CN115043935B - Nanometer antibody targeting novel coronavirus and preparation method and application thereof - Google Patents

Nanometer antibody targeting novel coronavirus and preparation method and application thereof Download PDF

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CN115043935B
CN115043935B CN202210637547.7A CN202210637547A CN115043935B CN 115043935 B CN115043935 B CN 115043935B CN 202210637547 A CN202210637547 A CN 202210637547A CN 115043935 B CN115043935 B CN 115043935B
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nanobody
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novel coronavirus
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CN115043935A (en
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杨传彬
王惠芳
郑六海
李志杰
耿庆山
钟晓茹
孙继超
戴凌云
王继刚
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Shenzhen Peoples Hospital
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The embodiment of the invention discloses a nano antibody for targeting a novel coronavirus and a preparation method and application thereof, wherein the nano antibody comprises at least one of a nano antibody NbN2, a nano antibody NbN3, a nano antibody NbN4, a nano antibody NbN5, a nano antibody NbN6 and a nano antibody NbN11, and the provided nano antibody has higher affinity to RBD structural domains of a novel coronavirus wild strain, a novel coronavirus variant Alpha (Alpha), a novel coronavirus Beta (Beta), a novel coronavirus Delta (Delta), a novel coronavirus omacron and subtype BA.2 thereof, can rapidly and specifically neutralize different novel coronaviruses, and effectively inhibit the activity of the novel coronavirus; meanwhile, the provided nano antibody has a longer CDR3 sequence, is beneficial to flexibly regulating the conformation of the nano antibody, improves the binding effect with viruses, and is beneficial to wide application.

Description

Nanometer antibody targeting novel coronavirus and preparation method and application thereof
The application is a divisional application of a nanometer antibody targeting a new coronavirus, a preparation method and application thereof, the application date of the original application is 2022, 5 and 23, the application number is 202210330386.7, and the invention creates the nanometer antibody targeting the new coronavirus, and the preparation method and application thereof.
Technical Field
The invention relates to the technical field of biology, in particular to a nano antibody targeting a novel coronavirus, a preparation method and application thereof.
Background
The novel coronavirus pneumonia (Corona Virus Disease 2019, covd-19) can be transmitted through the approaches of droplet transmission, contact with respiratory secretions, aerosol transmission and the like, and the consequences are serious. The acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus, SARS-CoV-2) causing the COVID-19 expresses mainly 4 structural proteins, spike (S), envelope (envelope), membrane (membrane) and nucleocapsid (nucleocapsid), respectively. Wherein the S protein comprises an N-terminal S1 subunit and a C-terminal S2 subunit, the S1 subunit comprising a receptor binding domain of about 200 amino acids (receptor binding domain, RBD) being responsible for binding of the virus to the host receptor and the S2 subunit being responsible for fusion of the virus to the host cell membrane. The S protein plays a vital role in virus attachment, infection and transmission, and is an important target for the research and development of anti-SARS-CoV-2 drugs by being combined with host cell angiotensin converting enzyme2 (angiotensin converting enzyme, ACE 2) to infect cells.
Neutralizing antibodies are an important therapy against covd-19. It has been found that clinical outcome can be significantly improved by delivering serum from a convalescent patient to a critically ill covd-19 patient, indicating that neutralizing antibody therapy also has better relief from new coronary critically ill patients. The mechanism of action is that the neutralizing antibody binds with spike glycoprotein on the surface of the novel coronavirus to further influence the binding of SARS-CoV-2 with the surface receptor of the host cell, thereby preventing the virus from infecting the target cell. There are a number of neutralizing antibodies currently marketed for the treatment of covd-19, and these monoclonal antibody targets are primarily focused on the RBD domain of the S protein. However, in the face of constantly mutated SARS-CoV-2, it is difficult for existing neutralizing antibodies to target new novel coronavariants. A recent study showed that omacron subtype BA.2 could evade almost all neutralizing antibody treatments. Thus, there is an urgent need to develop new neutralizing antibodies that can simultaneously target different novel crown variants, particularly omacron and its subtype ba.2.
Disclosure of Invention
The application aims to provide a nano antibody targeting a novel coronavirus, a preparation method and application thereof, and aims to solve the problem that a neutralizing antibody of a novel coronavirus variant Omicron and a subtype BA.2 thereof is lacking in the prior art.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the application discloses a nanobody for targeting a novel coronavirus, wherein the nanobody comprises at least one of nanobody NbN2, nanobody NbN3, nanobody NbN4, nanobody NbN5, nanobody NbN6 and nanobody NbN11, wherein the amino acid sequence of nanobody NbN2 is shown as seq. ID No.1, the amino acid sequence of nanobody NbN3 is shown as seq. ID No.2, the amino acid sequence of nanobody NbN4 is shown as seq. ID No.3, the amino acid sequence of nanobody NbN5 is shown as seq. ID No.4, and the amino acid sequence of nanobody NbN6 is shown as seq. ID No.5, and the amino acid sequence of nanobody NbN11 is shown as seq. ID No. 6.
In a second aspect, the present application discloses a method for preparing a nanobody targeting a novel coronavirus, comprising the steps of:
providing target proteins, coating the target proteins on an immune tube, and carrying out enrichment screening to obtain a phage library;
performing second generation sequencing on the eluent of the phage library, and synthesizing a gene sequence of the nano antibody with the original count ratio of 10 according to a sequencing result;
cloning the gene sequence of the nano antibody into an expression vector to obtain a recombinant plasmid, transferring the recombinant plasmid into a host cell to induce expression and purifying to obtain the nano antibody of the targeted novel coronavirus.
In a third aspect, the application discloses the use of nanobodies targeting novel coronaviruses in the preparation of a medicament for the prevention and/or treatment of novel coronavirus pneumonia.
In a fourth aspect, the application discloses application of a nanobody targeting a novel coronavirus in preparing a detection reagent or a detection kit for detecting the novel coronavirus.
The nanometer antibody of the novel coronavirus Omicron and subtype BA.2 provided in the first aspect of the application comprises at least one of a nanometer antibody NbN2, a nanometer antibody NbN3, a nanometer antibody NbN4, a nanometer antibody NbN5, a nanometer antibody NbN6 and a nanometer antibody NbN11, and the provided nanometer antibody has higher affinity to RBD domains of a wild strain of the novel coronavirus, a variant Alpha (Alpha) of the novel coronavirus, a Beta (Beta) of the novel coronavirus, delta (Delta) of the novel coronavirus, the Omicron of the novel coronavirus and subtype BA.2 thereof, can neutralize different novel coronaviruses rapidly and specifically, and effectively inhibit the activity of the novel coronavirus; meanwhile, the provided nano antibody has a longer CDR3 sequence, is beneficial to flexibly regulating the conformation of the nano antibody, improves the binding effect with viruses, has lower preparation cost and is beneficial to wide application.
