody and the nanobody fusion protein) can effectively inhibit the infection of SARS-CoV-2 and its mutant strains, can be administered by nebulization, can directly reach the lungs, has a faster onset and a longer half-life, and provides a more effective treatment strategy for the infection of the new coronavirus and its mutant strains.

[0011] Solution

[0012] To achieve the above-mentioned object, the present application provides the following technical solutions.

[0013] In a first aspect, the present application provides a multivalent nanobody comprising two or more nanobodies specifically binding to the RBD of SARS-CoV-2, wherein the nanobody specifically binding to the RBD of SARS-CoV-2 comprises the following CDRs:

[0014] a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1 (i.e., GFTLDYYAIG),

[0015] a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 2 (i.e., CISSSDGSTSYADSVKG), and

[0016] a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 3 (i.e., TPATYYSGRYYYQCPAGGMDY).

[0017] In a specific embodiment, the nanobody specifically binding to the RBD of SARS-CoV-2 further comprises four framework regions FR1-4, which are arranged in order interlaced with the CDR1, CDR2 and CDR3.

[0018] Preferably, the amino acid sequences of the FR1-4 are as set forth 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.

[0019] In preferred embodiments, the Nanobody specifically binding to SARS-CoV-2 RBD 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 as set forth in SEQ ID NO: 8; preferably, the amino acid sequence of the Nanobody is as set forth in SEQ ID NO: 8 below:

[0020] wherein the underlined parts are the framework regions FR1-4, respectively, and the blacked parts are CDR1, CDR2 and CDR3 of the heavy chain variable region, respectively.

[0021] In preferred embodiment I, the multivalent Nanobody is composed of two or more, preferably three, of said Nanobody specifically binding to SARS-CoV-2 RBD, connected by a Linker;

[0022] wherein the Linker is (GGGGS)n, wherein n = 1, 2, 3, or 4, preferably n = 2 or 3.

[0023] As a further preferred embodiment of embodiment I, the multivalent Nanobody is a trivalent Nanobody having an amino acid sequence as set forth in SEQ ID NO: 9:

[0024] QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSD GSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQC PAGGMDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGL VQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTSYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCPAGGMDYWGQGTQ VTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAVSG FTLDYYAIGWFRQAPGKEREGVSCISSSDGSTSYADSVKGRFTISRDNAKNTVYLQMN SLKPEDTALYYCAATPATYYSGRYYYQCPAGGMDYWGQGTQVTVSS (SEQ ID NO: 9).

[0025] In a preferred embodiment II, the multivalent Nanobody is an IgM pentamer formed by a fusion protein having the structure from N- to C-terminus as shown in formula (I):

[0026] A-L-B (I)

[0027] wherein,

[0028] A is a single, said Nanobody specifically binding to SARS-CoV-2 RBD, or a multivalent Nanobody as described in the preferred embodiment I above;

[0029] B is an Fc fragment of human IgM; preferably, the Fc fragment of human 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;

[0030] L is (GGGGS)m, wherein m = 0, 1, 2, 3, or 4.

[0031] As a preferred embodiment of the fusion protein described above, it has an amino acid sequence as set forth in SEQ ID NO: 11:

[0032] QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSD GSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQC PAGGMDYWGQGTQVTVSSVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQI QVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVD HRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWT RQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKY VTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGK PTLYNVSLVMSDTAGTCY (SEQ ID NO: 11).

[0033] In a second aspect, the present application provides a Nanobody fusion protein, the structure of which from N-terminal to C-terminal is shown as formula (I):

[0034] A-L-B (I)

[0035] wherein,

[0036] A is a single Nanobody specifically binding to SARS-CoV-2 RBD, which is defined as above in the first aspect; or, A is a multivalent Nanobody according to the preferred specific embodiment I of the first aspect, i.e. a multivalent Nanobody composed of two or more, preferably three, of said Nanobody specifically binding to SARS-CoV-2 RBD, which are connected by a Linker, wherein said Linker is (GGGGS)n, wherein n = 1, 2, 3, or 4, preferably n = 2 or 3.

[0037] B is an Fc fragment of human IgM; preferably, the Fc fragment of human 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 as set forth in SEQ ID NO: 10;

[0038] L is (GGGGS)m, wherein m = 0, 1, 2, 3, or 4.

[0039] As a preferred embodiment of the fusion protein described above, it has an amino acid sequence as set forth in SEQ ID NO: 11.

[0040] In a third aspect, the present application provides a polynucleotide encoding the multivalent nanobody according to the first aspect described above, or encoding the nanobody fusion protein according to the second aspect described above.

[0041] In specific embodiments, the polynucleotide is DNA or mRNA;

