CN115043937A - Nano antibody targeting new coronavirus, and preparation method and application thereof - Google Patents

Nano antibody targeting new coronavirus, and preparation method and application thereof Download PDF

Info

Publication number
CN115043937A
CN115043937A CN202210638231.XA CN202210638231A CN115043937A CN 115043937 A CN115043937 A CN 115043937A CN 202210638231 A CN202210638231 A CN 202210638231A CN 115043937 A CN115043937 A CN 115043937A
Authority
CN
China
Prior art keywords
nano antibody
seq
nanobody
omicron
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210638231.XA
Other languages
Chinese (zh)
Other versions
CN115043937B (en
Inventor
戴凌云
郑六海
李志杰
王惠芳
王继刚
耿庆山
钟晓茹
杨传彬
孙继超
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Peoples Hospital
Original Assignee
Shenzhen Peoples Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Peoples Hospital filed Critical Shenzhen Peoples Hospital
Priority to CN202210638231.XA priority Critical patent/CN115043937B/en
Publication of CN115043937A publication Critical patent/CN115043937A/en
Application granted granted Critical
Publication of CN115043937B publication Critical patent/CN115043937B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/567—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 targeting new 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 a new coronavirus wild strain, a new coronavirus variant strain Alpha (Alpha), a new coronavirus Beta (Beta), a new coronavirus Delta (Delta), a new coronavirus Omicron and an RBD structural domain of a subtype BA.2 thereof, can quickly and specifically neutralize different new coronaviruses and effectively inhibit the activity of the new coronaviruses; meanwhile, the provided nano antibody has a longer CDR3 sequence, is beneficial to flexibly adjusting the conformation of the nano antibody, improves the binding effect with viruses and is beneficial to wide application.