According to the preparation method of the nanometer antibody targeting the novel coronavirus Omicron and the subtype BA.2 thereof, which is provided by the second aspect of the application, a nanometer antibody library is constructed by taking RBD proteins of the novel coronavirus Omicron and the subtype BA.2 thereof as targets based on phage display screening technology, and the nanometer antibody targeting the novel coronavirus Omicron and the subtype BA.2 thereof is obtained by combining second-generation sequencing, sequence synthesis, cloning, expression and purification; the preparation method utilizes the biological characteristics of phage fast replication in host bacteria, can rapidly screen the nano antibody combined with the specific variant Omikovia and subtype BA.2RBD protein thereof with high flux, is rapid and simple, is favorable for large-scale screening, and improves the screening efficiency.
The application of the nanobody of the targeting novel coronavirus Omicron and subtype BA.2 thereof in preparing the medicine for preventing and/or treating the novel coronavirus pneumonia is provided in the third aspect of the application, and the nanobody of the targeting novel coronavirus Omicron and subtype BA.2 thereof obtained by screening comprises at least one of nanobody NbN2, nanobody NbN3, nanobody NbN4, nanobody NbN5, nanobody NbN6 and nanobody NbN11, has better neutralization activity capability and inhibition activity capability on viruses of wild strains and variant strains of the novel coronavirus, and is suitable for preparing related medicines for preventing and/or treating the novel coronavirus pneumonia.
The application of the nanometer antibody for targeting the novel coronavirus Omicron and the subtype BA.2 thereof in preparing the detection reagent or the detection kit for detecting the novel coronavirus is provided, the nanometer antibody for targeting the novel coronavirus Omicron and the subtype BA.2 thereof comprises at least one of nanometer antibody NbN2, nanometer antibody NbN3, nanometer antibody NbN4, nanometer antibody NbN5, nanometer antibody NbN6 and nanometer antibody NbN11, has higher binding capacity for the wild strain of the novel coronavirus and the viruses of various variants, and is suitable for preparing the detection reagent or the detection kit for detecting the novel coronavirus.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Wherein:
FIG. 1 is a schematic diagram of Omicron RBD protein nanobody screening.
FIG. 2 is a schematic representation of the sequence of third generation sequencing top 10 of phage eluate from Omicron RBD screening.
FIG. 3 is a schematic representation of Omicron RBD nanobody expression purification.
FIG. 4 is a schematic diagram of nanobody immunoblotting (Western Blot, WB) detection.
FIG. 5 is a schematic diagram of ELISA validation of Omacron RBD nanobodies.
FIG. 6 is a schematic diagram of ELISA validation of SARS-CoV-2 Wild-Type (Wild Type, WT) RBD nanobody.
FIG. 7 is a schematic of ELISA validation of Delta RBD nanobodies.
FIG. 8 is a schematic diagram of ELISA validation of nanobody and negative control protein bovine serum albumin (Bovine Serum Albumin, BSA).
FIG. 9 is a schematic of affinity constants of Biacore detection nanobody N2 and WT RBD proteins.
FIG. 10 is a schematic diagram showing affinity constants of the Biacore detection nanobody N2 and Alpha RBD proteins.
FIG. 11 is a schematic of the affinity constants of Biacore detection nanobody N2 and Beta RBD protein.
FIG. 12 is a schematic of the affinity constants of Biacore detection nanobody N2 and Delta RBD proteins.
FIG. 13 is a schematic diagram showing affinity constants of Biacore detection nanobody N2 and Omicron RBD protein.
FIG. 14 is a schematic representation of the affinity constants of Biacore detection nanobody N2 to Omicron BA.2RBD protein.
FIG. 15 is a schematic of the affinity constants of Biacore detection nanobody N3 and WT RBD proteins.
FIG. 16 is a schematic of the affinity constants of Biacore detection nanobody N3 and Alpha RBD protein.
FIG. 17 is a schematic of the affinity constants of Biacore detection nanobody N3 and Beta RBD protein.
FIG. 18 is a schematic of the affinity constants of Biacore detection nanobody N3 and Delta RBD protein.
FIG. 19 is a schematic of affinity constants of Biacore detection nanobody N3 and Omicron RBD protein.
FIG. 20 is a schematic representation of the affinity constants of Biacore detection nanobody N3 to Omicron BA.2RBD protein.
FIG. 21 is a schematic of the affinity constants of Biacore detection nanobody N4 to WT RBD protein.
FIG. 22 is a schematic of affinity constants of Biacore detection nanobody N4 and Alpha RBD protein.
FIG. 23 is a schematic representation of the affinity constants of Biacore detection nanobody N4 and Beta RBD protein.
FIG. 24 is a schematic of the affinity constants of Biacore detection nanobody N4 and Delta RBD protein.
FIG. 25 is a schematic representation of the affinity constants of Biacore detection nanobody N4 to Omicron RBD protein.
FIG. 26 is a schematic of the affinity constants of Biacore detection nanobody N4 with Omicron BA.2RBD protein.
FIG. 27 is a schematic of the affinity constants of Biacore detection nanobody N5 to WT RBD protein.
FIG. 28 is a schematic of the affinity constants of Biacore detection nanobody N5 and Alpha RBD protein.
FIG. 29 is a schematic of the affinity constants of Biacore detection nanobody N5 and Beta RBD protein.
FIG. 30 is a schematic diagram showing affinity of Biacore detection nanobody N5 with Delta RBD protein.
FIG. 31 is a schematic diagram showing affinity constants of Biacore detection nanobody N5 and Omicron RBD protein.
FIG. 32 is a schematic of affinity constants of Biacore detection nanobody N5 with Omicron BA.2RBD protein.
FIG. 33 is a schematic of the affinity constants of Biacore detection nanobody N6 to WT RBD protein.
FIG. 34 is a schematic of the affinity constants of Biacore detection nanobody N6 and Alpha RBD protein.
FIG. 35 is a schematic of the affinity constants of Biacore detection nanobody N6 and Beta RBD protein.
FIG. 36 is a schematic of the affinity constants of Biacore detection nanobody N6 and Delta RBD protein.
FIG. 37 is a schematic representation of the affinity constants of Biacore detection nanobody N6 to Omicron RBD protein.
FIG. 38 is a schematic of the affinity constants of Biacore detection nanobody N6 to Omicron BA.2RBD protein.
FIG. 39 is a schematic of the affinity constants of Biacore detection nanobody N11 to WT RBD proteins.
FIG. 40 is a schematic of the affinity constants of Biacore detection nanobody N11 and Alpha RBD protein.
FIG. 41 is a schematic of the affinity constants of Biacore detection nanobody N11 and Beta RBD protein.
FIG. 42 is a schematic of the affinity constants of Biacore detection nanobody N11 and Delta RBD protein.
FIG. 43 is a schematic of affinity constants of Biacore detection nanobody N11 with Omicron RBD protein.
FIG. 44 is a schematic of the affinity constants of Biacore detection nanobody N11 to Omicron BA.2RBD protein.
Figure 45 is a schematic of a nanobody and ACE2 competition ELISA.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only 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.