[0042] Preferably, the polynucleotide encodes a multivalent nanobody according to the preferred embodiment I of the first aspect described above, further preferably the polynucleotide comprises as SEQ ID NO: 12 (i.e., CAAGTGCAACTGCAGGAGAGCGGCGGAGGCCTGGTCCAACCTGGCGGCAGCCTG CGGCTGTCTTGTGCTGTGTCTGGATTCACCCTGGATTACTATGCCATCGGCTGGTTT AGACAGGCCCCTGGCAAGGAACGGGAAGGCGTTAGCTGCATCAGCTCTTCCGACG GCTCTACCAGCTACGCTGATTCTGTGAAGGGCCGCTTCACAATCAGCAGAGATAAT GCCAAAAACACGGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCC CTGTACTATTGCGCTGCCACACCCGCCACCTACTACAGCGGCAGATACTACTATCAG TGTCCTGCCGGAGGCATGGATTACTGGGGACAGGGCACCCAGGTGACAGTGAGCA GCGGAGGAGGCGGCAGCGGCGGAGGCGGCAGTGGTGGCGGCGGATCCGGCGGC GGAGGCAGCGGCGGCGGGGGCAGCCAGGTGCAGCTGCAGGAGAGCGGCGGCGG CCTGGTGCAGCCTGGAGGCAGCCTGAGACTGAGCTGTGCCGTGTCCGGTTTCACC CTGGACTACTACGCCATTGGATGGTTCAGACAGGCTCCAGGCAAGGAAAGAGAAG GCGTGTCCTGTATCAGCTCTTCTGATGGATCTACATCTTACGCCGACAGCGTGAAG GGCAGGTTCACCATCTCCAGAGACAATGCCAAGAACACCGTGTACCTGCAGATGA ACAGCCTGAAACCTGAGGATACCGCACTTTATTACTGCGCCGCCACCCCTGCTACA TACTACAGCGGAAGATACTACTACCAGTGCCCCGCCGGCGGCATGGACTACTGGG GCCAGGGCACCCAGGTCACAGTGAGCAGCGGCGGCGGCGGCTCCGGCGGAGGCGThe nucleotide sequence shown in (GCTCTGGTGGCGGCGGAAGCGGAGGCGGAGGCAGCGGCGGCGGAGGCTCTCAGG TGCAGCTGCAGGAGTCCGGCGGCGGGCTGGTGCAGCCAGGCGGCAGCCTGAGAC TGAGCTGCGCCGTGTCTGGCTTTACACTGGACTACTACGCCATCGGCTGGTTCCGG CAGGCCCCTGGCAAAGAGCGGGAAGGCGTGTCTTGCATCAGCAGCAGCGACGGC AGCACCAGCTACGCCGACAGCGTCAAGGGAAGATTCACCATCTCCCGGGACAACG CCAAGAACACAGTGTACCTGCAAATGAACAGCCTCAAGCCCGAGGACACCGCCCT GTACTACTGCGCCGCTACCCCTGCCACATACTACTCTGGCAGATACTACTACCAGTG CCCTGCCGGCGGCATGGACTACTGGGGCCAGGGCACACAGGTGACCGTGTCCAGC);

[0043] Preferably, the polynucleotide encodes a Nanobody fusion protein as described in the second aspect above, further preferably the polynucleotide comprises SEQ ID NO: 13 (i.e., CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTG AGACTCTCCTGTGCAGTCTCTGGATTTACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATTAGTAGTAGTGATG GTAGCACATCGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAC GCCAAAAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCC TTTATTACTGTGCAGCAACCCCTGCTACATACTATAGTGGACGTTACTACTACCAAT GTCCCGCGGGGGGCATGGACTACTGGGGCCAGGGGACCCAGGTGACCGTGAGCT CTGTGATCGCCGAGCTGCCCCCCAAGGTGAGCGTGTTCGTGCCCCCTAGAGACGG CTTCTTCGGCAACCCTAGAAAGAGCAAGCTGATCTGCCAAGCCACCGGCTTCTCC CCTAGACAGATCCAAGTGAGCTGGCTGAGAGAGGGCAAGCAAGTGGGCAGCGGC GTCACAACAGACCAAGTGCAAGCCGAGGCCAAGGAGAGCGGCCCCACCACCTAC AAGGTGACAAGCACCCTGACCATCAAGGAGAGCGACTGGCTGGGGCAGAGCATG TTCACCTGCAGAGTGGACCACAGAGGCCTGACCTTTCAGCAGAACGCTAGCAGCA TGTGCGTGCCCGACCAAGACACCGCCATCAGAGTGTTCGCCATCCCCCCTAGCTTC GCTAGCATCTTCCTGACCAAGAGCACCAAGCTGACCTGCCTCGTGACCGATCTGA CCACCTACGACAGCGTGACCATCAGCTGGACAAGACAGAACGGCGAGGCCGTGAAGACCCACACCAACATCAGCGAGAGCCACCCCAACGCCACCTTCAGCGCCGTGGGCGAGGCTAGCATCTGCGAGGACGACTGGAACAGCGGCGAGAGATTCACCTGCA CCACACCGACCTGCCTAGCCCCCTGAAGCAGACCATCAGCAGACCCAA GGGCGTGGCCCTGCACAGACCCGACGTGTACCTGCTGCCCCCCGCTAGAGAGCAG CTGAACCTGAGAGAGAGCGCCACCATCACCTGCCTGGTGACCGGCTTTAGCCCCG CTGACGTGTTCGTGCAGTGGATGCAGAGAGGGCAGCCCCTGAGCCCCGAGAAGTA CGTGACAAGCGCCCCCATGCCCGAGCCCCAAGCCCCCGGCAGATACTTCGCCCAC AGCATCCTGACCGTGAGCGAGGAAGAGTGGAACACCGGCGAGACCTACACCTGC GTGGTGGCCCACGAGGCCCTGCCCAACAGAGTGACCGAGAGAACCGTGGACAAG AGCACCGGCAAGCCCACCCTGTACAACGTGAGCCTGGTGATGAGCGACACCGCCG GCACCTGCTAC) as set forth in SEQ ID NO: 1.

[0044] In a fourth aspect, the present application provides a nucleic acid construct comprising the polynucleotide of the third aspect described above, and optionally, at least one expression regulatory element operably linked to the polynucleotide. For example, a histidine tag, a stop codon, and the like.

[0045] In a fifth aspect, the present application provides an expression vector comprising the nucleic acid construct of the fourth aspect described above.

[0046] In a sixth aspect, the present application provides a transformed cell comprising the polynucleotide of the third aspect described above, the nucleic acid construct of the fourth aspect described above, or the expression vector of the fifth aspect described above.