Description

Nano antibody of targeted new coronavirus, preparation method and application thereof
The application relates to a nano antibody targeting a new coronavirus, a preparation method thereof and a divisional application of the application, wherein the application date of the original application is 2022, 5 and 23 days, and the application number is 202210330386.7.
Technical Field
The invention relates to the technical field of biology, in particular to a nano antibody targeting a new coronavirus, and a preparation method and application thereof.
Background
The new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) can be transmitted through the ways of droplet transmission, respiratory tract secretion contact, aerosol transmission and the like, and the caused consequences are very serious. The coronavirus 2 (SARS-CoV-2) causing COVID-19 mainly expresses 4 structural proteins, namely spike protein (S), envelope protein (envelope), membrane protein (membrane) and nucleocapsid protein (nucleocapsid). Wherein the S protein comprises an N-terminal S1 subunit and a C-terminal S2 subunit, the S1 subunit comprises a Receptor Binding Domain (RBD) of about 200 amino acids responsible for binding of the virus to a host receptor, and the S2 subunit is responsible for fusion of the virus to a host cell membrane. The S protein plays a crucial role in virus attachment, infection and spread, and is combined with angiotensin converting enzyme2 (ACE 2) of host cells to infect the cells, so that the S protein is an important target for developing anti-SARS-CoV-2 medicaments.
Neutralizing antibodies are important therapeutics against COVID-19. It has been found that the clinical outcome can be significantly improved by delivering serum from convalescent patients with severe COVID-19, indicating that neutralizing antibody therapy also has a better relief effect in new coronary severe patients. The action mechanism is that the neutralizing antibody is combined with the spike glycoprotein on the surface of the new coronavirus to influence the combination of SARS-CoV-2 and the surface receptor of the host cell, so that the virus can not infect the target cell. A plurality of neutralizing antibodies are currently available on the market for COVID-19 therapy, and the monoclonal antibody targets are mainly focused on the RBD domain of the S protein. However, in the face of constantly mutating SARS-CoV-2, it is difficult for existing neutralizing antibodies to target new crown variants. A recent study showed that omacron subtype ba.2 can evade nearly all neutralizing antibody treatments. Therefore, there is an urgent need to develop new neutralizing antibodies that can simultaneously target different new crown variants, particularly omicron and its subtype ba.2.
Disclosure of Invention
The application aims to provide a nano antibody targeting a new corona virus, and a preparation method and application thereof, and aims to solve the problem that a neutralizing antibody of a new corona variant Omicron and a subtype BA.2 thereof is lacked in the prior art.
In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the application discloses a nanobody targeting a new coronavirus, which comprises at least one of a nanobody NbN2, a nanobody NbN3, a nanobody NbN4, a nanobody NbN5, a nanobody NbN6 and a nanobody NbN11, wherein an amino acid sequence of the nanobody NbN2 is shown in seq.id No.1, an amino acid sequence of the nanobody NbN3 is shown in seq.id No.2, an amino acid sequence of the nanobody NbN4 is shown in seq.id No.3, an amino acid sequence of the nanobody NbN5 is shown in seq.id No.4, an amino acid sequence of the nanobody NbN6 is shown in seq.id No.5, and an amino acid sequence of the nanobody NbN11 is shown in seq.id No. 6.
In a second aspect, the application discloses a method for preparing a nanobody targeting a novel coronavirus, comprising the following steps:
providing target protein, coating the target protein on an immune tube for 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 accounting for 10 th of the original count according to a sequencing result;
cloning the gene sequence of the nano antibody into an expression vector to obtain recombinant plasmids, transferring the recombinant plasmids into host cells for induced expression and purifying to obtain the nano antibody of the targeted new coronavirus.
In a third aspect, the application discloses an application of a nano antibody targeting a novel coronavirus in preparation of a medicine for preventing and/or treating novel coronavirus pneumonia.
In a fourth aspect, the application discloses an application of a nano antibody targeting a novel coronavirus in preparation of a detection reagent or a detection kit for detecting the novel coronavirus.
The nano antibody of the new crown virus Omicron and the subtype BA.2 thereof provided by the first aspect of the application 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 for RBD structural domains of a new crown virus wild strain, a new crown virus variant strain Alpha (Alpha), a new crown virus Beta (Beta), a new crown virus Delta (Delta), the new crown virus Omicron and the subtype BA.2 thereof, can quickly and specifically neutralize different new crown viruses and effectively inhibit the activity of the new crown viruses; meanwhile, the provided nano antibody has a longer CDR3 sequence, is beneficial to flexibly adjusting the conformation of the nano antibody and improving the binding effect with viruses, has lower preparation cost and is beneficial to wide application.
The preparation method of the nano antibody targeting the new coronavirus Omicron and the subtype BA.2 thereof is based on a phage display screening technology, a nano antibody library is constructed by taking RBD protein of the new coronavirus Omicron and the subtype BA.2 thereof as a target, and the nano antibody targeting the new 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 characteristic that the bacteriophage is quickly replicated in host bacteria, can quickly and high-flux screen the nano antibody combined with the specific variant strain Ormcken and the subtype BA.2RBD protein thereof, has quick and simple preparation method, is beneficial to large-scale screening, and improves the screening efficiency.
The application of the nano antibody targeting the new coronavirus Omicron and the subtype BA.2 thereof provided by the third aspect of the application in preparing the medicine for preventing and/or treating the novel coronavirus pneumonia, wherein the nano antibody targeting the new coronavirus Omicron and the subtype BA.2 thereof obtained by screening 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, has better neutralizing activity capability and inhibiting activity capability on viruses of a wild strain and various variant strains of the new coronavirus, and is suitable for preparing the related medicine for preventing and/or treating the novel coronavirus pneumonia.
The application of the nano antibody of the targeted new coronavirus Omicron and the subtype BA.2 thereof in preparing a detection reagent or a detection kit for detecting the novel coronavirus provided by the fourth aspect of the application provides that the nano antibody of the targeted new coronavirus Omicron and the subtype BA.2 thereof 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, has higher binding capacity for viruses of a wild strain and various variant strains of the new coronavirus, 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 present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Wherein:
FIG. 1 is a schematic diagram of Omicron RBD protein nanobody screening.
FIG. 2 is a schematic diagram of the third round of phage eluate second generation sequencing top 10 sequence by Omicron RBD screening.
FIG. 3 is a schematic diagram showing the expression and purification of Omicron RBD nanobody.
FIG. 4 is a diagram of detection of Nanobody immunoblotting (Western Blot, WB).
FIG. 5 is a schematic diagram of the Omicron RBD nanobody ELISA verification.
FIG. 6 is a diagram showing ELISA verification of SARS-CoV-2 Wild strain (Wild Type, WT) RBD nanobody.
FIG. 7 is a diagram of ELISA validation of Delta RBD nanobody.
FIG. 8 is a schematic diagram of the ELISA verification of the nanobody and the negative control protein Bovine Serum Albumin (BSA).
FIG. 9 is a diagram showing the affinity constants of Biacore detection nanobody N2 and WT RBD protein.
