CN114763380A - Construction body of nano antibody S43 and application thereof - Google Patents

Construction body of nano antibody S43 and application thereof Download PDF

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CN114763380A
CN114763380A CN202210375856.1A CN202210375856A CN114763380A CN 114763380 A CN114763380 A CN 114763380A CN 202210375856 A CN202210375856 A CN 202210375856A CN 114763380 A CN114763380 A CN 114763380A
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CN114763380B (en
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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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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/08—RNA viruses
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract

The invention provides a construct (comprising a multivalent nano antibody and a nano antibody fusion protein) based on a nano antibody S43 specifically combined with SARS-CoV-2RBD, a related product and application thereof; the construct (comprising multivalent nano antibody and nano antibody fusion protein) of the nano antibody S43 based on the specific binding of SARS-CoV-2RBD can effectively inhibit SARS-CoV-2 infection and the infection of variant strain thereof, can be atomized for administration, can directly reach the lung, has quick effect and long half-life period, and provides a more effective treatment strategy for clinically preventing or treating the infection of new coronavirus and variant strain thereof.

Description

Construction body of nano antibody S43 and application thereof
The application is a divisional application of an invention patent application with the application date of 2022, 3 and 21, the application number of 202210278937.X, and the name of the invention is 'a construct of a nano antibody S43 and application thereof'.
Technical Field
The invention relates to the field of biomedicine, in particular to a construct of a nano antibody S43 and application thereof, and more particularly relates to a multivalent nano antibody based on a nano antibody S43 specifically binding SARS-CoV-2RBD, a nano antibody fusion protein, a polynucleotide encoding the multivalent nano antibody, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a transformed cell containing the polynucleotide, the nucleic acid construct or the expression vector, a pharmaceutical composition containing any one of the above products, application of the pharmaceutical composition in preparation of a medicament for preventing or treating a novel coronavirus, and application of the pharmaceutical composition in preparation of a reagent or a kit for detecting the novel coronavirus or diagnosing the infection of the novel coronavirus.
Background
The novel coronavirus (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV) belong to the family Coronaviridae (family Coronaviridae) and are the main pathogens of human respiratory system, and are mainly transmitted by means of spray, aerosol and contact, and the like, and the infectivity is strong, so that the viruses causing respiratory system diseases seriously jeopardize public health safety, and particularly frequently cause respiratory infectious diseases and continuously mutate in recent years.
The current outbreak of the new coronary pneumonia outbreak promotes the development of various vaccines and antiviral drugs, wherein the vaccination can effectively prevent serious infectious diseases, but the applicable object of the vaccine is uninfected people, the research and development period is long, and the clinical research process is complex. For patients with confirmed diagnosis, treatment can be performed only by antiviral drugs, wherein one antiviral drug is a therapeutic antibody drug, mainly a neutralizing antibody; the neutralizing antibody medicine is mainly combined with the antigen on the surface of the pathogenic microorganism to prevent the specific molecule expressed by the pathogenic microorganism from being combined with a cell surface receptor, thereby achieving the effect of neutralizing. SARS-CoV-2 virus has a glycosylated spike protein (S) on its surface, which can interact with 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 new coronavirus infection.
Conventional monoclonal antibodies are generally administered by intravenous injection, however, the concentration of the drug entering the lung from the systemic circulation of the monoclonal antibody administered by intravenous injection is very low, greatly reducing the antiviral effect of the neutralizing antibody itself, resulting in failure to effectively reduce the viral load in the lung. The novel coronavirus initially infects the upper respiratory tract, and the first interaction with the immune system mainly occurs on the mucosal surface of the respiratory tract, so for the novel coronavirus infecting through the respiratory tract, while paying attention to serum antibodies, it is necessary to think, design and develop suitable antibody drugs from the viewpoint of mucosal immunity. For example, nebulization allows for higher local concentrations of antibody drug in the respiratory tract, which is more effective in blocking viral infection during viral entry.