The nanobody of the targeting novel coronavirus provided in the first aspect of the application includes at least one of nanobody NbN2, nanobody NbN3, nanobody NbN4, nanobody NbN5, nanobody NbN6 and nanobody NbN11, wherein the amino acid sequence of nanobody NbN2 is shown as seq.id No.1, the amino acid sequence of nanobody NbN3 is shown as seq.id No.2, the amino acid sequence of nanobody NbN4 is shown as seq.id No.3, the amino acid sequence of nanobody NbN5 is shown as seq.id No.4, the amino acid sequence of nanobody NbN6 is shown as seq.id No.5, and the amino acid sequence of nanobody NbN11 is shown as seq.id No. 6.
The nanometer antibody of the novel coronavirus Omicron and subtype BA.2 thereof provided in the first aspect of the application comprises at least one of nanometer antibody NbN2, nanometer antibody NbN3, nanometer antibody NbN4, nanometer antibody NbN5, nanometer antibody NbN6 and nanometer antibody NbN11, and the provided nanometer antibody has higher affinity to RBD domains of novel coronavirus wild strains, novel coronavirus variant Alpha (Alpha), novel coronavirus Beta (Beta), novel coronavirus Delta (Delta), novel coronavirus Omicron and subtype BA.2 thereof, can rapidly and specifically neutralize different novel coronaviruses, and effectively inhibit the activity of the novel coronavirus; meanwhile, the provided nano antibody has a longer CDR3 sequence, is beneficial to flexibly regulating the conformation of the nano antibody, improves the binding effect with viruses, has lower preparation cost and is beneficial to wide application.
The amino acid sequence of the nanobody NbN2 is shown as seq. ID No.1, and the seq. ID No.1 is as follows: MAVQLVESGGGLVQAGGSLRLSCAATGLTFTGYVMAWFRQAPGKNREFVAAVSRSGVVTRYADSVKGRFTVSRDNAKNTVYLQMNTLNPEDTALYYCAADWHPLTGIMTTDSTEYDYWGQGTQVTVSS.
The base sequence of nanobody NbN2 is shown as seq. ID No.44, and seq. ID No.44 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCACTGGACTCACCTTCACTGGTTATGTAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGAACCGTGAGTTTGTAGCGGCCGTTAGCCGGAGTGGTGTTGTTACACGCTATGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACACCCTGAATCCTGAGGACACGGCCCTTTATTACTGTGCAGCAGATTGGCATCCACTAACTGGAATAATGACGACTGACTCTACAGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
The amino acid sequence of the nanobody NbN3 is shown as seq. ID No.2, and the seq. ID No.2 is as follows: MAVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKERQFVAGISGSGIVTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAPLILARTVDMNHWGKGTQVTVSS.
The base sequence of nanobody NbN3 is shown as seq. ID No.45, and seq. ID No.45 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCCAGTTTGTAGCAGGTATTAGCGGGAGTGGTATAGTCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTACCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCATTTATTACTGTGCACCGTTGATCCTAGCACGAACTGTGGACATGAACCACTGGGGCAAAGGGACCCAGGTCACCGTCTCCTCA.
The amino acid sequence of the nanobody NbN4 is shown as seq. ID No.3, and the sequence of the seq. ID No.3 is as follows: MAVQLVESGGGVVQPGGSLRLSCTASGFGFSLYGMSWYRQAPGKERELVASIASGGDSTNYQDSVKGRFSMSRDNAKSTVYLQMNSLKPEDTAVYYCVAERVSSFLKIRTAYWGQGTQVTVSS.
The base sequence of nanobody NbN4 is shown as seq. ID No. 46. Seq. ID No.46 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGAGTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCGGCTTCAGTCTTTATGGCATGAGCTGGTACCGTCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCATCTATTGCCAGTGGTGGTGATAGTACAAACTATCAAGACTCCGTGAAGGGCCGATTCTCCATGTCCAGAGACAATGCCAAGAGCACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGCGTGGCAGAAAGAGTCTCGTCGTTCTTAAAAATTCGTACCGCCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
The amino acid sequence of the nanobody NbN5 is shown as seq. ID No.4, and the sequence of the seq. ID No.4 is as follows: MAVQLVESGGGLVQAGGSLRVSCEASGFLFNGDAYVLGWYRQVPGRQRELVATITSSGDTSYADSVKGRFTISRDNAKNTIFLQMSGLKPEDTAVYYCYAERWNGLLQRWGQGTQVTVSS.
The base sequence of nanobody NbN5 is shown as seq. ID No.47, and seq. ID No.47 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGAGTGTCCTGTGAAGCCTCTGGATTCCTTTTCAATGGCGATGCCTATGTCTTGGGCTGGTACCGCCAGGTTCCAGGTAGGCAGCGTGAATTGGTCGCAACTATTACGAGTAGCGGTGACACAAGCTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAATACAATTTTTCTGCAAATGAGCGGCCTGAAACCCGAGGACACGGCCGTCTATTACTGCTATGCAGAGAGGTGGAATGGGCTCCTTCAACGCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
The amino acid sequence of the nanobody NbN6 is shown as seq. ID No.5, and the sequence of the seq. ID No.5 is as follows: MAVQLVESGGGLVQPGGSLRLSCAASGIIFRFRTISWYRQAPGKQRELVASISGGSSTSYADTVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAASHTMVGWIYGMDYWGKGTQVTVSS.
The base sequence of nanobody NbN6 is shown as seq. ID No.48, and seq. ID No.48 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCATCTTCAGATTCAGAACCATATCCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAAGTATTAGTGGCGGCAGTAGCACGTCCTATGCAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGGCGTTTATTACTGTGCAGCGTCACATACTATGGTTGGGTGGATCTACGGCATGGACTACTGGGGCAAAGGGACCCAGGTCACCGTCTCCTCA.
The amino acid sequence of the nanobody NbN11 is shown as seq. ID No.6, and the sequence of the seq. ID No.6 is as follows: MAVQLVESGGGLVQAGGSLRLSCAASGSFFSINTMGWYRQAPGKQRELVAQITNAGNTNYADSVKGRFTISRDNAKNTIYLQMNSLKPEDTAVYYCNAGQLWGSYRSWGQGTQVTVSS.
The base sequence of nanobody NbN11 is shown as seq. ID No.49, and seq. ID No.49 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTTCTTCAGTATCAATACCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCACAAATTACTAATGCAGGTAACACAAACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACACGATATATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGCAATGCAGGACAGTTGTGGGGTAGTTACCGCTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
In some embodiments, nanobodies include 4 framework regions FR1, FR2, FR3, FR4, and 3 complementarity determining regions CDR1, CDR2, CDR3; the difference in amino acid sequences of the respective regions affects the binding ability of the nanobody to viruses.
In the nano antibody NbN2, the amino acid sequence of FR1 is shown as SEQ ID NO.7, and specifically comprises the following steps: AVQLVESGGGLVQAGGSLRLSCAATGLTFT; the amino acid sequence of FR2 is shown as SEQ ID NO.8, and specifically comprises the following steps: WFRQAPGKNREFVA; the amino acid sequence of FR3 is shown as SEQ ID NO.9, and specifically comprises the following steps: RFTVSRDNAKNTVYLQMNTLNPEDTALYYCAA; the amino acid sequence of FR4 is shown as SEQ ID NO.10, and specifically comprises the following steps: WGQGTQVTVSS; the amino acid sequence of CDR1 is shown in SEQ ID NO.11, specifically: GYVMA; the amino acid sequence of CDR2 is shown in SEQ ID NO.12, specifically: AVSRSGVVTRYADSVKG; the amino acid sequence of CDR3 is shown in SEQ ID NO.13, specifically: DWHPLTGIMTTDSTEYDY.