FIG. 10 is a diagram showing the affinity constants of Biacore detection nanobody N2 and Alpha RBD protein.
FIG. 11 is a diagram showing the affinity constants of Biacore detecting nanobody N2 and Beta RBD protein.
FIG. 12 is a diagram showing the affinity constants of Biacore detection nanobody N2 and Delta RBD protein.
FIG. 13 is a diagram showing the affinity constants of Biacore detection nanobody N2 and Omicron RBD protein.
FIG. 14 is a graph showing the affinity constants of Biacore detection nanobody N2 and Omicron BA.2RBD protein.
FIG. 15 is a diagram showing the affinity constants of Biacore detection nanobody N3 and WT RBD protein.
FIG. 16 is a diagram showing the affinity constants of Biacore detection nanobody N3 and Alpha RBD protein.
FIG. 17 is a diagram showing the affinity constants of Biacore detection nanobody N3 and Beta RBD protein.
FIG. 18 is a diagram showing the affinity constants of Biacore detection nanobody N3 and Delta RBD protein.
FIG. 19 is a diagram showing the affinity constants of Biacore detection nanobody N3 and Omicron RBD protein.
FIG. 20 is a diagram showing the affinity constants of Biacore detection nanobody N3 and Omicron BA.2RBD protein.
FIG. 21 is a diagram showing the affinity constants of Biacore detection nanobody N4 and WT RBD protein.
FIG. 22 is a diagram showing the affinity constants of Biacore detection nanobody N4 and Alpha RBD protein.
FIG. 23 is a diagram showing that Biacore detects the affinity constant of Nanobody N4 and Beta RBD protein.
FIG. 24 is a diagram showing the affinity constants of Biacore detection nanobody N4 and Delta RBD protein.
FIG. 25 is a graph showing the affinity constants of Biacore detection nanobody N4 and Omicron RBD protein.
FIG. 26 is a diagram showing the affinity constants of Biacore detection nanobody N4 and Omicron BA.2RBD protein.
FIG. 27 is a diagram showing the affinity constants of Biacore detection nanobody N5 and WT RBD protein.
FIG. 28 is a graph showing the affinity constants of Biacore for detecting Nanobody N5 and Alpha RBD protein.
FIG. 29 is a graph showing the affinity constants of Biacore for detecting Nanobody N5 and Beta RBD protein.
FIG. 30 is a general schematic diagram of Biacore detection of the affinity of Nanobody N5 with Delta RBD protein.
FIG. 31 is a diagram showing the affinity constants of Biacore detection nanobody N5 and Omicron RBD protein.
FIG. 32 is a graph showing the affinity constants of Biacore detection nanobody N5 and Omicron BA.2RBD protein.
FIG. 33 is a diagram showing the affinity constants of Biacore detection nanobody N6 and WT RBD protein.
FIG. 34 is a diagram showing the affinity constants of Biacore detection nanobody N6 and Alpha RBD protein.
FIG. 35 is a diagram showing the affinity constants of Biacore detecting nanobody N6 and Beta RBD protein.
FIG. 36 is a diagram showing the affinity constants of Biacore detection nanobody N6 and Delta RBD protein.
FIG. 37 is a graph showing the affinity constants of Biacore detection nanobody N6 and Omicron RBD protein.
FIG. 38 is a graph showing the affinity constants of Biacore detection nanobody N6 and Omicron BA.2RBD protein.
FIG. 39 is a diagram showing the affinity constants of Biacore detection nanobody N11 and WT RBD protein.
FIG. 40 is a graph showing the affinity constants of Biacore for detecting Nanobody N11 and Alpha RBD protein.
FIG. 41 is a diagram showing the affinity constants of Biacore detecting nanobody N11 and Beta RBD protein.
FIG. 42 is a diagram showing the affinity constants of Biacore detection nanobody N11 and Delta RBD protein.
FIG. 43 is a graph showing the affinity constants of Biacore detection nanobody N11 and Omicron RBD protein.
FIG. 44 is a graph showing the affinity constants of Biacore detection nanobody N11 and Omicron BA.2RBD protein.
Fig. 45 is a schematic diagram of a competitive ELISA of nanobodies with ACE 2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and 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.
The nano antibody provided by the first aspect of the embodiment of the present application includes at least one of nano antibody NbN2, nano antibody NbN3, nano antibody NbN4, nano antibody NbN5, nano antibody NbN6 and nano antibody NbN11, wherein an amino acid sequence of nano antibody NbN2 is shown in seq.id No.1, an amino acid sequence of nano antibody NbN3 is shown in seq.id No.2, an amino acid sequence of nano antibody NbN4 is shown in seq.id No.3, an amino acid sequence of nano antibody NbN5 is shown in seq.id No.4, an amino acid sequence of nano antibody NbN6 is shown in seq.id No.5, and an amino acid sequence of nano antibody NbN11 is shown in seq.id No. 6.
The nano antibody of the new crown virus Omicron and the subtype BA.2 thereof provided by the first aspect of the embodiment of the application 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 new crown virus wild strain, a new crown virus variant strain Alpha (Alpha), a new crown virus Beta (Beta), a new crown virus Delta (Delta), the new crown virus Omicron and the subtype BA.2 thereof, can quickly and specifically neutralize different new crown viruses and effectively inhibit the activity of the new crown viruses; meanwhile, the provided nano antibody has a longer CDR3 sequence, is beneficial to flexibly adjusting the conformation of the nano antibody and improving the binding effect with viruses, has lower preparation cost and is beneficial to wide application.
The amino acid sequence of the nano antibody NbN2 is shown in seq.ID No.1, and the seq.ID No.1 is as follows: MAVQLVESGGGLVQAGGSLRLSCAATGLTFTGYVMAWFRQAPGKNREFVAAV SRSGVVTRYADSVKGRFTVSRDNAKNTVYLQMNTLNPEDTALYYCAADWHP LTGIMTTDSTEYDYWGQGTQVTVSS are provided.
The base sequence of the nanobody NbN2 is shown in seq.ID No.44, and seq.ID No.44 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGG CTCTCTGAGACTCTCCTGTGCAGCCACTGGACTCACCTTCACTGGTTATGTA ATGGCCTGGTTCCGCCAGGCTCCAGGGAAGAACCGTGAGTTTGTAGCGGCC GTTAGCCGGAGTGGTGTTGTTACACGCTATGCAGACTCCGTGAAGGGCCGA TTCACCGTCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAAC ACCCTGAATCCTGAGGACACGGCCCTTTATTACTGTGCAGCAGATTGGCATC CACTAACTGGAATAATGACGACTGACTCTACAGAGTATGACTACTGGGGCC AGGGGACCCAGGTCACCGTCTCCTCA are provided.
The amino acid sequence of the nano antibody NbN3 is shown in seq.ID No.2, and the seq.ID No.2 is as follows: MAVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKERQFVAGI SGSGIVTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAPLILARTV DMNHWGKGTQVTVSS are provided.
The base sequence of the nanobody NbN3 is shown in seq.ID No.45, and seq.ID No.45 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGG CTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCC ATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCCAGTTTGTAGCAGGT ATTAGCGGGAGTGGTATAGTCACAAACTATGCAGACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTACCTGCAAATGAAC AGCCTGAAACCTGAGGACACGGCCATTTATTACTGTGCACCGTTGATCCTAG CACGAACTGTGGACATGAACCACTGGGGCAAAGGGACCCAGGTCACCGTC TCCTCA are provided.
The amino acid sequence of the nano antibody NbN4 is shown in seq.ID No.3, and the seq.ID No.3 is as follows: MAVQLVESGGGVVQPGGSLRLSCTASGFGFSLYGMSWYRQAPGKERELVASIA SGGDSTNYQDSVKGRFSMSRDNAKSTVYLQMNSLKPEDTAVYYCVAERVSSF LKIRTAYWGQGTQVTVSS are provided.
The base sequence of the nanobody NbN4 is shown in seq.ID No.46, and seq.ID No.46 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGAGTGGTGCAGCCTGGGGG GTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCGGCTTCAGTCTTTATGGC ATGAGCTGGTACCGTCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCATCT ATTGCCAGTGGTGGTGATAGTACAAACTATCAAGACTCCGTGAAGGGCCGA TTCTCCATGTCCAGAGACAATGCCAAGAGCACGGTGTATCTGCAAATGAAC AGCCTGAAACCTGAGGACACGGCCGTGTATTACTGCGTGGCAGAAAGAGT CTCGTCGTTCTTAAAAATTCGTACCGCCTACTGGGGCCAGGGGACCCAGGT CACCGTCTCCTCA are provided.
The amino acid sequence of the nano antibody NbN5 is shown in seq.ID No.4, and the amino acid sequence of seq.ID No.4 is as follows: MAVQLVESGGGLVQAGGSLRVSCEASGFLFNGDAYVLGWYRQVPGRQRELVA TITSSGDTSYADSVKGRFTISRDNAKNTIFLQMSGLKPEDTAVYYCYAERWNGL LQRWGQGTQVTVSS are provided.
The base sequence of the nanobody NbN5 is shown in seq.ID No.47, and seq.ID No.47 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGG GTCTCTGAGAGTGTCCTGTGAAGCCTCTGGATTCCTTTTCAATGGCGATGCC TATGTCTTGGGCTGGTACCGCCAGGTTCCAGGTAGGCAGCGTGAATTGGTC GCAACTATTACGAGTAGCGGTGACACAAGCTATGCGGACTCCGTGAAGGGC CGATTCACCATCTCCAGAGACAACGCCAAGAATACAATTTTTCTGCAAATG AGCGGCCTGAAACCCGAGGACACGGCCGTCTATTACTGCTATGCAGAGAGG TGGAATGGGCTCCTTCAACGCTGGGGCCAGGGGACCCAGGTCACCGTCTCC TCA are provided.
