CN116813758A - Construct of nanobody S43 and application thereof - Google Patents

Construct of nanobody S43 and application thereof Download PDF

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CN116813758A
CN116813758A CN202210278937.XA CN202210278937A CN116813758A CN 116813758 A CN116813758 A CN 116813758A CN 202210278937 A CN202210278937 A CN 202210278937A CN 116813758 A CN116813758 A CN 116813758A
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nanobody
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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 CN202210375856.1A priority Critical patent/CN114763380B/en
Priority to CN202210278937.XA priority patent/CN116813758A/en
Priority to CN202210374153.7A priority patent/CN116808200A/en
Priority to PCT/CN2023/082531 priority patent/WO2023179546A1/en
Priority to PCT/CN2023/082534 priority patent/WO2023179548A1/en
Publication of CN116813758A publication Critical patent/CN116813758A/en
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Abstract

The invention provides a construct (including multivalent nanobody and nanobody fusion protein) based on nanobody S43 which specifically binds SARS-CoV-2RBD, related products and applications thereof; the construct (comprising multivalent nanobody and nanobody fusion protein) based on the nanobody S43 specifically combined with 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

Construct of nanobody S43 and application thereof
Technical Field
The present invention relates to the field of biological medicine, in particular to a construct of a nanobody S43 and application thereof, and more particularly to a multivalent nanobody based on the nanobody S43 specifically binding SARS-CoV-2RBD, nanobody fusion protein, polynucleotide encoding the same, nucleic acid construct containing the polynucleotide, expression vector containing the nucleic acid construct, transformed cell containing the polynucleotide, nucleic acid construct or expression vector, and pharmaceutical composition containing any one of the above products, and application thereof in preparing a medicament for preventing or treating novel coronavirus, and application in preparing a reagent or kit for detecting novel coronavirus or diagnosing 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 transmitted mainly by means of spray, aerosol, contact and the like, and have strong infectivity.
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 S43 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) based on the nanobody S43 specifically combined with SARS-CoV-2RBD can effectively inhibit SARS-CoV-2 infection and variant strain infection thereof, can be directly delivered to 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., CISSNNSTYYADSVKG), and
the amino acid sequence is shown as CDR3 of SEQ ID NO. 3 (i.e., EPDYSGVYYYTCGWTDFGS). 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., QVQLQESGGGLVQPGGSLRLTCAPS), SEQ ID NO:5 (i.e., WFRQAPGKEREGVS), SEQ ID NO:6 (i.e., RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA) and SEQ ID NO:7 (i.e., WGQGTQVTVSS), respectively.
In a preferred embodiment, the nanobody specifically binds SARS-CoV-2RBDThe VHH chain 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; 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 darkened 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:
QVQLQESGGGLVQPGGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNN STYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGW TDFGSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQP GGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNNSTYYADSVKGRFTISRD NAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGWTDFGSWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLTCAPSGFTLDY YAIGWFRQAPGKEREGVSCISSNNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCAAEPDYSGVYYYTCGWTDFGSWGQGTQVTVSS(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:
QVQLQESGGGLVQPGGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNN STYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGW TDFGSWGQGTQVTVSSVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVS WLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRG LTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQN GEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPK GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTS APMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTL YNVSLVMSDTAGTCY(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., CAGGTCCAACTCCAAGAGAGCGGCGGCGGCCTCGTCCAACCCGGAGGATCACTC AGACTCACATGCGCCCCAAGCGGCTTCACACTCGACTACTACGCCATCGGCTGGTT CAGACAAGCCCCCGGCAAAGAGAGAGAAGGAGTGTCTTGCATTAGCAGCAACAA CAGCACCTACTACGCCGACAGTGTCAAAGGAAGATTCACCATCAGCAGGGACAAC GCTAAGAACACCGTGTATCTCCAGATGAACTCACTGAAGCCCGAGGACACCGCCG TGTACTACTGCGCCGCCGAGCCCGACTACAGCGGCGTTTACTACTACACCTGCGGA TGGACCGACTTCGGCAGCTGGGGCCAAGGAACCCAAGTCACCGTGAGCAGCGGA GGCGGAGGAAGCGGCGGTGGAGGAAGTGGCGGAGGCGGATCTGGGGGGGGAGG ATCAGGCGGAGGAGGAAGCCAGGTGCAGCTGCAGGAGAGCGGAGGAGGACTGG TGCAGCCAGGAGGAAGCCTGAGACTGACATGCGCACCAAGCGGATTCACACTGG ACTATTATGCTATCGGATGGTTCAGACAGGCCCCTGGAAAAGAGAGAGAGGGGGT GAGCTGCATCAGCAGCAATAACTCCACATACTACGCCGATAGCGTCAAGGGGAGG TTCACTATTAGCAGGGACAATGCAAAGAACACAGTGTACCTGCAGATGAACAGCC TGAAGCCCGAAGACACCGCCGTCTACTACTGCGCAGCCGAGCCCGATTACAGCGG CGTGTACTACTACACATGCGGATGGACAGACTTCGGCTCCTGGGGCCAAGGCACC CAAGTGACCGTGTCAAGCGGAGGCGGGGGGAGCGGAGGAGGTGGAAGTGGAGG GGGGGGATCTGGCGGGGGAGGAAGTGGAGGAGGAGGATCACAGGTGCAGCTCCA GGAGAGCGGGGGAGGACTGGTCCAGCCAGGAGGGAGCCTGAGACTCACATGTGC ACCCAGCGGATTTACACTGGATTATTACGCCATCGGATGGTTTAGGCAGGCACCCG GGAAAGAGAGAGAGGGCGTGAGCTGCATTAGCAGTAATAACAGCACCTATTACGC CGACTCAGTGAAGGGGCGGTTCACCATAAGCAGGGATAACGCCAAGAACACCGTC TACCTGCAGATGAATAGCCTGAAACCCGAAGACACAGCCGTGTACTATTGCGCCGC CGAACCCGACTACTCTGGAGTGTACTACTATACCTGCGGCTGGACCGACTTTGGCA GCTGGGGGCAAGGCACCCAGGTGACCGTGAGCAGT);
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., CAGGTGCAGCTGCAGGAGAGCGGAGGAGGGCTGGTGCAGCCCGGAGGAAGCCTG AGACTGACCTGCGCCCCCAGCGGATTCACCCTGGATTATTATGCTATTGGCTGGTTT AGGCAGGCTCCCGGCAAAGAGAGAGAGGGGGTGTCATGCATTAGCAGCAATAACT CAACCTACTACGCCGACAGCGTCAAGGGACGCTTCACCATTTCCAGGGACAACGC TAAGAACACCGTGTATCTCCAGATGAATAGCCTGAAGCCCGAGGACACCGCAGTG TACTACTGCGCCGCCGAGCCCGACTACAGCGGTGTGTATTACTACACCTGCGGATG GACCGACTTCGGCAGCTGGGGCCAGGGAACCCAGGTGACAGTGAGCAGCGTGAT CGCCGAGCTGCCCCCCAAGGTGAGCGTGTTCGTGCCCCCTAGAGACGGCTTCTTC GGCAACCCTAGAAAGAGCAAGCTGATCTGCCAAGCCACCGGCTTCTCCCCTAGAC AGATCCAAGTGAGCTGGCTGAGAGAGGGCAAGCAAGTGGGCAGCGGCGTCACAA CAGACCAAGTGCAAGCCGAGGCCAAGGAGAGCGGCCCCACCACCTACAAGGTGA CAAGCACCCTGACCATCAAGGAGAGCGACTGGCTGGGGCAGAGCATGTTCACCTG CAGAGTGGACCACAGAGGCCTGACCTTTCAGCAGAACGCTAGCAGCATGTGCGTG CCCGACCAAGACACCGCCATCAGAGTGTTCGCCATCCCCCCTAGCTTCGCTAGCAT CTTCCTGACCAAGAGCACCAAGCTGACCTGCCTCGTGACCGATCTGACCACCTAC GACAGCGTGACCATCAGCTGGACAAGACAGAACGGCGAGGCCGTGAAGACCCAC ACCAACATCAGCGAGAGCCACCCCAACGCCACCTTCAGCGCCGTGGGCGAGGCTA GCATCTGCGAGGACGACTGGAACAGCGGCGAGAGATTCACCTGCACCGTGACCCA CACCGACCTGCCTAGCCCCCTGAAGCAGACCATCAGCAGACCCAAGGGCGTGGCC CTGCACAGACCCGACGTGTACCTGCTGCCCCCCGCTAGAGAGCAGCTGAACCTGA GAGAGAGCGCCACCATCACCTGCCTGGTGACCGGCTTTAGCCCCGCTGACGTGTT CGTGCAGTGGATGCAGAGAGGGCAGCCCCTGAGCCCCGAGAAGTACGTGACAAG CGCCCCCATGCCCGAGCCCCAAGCCCCCGGCAGATACTTCGCCCACAGCATCCTG ACCGTGAGCGAGGAAGAGTGGAACACCGGCGAGACCTACACCTGCGTGGTGGCC CACGAGGCCCTGCCCAACAGAGTGACCGAGAGAACCGTGGACAAGAGCACCGGC AAGCCCACCCTGTACAACGTGAGCCTGGTGATGAGCGACACCGCCGGCACCTGCT AC).