Nanobodies have been of great interest as therapeutic drugs, for example, the Nanobody drug Capracizumab (Cablivi) developed by the company AblynxTM) Is used for treating acquired thrombotic thrombocytopenic purpura and is the first approved nano antibody medicine; as another example, Nanobody candidate drug ALX-0171 is a trivalent form of Nanobody used in the treatment of pediatric Respiratory Syncytial Virus (RSV) infection, which has entered clinical phase II (https:// clinical trials. gov), suggesting: the nano antibody medicine has safety and feasibility.
Therefore, the development of the nano antibody medicine aiming at the novel coronavirus and suitable for respiratory tract mucosa immunity has potential clinical application value and prospect.
Disclosure of Invention
Object of the Invention
The invention aims to provide a construct (comprising a multivalent nanobody and a nanobody fusion protein) based on a nanobody S43 specifically binding SARS-CoV-2RBD, 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, a pharmaceutical composition comprising any one of the above products, an application of the pharmaceutical composition in preparation of a medicament for preventing or treating novel coronavirus, and an application of the pharmaceutical composition in preparation of a reagent or a kit for detecting novel coronavirus or diagnosing novel coronavirus infection.
The construct (comprising multivalent nano antibody and nano antibody fusion protein) of the nano antibody S43 based on specific binding SARS-CoV-2RBD can effectively inhibit SARS-CoV-2 infection and SARS-CoV-2 variant strain infection, can be atomized for administration, can directly reach the lung, has quick effect and long half-life period, and provides a more effective treatment strategy for new coronavirus and SARS-CoV-2 variant strain infection.
Solution scheme
In order to realize the purpose, the invention provides the following technical scheme:
in a first aspect, the present invention provides a multivalent nanobody, which comprises two or more nanobodies specifically binding to SARS-CoV-2RBD, wherein the nanobody specifically binding to SARS-CoV-2RBD comprises the following CDRs:
CDR1 having an amino acid sequence shown in SEQ ID NO:1 (i.e., GFTLDYYAIG),
CDR2 having an amino acid sequence as set forth in SEQ ID NO:2 (i.e., CISSNNSTYYADSVKG), and
the amino acid sequence is CDR3 as shown in SEQ ID NO. 3 (i.e., EPDYSGVYYYTCGWTDFGS).
In a specific embodiment, the nanobody that specifically binds to SARS-CoV-2RBD further comprises 4 framework regions FR1-4, the FR1-4 is staggered in order from the CDR1, CDR2 and CDR 3;
preferably, the amino acid sequence of FR1-4 is shown as 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 that specifically binds to SARS-CoV-2RBD has the amino acid sequence 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 nanobody is shown as the following SEQ ID NO: 8:
Figure BDA0003590710130000031
CDR1, CDR2, and CDR3 for the heavy chain variable region.
In a preferred embodiment I, the multivalent nanobody consists of two or more, preferably three, nanobodies specifically binding to SARS-CoV-2RBD linked by Linker;
wherein the Linker is (GGGGS) n, wherein n is 1,2,3, or 4, preferably n is 2 or 3.
As a further preference of the specific embodiment I, the multivalent nanobody is a trivalent nanobody having an 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 the structure from N-terminus to C-terminus as shown in formula (I):
A-L-B (I)
wherein, the first and the second end of the pipe are connected with each other,
a is a single strip, the nanobody that specifically binds SARS-CoV-2RBD, or a multivalent nanobody as described in preferred embodiment I above;
b is Fc fragment of human IgM; preferably, the Fc fragment of the human IgM has the amino acid sequence shown as 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 shown as SEQ ID NO: 10;
l is (GGGGS) m, wherein m is 0,1,2,3, or 4.