The amino acid sequence of the nano antibody NbN6 is shown in seq.ID No.5, and the amino acid sequence of seq.ID No.5 is as follows: MAVQLVESGGGLVQPGGSLRLSCAASGIIFRFRTISWYRQAPGKQRELVASISG GSSTSYADTVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAASHTMVGWI YGMDYWGKGTQVTVSS are provided.
The base sequence of the nanobody NbN6 is shown in seq.ID No.48, and seq.ID No.48 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGG GTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCATCTTCAGATTCAGAACC ATATCCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAAGT ATTAGTGGCGGCAGTAGCACGTCCTATGCAGACACTGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAG CCTGAAACCTGAGGACACAGGCGTTTATTACTGTGCAGCGTCACATACTATG GTTGGGTGGATCTACGGCATGGACTACTGGGGCAAAGGGACCCAGGTCACC GTCTCCTCA are provided.
The amino acid sequence of the nano antibody NbN11 is shown in seq.ID No.6, and the seq.ID No.6 is as follows: MAVQLVESGGGLVQAGGSLRLSCAASGSFFSINTMGWYRQAPGKQRELVAQIT NAGNTNYADSVKGRFTISRDNAKNTIYLQMNSLKPEDTAVYYCNAGQLWGSY RSWGQGTQVTVSS are provided.
The base sequence of the nanobody NbN11 is shown in seq.ID No.49, and seq.ID No.49 is as follows: ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGG GTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTTCTTCAGTATCAATACC ATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCACA AATTACTAATGCAGGTAACACAAACTATGCAGACTCCGTGAAGGGCCGGTT CACCATCTCCAGAGACAACGCCAAGAACACGATATATCTGCAAATGAACAG CCTGAAACCTGAGGACACGGCCGTCTATTACTGCAATGCAGGACAGTTGTG GGGTAGTTACCGCTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA are provided.
In some embodiments, the nanobody comprises 4 framework regions FR1, FR2, FR3, FR4 and 3 complementarity determining regions CDR1, CDR2, CDR 3; the difference of amino acid sequences of the various regions can affect the binding capacity of the nanobody to the virus.
In the nano antibody NbN2, the amino acid sequence of FR1 is shown as SEQ ID NO.7, and specifically comprises the following components: AVQLVESGGGLVQAGGSLRLSCAATGLTFT, respectively; the amino acid sequence of FR2 is shown in SEQ ID NO.8, and specifically comprises: WFRQAPGKNREFVA, respectively; the amino acid sequence of FR3 is shown as SEQ ID NO.9, and specifically comprises: RFTVSRDNAKNTVYLQMNTLNPEDTALYYCAA, respectively; the amino acid sequence of FR4 is shown in SEQ ID NO.10, and specifically comprises: WGQGTQVTVSS, respectively; the amino acid sequence of CDR1 is shown in SEQ ID NO.11, and specifically comprises: GYVMA; the amino acid sequence of CDR2 is shown in SEQ ID NO.12, and specifically comprises: AVSRSGVVTRYADSVKG, respectively; the amino acid sequence of CDR3 is shown in SEQ ID NO.13, and specifically comprises: DWHPLTGIMTTDSTEYDY are provided.
In the nano antibody NbN3, the amino acid sequence of FR1 is shown as SEQ ID NO.14, and specifically comprises the following components: AVQLVESGGGLVQAGGSLRLSCAASGRTFS, respectively; the amino acid sequence of FR2 is shown in SEQ ID NO.15, and specifically comprises: WFRQAPGKERQFVA, respectively; the amino acid sequence of FR3 is shown in SEQ ID NO.16, and specifically comprises: RFT ISRDNAKNTVYLQMNSLKPEDTA I YYCAP, respectively; the amino acid sequence of FR4 is shown as SEQ ID NO.17, specifically WGKGTQVTVSS; the amino acid sequence of CDR1 is shown in SEQ ID NO.18, and specifically comprises: RYAMG; the amino acid sequence of CDR2 is shown in SEQ ID NO.19, and specifically comprises: GI SGSGI VTNYADSVKG, respectively; the amino acid sequence of CDR3 is shown in SEQ ID NO.20, and specifically comprises: LILARTVDMNH is added.
In the nanometer antibody NbN4, the amino acid sequence of FR1 is shown as SEQ ID NO.21, and specifically comprises the following components: AVQLVESGGGVVQPGGSLRLSCTASGFGFS, respectively; the amino acid sequence of FR2 is shown in SEQ ID NO.22, and specifically comprises: WYRQAPGKERELVA, respectively; the amino acid sequence of FR3 is shown in SEQ ID NO.23, and specifically comprises: RFSMSRDNAKSTVYLQMNSLKPEDTAVYYCVA, respectively; the amino acid sequence of FR4 is shown in SEQ ID NO.10, and specifically comprises: WGQGTQVTVSS; the amino acid sequence of CDR1 is shown in SEQ ID NO.24, and specifically comprises: LYGMS; the amino acid sequence of CDR2 is shown in SEQ ID NO.25, and specifically comprises: SI ASGGDSTNYQDSVKG, respectively; the amino acid sequence of CDR3 is shown in SEQ ID NO.26, and specifically comprises: ERVSSFL K I RTAY are provided.
In the nanometer antibody NbN5, the amino acid sequence of FR1 is shown as SEQ ID NO.27, and specifically comprises the following components: AVQLVESGGGLVQAGGSLRVSCEASGFLFN, respectively; the amino acid sequence of FR2 is shown in SEQ ID NO.28, and specifically comprises: WYRQVPGRQRELVA, respectively; the amino acid sequence of FR3 is shown in SEQ ID NO.29, and specifically comprises: RFTISRDNAKNTIFLQMSGLKPEDTAVYYCYA, respectively; the amino acid sequence of FR4 is shown as SEQ ID NO.10, and specifically comprises: WGQGTQVTVSS, respectively; the amino acid sequence of CDR1 is shown in SEQ ID NO.30, and specifically comprises: GDAYVLG; the amino acid sequence of CDR2 is shown in SEQ ID NO.31, and specifically comprises: TITSSGDTSYADSVKG, respectively; the amino acid sequence of CDR3 is shown in SEQ ID NO.32, and specifically comprises: ERWNGLLQR are provided.
In the nano antibody NbN6, the amino acid sequence of FR1 is shown as SEQ ID NO.33, and specifically comprises the following components: AVQLVESGGGLVQPGGSLRLSCAASG I IFR, respectively; the amino acid sequence of FR2 is shown in SEQ ID NO.34, and specifically comprises: WYRQAPGKQRELVA, respectively; the amino acid sequence of FR3 is shown in SEQ ID NO.35, and specifically comprises: RFTISRDNAKNTVYLQMNSLKPEDTGVYYCAA, respectively; the amino acid sequence of FR4 is shown in SEQ ID NO.17, and specifically comprises: WGKGTQVTVSS, respectively; the amino acid sequence of CDR1 is shown in SEQ ID NO.36, and specifically comprises: FRT I S; the amino acid sequence of CDR2 is shown in SEQ ID NO.37, and specifically comprises: SI SGGSSTSYADTVKG, respectively; the amino acid sequence of CDR3 is shown in SEQ ID NO.38, and specifically comprises: SHTMVGWIYGMDY are provided.
In the nanometer antibody NbN11, the amino acid sequence of FR1 is shown as SEQ ID NO.39, and specifically comprises the following components: AVQLVESGGGLVQAGGSLRLSCAASGSFFS, respectively; the amino acid sequence of FR2 is shown in SEQ ID NO.34, and specifically comprises: WYRQAPGKQRELVA; the amino acid sequence of FR3 is shown in SEQ ID NO.40, and specifically comprises: RFTISRDNAKNTIYLQMNSLKPEDTAVYYCNA, respectively; the amino acid sequence of FR4 is shown in SEQ ID NO.10, and specifically comprises: WGQGTQVTVSS, respectively; the amino acid sequence of CDR1 is shown in SEQ ID NO.41, and specifically comprises: INTMG; the amino acid sequence of CDR2 is shown in SEQ ID NO.42, and specifically comprises: QITNAGNTNYADSVKG, respectively; the amino acid sequence of CDR3 is shown in SEQ ID NO.43, and specifically comprises: GQLWGSYRS are provided.
The second aspect of the embodiments of the present application provides a method for preparing a nanobody targeting a new coronavirus, comprising the following steps:
s01, providing target protein, coating the target protein on an immune tube for enrichment screening to obtain a phage library;
s02, performing second-generation sequencing on the eluate of the phage library, and synthesizing a gene sequence of the nano antibody which accounts for 10 times of the original before counting according to a sequencing result;
s03, cloning the gene sequence of the nano antibody into an expression vector to obtain recombinant plasmids, transferring the recombinant plasmids into host cells for induced expression and purifying to obtain the nano antibody targeting the new coronavirus.
The preparation method of the nano antibody targeting the new coronavirus Omicron and the subtype BA.2 thereof is based on a phage display screening technology, a nano antibody library is constructed by taking RBD protein of the new coronavirus Omicron and the subtype BA.2 thereof as a target, and the nano antibody targeting the new 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 characteristic that the bacteriophage is quickly replicated in host bacteria, can quickly and high-flux screen the nano antibody combined with the specific variant strain Ormcken and the subtype BA.2RBD protein thereof, has quick and simple preparation method, is beneficial to large-scale screening, and improves the screening efficiency.