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; further preferably, the SARS-CoV-2 variant strain is a Delta (B.1.617.2) strain, an Omicron (B.1.1.529) subtype BA.1 strain or an 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; further preferably, the SARS-CoV-2 variant strain is a Delta (B.1.617.2) strain, an Omicron (B.1.1.529) subtype BA.1 strain or an 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 S43-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 S43-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 nano antibody (TS 43) and IgM pentamer form (MS 43) based on the nano antibody S43 have obviously improved neutralization activity and obviously prolonged half-life compared with the monomer (namely, the nano antibody S43), realize mucosal immunity, limit virus propagation and further cross mucosal barrier, control virus mucosal propagation, provide potential novel antibody medicine capable of realizing atomized administration for clinical prevention and treatment of novel coronaviruses, and 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 TS43 and MS43 constructed in example 1 of the invention;
FIG. 2 is a diagram showing the results of molecular sieve chromatography and SDS-PAGE identification of the S43 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 TS43 protein according to example 1 of the present invention;
FIG. 4 is a graph showing the results of molecular sieve chromatography and SDS-PAGE identification of MS43 protein according to 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 S43 on the SARS-CoV-2 prototype strain pseudovirus before and after atomization, B is the neutralization activity result of the nano antibody construct TS43 on the SARS-CoV-2 prototype strain pseudovirus, and C is the neutralization activity result of the nano antibody construct MS43 on the SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus.
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-2RBD 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 synthesis by synthesis companies, e.g., the coding sequence of TS43 in the present invention was synthesized by Beijing, biotechnology, inc.
Example 1: construction, expression and purification of antibodies based on trivalent form of nanobody S43 (TS 43) and IgM pentameric form (MS 43)
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 S43 is obtained by immunizing alpaca with SARS-CoV-2S protein, constructing antibody library and screening by phage display technology in the laboratory; the VHH chain of the single-chain nanobody S43 has the amino acid sequence shown in SEQ ID NO. 8, which can specifically bind SARS-CoV-2RBD with high affinity (binding constant 1.2E-10.+ -. 1.4E-11M), and can neutralize SARS-CoV-2 pseudovirus with high neutralization activity in pseudovirus neutralization experiments, which all indicate: the S43 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 neutralization activity.