As a preferred embodiment of the above fusion protein, it has an amino acid sequence 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 of the nanobody fusion protein from N-terminus to C-terminus is represented by formula (I):
A-L-B (I)
wherein the content of the first and second substances,
a is a single nanobody that specifically binds to SARS-CoV-2RBD as defined above in the first aspect; alternatively, a is the multivalent nanobody according to preferred embodiment I of the above first aspect, i.e., a multivalent nanobody composed of two or more, preferably three, nanobodies specifically binding to SARS-CoV-2RBD linked by a Linker, wherein the Linker is (GGGGS) n, wherein n is 1,2,3, or 4, preferably n is 2 or 3;
b is Fc fragment of human IgM; preferably, the Fc fragment of the human IgM has the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence with at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO. 10;
l is (GGGGS) m, wherein m is 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 particular embodiments, the polynucleotide is DNA or mRNA;
preferably, the polynucleotide encodes a multivalent nanobody according to preferred embodiment I of the above first aspect, 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 a nucleotide sequence as set forth in 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 a histidine tag, stop codon, etc.
In a fifth aspect, the present invention provides an expression vector comprising the nucleic acid construct according to the fourth aspect above.
In a sixth aspect, the present invention provides a transformed cell comprising a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above or an expression vector as described in the fifth aspect above.
In a seventh aspect, the present invention provides a pharmaceutical composition comprising a multivalent nanobody according to the first aspect described above, a nanobody fusion protein according to the second aspect described above, a polynucleotide according to the third aspect described above, a nucleic acid construct according to the fourth aspect described above, an expression vector according to the fifth aspect described above or a transformed cell according to the sixth aspect described above, 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 an aerosol, a spray and a powder spray;
preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets and ointments;
preferably, the parenteral formulation is a transdermal agent, an ointment, a plaster, a topical liquid, an injectable or a bolus formulation.
The dose of the active ingredient of the pharmaceutical composition of the present invention varies depending on the subject, the target organ, the symptom, the administration method, and the like, and can be determined by the judgment of the doctor in consideration of the type of the formulation, the administration method, the age and weight of the patient, the symptom of the patient, and the like.
In an eighth aspect, the present invention provides a multivalent nanobody according to the first aspect, a nanobody fusion protein according to the second aspect, a polynucleotide according to the third aspect, a nucleic acid construct according to the fourth aspect, an expression vector according to the fifth aspect, a transformed cell according to the sixth aspect, or a pharmaceutical composition according to the seventh aspect, for use in 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) strain, 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 Delta (B.1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain.
In a ninth aspect, the present invention provides a multivalent nanobody according to the first aspect above, a nanobody fusion protein according to the second aspect above, a polynucleotide according to the third aspect above, a nucleic acid construct according to the fourth aspect above, an expression vector according to the fifth aspect above or a transformed cell according to the sixth aspect above for use in the preparation of a reagent or kit for the detection of a novel coronavirus or for the diagnosis 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) strain, 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 Delta (B.1.617.2) strain, Omicron (B.1.1.529) subtype BA.1 strain or Omicron (B.1.1.529) subtype BA.2 strain.
In a tenth aspect, the present invention provides a novel coronavirus detection kit comprising a multivalent nanobody according to the first aspect, a nanobody fusion protein according to the second aspect, a polynucleotide according to the third aspect, a nucleic acid construct according to the fourth aspect, an expression vector according to the fifth aspect, or a transformed cell according to the sixth aspect.
Advantageous effects
The invention carries on the medicine development of the construct of nanometer antibody to the new coronavirus, the construct based on nanometer antibody S43 of the invention can combine with SARS-CoV-2RBD with high affinity, can neutralize the false virus and live virus of SARS-CoV-2 prototype strain and a series of variant strains with high neutralizing activity, these show: the construct based on nanobody S43 is a novel coronavirus (SARS-CoV-2) nanobody that can bind to SARS-CoV-2RBD with high affinity and has high neutralizing activity.