In the nano antibody NbN3, the amino acid sequence of FR1 is shown as SEQ ID NO.14, and specifically comprises the following steps: AVQLVESGGGLVQAGGSLRLSCAASGRTFS; the amino acid sequence of FR2 is shown as SEQ ID NO.15, and specifically comprises the following steps: WFRQAPGKERQFVA; the amino acid sequence of FR3 is shown as SEQ ID NO.16, and specifically comprises the following steps: RFT ISRDNAKNTVYLQMNSLKPEDTA I YYCAP; the amino acid sequence of FR4 is shown as SEQ ID NO.17, specifically WGKGTQVTVSS; the amino acid sequence of CDR1 is shown as SEQ ID NO.18, specifically: RYAMG; the amino acid sequence of CDR2 is shown in SEQ ID NO.19, specifically: GI SGSGI VTNYADSVKG; the amino acid sequence of CDR3 is shown in SEQ ID NO.20, specifically: LILARTVDMNH.
In the nano antibody NbN4, the amino acid sequence of FR1 is shown as SEQ ID NO.21, and specifically comprises the following steps: AVQLVESGGGVVQPGGSLRLSCTASGFGFS; the amino acid sequence of FR2 is shown as SEQ ID NO.22, and specifically comprises the following steps: WYRQAPGKERELVA; the amino acid sequence of FR3 is shown as SEQ ID NO.23, and specifically comprises the following steps: RFSMSRDNAKSTVYLQMNSLKPEDTAVYYCVA; the amino acid sequence of FR4 is shown as SEQ ID NO.10, and specifically comprises the following steps: WGQGTQVTVSS; the amino acid sequence of CDR1 is shown in SEQ ID NO.24, specifically: LYGMS; the amino acid sequence of CDR2 is shown in SEQ ID NO.25, specifically: SI ASGGDSTNYQDSVKG; the amino acid sequence of CDR3 is shown in SEQ ID NO.26, specifically: ERVSSFL K I RTAY.
In the nano antibody NbN5, the amino acid sequence of FR1 is shown as SEQ ID NO.27, and specifically comprises the following steps: AVQLVESGGGLVQAGGSLRVSCEASGFLFN; the amino acid sequence of FR2 is shown as SEQ ID NO.28, and specifically comprises the following steps: WYRQVPGRQRELVA; the amino acid sequence of FR3 is shown as SEQ ID NO.29, and specifically comprises the following steps: RFTISRDNAKNTIFLQMSGLKPEDTAVYYCYA; the amino acid sequence of FR4 is shown as SEQ ID NO.10, and specifically comprises the following steps: WGQGTQVTVSS; the amino acid sequence of CDR1 is shown as SEQ ID NO.30, specifically: GDAYVLG; the amino acid sequence of CDR2 is shown in SEQ ID NO.31, specifically: TITSSGDTSYADSVKG; the amino acid sequence of CDR3 is shown in SEQ ID NO.32, specifically: ERWNGLLQR.
In the nano antibody NbN6, the amino acid sequence of FR1 is shown as SEQ ID NO.33, and specifically comprises the following steps: AVQLVESGGGLVQPGGSLRLSCAASG I IFR; the amino acid sequence of FR2 is shown as SEQ ID NO.34, specifically: WYRQAPGKQRELVA; the amino acid sequence of FR3 is shown as SEQ ID NO.35, and specifically comprises the following steps: RFTISRDNAKNTVYLQMNSLKPEDTGVYYCAA; the amino acid sequence of FR4 is shown as SEQ ID NO.17, and specifically comprises the following steps: WGKGTQVTVSS; the amino acid sequence of CDR1 is shown in SEQ ID NO.36, specifically: FRT I S; the amino acid sequence of CDR2 is shown in SEQ ID NO.37, specifically: SI SGGSSTSYADTVKG; the amino acid sequence of CDR3 is shown in SEQ ID NO.38, specifically: SHTMVGWIYGMDY.
In the nano antibody NbN11, the amino acid sequence of FR1 is shown as SEQ ID NO.39, and specifically comprises the following steps: AVQLVESGGGLVQAGGSLRLSCAASGSFFS; the amino acid sequence of FR2 is shown as SEQ ID NO.34, specifically: WYRQAPGKQRELVA; the amino acid sequence of FR3 is shown as SEQ ID NO.40, and specifically comprises the following steps: RFTISRDNAKNTIYLQMNSLKPEDTAVYYCNA; the amino acid sequence of FR4 is shown as SEQ ID NO.10, and specifically comprises the following steps: WGQGTQVTVSS; the amino acid sequence of CDR1 is shown in SEQ ID NO.41, specifically: INTMG; the amino acid sequence of CDR2 is shown in SEQ ID NO.42, specifically: QITNAGNTNYADSVKG; the amino acid sequence of CDR3 is shown in SEQ ID NO.43, specifically: GQLWGSYRS.
In a second aspect, an embodiment of the present application provides a method for preparing a nanobody targeting a novel coronavirus, including the steps of:
s01, providing target proteins, coating the target proteins on an immune tube, and carrying out enrichment screening to obtain a phage library;
s02, performing second generation sequencing on eluent of the phage library, and synthesizing a gene sequence of the nano antibody with the initial counting front ratio of 10 according to a sequencing result;
s03, cloning the gene sequence of the nano antibody into an expression vector to obtain a recombinant plasmid, transferring the recombinant plasmid into a host cell to induce expression and purifying to obtain the nano antibody of the targeted novel coronavirus.
According to the preparation method of the nano antibody targeting the novel coronavirus Omicron and the subtype BA.2 thereof, which is provided by the second aspect of the embodiment of the application, a nano antibody library is constructed by taking RBD proteins of the novel coronavirus Omicron and the subtype BA.2 thereof as targets based on phage display screening technology, and the nano antibody targeting the novel coronavirus Omicron and the subtype BA.2 thereof is obtained by combining second-generation sequencing, sequence synthesis, cloning, expression and purification; the preparation method utilizes the biological characteristics of phage fast replication in host bacteria, can rapidly screen the nano antibody combined with the specific variant Omikovia and subtype BA.2RBD protein thereof with high flux, is rapid and simple, is favorable for large-scale screening, and improves the screening efficiency.
In step S01, target proteins are provided, and the target proteins are coated on an immune tube for enrichment screening to obtain a phage library.
In some embodiments, the protein of interest is provided as the spinous process protein RBD domain of the novel coronavirus variant, omicron (Omicron), and the concentration of the protein of interest is provided at a concentration of 10-12 μg/mL. In a specific embodiment, the protein of interest is coated onto the immune tube at a concentration of 10. Mu.g/mL.