[0047] In a seventh aspect, the present application provides a pharmaceutical composition comprising the multivalent nanobody according to the first aspect described above, the nanobody fusion protein according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above, or the transformed cell according to the sixth aspect described above, and a pharmaceutically acceptable carrier and / or excipient.

[0048] In a specific embodiment, the pharmaceutical composition can be in the form of a nasal spray, an oral preparation, a suppository, or a parenteral preparation.

[0049] Preferably, the nasal spray is selected from the group consisting of an aerosol, a spray, and a powder spray.

[0050] Preferably, the oral preparation is selected from the group consisting of a tablet, a powder, a pill, a granule, a fine granule, a soft / hard capsule, a film-coated agent, a pellet, a sublingual tablet, and a paste.

[0051] Preferably, the parenteral preparation is a transdermal agent, an ointment, a plaster, a topical liquid agent, an injectable or bolus preparation.

[0052] The amount of the effective component of the pharmaceutical composition of the present application to be administered varies depending on the subject of administration, the organ of the subject, the symptoms, the method of administration, etc., and can be determined according to the judgment of a doctor, taking into account the type of dosage form, the method of administration, the age and weight of the patient, the symptoms of the patient, etc.

[0053] In an eighth aspect, the present application provides use of the multivalent nanobody according to the first aspect described above, the nanobody fusion protein according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above, the transformed cell according to the sixth aspect described above, or the pharmaceutical composition according to the seventh aspect described above, in the preparation of a medicament for preventing and / or treating a novel coronavirus infection.

[0054] In a specific embodiment, the novel coronavirus can be a SARS-CoV-2 original strain and / or a SARS-CoV-2 variant strain.

[0055] 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) strain, Omicron (B.1.1.529) sub-lineage BA.1 strain or Omicron (B.1.1.529) sub-lineage BA.2 strain, further preferably Delta (B.1.617.2) strain, Omicron (B.1.1.529) sub-lineage BA.1 strain or Omicron (B.1.1.529) sub-lineage BA.2 strain.

[0056] In a ninth aspect, the present application provides use of the multivalent nanobody according to the first aspect described above, the nanobody fusion protein according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above or the transformed cell according to the sixth aspect described above in the preparation of a reagent or kit for detecting a novel coronavirus or for diagnosing a novel coronavirus infection.

[0057] In specific embodiments, the novel coronavirus can be SARS-CoV-2 original strain and / or SARS-CoV-2 variant strain.

[0058] 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) strain, Omicron (B.1.1.529) sub-lineage BA.1 strain or Omicron (B.1.1.529) sub-lineage BA.2 strain, further preferably Delta (B.1.617.2) strain, Omicron (B.1.1.529) sub-lineage BA.1 strain or Omicron (B.1.1.529) sub-lineage BA.2 strain.

[0059] In a tenth aspect, the present application provides a novel coronavirus detection kit comprising the multivalent nanobody according to the first aspect described above, the nanobody fusion protein according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above or the transformed cell according to the sixth aspect described above.

[0060] Advantages

[0061] The present application is directed to the development of nanobody constructs for the new coronavirus, and the constructs based on nanobody R14 of the present application can all bind to SARS-CoV-2 RBD with high affinity, and can neutralize SARS-CoV-2 prototype strains and a series of mutant strains of pseudovirus and live virus with high neutralization activity, which all indicate that the constructs based on nanobody R14 are novel coronavirus (SARS-CoV-2) nanobodies that can bind to SARS-CoV-2 RBD with high affinity and have high neutralization activity.

[0062] In particular, the inventors have demonstrated through a series of experiments that the trivalent nanobody (TR14) and IgM pentamer form (MR14) based on nanobody R14 of the present application have significantly improved neutralization activity and significantly prolonged half-life compared to their monomer (i.e., nanobody R14), which achieves mucosal immunity, can limit the reproduction and further crossing of the mucosal barrier of the virus, controls the mucosal transmission of the virus, provides a potential nebulizable antibody drug for the clinical prevention and treatment of the new coronavirus, and can achieve sensitive and reliable detection of the new coronavirus. BRIEF DESCRIPTION OF DRAWINGS

[0063] One or more embodiments are illustrated by way of example in the figures that form a part of this patent document. This illustration is not necessarily drawn to scale. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0064] Figure 1 is a structural schematic diagram of the nanobody constructs TR14 and MR14 constructed in embodiment 1 of the present application;

[0065] Figure 2 is a protein molecular sieve chromatography and SDS-PAGE identification result graph of the R14 protein described in embodiment 1 of the present application;

[0066] Figure 3 is a protein molecular sieve chromatography and SDS-PAGE identification result graph of the TR14 protein described in embodiment 1 of the present application;

[0067] Figure 4 is a protein molecular sieve chromatography and SDS-PAGE identification result graph of the MR14 protein described in embodiment 1 of the present application;

[0068] Figure 5Figure is a SDS-PAGE identification result diagram of SARS-CoV-2 RBD-his protein (A), RBD-his protein of variant strain Omicron (B.1.1.529) subtype BA.1 (B) and RBD-his protein of variant strain Omicron (B.1.1.529) subtype BA.2 (C) described in embodiment 2 of the present application.

[0069] Figure 6 Figure is a schematic diagram of the effect of neutralizing SARS-CoV-2 prototype strain pseudovirus infection by three antibodies determined in embodiment 5 of the present application.

[0070] Figure 7 Figure is a schematic diagram of the effect of neutralizing SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus infection by three antibodies determined in embodiment 5 of the present application.

[0071] Figure 8 Figure is a schematic diagram of the effect of neutralizing SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 pseudovirus infection by three antibodies determined in embodiment 5 of the present application.