In step S01, a target protein is provided, and the target protein is coated on an immune tube for enrichment and screening to obtain a phage library.
In some embodiments, the protein of interest is provided as the spinous process protein RBD domain of Ormcknon (Omicron) a novel variant of coronavirus, and the concentration of the protein of interest is provided at 10-12. mu.g/mL. In a specific example, the protein of interest is coated on an 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 has large capacity, and the screening of positive clones is facilitated.
In step S02, the eluate of the phage library is subjected to second-generation sequencing, and the gene sequence of the nanobody that accounts for the first 10 before the original count is synthesized according to the sequencing result.
In step S03, the gene sequence of the nanobody is cloned into an expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transferred into a host cell for inducible expression and purification to obtain the nanobody targeting the new coronavirus.
In some embodiments, the expression vector is selected from a pColdII vector, and the gene sequence of the nano-antibody is cloned into the pColdII vector to obtain a recombinant plasmid; furthermore, a hemagglutinin tag (hemagglutinin HA tag) is simultaneously expressed in a fusion way in the cloning process for subsequent detection.
In some embodiments, the host cell is selected from E.coli, and the recombinant plasmid is transformed into E.coli cells to induce expression and purified.
In some embodiments, the recombinant plasmid is transferred into E.coli cells for induction expression and purification, as follows: (1) the induction expression is carried out at the low temperature of 16 ℃ by using IPTG with the concentration of 0.2mM, so that the formation of inclusion bodies and the degradation of protein can be prevented; (2) carrying out mass induction expression according to the induction conditions of the preliminary experiment, and carrying out bacterium breaking under the working condition of a high-pressure bacterium breaking instrument of 1000W; (3) centrifuging at the rotation speed of 17000g at 4 ℃ for 30min, taking the supernatant and the Ni filler to incubate for 1 hour at 4 ℃, and then eluting and purifying; (4) and (4) carrying out molecular sieve separation after the Ni column is purified, setting the AKATA parameter to be 0.5mL flow rate/min, and collecting every 1mL to obtain the nano antibody.
The third aspect of the embodiment of the application discloses the application of the nano antibody targeting the novel coronavirus in the preparation of the medicine for preventing and/or treating the novel coronavirus pneumonia.
The third aspect of the embodiment of the application provides an application of the nano antibody targeting the new coronavirus Omicron and the subtype BA.2 thereof in preparing a medicament for preventing and/or treating the novel coronavirus pneumonia, wherein the nano antibody targeting the new coronavirus Omicron and the subtype BA.2 thereof obtained by screening 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, has better neutralizing activity and inhibiting activity on viruses of a wild strain and various variant strains of the new 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 the application of the nano antibody targeting the novel coronavirus in the preparation of a detection reagent or a detection kit for detecting the novel coronavirus.
The application of the nano antibody targeting the new coronavirus Omicron and the subtype BA.2 thereof provided in the fourth aspect of the embodiment of the application in preparing a detection reagent or a detection kit for detecting the new coronavirus, wherein the provided nano antibody targeting the new coronavirus Omicron and the subtype BA.2 thereof 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, has higher binding capacity for viruses of a wild strain and various variant strains of the new coronavirus, and is suitable for preparing the detection reagent or the detection kit for detecting the new coronavirus.
The following description will be given with reference to specific examples.
Example 1
Nano antibody targeting new coronavirus Omicron and subtype BA.2 thereof and preparation method thereof
The test steps are as follows:
(1) coating the target protein Omicron RBD on an immune tube according to the concentration of 10 mu g/mL, and carrying out 3-round enrichment screening; (2) sending the third round of phage eluate to a company for second-generation sequencing to determine sequence information; (3) designing and synthesizing the screened nano antibody according to the sequencing information; (4) cloning a gene sequence of the nano antibody into a pColdII vector, and fusing and expressing a hemagglutinin tag (hemagglutinin HA tag) for subsequent detection; (5) inducing at low temperature of 16 ℃ by using IPTG (isopropyl thiogalactoside) with concentration of 0.2mM (6), carrying out mass induction expression according to the induction conditions of the preliminary experiment, and carrying out bacterium breaking under the working condition of a high-pressure bacterium breaker of 1000W; (7)17000g, centrifuging at 4 ℃ for 30min, taking the supernatant and incubating with Ni filler at 4 ℃ for 1 h; (8) and (3) carrying out molecular sieve separation after Ni column purification, setting the AKATA parameter at the flow rate of 0.5 mL/min, and collecting every 1mL to obtain the nano antibody targeting the new coronavirus Omicron and the subtype BA.2 thereof.
And (4) analyzing results:
screening and identification of Omicron RBD nano antibody
Screening a phage nanobody library on Omicron RBD, wherein after three rounds of screening, as shown in figure 1, the library is enriched by nearly 3000 times, the third round of phage eluate is sent to second-generation sequencing, as shown in figure 2, and sequences with the top 10 content are selected for expression, wherein the sequences are respectively as follows: NbN2, NbN3, NbN4, NbN5, NbN6, NbN7, NbN8, NbN9, NbN10, NbN 11.
Further purification was carried out to obtain 10 nanobodies of high purity, and the protein results of the 10 nanobodies obtained by purification were as shown in FIG. 3, with a size of about 15 kD. The correctness of the protein is verified by an anti-HA WB test, and 10 screened clones are expressed correctly as shown in FIG. 4.
(II) ELISA preliminary identification of the binding of the Nanobody and the RBD of each SARS-CoV-2 variant
The test steps are as follows:
the HA tag is fused into a gene coding sequence of a nano antibody to express 10 nano antibodies NbN2, NbN3, NbN4, NbN5, NbN6, NbN7, NbN8, NbN9, NbN10 and NbN11 with the HA tag, Omicron RBD protein is coated by an ELISA plate and sealed, then the nano antibodies with various concentrations are added to incubate for 1 hour at room temperature, PBS is rinsed for 3 times, and after the anti-HA antibody is incubated for 1 hour at room temperature, the anti-HA antibody marked by horseradish peroxidase amplifies signals, TMB is developed, and meanwhile, the control of irrelevant nano antibodies and the blank control of irrelevant protein antigens are made.
Analysis of results
ELISA verification is carried out on the 10 nanobodies which are purified and expressed and verified to be correct in the above step, SARS-CoV-2 WT, Delta (B.1.617.2), Omicron (B.1.1.529) RBD protein and negative control protein Bovine Serum Albumin (BSA), wherein the corresponding legends of the 10 nanobodies NbN2, NbN3, NbN4, NbN5, NbN6, NbN7, NbN8, NbN9, NbN10 and NbN11 in the figure are as follows: NGS2, NGS3, NGS4, NGS5, NGS6, NGS7, NGS8, NGS9, NGS10, NGS 11.
The analysis results of the binding to Omicron RBD protein are shown in fig. 5, and it can be seen that six nanobodies, NbN2, NbN3, NbN4, NbN5, NbN6, and NbN11, among them showed binding activity to Omicron RBD.
As a result of analysis of binding to SARS-CoV-2 Wild Type (Wild Type, WT) RBD protein, it can be seen from FIG. 6 that six nanobodies, NbN2, NbN3, NbN4, NbN5, NbN6 and NbN11, showed binding activity to Omicron RBD.
The analysis results of the binding to Delta RBD protein are shown in FIG. 7, and it can be seen that six nanobodies, NbN2, NbN3, NbN4, NbN5, NbN6 and 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 10 nanobodies among them were found to have no binding activity to the BSA protein.
Thus, it was confirmed that 6 nanobodies (Nb N2, NbN3, NbN4, NbN5, NbN6, and NbN11) had binding activity to the RBD of each SARS-CoV-2 variant and did not bind to BSA, a negative control protein (FIGS. 5 to 8).