In particular, the inventors have proved through a series of experiments that the trivalent nanobody (TS43) and the IgM pentamer form (MS43) based on the nanobody S43 of the present invention have significantly improved neutralizing activity and significantly prolonged half-life compared to the monomer thereof (i.e., the nanobody S43), which realizes mucosal immunity, can limit the propagation of viruses and further cross mucosal barriers, controls mucosal transmission of viruses, provides a potential new antibody drug capable of being administered by atomization for clinical prevention and treatment of novel coronaviruses, and can realize sensitive and reliable detection of novel coronaviruses.
Drawings
One or more embodiments are illustrated by the corresponding figures in the drawings, which are not meant to be limiting. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
FIG. 1 is a schematic structural diagram of nanobody constructs TS43 and MS43 constructed in example 1 of the present invention;
FIG. 2 is a graph 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 described in 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 described in example 1 of the present invention;
FIG. 5 is a SDS-PAGE result of the SARS-CoV-2RBD-his protein (A), the RBD-his protein (B) of the variant strain Omicron (B.1.1.529) subtype BA.1 and the RBD-his protein (C) of the variant strain Omicron (B.1.1.529) subtype BA.2 described in example 2 of the present invention.
FIG. 6 is a graphical representation of the effect of three antibodies on the neutralization of pseudoviral infection of the SARS-CoV-2 prototype strain as determined in example 5 of the present invention.
FIG. 7 is a graph showing the effect of three antibodies tested in example 5 of the present invention in neutralizing the infection of the SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus.
FIG. 8 is a graph showing the effect of neutralizing infection with the SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 pseudovirus by three antibodies measured in example 5 of the present invention.
FIG. 9 is a graph showing the effect of neutralizing infection with the SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.2 pseudovirus by three antibodies measured in example 5 of the present invention.
FIG. 10 is a graph showing the neutralizing activity of the three antibodies against pseudoviruses before and after nebulization, measured in example 7 of the present invention; wherein, A is the result of the neutralization activity of the nano antibody S43 on SARS-CoV-2 prototype strain pseudovirus before and after atomization, B is the result of the neutralization activity of the nano antibody construct TS43 on SARS-CoV-2 prototype strain pseudovirus, and C is the result of the neutralization activity of the nano antibody construct MS43 on SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present 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, and the like that are well known to those skilled in the art are not described in detail in order to not unnecessarily obscure the present invention.
The present invention will be described in detail below.
Definition of
"Nanobodies", i.e., "heavy chain single domain antibodies", comprise only one heavy chain variable region (VHH), from which a light chain is naturally deleted compared to other antibodies.
Due to its biophysical advantages, nanobodies can be easily aerosolized and delivered directly to the lungs via an inhaler to treat respiratory virus-induced infections, and are considered to be very potential antibody-based drugs.
"specific" binding, when referring to ligand/receptor, antibody/antigen or other binding pairs, refers to determining the presence or absence of a binding reaction of a protein, such as a nanobody of the invention, to a SARS-CoV-2RBD protein in a heterogeneous population of proteins and/or other biological agents. Thus, under the conditions specified, 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, and milk used in the following examples of the present invention are commercially available, and for example, TRIzol is purchased from Invitrogen, and Superscript II First-Strand Synthesis System for RT-PCR kit is purchased from Invitrogen.
Some commonly used biological materials, such as competent cells, vectors, helper phages, cells to be transformed, etc., are also commercially available products, e.g., pCAGGS vectors are available from miaolingplasmds; 293F cells, HEK293T cells and the like were purchased from ATCC; series Sensor Chip SA chips were purchased from GE Healthcare; vero cells were purchased from ATCC CCL 81.
Some synthetic biomaterials, such as primers, sequences, etc., requiring artificial synthesis are prepared by synthetic companies, for example, the coding sequence of TS43 in the present invention is synthesized by Beijing Optimalaceae Biotechnology, Inc.
Example 1: construction, expression and purification of trivalent form (TS43) and IgM pentamer form (MS43) antibodies based on nano antibody S43
The schematic structure of the monovalent nanobody and its trivalent form and IgM pentamer form in this example is shown in fig. 1.