In some embodiments, in the step of enrichment screening, 2-3 rounds of enrichment screening are performed. Through multiple rounds of enrichment, the obtained library is ensured to have large capacity, and the screening of positive clones is facilitated.
In step S02, the eluent of the phage library is subjected to second generation sequencing, and the gene sequence of the nanobody with the initial pre-count ratio of 10 is synthesized according to the sequencing result.
In the step S03, the gene sequence of the nano antibody is cloned into an expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transferred into a host cell to induce expression and purify to obtain the nano antibody of the targeted novel coronavirus.
In some embodiments, the expression vector is selected from the group consisting of pcoldi vectors, and the gene sequences of the nanobodies are cloned into pcoldi vectors to obtain recombinant plasmids; further, the hemagglutinin tag (hemagglutinin HA tag) was fusion expressed simultaneously during cloning for subsequent detection.
In some embodiments, the host cell is selected from E.coli, and the recombinant plasmid is transferred into E.coli cells to induce expression and purified.
In some embodiments, the recombinant plasmid is transferred into E.coli cells to induce expression and purified as follows: (1) The IPTG with the concentration of 0.2mM is used for induction expression at the low temperature of 16 ℃, so that inclusion bodies and protein degradation can be prevented from being formed; (2) Performing a large amount of induction expression according to the pre-experiment induction conditions, and performing bacteria breaking under the working condition of 1000W of a high-pressure bacteria breaker; (3) Centrifuging at 17000g and 4deg.C for 30min, incubating supernatant with Ni filler at 4deg.C for 1 hr, and eluting; (4) And (3) carrying out molecular sieve separation after Ni column purification, wherein AKATA parameters are set at a flow rate of 0.5 mL/min, and the nano antibody is obtained after collecting once every 1 mL.
In a third aspect, embodiments of the present application disclose the use of nanobodies targeting novel coronaviruses in the preparation of a medicament for the prevention and/or treatment of novel coronavirus pneumonia.
The application of the nanobody of the targeting novel coronavirus Omicron and subtype BA.2 thereof in preparing the medicament for preventing and/or treating the novel coronavirus pneumonia provided by the third aspect of the application is that the nanobody of the targeting novel coronavirus Omicron and subtype BA.2 thereof obtained by screening comprises at least one of nanobody NbN2, nanobody NbN3, nanobody NbN4, nanobody NbN5, nanobody NbN6 and nanobody NbN11, has better neutralization activity capability and inhibition activity capability on viruses of wild strains and variant strains of the novel coronavirus, and is suitable for preparing related medicaments for preventing and/or treating the novel coronavirus pneumonia.
The fourth aspect of the embodiment of the application discloses application of a nanobody targeting a novel coronavirus in preparation of a detection reagent or a detection kit for detecting the novel coronavirus.
The application of the nanobody for targeting the novel coronavirus Omicron and the subtype BA.2 thereof in preparing the detection reagent or the detection kit for detecting the novel coronavirus provided by the fourth aspect of the application provides the nanobody for targeting the novel coronavirus Omicron and the subtype BA.2 thereof, which comprises at least one of the nanobody NbN2, the nanobody NbN3, the nanobody NbN4, the nanobody NbN5, the nanobody NbN6 and the nanobody NbN11, has higher binding capacity for the wild strain of the novel coronavirus and the viruses of various variants, and is suitable for preparing the detection reagent or the detection kit for detecting the novel coronavirus.
The following description is made with reference to specific embodiments.
Example 1
Nano antibody for targeting novel coronavirus Omicron and subtype BA.2 and preparation method thereof
The test steps are as follows:
(1) Coating target protein Omicron RBD on an immune tube according to the concentration of 10 mug/mL, and carrying out 3 rounds of enrichment screening; (2) Second generation sequencing using third round phage eluate to determine sequence information; (3) Designing and synthesizing the screened nano antibody according to the sequencing information; (4) Cloning the nano antibody gene sequence into a pColdII vector, and simultaneously fusion-expressing a hemagglutinin tag (hemagglutinin HA tag) for subsequent detection; (5) Inducing by using IPTG with the concentration of 0.2mM at the low temperature of 16 ℃ (6) carrying out a large amount of induced expression according to the pre-experimental inducing conditions, and carrying out bacteria breaking under the working condition of a high-pressure bacteria breaker of 1000W; (7) 17000g, centrifuging at 4deg.C for 30min, taking supernatant, and incubating with Ni filler at 4deg.C for 1 hr; (8) And (3) carrying out molecular sieve separation after Ni column purification, wherein AKATA parameters are set at a flow rate of 0.5 mL/min, and collecting once every 1mL to obtain the nanobody targeting the novel coronavirus Omicron and the subtype BA.2 thereof.
Analysis of results:
screening and identification of Omicron RBD nanobodies
Screening of phage nanobody library was performed on omicon RBD, after three rounds of screening, as shown in fig. 1, the library was enriched approximately 3000 times, and the third round phage eluate was subjected to second generation sequencing, as shown in fig. 2, and the sequences with the first 10 content were selected for expression, respectively: nbN2, nbN3, nbN4, nbN5, nbN6, nbN7, nbN8, nbN9, nbN10, nbN11.
Further purifying to obtain 10 high-purity nano antibodies, wherein the protein results of the 10 nano antibodies obtained by purifying are shown in figure 3, and the size of the protein is about 15 kD. And the correctness of the protein is verified by anti-HA WB detection, as shown in figure 4, 10 clones obtained by screening are correctly expressed.
(II) preliminary ELISA identification of nanobody binding to RBD of each variant strain of SARS-CoV-2
The test steps are as follows:
the HA tag is fused into a nanobody gene coding sequence, 10 nanobodies NbN2, nbN3, nbN4, nbN5, nbN6, nbN7, nbN8, nbN9, nbN10 and NbN11 with the HA tag are expressed, an Omicron RBD protein is coated and blocked by ELISA plates, then nanobodies with various concentrations are added for incubation for 1 hour at room temperature, PBS is used for rinsing 3 times, the anti-HA antibody is incubated for 1 hour at room temperature, and then the horseradish peroxidase labeled anti-HA antibody amplifies the signal, TMB color is developed, and meanwhile, the control of irrelevant nanobodies and the blank control of irrelevant protein antigens are made.
Analysis of results
ELISA was performed on 10 nanobodies whose expression was confirmed to be correct by the purification of the above step, and SARS-CoV-2WT, delta (B.1.617.2), omicron (B.1.1.529) RBD protein and bovine serum albumin (Bovine Serum Albumin, BSA), wherein the corresponding legends of 10 nanobodies NbN2, nbN3, nbN4, nbN5, nbN6, nbN7, nbN8, nbN9, nbN10, nbN11 in the figures are as follows: NGS2, NGS3, NGS4, NGS5, NGS6, NGS7, NGS8, NGS9, NGS10, NGS11.
The results of the analysis of the binding to Omicron RBD protein are shown in fig. 5, and it can be seen that, among them, six nanobodies of NbN2, nbN3, nbN4, nbN5, nbN6, and NbN11 showed binding activity to Omicron RBD.