Affinity of six positive nano antibodies and SARS-CoV-2 variant RBD protein
The testing steps are as follows:
6 nanobodies (Nb N2, NbN3, NbN4, NbN5, NbN6 and NbN11) were provided, and the affinity of the nanobodies with the RBD protein of each SARS-CoV-2 variant (WT, Alpha, Beta, Delta, Omicron and Omicron BA.2) was analyzed and examined on the basis of Biacore, a molecular interaction analysis platform.
Analysis of results
The affinities of 6 positive nanobodies Nb N2, NbN3, NbN4, NbN5, NbN6 and NbN11 to the RBD proteins of the SARS-CoV-2 variants (WT, Alpha, Beta, Delta, Omicron and Omicron ba.2) are shown in table 1, and it can be seen that the affinities are all at nanomolar level and have high affinity.
TABLE 1
Figure BDA0003681360740000161
(IV) 5 positive nanobodies and Surface Plasmon Resonance (SPR) analysis
The test steps are as follows:
experiments are used for verifying that the in vitro expressed and purified nano antibody directly interacts with the in vitro purified antigen protein, and the equilibrium constant of the two is calculated. The multi-cycle kinetics were run on Biacore S200 to study the binding kinetics of nanobodies and Omicron RBD proteins. 5 positive nanobodies Nb D2, NbE8, NbF2, NbG1 and NbG4 were provided, respectively, Neocrown wild strain (WT), variant strain Alpha (Alpha), Beta (Beta), Deltay (Delta) and Ormcron (Omicron) and its subtype BA.2RBD protein were immobilized on the surface of CM5 Series S sensor chip at 25 ℃ operating temperature, double diluted nanobodies were flowed over the chip surface at 30. mu.L/min for 120 sec followed by 240 sec of dissociation, kinetic curves were made and the relevant parameters were calculated.
Analysis of results
(1) Surface Plasmon Resonance (SPR) analysis of Nb N2 nanobody
The SPR result shows that the Nb N2 nano antibody is combined with the RBD protein of each variant strain (WT, Alpha, Beta, Delta, Omicron and Omicron BA.2) of SARS-CoV-2, and as shown in FIG. 9, the affinity of Nb N2 and the WT RBD protein is 24.45 nM; as shown in fig. 10, Nb N2 had an affinity for Alpha RBD protein of 43 nM; as shown in fig. 11, Nb N2 had an affinity for Beta RBD protein of 28.87 nM; as shown in FIG. 12, the affinity of Nb N2 for Delta RBD protein was 97.75 nM; as shown in fig. 13, the affinity of Nb N2 for Omicron RBD protein was 55.44 nM; as shown in fig. 14, the affinity of Nb N2 for Omicron ba.2rbd protein was 8.33 nM.
(2) Surface Plasmon Resonance (SPR) analysis of Nb N3 nanobody
The SPR result shows that the Nb N3 nano antibody is combined with the RBD protein of each variant strain of SARS-CoV-2 (WT, Alpha, Beta, Delta, Omicron and Omicron BA.2), and as shown in FIG. 15, the affinity of Nb N3 and the WT RBD protein is 12.09 nM; as shown in fig. 16, Nb N3 had an affinity for Alpha RBD protein of 21.24 nM; as shown in fig. 17, the affinity of Nb N3 for Beta RBD protein was 16.92 nM; as shown in FIG. 18, the affinity of Nb N3 for Delta RBD protein was 18.65 nM; as shown in fig. 19, the affinity of Nb N3 for Omicron RBD protein was 4.79 nM; as shown in fig. 20, the affinity of Nb N3 for Omicron ba.2rbd protein was 8.77 nM.
(3) Surface Plasmon Resonance (SPR) analysis of Nb N4 nanobody
The SPR result shows that the Nb N4 nano antibody is combined with the RBD protein of each variant strain of SARS-CoV-2 (WT, Alpha, Beta, Delta, Omicron and Omicron BA.2), and as shown in FIG. 21, the affinity of Nb N4 and the WT RBD protein is 2.89 pM; as shown in fig. 22, the affinity of Nb N4 for Alpha RBD protein was 73.09 nM; as shown in fig. 23, Nb N4 had an affinity for Beta RBD protein of 46.4 nM; as shown in FIG. 24, the affinity of Nb N4 for Delta RBD protein was 23.42 nM; as shown in fig. 25, the affinity of Nb N4 for Omicron RBD protein was 5.09 nM; as shown in fig. 26, Nb N4 had an affinity for Omicron ba.2rbd protein of 38.93 nM.
(4) Surface Plasmon Resonance (SPR) analysis of Nb N5 nanobody
The SPR result shows that the Nb N5 nano antibody is combined with the RBD protein of each variant strain of SARS-CoV-2 (WT, Alpha, Beta, Delta, Omicron and Omicron BA.2), and as shown in FIG. 27, the affinity of Nb N5 and the WT RBD protein is 0.21 nM; nb N5 had an affinity for Alpha RBD protein 80.48nM as shown in figure 28; as shown in fig. 29, Nb N5 has an affinity for Beta RBD protein of 37.22 nM; as shown in FIG. 30, the affinity of Nb N5 for Delta RBD protein was 24.46 nM; as shown in fig. 31, the affinity of Nb N5 for Omicron RBD protein was 90.88 nM; as shown in fig. 32, Nb N5 had an affinity for Omicron ba.2rbd protein of 20.38 nM.
(5) Surface Plasmon Resonance (SPR) analysis of Nb N6 nanobody
The SPR result shows that the Nb N6 nano antibody is combined with the RBD protein of each variant strain of SARS-CoV-2 (WT, Alpha, Beta, Delta, Omicron and Omicron BA.2), and as shown in FIG. 33, the affinity of Nb N6 and the WT RBD protein is 38.65 nM; as shown in fig. 34, Nb N6 had an affinity for Alpha RBD protein of 101.3 nM; as shown in fig. 35, Nb N6 has an affinity for Beta RBD protein of 65.36 nM; as shown in FIG. 36, the affinity of Nb N6 for Delta RBD protein was 26.91 nM; as shown in fig. 37, Nb N6 has an affinity for Omicron RBD protein of 78.77 nM; as shown in fig. 38, Nb N6 had an affinity for Omicron ba.2rbd protein of 89.52 nM.
(6) Surface Plasmon Resonance (SPR) analysis of Nb N11 nanobody
The SPR result shows that the Nb N11 nano antibody is combined with the RBD protein of each variant strain of SARS-CoV-2 (WT, Alpha, Beta, Delta, Omicron and Omicron BA.2), and as shown in FIG. 39, the affinity of Nb N11 and the WT RBD protein is 36.06 nM; as shown in fig. 40, Nb N11 had an affinity for Alpha RBD protein of 26.18 nM; as shown in fig. 41, Nb N11 has an affinity for Beta RBD protein of 19.17 nM; as shown in FIG. 42, the affinity of Nb N11 for Delta RBD protein was 28.66 nM; as shown in fig. 43, Nb N11 had an affinity for Omicron RBD protein of 47.39 nM; as shown in fig. 44, Nb N11 had an affinity for Omicron ba.2rbd protein of 105 nM.
(V) six positive nanobodies and ACE2 competitive ELISA experiment
Test procedure
And according to the SPR result, selecting the nano antibody with better affinity to perform competitive ELISA experimental verification. Omicron RBD protein was coated onto ELISA plates at a concentration of 0.5. mu.g/mL overnight at 4 ℃. The next day was washed once with PBS and blocked with 3% BSA at room temperature for 2 hours. The nanobody fold-rate dilution was performed according to the lowest saturation concentration of ACE2, i.e. the dilution was 0.1% PBST solution containing 0.5 μ g/mL ACE2 and incubated for 2.5 hours at room temperature. 0.1% PBST rinse.
Analysis of results
As shown in fig. 45, it was examined whether these 6 nanobodies competed with ACE2 for binding to omicron RBD protein, diluted with 0.1% PBST solution containing ACE2 at the lowest saturation concentration, and incubated with ELISA plates previously coated with omicron RBD protein at room temperature for 2.5 hours. 0.1% PBST is rinsed 3 times, anti-ACE2 antibody is used for detecting the content of ACE2, and blank control without nanometer antibody and ACE2 protein is made. The results show that Nb N2 can significantly competitively inhibit the binding of ACE2 to the omicron RBD protein with a half inhibitory concentration (IC 50) of 0.09 μ M. The results show that the Nb N2 nanobody can significantly competitively inhibit the binding of ACE2 to omicron RBD protein.
Therefore, the nano antibody of the new crown virus Omicron and the subtype BA.2 thereof provided by the application comprises at least one of nano antibody NbN2, nano antibody NbN3, nano antibody NbN4, nano antibody NbN5, nano antibody NbN6 and nano antibody NbN11, and the provided nano antibody has higher affinity for RBD structural domains of a new crown virus wild strain, a new crown virus variant strain (Alpha), a new crown virus Beta (Beta), a new crown virus Delta (Delta), the new crown virus Omicron and the subtype BA.2 thereof, can quickly and specifically neutralize different new crown viruses and effectively inhibit the activity of the new crown viruses; meanwhile, the provided nano antibody has a longer CDR3 sequence, is beneficial to flexibly adjusting the conformation of the nano antibody and improving the binding effect with viruses, has lower preparation cost and is beneficial to wide application.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.