[0072] Figure 9 Figure is a schematic diagram of the effect of neutralizing SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.2 pseudovirus infection by three antibodies determined in embodiment 5 of the present application.

[0073] Figure 10 Figure is the neutralization activity of three antibodies on pseudovirus before and after atomization determined in embodiment 7 of the present application; wherein A is the neutralization activity result of nanobody R14 on SARS-CoV-2 prototype strain pseudovirus before and after atomization, B is the neutralization activity result of nanobody construct TR14 on SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus, and C is the neutralization activity result of nanobody construct MR14 on SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus.

[0074] Figure 11 Figure is the half-life of three antibodies in blood determined in embodiment 8 of the present application.

[0075] Figure 12 Figure is the atomization system used in embodiment 8 of the present application.

[0076] Figure 13 Figure is the half-life of three antibodies in lung determined in embodiment 8 of the present application. DETAILED DESCRIPTION

[0077] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application. Unless otherwise clearly indicated, in the entire specification and claims, the term "comprise" or its variants such as "contain" or "include" and the like are understood to include the stated element or component without excluding other elements or components.

[0078] In addition, in order to better illustrate the present application, a large number of specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means and the like which are well known to those skilled in the art are not described in detail, so as to highlight the main idea of the present application.

[0079] Hereinafter, the present application will be described in detail.

[0080] Definitions

[0081] "Nanobody", i.e. "heavy-chain single-domain antibody", this kind of antibody only contains one heavy chain variable region (VHH, variable domain of heavy chain of heavy-chain antibody), compared with other antibodies, the light chain is naturally missing.

[0082] Due to the biophysical advantages of nanobodies themselves, they can be easily atomized and directly delivered to the lungs through inhalers to treat respiratory viral infections, and are considered to be very potential antibody drugs.

[0083] When referring to ligand / receptor, antibody / antigen or other binding pairs, "specific" binding refers to the determination of the presence or absence of a binding reaction between the protein, e.g. the nanobody of the present application, and the SARS-CoV-2 RBD protein in a heterogeneous population of proteins and / or other biological reagents. Therefore, under the specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind to other proteins present in the sample in a significant amount.

[0084] The chemical materials such as reagents, enzymes, culture media, antibiotics and milk used in the following embodiments of the present application are commercially available products, for example, TRIzol is purchased from Invitrogen, and Superscript II First-Strand Synthesis System for RT-PCR kit is purchased from Invitrogen.

[0085] Some commonly used biomaterials, such as competent cells, vectors, helper phages, cells to be transformed, etc., are also commercially available products, for example, pCAGGS vectors are purchased from MiaoLing Plasmid; 293F cells, HEK293T cells, etc. are purchased from ATCC; Series Sensor Chip SA chips are purchased from GE Healthcare; Vero cells are purchased from ATCC CCL81.

[0086] Some synthetic biomaterials, such as primers, sequences, etc., require artificially synthesized materials, which are all entrusted to synthesis companies to complete, for example, the coding sequence of TR14 in the present application is synthesized by Nanjing Kingsrui Biotechnology Co., Ltd.

[0087] Example 1: Construction, expression and purification of trivalent form (TR14) and IgM pentamer form (MR14) antibodies based on nanobody R14

[0088] The structural schematic diagram of the monovalent nanobody and its trivalent form and IgM pentamer form in the present embodiment is shown in Figure 1 .

[0089] The basic nanobody R14 used is obtained by simultaneously immunizing a llama with SARS-CoV-2 RBD protein and SARS-CoV-2 NTD protein, constructing an antibody library, and screening using phage display technology in the laboratory; the amino acid sequence of the monovalent nanobody R14 is shown in SEQ ID NO: 8, which can specifically bind to SARS-CoV-2 RBD with high affinity (binding constant less than 1E-10M), and can neutralize SARS-CoV-2 pseudovirus with high neutralization activity in the pseudovirus neutralization experiment, all of which indicate that R14 nanobody is a new coronavirus (SARS-CoV-2) llama-derived nanobody that can bind to SARS-CoV-2 RBD with high affinity and has high neutralization activity.

[0090] The coding sequence of the monovalent nanobody R14 (shown in SEQ ID NO: 14) is connected with a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15) at the 5' end, the coding sequence of a hexa-His-tag and a translation termination codon TGA at the 3' end, which is constructed into a 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) to express R14-his protein. The cell culture solution containing the target protein is subjected to nickel ion affinity chromatography (HisTrapTM excel ((GE Healthcare)) and gel filtration chromatography (Superdex TM After purification by two steps (GE Healthcare), the relatively pure target protein can be obtained. The SDS-PAGE identification size of R14-his protein is about 15KD, and the result is shown in Figure 2

[0091] The coding sequence of three nanobodies R14 as shown in SEQ ID NO: 14 was connected in a head-to-tail form by two (GGGGS)3 sequences (directly synthesized by Nanjing Kings River Biotech Co., Ltd.), and the coding sequence of a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15) was connected at its 5' end, and the coding sequence of a hexa-His-tag and a translation termination codon TGA was connected at its 3' end. Through restriction enzyme sites EcoRI and XhoI, it was constructed into a pCAGGS vector (purchased from Invitrogen), and then the obtained recombinant vector was transfected into 293F cells (purchased from Invitrogen) to express TR14-his protein. The cell culture solution containing the target protein was purified by nickel ion affinity chromatography (HisTrap TM excel ((GE Healthcare)) and gel filtration chromatography (Superdex TM After purification by two steps (GE Healthcare), the relatively pure target protein can be obtained. The SDS-PAGE identification size of R14-his protein is about 15KD, and the result is shown in Figure 3