The 5 'end of the coding sequence (shown as SEQ ID NO: 14) of the single-chain nano antibody S43VHH chain is connected with a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15), and the 3' end is connected with 6 groupsThe coding sequence for the amino acid tag (hexa-His-tag) and the translation termination codon TGA were constructed into the pCAGGS vector (available from Invitrogen) via restriction enzyme sites EcoRI and XhoI, and the resulting recombinant vector was transfected into 293F cells (available from Invitrogen) for expression of the S43-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 S43-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 S43VHH chain of three nanobodies shown as SEQ ID NO. 14 was concatenated end-to-end (synthesized directly by Beijing engine technology 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 the 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 the TS43-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. SDS-PAGE of TS43-his protein identified a size of about 50KD, and the results are shown in FIG. 3.
The coding sequence of the nanobody S43VHH chain (shown as SEQ ID NO: 14) was ligated to the coding sequence of the Fc of the human IgM antibody (shown as SEQ ID NO: 16) by homologous recombination, a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15) was ligated to the 5 'end thereof, and a translation termination codon TGA was ligated to the 3' end thereof, which was constructed into a pCAGGS vector (purchased from Invitrogen) by restriction enzyme sites EcoRI and XhoI, to obtain a pCAGGS-S43-IgM Fc recombinant expression vector. The 3' -end of the coding sequence of the J chain (coding chain) as shown in SEQ ID NO:17 is connected with a translation termination codon TGA, and thenThe restriction enzyme sites EcoRI and XhoI were used and constructed into the pCAGGS vector (purchased from Invitrogen) to obtain the pCAGGS-J chain recombinant expression vector. The two recombinant expression vectors pCAGGS-S43-IgM Fc and pCAGGS-J chain were co-transfected into 293F cells (purchased from Invitrogen), and the S43-IgM Fc fusion protein and J chain were expressed, and self-assembled to form the IgM-form MS43 protein. The MS43 protein is obtained by HiTrap TM IgM Purification HP (GE Healthcare) and Superose TM 6 incrustase 10/300GL (GE Healthcare) was purified and identified by SDS-PAGE. SDS-PAGE of MS43 protein identified 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. Recombinant bacmid was transfected into Sf9 cells for viral expansion and protein expression 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 is provided withGel 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 S43 prepared in the above example and its constructs TS43 and MS43 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: the single-chain nano antibody S43 and constructs TS43 and MS43 thereof 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 S43 and results of affinity between MS43 and RBD of SARS-CoV-2 original strain and its constructs TS43 and MS43
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 koku science and technology 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 culturing 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
Purified single chain nanobody S43 and constructs TS43 and MS43 (prepared in example 1)Obtained) was diluted 5-fold to the 9 th gradient (2.56 pg/mL), and the diluted solution was mixed with 1.6X10-fold 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 CQ1Confocal 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 constructs TS43 and MS43 is improved relative to single-chain nanobody S43.
TABLE 2 Single chain nanobody S43 and neutralizing Capacity of its constructs TS43 and MS43 to pseudoviruses of SARS-CoV-2 original and variant strains
Note that: IC (integrated circuit) 50 (μg/mL) is the half inhibitory concentration of the antibody. * Indicating that the assay at the highest concentration of 10mg/mL still did not reach 100% inhibition.
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 S43 and its TS43 and MS43 (prepared in example 1) were diluted 2-fold to 11 th gradient, 4 duplicate wells per gradient, 50. Mu.L per well, respectively, and the dilutions were combined with an equal volume of 100 TCID 50 Is incubated at 37℃with SARS-CoV-2 original strain or variant strain Delta, omicron subtype BA.1. 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 . ExperimentAll were 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 S43 and the TS43 and MS43 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: TS43 and MS43 have good inhibiting effect on the live virus of the original strain and the variant strain of the new coronavirus.