Claims (5)

1. A nanobody targeting a new coronavirus comprises a nanobody NbN4, wherein the amino acid sequence of the nanobody NbN4 is shown in seq.ID No. 3.
2. The nanobody targeting neocoronaviruses according to claim 1, which comprises 4 framework regions FR1, FR2, FR3, FR4 and 3 complementarity determining regions CDR1, CDR2, CDR 3;
in the nano antibody NbN4, the amino acid sequence of FR1 is shown as SEQ ID NO.21, the amino acid sequence of FR2 is shown as SEQ ID NO.22, the amino acid sequence of FR3 is shown as SEQ ID NO.23, 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.24, the amino acid sequence of CDR2 is shown as SEQ ID NO.25, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 26.
3. The nanobody targeting the neocoronaviruses as claimed in claim 1, wherein the base sequence of the nanobody NbN4 is shown in seq.id No. 46.
4. Use of the nanobody targeting a novel coronavirus according to any one of claims 1 to 3 in the preparation of a medicament for the prevention and/or treatment of a novel coronavirus pneumonia.
5. Use of the nanobody targeting a novel coronavirus according to any one of claims 1 to 3 in the preparation of a detection reagent or a detection kit for detecting a novel coronavirus.
CN202210638231.XA 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof Active CN115043937B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210638231.XA CN115043937B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210330386.7A CN114478757B (en) 2022-03-31 2022-03-31 Nano antibody targeting new coronavirus, and preparation method and application thereof
CN202210638231.XA CN115043937B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN202210330386.7A Division CN114478757B (en) 2022-03-31 2022-03-31 Nano antibody targeting new coronavirus, and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN115043937A true CN115043937A (en) 2022-09-13
CN115043937B CN115043937B (en) 2023-06-02