[0092] ​​The coding sequence of Nanobody R14 (as shown in SEQ ID NO: 14) was connected to the coding sequence of Fc of human IgM antibody (as shown in SEQ ID NO: 16) by homologous recombination, a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15) was connected at the 5' end, a translation termination codon TGA was connected at the 3' end, and the same was constructed into a pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI to obtain a pCAGGS-R14-IgM Fc recombinant expression vector. The coding sequence of J chain (as shown in SEQ ID NO: 17) was connected to a translation termination codon TGA at the 3' end, and the same was constructed into a pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI to obtain a pCAGGS-J chain recombinant expression vector. The above 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 the two were self-assembled to form MR14 protein in the form of IgM. The obtained MR14 protein was purified by HiTrap TM IgM Purification HP (GE Healthcare) and Superose TM 6 increase 10 / 300GL (GE Healthcare) and identified by SDS-PAGE. The SDS-PAGE identification size of the MR14 protein was about 70KD, and the results are shown in Figure 4

[0093] Example 2: Expression and purification of SARS-CoV-2 and its variant RBD

[0094] The coding sequence of the RBD protein of the original strain of SARS-CoV-2 (the amino acid sequence is shown in SEQ ID NO: 18) was connected to the coding sequence of a hexa-His-tag and a translation termination codon TGA at the 3' end, and the same was constructed into a pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, and then the obtained recombinant vector was transfected into 293F cells (purchased from Invitrogen) to express SARS-CoV-2 RBD-his protein.

[0095] ​The coding sequence of the RBD protein of SARS-CoV-2 mutant strain Omicron (B.1.1.529) subtype BA.1 (the amino acid sequence of which is shown as SEQ ID NO: 19) and the coding sequence of the RBD protein of Omicron (B.1.1.529) subtype BA.2 (the amino acid sequence of which is shown as SEQ ID NO: 20) were respectively connected to the 3' end of the coding sequence of the hexa-His-tag and the translation termination codon TGA, and expressed by the bac-to-bac baculovirus expression system (Invitrogen). The pFastbac1 plasmid containing the target gene was transformed into DH10Bac competent cells to generate recombinant bacmids. The recombinant bacmids were transfected into Sf9 cells to amplify the virus, and protein expression was performed in Hi5 cells. After 48h of expression, the Hi5 cell supernatant was collected and the soluble protein was purified by nickel affinity chromatography using HisTrap TM excel (GE Healthcare) through nickel affinity chromatography.

[0096] The cell culture solution containing the target protein was passed through a nickel ion affinity chromatography column HisTrap TM excel (GE Healthcare) and gel filtration chromatography Superdex TM 200Increase 10 / 300GL column (GE Healthcare). After purification, the target protein can be obtained in a relatively pure form. The SDS-PAGE identification size of SARS-CoV-2 RBD-his protein, RBD-his protein of mutant strain Omicron (B.1.1.529) subtype BA.1 and RBD-his protein of Omicron (B.1.1.529) subtype BA.2 is about 30KD, as shown in Figure 5 A-C, respectively.

[0097] Example 3: Surface plasmon resonance technology for detecting the binding ability of each antibody to the RBD protein of SARS-CoV-2 original strain and its mutant strain

[0098] Surface plasmon resonance analysis was performed using Biacore 8K (Biacore Inc.). The specific steps are as follows:

[0099] The monovalent nanobody R14 and its constructs TR14 and MR14 prepared in the above examples were biotinylated and then immobilized on a Series Sensor Chip SA chip (Cytiva Life Sciences); the RBD proteins of the SARS-CoV-2 original strain and its variants prepared in the above examples were diluted by 2 times with PBST buffer (2.7 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 0.05% Tween) respectively, and then loaded onto the chip from low concentration to high concentration. The calculation of the binding kinetic constant was performed by using BIAevaluation software 8K (Biacore, Inc.) software. The equilibrium dissociation constant (Kd) between each antibody and each RBD was calculated by using the following equation: Kd = (Rmax - Bmax) / Kd D As shown in Table 1, the results of Table 1 show that the monovalent nanobody R14 and its constructs TR14 and MR14 can bind to the RBD proteins of the SARS-CoV-2 original strain and the Omicron subtypes BA.1 and BA.2 of the variant strain with high affinity.

[0100] Table 1, the affinity results between the monovalent nanobody R14 and its constructs TR14 and MR14 and the RBD of the SARS-CoV-2 original strain and the variant strain

[0101]

[0102] Example 4: Packaging of SARS-CoV-2 original strain and variant strain pseudovirus

[0103] 1) The genes encoding the 18th amino acids after the S protein of the SARS-CoV-2 original strain (WT) and the variant strains Delta (B.1.617.2), Omicron (B.1.1.529) subtype BA.1 and Omicron (B.1.1.529) subtype BA.2 were removed, and the remaining sequence of the S protein was synthesized (synthesis service was provided by Suzhou Jinyuzhi), obtaining 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, the sequences are shown in SEQ ID NO: 21-24, respectively.

[0104] 2) The protein gene obtained in 1) was respectively cloned into pCAGGS vector to obtain expression plasmids pCAGGS-SARS-CoV-2-WT-S-del18, pCAGGS-B.1.617.2-S-del18, pCAGGS-B.1.1.529-BA.1-S-del18 and pCAGGS-B.1.1.529-BA.2-S-del18.

[0105] The packaging steps of SARS-CoV-2 original strain and variant strain pseudovirus are as follows:

[0106] a. Cell preparation: HEK293T cells (purchased from ATCC CRL-3216) were plated in a 10 cm cell culture dish, and the cell confluence density was about 80% the next day. The culture medium was DMEM medium containing 10% FBS.