The results of the analysis of binding to SARS-CoV-2 Wild-Type (WT) RBD protein are shown in FIG. 6, wherein six nanobodies of NbN2, nbN3, nbN4, nbN5, nbN6 and NbN11 exhibit binding activity to Omicron RBD.
The results of the analysis of binding to Delta RBD protein are shown in FIG. 7, and it can be seen that, among them, six nanobodies of NbN2, nbN3, nbN4, nbN5, nbN6, nbN11 showed binding activity to Omicron RBD.
The results of the analysis of binding to the negative control protein bovine serum albumin are shown in FIG. 8, and it can be seen that, among them, 10 nanobodies were found to have no binding activity to BSA protein.
Therefore, it was confirmed that 6 nanobodies (Nb N2, nbN3, nbN4, nbN5, nbN6, nbN 11) had binding activity to each variant RBD of SARS-CoV-2, but did not bind to the negative control protein BSA (FIGS. 5-8).
Affinity of six positive nano antibodies with RBD protein of each variant strain SARS-CoV-2
The test steps are as follows:
6 nanobodies (Nb N2, nbN3, nbN4, nbN5, nbN6, nbN 11) were provided, and the affinity of each nanobody to each variant SARS-CoV-2 (WT, alpha, beta, delta, omicron and Omicron BA.2) RBD protein was analyzed and detected based on the molecular interaction analysis platform Biacore.
Analysis of results
The affinities of the 6 positive nanobodies Nb N2, nbN3, nbN4, nbN5, nbN6 and NbN11 with the RBD proteins of each variant strain (WT, alpha, beta, delta, omicron and Omicron BA.2) of SARS-CoV-2 are shown in Table 1, and the affinities are at the nanomolar level, so that the affinity is higher.
TABLE 1
Figure BDA0003681117590000161
(IV) 5 positive nanobody and surface plasmon resonance (Surface Plasmon Resonance, SPR) analysis
The test steps are as follows:
experiments were used to verify the direct interaction of the in vitro purified nanobody expressed in vitro with the in vitro purified antigen protein and to calculate the equilibrium constants of the two. Multicycle kinetics were run on Biacore S200 to study the binding kinetics of nanobodies and omacron RBD proteins. 5 positive nanobodies Nb D2, nbE8, nbF2, nbG and NbG were provided, and new crown wild strain (WT), variant Alpha (Alpha), beta (Beta), delta (Delta) and Omicron (Omicron) and their sub-BA.2 RBD proteins were immobilized on the surface of a CM5 Series S sensor chip at an operating temperature of 25℃respectively, and the nanobodies diluted in a double ratio were flowed over the chip surface at a rate of 30. Mu.L/min for 120 seconds followed by dissociation for 240 seconds to prepare a kinetic curve, and each relevant parameter was calculated.
Analysis of results
(1) Nb N2 nanobody surface plasmon resonance (Surface Plasmon Resonance, SPR) analysis
SPR results showed that Nb N2 nanobody was bound to each variant (WT, alpha, beta, delta, omicron and Omicron BA.2) RBD protein of SARS-CoV-2, and that Nb N2 had an affinity of 24.45nM to WT RBD protein as shown in FIG. 9; as shown in FIG. 10, nb N2 has an affinity of 43nM with Alpha RBD protein; as shown in FIG. 11, nb N2 has an affinity of 28.87nM for Beta RBD protein; as shown in FIG. 12, nb N2 has an affinity of 97.75nM for the Delta RBD protein; as shown in FIG. 13, nb N2 has an affinity of 55.44nM with Omicron RBD protein; as shown in FIG. 14, nb N2 has an affinity of 8.33nM with Omicron BA.2RBD protein.
(2) Nb N3 nanobody surface plasmon resonance (Surface Plasmon Resonance, SPR) analysis
SPR results showed that Nb N3 nanobody was bound to each variant (WT, alpha, beta, delta, omicron and Omicron BA.2) RBD protein of SARS-CoV-2, as shown in FIG. 15, the affinity of Nb N3 to WT RBD protein was 12.09nM; as shown in FIG. 16, nb N3 has an affinity of 21.24nM with Alpha RBD protein; as shown in FIG. 17, nb N3 has an affinity of 16.92nM for Beta RBD protein; as shown in FIG. 18, nb N3 has an affinity of 18.65nM for the Delta RBD protein; as shown in FIG. 19, nb N3 has an affinity of 4.79nM with Omicron RBD protein; as shown in FIG. 20, nb N3 had an affinity of 8.77nM for Omicron BA.2RBD protein.
(3) Nb N4 nanobody surface plasmon resonance (Surface Plasmon Resonance, SPR) analysis
SPR results showed that Nb N4 nanobody was bound to each variant (WT, alpha, beta, delta, omicron and Omicron BA.2) RBD protein of SARS-CoV-2, and that Nb N4 had an affinity of 2.89pM with WT RBD protein as shown in FIG. 21; as shown in FIG. 22, nb N4 has an affinity of 73.09nM for Alpha RBD protein; as shown in FIG. 23, nb N4 has an affinity of 46.4nM for Beta RBD protein; as shown in FIG. 24, nb N4 has an affinity of 23.42nM for the Delta RBD protein; as shown in FIG. 25, nb N4 had an affinity of 5.09nM for Omicron RBD protein; as shown in FIG. 26, nb N4 had an affinity of 38.93nM for the Omacron BA.2RBD protein.
(4) Nb N5 nanobody surface plasmon resonance (Surface Plasmon Resonance, SPR) analysis
SPR results showed that Nb N5 nanobody was bound to each variant (WT, alpha, beta, delta, omicron and Omicron BA.2) RBD protein of SARS-CoV-2, as shown in FIG. 27, nb N5 had an affinity of 0.21nM to WT RBD protein; as shown in FIG. 28, nb N5 has an affinity of 80.48nM for Alpha RBD protein; as shown in FIG. 29, nb N5 has an affinity of 37.22nM for Beta RBD protein; as shown in FIG. 30, nb N5 has an affinity of 24.46nM for the Delta RBD protein; as shown in FIG. 31, nb N5 has an affinity of 90.88nM with Omicron RBD protein; as shown in FIG. 32, nb N5 had an affinity of 20.38nM for the Omacron BA.2RBD protein.
(5) Nb N6 nanobody surface plasmon resonance (Surface Plasmon Resonance, SPR) analysis
SPR results showed that Nb N6 nanobody was bound to each variant (WT, alpha, beta, delta, omicron and Omicron BA.2) RBD protein of SARS-CoV-2, as shown in FIG. 33, nb N6 has an affinity of 38.65nM to WT RBD protein; as shown in FIG. 34, nb N6 has an affinity of 101.3nM with Alpha RBD protein; as shown in FIG. 35, nb N6 has an affinity of 65.36nM for Beta RBD protein; as shown in FIG. 36, nb N6 has an affinity of 26.91nM for the Delta RBD protein; as shown in FIG. 37, nb N6 has an affinity of 78.77nM with Omicron RBD protein; as shown in FIG. 38, nb N6 had an affinity of 89.52nM for the Omacron BA.2RBD protein.