The adopted basic nano antibody S43 is obtained by using SARS-CoV-2S protein to immunize alpaca, constructing an antibody library and screening by using a phage display technology in the laboratory; the amino acid sequence of the monovalent nanometer antibody S43 is shown in SEQ ID NO:8, and the monovalent nanometer antibody S43 can be specifically combined with SARS-CoV-2RBD (the combination constant is 1.2E-10 +/-1.4E-11M) with high affinity, and can neutralize SARS-CoV-2 pseudovirus with high neutralization activity in a pseudovirus neutralization experiment, which shows that: the S43 nanometer antibody is a novel coronavirus (SARS-CoV-2) alpaca source nanometer antibody which can be combined with SARS-CoV-2RBD with high affinity and has high neutralization activity.
The 5 'end of the coding sequence of the above monovalent nanobody S43 (shown in SEQ ID NO: 14) was ligated with a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO:15), the 3' end was ligated with a coding sequence of 6 histidine tags (hexa-His-tag) and the translation termination codon TGA, which were constructed into pCAGGS vector (purchased from Invitrogen) via restriction enzyme sites EcoRI and XhoI, and the resulting recombinant vector was transfected into 293F cells (purchased from Invitrogen) to express S43-His protein. The cell culture fluid containing the target protein is subjected to nickel ion affinity chromatography (HisTrap)TMexcel (GE Healthcare) and gel filtration chromatography (Superdex)TM75 Increate 10/300GL column (GE Healthcare)) can obtain purer target protein after purification. The size of the S43-his protein was identified to be about 15KD by SDS-PAGE, and the results are shown in FIG. 2.
By two stages (GGGGS)3The sequence, three of the coding sequences of the Nanobody S43 shown in SEQ ID NO:14 were connected in series by head-to-tail connection (directly synthesized by Biotech, Inc., of Ongjingkidaceae), the 5 'end was connected with a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO:15), the 3' end was connected with a coding sequence of 6 histidine tags (hexa-His-tag) and a translation termination codon TGA, and the coding sequences were constructed into pCAGGS vectors (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, and then the resulting recombinant vectors were transfected into 293F cells (purchased from Invitrogen) to express the TS43-His protein. The cell culture fluid containing the target protein is subjected to nickel ion affinity chromatography (HisTrap)TMexcel ((GE Healthcare)) and gel filtration chromatography (Superdex)TM200 Increate 10/300GL column (GE Healthcare)) can obtain purer target protein after purification. The size of the TS43-his protein was identified to be around 50kD by SDS-PAGE, and the results are shown in FIG. 3.
Connecting the coding sequence of the nano antibody S43 (shown as SEQ ID NO: 14) with the coding sequence of Fc of the human IgM antibody (shown as SEQ ID NO: 16) by means of homologous recombination, connecting a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO:15) at the 5 'end of the nano antibody S43 and a translation stop codon TGA at the 3' end of the nano antibody S43, and passing through restriction enzymesSites EcoRI and XhoI, which were constructed into pCAGGS vector (purchased from Invitrogen) to obtain pCAGGS-S43-IgM Fc recombinant expression vector. The 3' -end of the coding sequence (shown in SEQ ID NO: 17) of J chain (Joining chain) was ligated with translation termination codon TGA, which was constructed into pCAGGS vector (purchased from Invitrogen) via restriction enzyme sites EcoRI and XhoI, to obtain pCAGGS-J chain recombinant expression vector. The two recombinant expression vectors pCAGGS-S43-IgM Fc and pCAGGS-J chain are co-transfected into 293F cells (purchased from Invitrogen), and S43-IgM Fc fusion protein and J chain are expressed and self-assembled to form IgM-form MS43 protein. The obtained MS43 protein passes through HiTrapTMIgM Purification HP (GE Healthcare) and Superose TM6 incrase 10/300GL (GE healthcare) was purified and characterized by SDS-PAGE. The size of the MS43 protein was about 70kD as determined by SDS-PAGE, and the results are shown in FIG. 4.