[0107] b. Transfection: 30 μg of the expression plasmid of each S protein in step 2) above was transfected with PEI per 10 cm cell culture dish. The target plasmid was mixed with PEI at a ratio of 1:3 before transfection. The culture medium (DMEM medium containing 10% FBS) was changed 4-6 h after transfection, and the cells were cultured at 37°C for 24 h.

[0108] c. Virus addition: The pseudovirus packaging backbone virus G*VSV-delG (purchased from Wuhan Privy Brain Science and Technology Co., Ltd.) was added to the HEK293T cells after transfection above, and incubated at 37°C for 2 h. The culture medium (DMEM medium containing 10% FBS) was changed, and VSV-G antibody (hybridoma cells expressing the antibody were purchased from ATCC cell bank) was added. The cells were further cultured in the incubator for 30 h.

[0109] d. Virus collection: The supernatant was centrifuged at 3000 rpm for 10 min, filtered through a 0.45 μm sterile filter in a clean bench to remove cell debris, aliquoted, and stored in a -80°C refrigerator.

[0110] Pseudovirus of SARS-CoV-2 original strain (SARS-CoV-2 WT) and variant strains 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.

[0111] Example 5: Detection of antibody neutralization of pseudovirus infection

[0112] The purified monovalent nanobody R14 and its constructs TR14 and MR14 (prepared in Example 1) were diluted by 5 times to the 9th gradient (2.56 pg / mL), respectively. The diluent was mixed with 1.6 x 10 4 TCID50 The pseudoviruses of a series of SARS-CoV-2 original strains and variant strains obtained in Example 4 were mixed respectively, and incubated at 37°C for 1 h, and then added to a 96-well plate pre-seeded with Vero cells (purchased from ATCC CCL81). After 18-20 hours of incubation, detection was performed by CQ1 Confocal Quantitative Image Cytometer (Yokogawa). According to the number of cells with GFP fluorescence, the neutralization ability of the antibodies to the pseudoviruses of the above-mentioned series of SARS-CoV-2 original strains and variant strains Delta, BA.1 and BA.2 was calculated, and the results are shown in FIGS. 1-3, respectively. The results show that the neutralization effect of the constructs TR14 and MR14 of the monovalent nanobody R14 is improved. Figures 6-9 The results are shown in Table 2; the results show that the neutralization effect of the constructs TR14 and MR14 of the monovalent nanobody R14 is improved.

[0113] Table 2, Neutralization ability of monovalent nanobody R14 and its constructs TR14 and MR14 to pseudoviruses of SARS-CoV-2 original strains and variant strains

[0114]

[0115] Note: IC 50 (μg / mL) is the half-inhibitory concentration of the antibody.

[0116] Example 6: Detection of antibody neutralization of live virus infection

[0117] In this example, the neutralization effect of each antibody on live coronavirus was determined by a live virus neutralization test based on cytopathic effect (CPE). The specific steps are as follows:

[0118] The purified monovalent nanobody R14 and its TR14 and MR14 (prepared in Example 1) were diluted by 2-fold ratio to the 11th gradient, 4 repeated wells for each gradient, and 50 μL for each well. The dilution was incubated with an equal volume of 100 TCID 50 of SARS-CoV-2 original strain or its variant strain Delta, Omicron subtype BA.1 at 37°C. After 1 hour, the mixture was added to the suspended Vero cells and incubated at 37°C for 3 days. The cytopathic effect was observed and recorded. The IC 50 of the nanobody and its constructs was calculated using GraphPad Prism 7.0. The experiments were all carried out in a biosafety level 3 laboratory (BSL3) of the China Disease Prevention and Control Center.

[0119] The neutralization effect of monovalent nanobody R14 and its TR14 and MR14 on live viruses of the original strain and variant strains of the new coronavirus is shown in Table 3, and the results in Table 3 show that TR14 and MR14 have good inhibitory effect on live viruses of the original strain and variant strains of the new coronavirus.

[0120] Table 3, neutralization ability of monovalent nanobody R14 and its constructs TR14 and MR14 on live viruses of SARS-CoV-2 original strain and variant strains

[0121]

[0122] Note: IC 50 (μg / mL) is the half-inhibitory concentration of the antibody. # indicates that at the lowest concentration of 0.001 μg / mL in the test, it still has 100% inhibitory effect on live viruses.

[0123] Example 7: Detection of the stability of the antibody before and after nebulization

[0124] Using an Aerogen Solo (Aerogen Inc., Chicago, USA) nebulizer, monovalent nanobody R14 and its constructs TR14 and MR14 were nebulized respectively, and then the nebulized antibodies were collected using a full-glass SKC (Eighty Four, PA, USA) containing 20 mL of PBS, and the pseudovirus neutralization test was performed as described in Example 5. The results are shown in Figure 10 , wherein A is the neutralization activity results of nanobody R14 before and after nebulization on SARS-CoV-2 prototype strain pseudovirus, B is the neutralization activity results of nanobody construct TR14 on SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus, and C is the neutralization activity results of nanobody construct MR14 on SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus. Figure 10 The results show that the nanobody constructs TR14 and MR14 of the application maintain stable neutralization activity on pseudoviruses of the original strain or variant strains of the new coronavirus before and after nebulization, suggesting that they are both suitable for administration by the nebulization route.