TABLE 3 neutralization Capacity of Single-chain nanobody S43 and its constructs TS43 and MS43 for live virus 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 7: detection of stability of antibodies before and after nebulization
Single-chain nanobody S43 and its constructs TS43 and MS43 were aerosolized using an Aerogen Solo (Aerogen Inc., chicago, USA) nebulizer, respectively, and the aerosolized antibodies were then collected using a full glass SKC (Eight Four, 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 neutralization activity of nanobody S43 on SARS-CoV-2 prototype strain pseudovirus before and after atomization, B is the result of neutralization activity of nanobody construct TS43 on SARS-CoV-2 prototype strain pseudovirus, and C is the result of neutralization activity of nanobody construct MS43 on SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus; the results in fig. 10 show that: the nanobody constructs TS43 and MS43 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 via the aerosolization route.
The above results indicate that the nanobody S43-based constructs TS43 and MS43 of the present invention have potential to be developed into highly neutralizing active antibody drugs, particularly aerosolized drugs, for the treatment of novel coronavirus and variant strains thereof infections.
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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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
370 375 380
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
385 390 395 400
Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr
405 410 415
Asp Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
420 425 430
<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> 476
<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 Thr Cys Ala Pro 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 Asn Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr
100 105 110
Asp Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Val
115 120 125
Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp
130 135 140
Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala Thr
145 150 155 160
Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys
165 170 175
Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys
180 185 190
Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys
195 200 205
Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp His
210 215 220
Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp
225 230 235 240
Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala Ser
245 250 255
Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp Leu
260 265 270
Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly Glu
275 280 285
Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala Thr
290 295 300
Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn Ser
305 310 315 320
Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser Pro
325 330 335
Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val Ala Leu His Arg Pro
340 345 350
Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu
355 360 365
Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp Val
370 375 380
Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys Tyr
385 390 395 400
Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr Phe
405 410 415
Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp Asn Thr Gly Glu
420 425 430
Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val Thr
435 440 445
Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn Val
450 455 460
Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr
465 470 475
<210> 12
<211> 1293
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> coding sequence of trivalent nanobody
<400> 12
caggtccaac tccaagagag cggcggcggc ctcgtccaac ccggaggatc actcagactc 60
acatgcgccc caagcggctt cacactcgac tactacgcca tcggctggtt cagacaagcc 120
cccggcaaag agagagaagg agtgtcttgc attagcagca acaacagcac ctactacgcc 180