Example 2: expression and purification of SARS-CoV-2 and its variant strain RBD
The 3' end of the coding sequence of the RBD protein of SARS-CoV-2 original strain (the amino acid sequence of which is shown in SEQ ID NO: 18) is linked with the coding sequence of 6 histidine-tag (hexa-His-tag) and the translation termination codon TGA, which are constructed into pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, and then the resulting recombinant vector is transfected into 293F cells (purchased from Invitrogen) for expression of SARS-CoV-2RBD-His protein.
The coding sequence of 6 histidine tags (hexa-His-tag) and the translation termination codon TGA were ligated to the 3' end of the coding sequence of the RBD protein of SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 (the amino acid sequence of which is shown in SEQ ID NO: 19) and Omicron (B.1.1.529) subtype BA.2 (the amino acid sequence of which is shown in SEQ ID NO: 20), respectively, and expressed by a bac-to-bac baculovirus expression system (Invitrogen). The pFastbac1 plasmid containing the gene of interest was transformed into DH10Bac competent cells to generate recombinant bacmids (bacmids). Recombinant bacmids were transfected into Sf9 cells for viral expansion and protein expression in Hi5 cells. After 48h expression, Hi5 cell supernatants were collected using HisTrapTMexcel (GE healthcare) soluble proteins were purified by nickel affinity chromatography.
Passing cell culture solution containing target protein through nickel ion affinity chromatography column HisTrapTMexcel (GE healthcare) and gel filtration chromatography SuperdexTMAfter purification with 200 Increatase 10/300GL column (GE Healthcare), a purer target protein can be obtained. The SDS-PAGE identification size of the SARS-CoV-2RBD-his protein, the RBD-his protein of the variant strain Omicron (B.1.1.529) subtype BA.1 and the RBD-his protein of the variant strain Omicron (B.1.1.529) subtype BA.2 is about 30KD, which is respectively shown in FIGS. 5A-C.
Example 3: surface plasma resonance technology for detecting the binding capacity of each antibody and the RBD protein of SARS-CoV-2 original strain and its variant
Surface plasmon resonance analysis was performed using Biacore 8K (Biacore Inc.). The method comprises the following specific steps:
the monovalent nanobody S43 prepared in the above example and its constructs TS43 and MS43 were biotinylated and then immobilized on Series Sensor Chip SA chips (Cytiva Life Sciences), respectively; using PBST buffer (2.7 mM KCl, 137mM NaCl, 4.3mM Na)2HPO4，1.4mM KH2PO40.05% tween) the RBD proteins of the original strain of SARS-CoV-2 and its variant prepared in the above examples were diluted in multiple ratios, and loaded one by one onto the chip from low to high concentrations. The calculation of binding kinetic constants was performed using BIAevaluation software 8K (Biacore, Inc.). Equilibrium dissociation constant (K) between antibodies and RBDsD) As shown in table 1, the results in table 1 indicate: the monovalent nanometer antibody S43 and the constructs TS43 and MS43 thereof can be combined with RBD proteins of SARS-CoV-2 prototype strain and variant strain Omicron subtype BA.1 and Omicron subtype BA.2 with higher affinity.
TABLE 1 results of the affinity between monovalent nanobody S43 and its constructs TS43 and MS43 and RBD of SARS-CoV-2 original strain and variant strain
Figure BDA0003590710130000131
Example 4: SARS-CoV-2 original strain and variant strain pseudovirus package
1) The 18 th amino acid gene of 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 is removed, and the rest sequence of S protein is synthesized (providing synthesis service by Jinweizhi, Suzhou) to obtain 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, whose sequences are respectively shown in SEQ ID NO: 21-24.
2) The protein gene obtained in 1) was 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-del 18 and pCAGGS-B.1.1.529-BA.2-S-del18, respectively.