[0125] Example 8: Determination of the half-life of the antibody

[0126] The half-lives of the nanobody R14 and its constructs TR14 and MR14 in blood were studied by intraperitoneal injection, and the specific procedures were as follows: 6-8-week-old female SPF BALB / c mice (purchased from Vital River) were anesthetized with isoflurane and slowly injected with 200 μl of 2 mg / mL (20 mg / kg body weight) of R14, TR14 and MR14, respectively, by intraperitoneal injection. Blood samples were collected by orbital blood collection at 1 hour, 6 hours, 10 hours, 24 hours, 48 hours, 72 hours and 96 hours after injection, respectively. After centrifugation, the supernatant was taken and the concentration of antibodies in the serum was determined by ELISA. The half-lives (t 1 / 2 ) of the nanobody R14 and its constructs TR14 and MR14 were calculated using the software PKSolver. Figure 11 The results showed that, compared with the monovalent antibody R14, the constructs TR14 and MR14 exhibited significantly prolonged half-lives, which was beneficial to improve the antiviral effect of the antibody in vivo. Figure 11

[0127] Next, the residence time of the nanobody R14 and its constructs TR14 and MR14 in the lung was also studied by nebulization administration. Using a nebulization system (as shown in Figure 12 ), 20 mg / ml of the three antibodies R14, TR14 and MR14 were nebulized, respectively, and the mice were exposed to the nebulization chamber for 10 minutes. Then the mice were anesthetized with tribromoethanol by intraperitoneal injection, and the bronchoalveolar lavage fluid was collected at 0 hour, 1 hour, 6 hours and 24 hours, respectively. The concentration of antibodies in the lavage fluid was determined by ELISA, and the half-life (t 1 / 2 ) of the nanobody in the lung was calculated using PKSolver. The results of the half-lives of R14, TR14 and MR14 in the lung are shown in Figure 13 , Figure 13 The results showed that, compared with the monovalent antibody R14, the constructs TR14 and MR14 exhibited significantly prolonged half-lives, which was beneficial to improve the antiviral effect of the antibody in vivo after administration by nebulization.

[0128] The above results showed that the constructs TR14 and MR14 based on the nanobody R14 of the present application had the potential to develop into high neutralization activity antibody drugs, especially nebulization drugs, for treating infection of the novel coronavirus and its mutant strains.