gacagtgtca aaggaagatt caccatcagc agggacaacg ctaagaacac cgtgtatctc 240
cagatgaact cactgaagcc cgaggacacc gccgtgtact actgcgccgc cgagcccgac 300
tacagcggcg tttactacta cacctgcgga tggaccgact tcggcagctg gggccaagga 360
acccaagtca ccgtgagcag cggaggcgga ggaagcggcg gtggaggaag tggcggaggc 420
ggatctgggg ggggaggatc aggcggagga ggaagccagg tgcagctgca ggagagcgga 480
ggaggactgg tgcagccagg aggaagcctg agactgacat gcgcaccaag cggattcaca 540
ctggactatt atgctatcgg atggttcaga caggcccctg gaaaagagag agagggggtg 600
agctgcatca gcagcaataa ctccacatac tacgccgata gcgtcaaggg gaggttcact 660
attagcaggg acaatgcaaa gaacacagtg tacctgcaga tgaacagcct gaagcccgaa 720
gacaccgccg tctactactg cgcagccgag cccgattaca gcggcgtgta ctactacaca 780
tgcggatgga cagacttcgg ctcctggggc caaggcaccc aagtgaccgt gtcaagcgga 840
ggcgggggga gcggaggagg tggaagtgga ggggggggat ctggcggggg aggaagtgga 900
ggaggaggat cacaggtgca gctccaggag agcgggggag gactggtcca gccaggaggg 960
agcctgagac tcacatgtgc acccagcgga tttacactgg attattacgc catcggatgg 1020
tttaggcagg cacccgggaa agagagagag ggcgtgagct gcattagcag taataacagc 1080
acctattacg ccgactcagt gaaggggcgg ttcaccataa gcagggataa cgccaagaac 1140
accgtctacc tgcagatgaa tagcctgaaa cccgaagaca cagccgtgta ctattgcgcc 1200
gccgaacccg actactctgg agtgtactac tatacctgcg gctggaccga ctttggcagc 1260
tgggggcaag gcacccaggt gaccgtgagc agt 1293
<210> 13
<211> 1428
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> coding sequence of nanobody fusion protein
<400> 13
caggtgcagc tgcaggagag cggaggaggg ctggtgcagc ccggaggaag cctgagactg 60
acctgcgccc ccagcggatt caccctggat tattatgcta ttggctggtt taggcaggct 120
cccggcaaag agagagaggg ggtgtcatgc attagcagca ataactcaac ctactacgcc 180
gacagcgtca agggacgctt caccatttcc agggacaacg ctaagaacac cgtgtatctc 240
cagatgaata gcctgaagcc cgaggacacc gcagtgtact actgcgccgc cgagcccgac 300
tacagcggtg tgtattacta cacctgcgga tggaccgact tcggcagctg gggccaggga 360
acccaggtga cagtgagcag cgtgatcgcc gagctgcccc ccaaggtgag cgtgttcgtg 420
ccccctagag acggcttctt cggcaaccct agaaagagca agctgatctg ccaagccacc 480
ggcttctccc ctagacagat ccaagtgagc tggctgagag agggcaagca agtgggcagc 540
ggcgtcacaa cagaccaagt gcaagccgag gccaaggaga gcggccccac cacctacaag 600
gtgacaagca ccctgaccat caaggagagc gactggctgg ggcagagcat gttcacctgc 660
agagtggacc acagaggcct gacctttcag cagaacgcta gcagcatgtg cgtgcccgac 720
caagacaccg ccatcagagt gttcgccatc ccccctagct tcgctagcat cttcctgacc 780
aagagcacca agctgacctg cctcgtgacc gatctgacca cctacgacag cgtgaccatc 840
agctggacaa gacagaacgg cgaggccgtg aagacccaca ccaacatcag cgagagccac 900
cccaacgcca ccttcagcgc cgtgggcgag gctagcatct gcgaggacga ctggaacagc 960
ggcgagagat tcacctgcac cgtgacccac accgacctgc ctagccccct gaagcagacc 1020
atcagcagac ccaagggcgt ggccctgcac agacccgacg tgtacctgct gccccccgct 1080
agagagcagc tgaacctgag agagagcgcc accatcacct gcctggtgac cggctttagc 1140
cccgctgacg tgttcgtgca gtggatgcag agagggcagc ccctgagccc cgagaagtac 1200
gtgacaagcg cccccatgcc cgagccccaa gcccccggca gatacttcgc ccacagcatc 1260
ctgaccgtga gcgaggaaga gtggaacacc ggcgagacct acacctgcgt ggtggcccac 1320
gaggccctgc ccaacagagt gaccgagaga accgtggaca agagcaccgg caagcccacc 1380
ctgtacaacg tgagcctggt gatgagcgac accgccggca cctgctac 1428
<210> 14
<211> 381
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Polynucleotide
<220>
<221> misc_feature
<223> coding sequence of nanobody S43VHH chain
<400> 14
caggtgcagc tgcaggagag cggaggaggg ctggtgcagc ccggaggaag cctgagactg 60
acctgcgccc ccagcggatt caccctggat tattatgcta ttggctggtt taggcaggct 120
cccggcaaag agagagaggg ggtgtcatgc attagcagca ataactcaac ctactacgcc 180
gacagcgtca agggacgctt caccatttcc agggacaacg ctaagaacac cgtgtatctc 240
cagatgaata gcctgaagcc cgaggacacc gcagtgtact actgcgccgc cgagcccgac 300
tacagcggtg tgtattacta cacctgcgga tggaccgact tcggcagctg gggccaggga 360
acccaggtga cagtgagcag c 381
<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.
CN202210278937.XA 2022-03-21 2022-03-21 Construct of nanobody S43 and application thereof Pending CN116813758A (en)

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