(6) Nb N11 nanobody surface plasmon resonance (Surface Plasmon Resonance, SPR) analysis
SPR results showed that Nb N11 nanobody was bound to each variant (WT, alpha, beta, delta, omicron and Omicron BA.2) RBD protein of SARS-CoV-2, and that Nb N11 had an affinity of 36.06nM to WT RBD protein as shown in FIG. 39; as shown in FIG. 40, nb N11 has an affinity of 26.18nM with Alpha RBD protein; as shown in FIG. 41, nb N11 has an affinity of 19.17nM for Beta RBD protein; as shown in FIG. 42, nb N11 has an affinity of 28.66nM for the Delta RBD protein; as shown in FIG. 43, nb N11 has an affinity of 47.39nM for omacron RBD protein; as shown in FIG. 44, nb N11 had an affinity of 105nM for Omicron BA.2RBD protein.
(fifth) six Positive nanobodies competitive ELISA experiments with ACE2
Test procedure
According to SPR results, selecting nano antibodies with better affinity for competitive ELISA experiment verification. Omicron RBD protein was coated on ELISA plates at a concentration of 0.5. Mu.g/mL overnight at 4 ℃. The next day, PBS was washed once, and blocked with 3% BSA for 2 hours at room temperature. Nanobody fold dilution was performed according to the lowest saturation concentration of ACE2, i.e. the dilution was 0.1% pbst solution containing 0.5 μg/mL ACE2, and incubated for 2.5 hours at room temperature. Rinse with 0.1% pbst.
Analysis of results
As shown in fig. 45, it was examined whether or not these 6 nanobodies compete with ACE2 for binding to omicron RBD protein, nanobody dilution was performed with 0.1% pbst solution containing the lowest saturation concentration of ACE2, and incubated with ELISA plates coated with omicron RBD protein in advance for 2.5 hours at room temperature. 0.1% PBST was rinsed 3 times, and the anti-ACE2 antibody was used to detect the ACE2 content, while a blank control without nanobody and without ACE2 protein was made. The results show that Nb N2 can significantly competitively inhibit ACE2 binding to omicron RBD protein at a half inhibitory concentration (half maximum inhibitory concentration, IC 50) of 0.09 μm. The results show that Nb N2 nanobodies can significantly competitively inhibit ACE2 binding to omicron RBD protein.
Therefore, the nanometer antibody of the novel coronavirus Omicron and subtype BA.2 thereof provided by the application comprises at least one of nanometer antibody NbN2, nanometer antibody NbN3, nanometer antibody NbN4, nanometer antibody NbN5, nanometer antibody NbN6 and nanometer antibody NbN11, and the provided nanometer antibody has higher affinity to RBD domains of novel coronavirus wild strains, novel coronavirus variant Alpha (Alpha), novel coronavirus Beta (Beta), novel coronavirus Delta (Delta), novel coronavirus Omicron and subtype BA.2 thereof, can neutralize different novel coronaviruses rapidly and specifically, and effectively inhibit the activity of the novel coronavirus; meanwhile, the provided nano antibody has a longer CDR3 sequence, is beneficial to flexibly regulating the conformation of the nano antibody, improves the binding effect with viruses, has lower preparation cost and is beneficial to wide application.
The foregoing disclosure is illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Sequence listing
<110> Shenzhen City people Hospital
<120> nanobody targeting novel coronavirus, preparation method and application thereof
<140> 2022106375477
<141> 2022-03-31
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Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys
85 90 95
Ala Ala Ser His Thr Met Val Gly Trp Ile Tyr Gly Met Asp Tyr Trp
100 105 110
Gly Lys Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 6
<211> 118
<212> PRT
<213> Artificial Sequence
<400> 6
Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Phe Phe Ser Ile
20 25 30
Asn Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu
35 40 45
Val Ala Gln Ile Thr Asn Ala Gly Asn Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Gly Gln Leu Trp Gly Ser Tyr Arg Ser Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 7
<211> 30
<212> PRT
<213> Artificial Sequence
<400> 7
Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Thr Gly Leu Thr Phe Thr
20 25 30
<210> 8
<211> 14
<212> PRT
<213> Artificial Sequence
<400> 8
Trp Phe Arg Gln Ala Pro Gly Lys Asn Arg Glu Phe Val Ala
1 5 10
<210> 9
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 9
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
1 5 10 15
Met Asn Thr Leu Asn Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala
20 25 30
<210> 10
<211> 11
<212> PRT
<213> Artificial Sequence
<400> 10
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 11
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 11
Gly Tyr Val Met Ala
1 5
<210> 12
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 12
Ala Val Ser Arg Ser Gly Val Val Thr Arg Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 13
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 13
Asp Trp His Pro Leu Thr Gly Ile Met Thr Thr Asp Ser Thr Glu Tyr
1 5 10 15
Asp Tyr
<210> 14
<211> 30
<212> PRT
<213> Artificial Sequence
<400> 14
Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
20 25 30
<210> 15
<211> 14
<212> PRT
<213> Artificial Sequence
<400> 15
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val Ala
1 5 10
<210> 16
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 16
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Pro
20 25 30
<210> 17
<211> 11
<212> PRT
<213> Artificial Sequence
<400> 17
Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 18
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 18
Arg Tyr Ala Met Gly
1 5
<210> 19
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 19
Gly Ile Ser Gly Ser Gly Ile Val Thr Asn Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 20
<211> 11
<212> PRT
<213> Artificial Sequence
<400> 20
Leu Ile Leu Ala Arg Thr Val Asp Met Asn His
1 5 10
<210> 21
<211> 30
<212> PRT
<213> Artificial Sequence
<400> 21
Ala Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Gly Phe Ser
20 25 30
<210> 22
<211> 14
<212> PRT
<213> Artificial Sequence
<400> 22
Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala
1 5 10
<210> 23
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 23
Arg Phe Ser Met Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala
20 25 30
<210> 24
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 24
Leu Tyr Gly Met Ser
1 5
<210> 25
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 25