[0129] ​It should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. SEQUENCE LISTING <110> Institute of Microbiology, Chinese Academy of Sciences <120> Construct of Nanobody R14 and its application <130> 1087-220047F-D2 <160> 24 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> CDR1 sequence of VHH chain of Nanobody R14 <400> 1 Gly Phe Thr Leu Asp Tyr Tyr Ala Ile Gly 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> CDR2 sequence of VHH chain of Nanobody R14 <400> 2 Cys lie Ser Ser Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> CDR3 sequence of VHH chain of Nanobody R14 <400> 3 Thr Pro Ala Thr Tyr Tyr Ser Gly Arg Tyr Tyr Tyr Gin Cys Pro Ala 1 5 10 15 Gly Gly Met Asp Tyr 20 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR1 sequence of VHH chain of Nanobody R14 <400> 4 Gln Val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser 20 25 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR2 sequence of VHH chain of Nanobody R14 <400> 5 Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Gly Val Ser 1 5 10 <210> 6 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR3 sequence of VHH chain of Nanobody R14 <400> 6 Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gin 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala 20 25 30 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR4 sequence of VHH chain of Nanobody R14 <400> 7 Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of VHH chain of Nanobody R14 <400> 8 Gln Val Gin Leu Gin Glu Ser Gly Gly Gly Leu Val Gin 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 Gin 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 Gin 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 Gin Phe 100 105 110 Pro Ala Gly Gly Met Asp Tyr Trp Gly Gin Gly Thr Gin Val Thr Val 115 120 125 Ser Ser 130 <210> 9 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of a trivalent nanobody <400> 9 Gln Val Gin Leu Gin Gin Ser Gly Gly Gly Leu Val Gin 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 Gin 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 Gin 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 Gin Cys 100 105 110 Pro Ala Gly Gly Met Asp Tyr Trp Gly Gin Gly Thr Gin 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 Gin Val Gin Leu Gin Gin Gin Gin Gin Gin 145 150 155 160 Glu Ser Gly Gly Gly Leu Val Gin 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 Gin 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 Gin 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> Human IgM Fc fragment <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 Gin Leu Asn Leu Arg 225 230 235 240 Glu Ser Ala Thr lie Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp 245 250 255 Val Phe Val Gin Trp Met Gin Arg Gly Gin Pro Leu Ser Pro Glu Lys 260 265 270 Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gin Ala Pro Gly Arg Tyr 275 280 285 Phe Ala His Ser lie 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 a 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 Gin 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 Gin Trp Met Gin Arg Gly Gin Pro Leu Ser Pro 385 390 395 400 Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gin 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 a 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 caaggaaga gaaggcgtgt cctgtatcag ctcttctgat ggatctacat cttacgccga cagcgtgaag ggcaggttca ccatctccag agacaatgcc aagaacaccg tgtacctgca gatgaacagc ctgaaacctg aggataccgc actttattac tgcgccgcca cccctgctac attackcagc 840. ggagatact actaccagtg ccccgccggc ggcatggact actggggcca gggcacccag 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 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 a 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 for 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 for 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 GAGAAGAAGA AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA AGAAG 48 TGGATGCAGA GAGGGCAGCC CCTGAGCCCC GAGAAGTACG TGACAAGCGC CCCATGCCC 840 GAGCCCCAAG CCCCCGGCAG ATACTTCGCC CACAGCATCC TGACCCTGAG CGAGGAAGAG 900 TGGAACACCG GCAGACCTAC ACCTGCCTGG TGGCCCACGA GGCCCTGCCT CAACAGAGTG 960 ACCAGAAGAA CCCTGGACAA GAGCACCAGC AAGCCCACCT GTACAACCTG AGCCTGGTG 1020 ATGAGCGACA CCAGAGGCAC CTGCTAC 1047 <210> 17 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence for J chain <400> 17 ATGAAGAACC ACCCTGCTGT TCTGGGGCGT GCTGGCCGTG TTTCATCAAG GCCTGCACGT G 60 AAGGCCCAAG AGGACGAGAG AATCGTGCTG GTGGACAACA AGTGCAAATG CGCTAGAATC 120 ACAAGCAGAA TCTTCAGAAG CAGCGAGGCC CAACGAGGAC ATCGTGGAAG AAACATCTC 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 lie 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 lie Arg Gly Asp Glu Val Arg Gin lie Ala Pro Gly Gin Thr 100 105 110 Gly Lys lie Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 115 120 125 Cys Val lie 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 lie Ser Thr Glu lie Tyr Gin Ala Gly Ser Thr Pro 165 170 175 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr 180 185 190 Gly Phe Gin Pro Thr Asn Gly Val Gly Tyr Gin 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) sublineage 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) sublineage 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 GAGCCTGGAG GAGGACGGAGT GGGAGAGGAG T GAGT GAGT G 20 CTGGCCGCCA CCAAGATGAG CGAGTGCGTG CTGGGGCAGA GCAAGAGAGT GGACTTCTGC 3120 AGCGGGTGGAC CTTTGGCGCC GGGGGCGCCC CTGCAGATCC CCTTCGCCAT GCAGATGGCC 2700 TACAGATTC A AC GGC ATC GGC GTG AC AC AG AAC GTGCTGT ACGAGAAC T C AAGCTGATC 2760 GCCAATCAGT TCAACAGCGC CATC GGC AAG ATC C AAGAC AGCCTGAGC AG C ACCGCTAGC 2820 GCCCTGGGCA AGCTGCAAGA CGTG GTG AAT C AGAAC GCC C AAGCCCTG A AC ACCCTGGTG 2880 AAGCAGCTGA GCAGC AACTT C GGC GCC ATC AGC AGC GTG CTG AAC GAC ATC CTGGCTAGA 2940 CTGGACAAGG TGGAGGCCGA GGTGCAGATC GATAGACTGA TC ACCGGCAG ACTGCAGAGC 3000 CTGCAGACCT AC GTG AC AC AGC AGCTGATC AGAGCCGCCG AGATC AGAGC TAGCGCCAAC 3060 CTGGCCGCCA CCAAGATGAG CGAGTGCGTG CTGGGGCAGA GCAAGAGAGT GGACTTCTGC 3120 GGC AAGGGCT ACC ACC TGA TGAGCTTCCCT CAGAGCGCCCC CCCACGGCGT GGTGTT CCTG 3180 CACGTGACCT AC GTG CCCGC C AAGAGAAG AACTT C ACC ACC GCC CCCGC CATCTGCCAC 3240 GACGGCAAGGC CCACCTTCCC TAGAGAGGGC GTGTTCGTGA GCAACGGCAC CCACGTGTTC 3300 GTGACACAGA GAAACTTCTA CGAGCCTCAG ATCATCACCA CCCACAACAC CTTCGTGAGC 3360 GGCAACTGCG ACGTGGTGAT CGGCGTCGTG AACAACACGT GTACGACCCC TCTGCAGCCC 3420 GAGCTGGACA GCTTCAAGGA GGAGCTGGAC AAGTACTTCA AGAACCACAC AAGCCCCGAC 3480 GTGGACCTGG GCGACATCAG CGGGATCAAC GCTAGCGTGG TGAACATTC AAGGAAATC 3540 GACAGACTGA ATGAGGTGGC CAAGAACCCT 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> B.1.617.2-S-del18 gene nucleotide sequence <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 the 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

1. The application of a multivalent nanobody in the preparation of a drug for the prevention or treatment of SARS-CoV-2 infection, characterized in that, The multivalent nanobody is composed of three nanobodies that specifically bind to SARS-CoV-2 RBD linked together by a linker; The nanobody specifically binding to SARS-CoV-2 RBD includes the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO: 1, CDR2 with the amino acid sequence shown in SEQ ID NO: 2, and CDR3 with the amino acid sequence shown in SEQ ID NO: 3; and, The Linker is (GGGGS)n, where n = 1, 2, 3, or 4.

2. The application according to claim 1, characterized in that, n = 2 or 3.

3. The application according to claim 1, characterized in that, The nanobody that specifically binds to SARS-CoV-2 RBD also includes four framework regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

4. The application according to claim 3, characterized in that, The amino acid sequences of FR1-4 are shown in SEQ ID NO:4, 5, 6, and 7, respectively.

5. The application according to claim 1, characterized in that, The nanobody that specifically binds to SARS-CoV-2 RBD has an amino acid sequence as shown in SEQ ID NO:

8.

6. The application according to any one of claims 1-5, characterized in that, The SARS-CoV-2 mentioned refers to the original SARS-CoV-2 strain and / or a variant of SARS-CoV-2.

7. The application according to claim 6, characterized in that, The SARS-CoV-2 variant strains mentioned are Alpha, Beta, Gamma, Kappa, Delta strains, Omicron subtype BA.1 strain and / or Omicron subtype BA.2 strain.

8. The application according to any one of claims 1-5, characterized in that, The drug is in the form of nasal spray, oral preparation, suppository, transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

9. The application according to claim 8, characterized in that, The nasal spray is selected from aerosols, sprays, and powders; the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, and film-coated formulations; the injectable formulation is a push-in formulation.

10. The application according to claim 9, characterized in that, The tablets are sublingual tablets; The powder is a granule; The pills are small pills; The granules are fine granules.

Images