The SARS-CoV-2 original strain and variant strain pseudovirus are packaged by the following steps:
a. cell preparation: HEK293T cells (purchased from ATCC CRL-3216) were plated in 10cm cell culture dishes to reach a cell confluency density of about 80% the next day. The culture solution is DMEM medium containing 10% FBS.
b. Transfection: and (3) taking the expression plasmids of each S protein in the step 2), transfecting 30 mu g of plasmids/10 cm of cell culture dishes by using PEI, uniformly mixing the target plasmids and PEI according to the ratio of 1:3, transfecting, replacing a culture solution (DMEM medium containing 10% FBS) for 4-6h, and culturing for 24h at 37 ℃.
c. Adding poison: pseudovirus packaging frame virus G.VSV-delG (purchased from Wuhan Shu Ministry of encyclopedia scientific and technology Co., Ltd.) was added to the above transfected HEK293T cells, incubated at 37 ℃ for 2 hours, 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 and the culture was continued in the incubator for 30 hours.
d. And (3) toxin collection: collecting supernatant, centrifuging at 3000rpm for 10min, filtering in a 0.45 μm sterile filter in an ultra-clean bench, removing cell debris, packaging, and freezing at-80 deg.C in a refrigerator.
The pseudoviruses of SARS-CoV-2 prototype strain (SARS-CoV-2WT) 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 obtained respectively.
Example 5: detection of antibody-neutralizing pseudoviral infection
The purified monovalent nanobody S43 and its constructs TS43 and MS43 (prepared from example 1) were diluted 5-fold to the 9 th gradient (2.56pg/mL), respectively, and the dilutions were mixed with 1.6X 104TCID50A series of pseudoviruses of the original strain and the variant strain of SARS-CoV-2 obtained in example 4 were separately mixed, incubated at 37 ℃ for 1 hour, and then added to a 96-well plate previously inoculated with Vero cells (purchased from ATCC CCL 81). After incubation for 18-20 hours, the cells were tested by CQ1 capacitive Quantitative Image Cytometer (Yokogawa). According to the number of cells with GFP fluorescence, the neutralizing capacity of the antibody to the pseudoviruses of the series of SARS-CoV-2 prototype strains and variant strains Delta, BA.1 and BA.2 is calculated, the results are respectively shown in FIGS. 6-9, and the statistics of the results are shown in Table 2; the results show that the pseudovirus neutralization effect of the constructs TS43 and MS43 is improved relative to the univalent nanobody S43.
TABLE 2 neutralizing ability of monovalent nanobody S43 and its constructs TS43 and MS43 against pseudovirus of SARS-CoV-2 original strain and variant strain
Figure BDA0003590710130000141
Note: IC (integrated circuit)50(μ g/mL) is the half inhibitory concentration of the antibody. Indicated by an indication that 100% inhibition was not achieved at the highest concentration of 10 mg/mL.
Example 6: detection of antibody-neutralized live virus infection
In this example, the neutralizing effect of each antibody on live coronavirus was determined by a live virus neutralizing assay based on cytopathic effect (CPE). The method comprises the following specific steps:
purified monovalent nanobody S43 and TS43 and MS43 (made from example 1) thereof were diluted 2-fold to 11 th gradient with 4 replicate wells per gradient, 50 μ L per well, and the dilutions were mixed with equal volume of 100 TCID50The SARS-CoV-2 original strain or its variant strain Delta, Omicron subtype BA.1Incubation was performed at 37 ℃. After 1 hour, the mixture was added to the suspended Vero cells and incubation was continued for 3 days at 37 ℃. Cytopathic conditions were observed and recorded. Calculation of IC of Nanobodies and constructs Using GraphPad Prism 7.050. The experiments were all performed in the biosafety third-level laboratory (BSL3) of the chinese centers for disease prevention and control.