Ser Ile Ala Ser Gly Gly Asp Ser Thr Asn Tyr Gln Asp Ser Val Lys
1 5 10 15
Gly
<210> 26
<211> 13
<212> PRT
<213> Artificial Sequence
<400> 26
Glu Arg Val Ser Ser Phe Leu Lys Ile Arg Thr Ala Tyr
1 5 10
<210> 27
<211> 30
<212> PRT
<213> Artificial Sequence
<400> 27
Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Val Ser Cys Glu Ala Ser Gly Phe Leu Phe Asn
20 25 30
<210> 28
<211> 14
<212> PRT
<213> Artificial Sequence
<400> 28
Trp Tyr Arg Gln Val Pro Gly Arg Gln Arg Glu Leu Val Ala
1 5 10
<210> 29
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 29
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Phe Leu Gln
1 5 10 15
Met Ser Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala
20 25 30
<210> 30
<211> 7
<212> PRT
<213> Artificial Sequence
<400> 30
Gly Asp Ala Tyr Val Leu Gly
1 5
<210> 31
<211> 16
<212> PRT
<213> Artificial Sequence
<400> 31
Thr Ile Thr Ser Ser Gly Asp Thr Ser Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 32
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 32
Glu Arg Trp Asn Gly Leu Leu Gln Arg
1 5
<210> 33
<211> 30
<212> PRT
<213> Artificial Sequence
<400> 33
Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Ile Phe Arg
20 25 30
<210> 34
<211> 14
<212> PRT
<213> Artificial Sequence
<400> 34
Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala
1 5 10
<210> 35
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 35
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala Ala
20 25 30
<210> 36
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 36
Phe Arg Thr Ile Ser
1 5
<210> 37
<211> 16
<212> PRT
<213> Artificial Sequence
<400> 37
Ser Ile Ser Gly Gly Ser Ser Thr Ser Tyr Ala Asp Thr Val Lys Gly
1 5 10 15
<210> 38
<211> 13
<212> PRT
<213> Artificial Sequence
<400> 38
Ser His Thr Met Val Gly Trp Ile Tyr Gly Met Asp Tyr
1 5 10
<210> 39
<211> 30
<212> PRT
<213> Artificial Sequence
<400> 39
Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Phe Phe Ser
20 25 30
<210> 40
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 40
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala
20 25 30
<210> 41
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 41
Ile Asn Thr Met Gly
1 5
<210> 42
<211> 16
<212> PRT
<213> Artificial Sequence
<400> 42
Gln Ile Thr Asn Ala Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 43
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 43
Gly Gln Leu Trp Gly Ser Tyr Arg Ser
1 5
<210> 44
<211> 384
<212> DNA
<213> Artificial Sequence
<400> 44
atggcggtgc agctggtgga gtctggggga ggattggtgc aggctggggg ctctctgaga 60
ctctcctgtg cagccactgg actcaccttc actggttatg taatggcctg gttccgccag 120
gctccaggga agaaccgtga gtttgtagcg gccgttagcc ggagtggtgt tgttacacgc 180
tatgcagact ccgtgaaggg ccgattcacc gtctccagag acaacgccaa gaacacggtg 240
tatctgcaaa tgaacaccct gaatcctgag gacacggccc tttattactg tgcagcagat 300
tggcatccac taactggaat aatgacgact gactctacag agtatgacta ctggggccag 360
gggacccagg tcaccgtctc ctca 384
<210> 45
<211> 363
<212> DNA
<213> Artificial Sequence
<400> 45
atggcggtgc agctggtgga gtctggggga ggattggtgc aggctggggg ctctctgaga 60
ctctcctgtg cagcctctgg acgcaccttc agtaggtatg ccatgggctg gttccgccag 120
gctccaggga aggagcgcca gtttgtagca ggtattagcg ggagtggtat agtcacaaac 180
tatgcagact ccgtgaaggg ccgattcacc atctccagag acaacgccaa gaacacggtg 240
tacctgcaaa tgaacagcct gaaacctgag gacacggcca tttattactg tgcaccgttg 300
atcctagcac gaactgtgga catgaaccac tggggcaaag ggacccaggt caccgtctcc 360
tca 363
<210> 46
<211> 369
<212> DNA
<213> Artificial Sequence
<400> 46
atggcggtgc agctggtgga gtctggggga ggagtggtgc agcctggggg gtctctgaga 60
ctctcctgta cagcctctgg attcggcttc agtctttatg gcatgagctg gtaccgtcag 120
gctccaggga aggagcgcga gttggtcgca tctattgcca gtggtggtga tagtacaaac 180
tatcaagact ccgtgaaggg ccgattctcc atgtccagag acaatgccaa gagcacggtg 240
tatctgcaaa tgaacagcct gaaacctgag gacacggccg tgtattactg cgtggcagaa 300
agagtctcgt cgttcttaaa aattcgtacc gcctactggg gccaggggac ccaggtcacc 360
gtctcctca 369
<210> 47
<211> 360
<212> DNA
<213> Artificial Sequence
<400> 47
atggcggtgc agctggtgga gtctggggga ggcttggtgc aggctggggg gtctctgaga 60
gtgtcctgtg aagcctctgg attccttttc aatggcgatg cctatgtctt gggctggtac 120
cgccaggttc caggtaggca gcgtgaattg gtcgcaacta ttacgagtag cggtgacaca 180
agctatgcgg actccgtgaa gggccgattc accatctcca gagacaacgc caagaataca 240
atttttctgc aaatgagcgg cctgaaaccc gaggacacgg ccgtctatta ctgctatgca 300
gagaggtgga atgggctcct tcaacgctgg ggccagggga cccaggtcac cgtctcctca 360
<210> 48
<211> 366
<212> DNA
<213> Artificial Sequence
<400> 48
atggcggtgc agctggtgga gtctggggga ggcttggtgc aacctggggg gtctctgaga 60
ctctcctgtg cagcctctgg aatcatcttc agattcagaa ccatatcctg gtaccgccag 120
gctccaggga agcagcgcga gttggtcgca agtattagtg gcggcagtag cacgtcctat 180
gcagacactg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acaggcgttt attactgtgc agcgtcacat 300
actatggttg ggtggatcta cggcatggac tactggggca aagggaccca ggtcaccgtc 360
tcctca 366
<210> 49
<211> 354
<212> DNA
<213> Artificial Sequence
<400> 49
atggcggtgc agctggtgga gtctggggga ggcttggtgc aggctggggg gtctctgaga 60
ctctcctgtg cagcctctgg aagcttcttc agtatcaata ccatgggctg gtaccgccag 120
gctccaggga agcagcgcga gttggtcgca caaattacta atgcaggtaa cacaaactat 180
gcagactccg tgaagggccg gttcaccatc tccagagaca acgccaagaa cacgatatat 240
ctgcaaatga acagcctgaa acctgaggac acggccgtct attactgcaa tgcaggacag 300
ttgtggggta gttaccgctc ctggggccag gggacccagg tcaccgtctc ctca 354

Claims (4)

1. A nanobody targeting a novel coronavirus, wherein said nanobody comprises nanobody NbN5, wherein the amino acid sequence of nanobody NbN5 is as shown in seq.id No. 4.
2. The nanobody of claim 1, wherein said nanobody comprises 4 framework regions FR1, FR2, FR3, FR4 and 3 complementarity determining regions CDR1, CDR2, CDR3;
in the nano antibody NbN5, the amino acid sequence of FR1 is shown as SEQ ID NO.27, the amino acid sequence of FR2 is shown as SEQ ID NO.28, the amino acid sequence of FR3 is shown as SEQ ID NO.29, the amino acid sequence of FR4 is shown as SEQ ID NO.10, the amino acid sequence of CDR1 is shown as SEQ ID NO.30, the amino acid sequence of CDR2 is shown as SEQ ID NO.31, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 32.
3. The nanobody for targeting a novel coronavirus according to claim 1, wherein the base sequence of the nanobody NbN5 is represented by seq.id No. 47.
4. Use of a nanobody of any one of claims 1 to 3 for targeting a novel coronavirus for the preparation of a detection reagent or a detection kit for detecting a novel coronavirus.
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