The neutralizing effect of the monovalent nanobody S43, TS43 and MS43 on live viruses of the original strain of the new coronavirus and the variant strain thereof is shown in table 3, and the results in table 3 show that: TS43 and MS43 have good inhibition effects on live viruses of original strains and variant strains of the new coronavirus.
TABLE 3 neutralizing ability of monovalent nanobody S43 and its constructs TS43 and MS43 against live virus of SARS-CoV-2 original strain and variant strain
Figure BDA0003590710130000151
Note: IC (integrated circuit)50(μ g/mL) is the half inhibitory concentration of the antibody.
Example 7: detection of stability before and after antibody atomization
Monovalent nanobodies S43 and its constructs TS43 and MS43 were separately nebulized using an Aerogen Solo (Aerogen inc., Chicago, USA) nebulizer, and the nebulized antibodies were collected using full glass SKC (origin 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 the neutralization activity of the nanobody S43 on the SARS-CoV-2 prototype strain pseudovirus before and after atomization, B is the result of the neutralization activity of the nanobody construct TS43 on the SARS-CoV-2 prototype strain pseudovirus, and C is the result of the neutralization activity of the nanobody construct MS43 on the 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 present invention remained stable against the neutralizing activity of pseudoviruses of the prototype strain of the new coronavirus or its variant strain before and after nebulization, suggesting that they are both suitable for administration by nebulization route.
The results show that the constructs TS43 and MS43 based on the nano antibody S43 have the potential of being developed into high-neutralization-activity antibody medicines, particularly atomized medicines, for treating the infection of novel coronaviruses and variant strains thereof.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
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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 nano antibody 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 nanometer antibody
<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 nano antibody 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 nano antibody S43 VHH 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> Fc coding sequence 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 (8)

1. The application of IgM pentamer formed by the following nano antibody fusion protein in preparing a medicament for preventing or treating new coronavirus infection is characterized in that the structure of the nano antibody fusion protein from N end to C end is shown as the formula (I):
A-L-B (I)
wherein the content of the first and second substances,
a is a nano antibody specifically combined with SARS-CoV-2 RBD;
b is Fc fragment of human IgM;
l is (GGGGS) m, wherein m is 0,1,2,3, or 4.
2. The use of claim 1, wherein the nanobody that specifically binds to SARS-CoV-2RBD comprises the following CDRs: CDR1 with an amino acid sequence shown as SEQ ID NO. 1, CDR2 with an amino acid sequence shown as SEQ ID NO. 2 and CDR3 with an amino acid sequence shown as SEQ ID NO. 3.
3. The use of claim 2, wherein said nanobody that specifically binds to SARS-CoV-2RBD further comprises 4 framework regions FR1-4, said FR1-4 being staggered in sequence from said CDR1, CDR2 and CDR 3;
preferably, the FR1-4 is shown as SEQ ID NO. 4, 5, 6 and 7 respectively.
4. The use of any one of claims 1-3, wherein the nanobody that specifically binds to SARS-CoV-2RBD has an amino acid sequence as shown in SEQ ID NO. 8.
5. The use of any one of claims 1-4, wherein the Fc fragment of human IgM has the amino acid sequence shown in SEQ ID NO 10.
6. The use of any one of claims 1-5, wherein the nanobody fusion protein has the amino acid sequence shown in SEQ ID NO. 11.
7. The use of any one of claims 1 to 6, 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) strain, Omicron (B.1.1.529) subtype BA.1 strain and/or Omicron (B.1.1.529) subtype BA.2 strain.
8. The use according to any one of claims 1 to 7, wherein the medicament is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation;
preferably, the nasal spray is selected from the group consisting of an aerosol, a spray and a powder spray;
preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets and ointments;
preferably, the parenteral formulation is a transdermal agent, ointment, plaster, topical liquid, injectable or bolus formulation.
CN202210375856.1A 2022-03-21 2022-03-21 Construction body of nano antibody S43 and application thereof Active CN114763380B (en)

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