Family

ID=81488870

Family Applications (6)

Application Number Title Priority Date Filing Date
CN202210637519.5A Active CN115043933B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof
CN202210638231.XA Active CN115043937B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof
CN202210637521.2A Active CN115043934B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof
CN202210638220.1A Active CN115043936B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof
CN202210637547.7A Active CN115043935B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof
CN202210330386.7A Active CN114478757B (en) 2022-03-31 2022-03-31 Nano antibody targeting new coronavirus, and preparation method and application thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202210637519.5A Active CN115043933B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof

Family Applications After (4)

Application Number Title Priority Date Filing Date
CN202210637521.2A Active CN115043934B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof
CN202210638220.1A Active CN115043936B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof
CN202210637547.7A Active CN115043935B (en) 2022-03-31 2022-03-31 Nanometer antibody targeting novel coronavirus and preparation method and application thereof
CN202210330386.7A Active CN114478757B (en) 2022-03-31 2022-03-31 Nano antibody targeting new coronavirus, and preparation method and application thereof

Country Status (1)

Country Link
CN (6) CN115043933B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114773459B (en) * 2022-06-16 2022-09-09 深圳大学 Nano antibody for resisting novel coronavirus and variant thereof, preparation method and application thereof
CN114773464B (en) * 2022-06-20 2022-08-26 北京市疾病预防控制中心 Single-domain antibody VHH-2 aiming at new coronavirus omicron strain S protein, coding sequence and application
CN115772218B (en) * 2022-11-04 2023-08-29 四川大学 Broad-spectrum neutralizing nanobody of two pan-variant strains targeting novel coronavirus S protein receptor binding region and potential application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021224606A1 (en) * 2020-05-04 2021-11-11 The Rosalind Franklin Institute Single domain antibodies binding to sars-cov-2 spike protein
WO2022020668A1 (en) * 2020-07-23 2022-01-27 The United State Of America, As Represented By The Secretary, Department Of Health And Human Services Nanobodies directed to coronavirus spike protein receptor binding domain and uses thereof
CN114031685A (en) * 2022-01-10 2022-02-11 中国人民解放军军事科学院军事医学研究院 Fully human anti-new coronavirus broad-spectrum neutralizing antibody ZW2G10 and application thereof
WO2022040423A2 (en) * 2020-08-19 2022-02-24 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Coronavirus nanobodies and methods for their use and identification
CN114106166A (en) * 2022-01-29 2022-03-01 中国疾病预防控制中心病毒病预防控制所 Humanized anti-neocoronavirus neutralizing antibody D2 and application thereof

Family Cites Families (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3946256B2 (en) * 1995-09-11 2007-07-18 協和醗酵工業株式会社 Antibody to human interleukin 5 receptor α chain
AU2007331672A1 (en) * 2006-12-15 2008-06-19 Ablynx N.V. Amino acid sequences that modulate the interaction between cells of the immune system
GB201115214D0 (en) * 2011-09-02 2011-10-19 Health Prot Agency Influenza virus antibody compositions
EP2992101B1 (en) * 2013-04-29 2018-10-10 Agrosavfe N.V. Agrochemical compositions comprising antibodies binding to sphingolipids
CA2948812A1 (en) * 2014-04-10 2015-10-15 Stichting Vumc Immunoglobulins binding human v.gamma.9v.delta.2 t cell receptors
CN111434692B (en) * 2019-01-15 2021-12-31 浙江道尔生物科技有限公司 anti-CLD18A2 nano antibody and application thereof
CN111116752B (en) * 2019-12-24 2021-09-03 北京纽安博生物技术有限公司 Immunoglobulin-binding single domain antibody, anti-avian influenza single domain antibody, bifunctional antibody and application thereof
WO2021156490A2 (en) * 2020-02-06 2021-08-12 Vib Vzw Corona virus binders
AU2021224851A1 (en) * 2020-02-21 2022-09-15 Harpoon Therapeutics, Inc. FLT3 binding proteins and methods of use
CN111592595B (en) * 2020-04-27 2021-02-19 南京医科大学 Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof
CN111647076B (en) * 2020-04-27 2021-02-26 南京医科大学 Neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof
US20220074938A1 (en) * 2020-05-07 2022-03-10 Senseutics Limited Method of detecting pathogens and/or antigens in samples
KR20230065931A (en) * 2020-05-15 2023-05-12 우니베르시다드 오스뜨랄 데 칠레 RAPID SINGLE-STEP GRADIENT METHOD FOR THE GENERATION OF NANOANTIBODIES
CN113754739A (en) * 2020-06-03 2021-12-07 中国人民解放军军事科学院军事医学研究院 Preparation method and application of coronavirus S protein RBD glycoprotein
WO2022015573A2 (en) * 2020-07-13 2022-01-20 President And Fellows Of Harvard College Sars-cov-2 antigen-binding proteins and uses thereof
WO2022015668A1 (en) * 2020-07-15 2022-01-20 Regents Of The University Of Minnesota SARS-CoV-2 NANOBODIES AND METHODS OF USE THEREOF
WO2022011717A1 (en) * 2020-07-17 2022-01-20 上海洛启生物医药技术有限公司 Nanobody against novel coronavirus, and use thereof
KR20220012810A (en) * 2020-07-23 2022-02-04 (주)셀트리온 SARS-CoV-2 Virus Neutralizing Binding Molecule Binding To A Epitopes Of Spike Protein Of SARS-CoV-2 Virus
EP3944877A1 (en) * 2020-07-29 2022-02-02 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Nanobodies sars-cov2
US20230416343A1 (en) * 2020-08-07 2023-12-28 Sorrento Therapeutics, Inc. Neutralizing Antibodies that Bind the SARS-COV-2 S Protein
WO2022040603A2 (en) * 2020-08-21 2022-02-24 The Rockefeller University Single-domain antibodies that bind sars-cov-2
CN112010967B (en) * 2020-09-07 2021-03-23 康维众和(中山)生物药业有限公司 Novel nano antibody for neutralizing toxicity of coronavirus SARS-Cov-2 and preparation method and application thereof
EP3970798A1 (en) * 2020-09-18 2022-03-23 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Sars-cov-2-nanobodies
CN112062840A (en) * 2020-09-22 2020-12-11 石河子大学 Nano antibody based on novel coronavirus S protein and application thereof
CN112062839A (en) * 2020-09-22 2020-12-11 石河子大学 Nano antibody based on novel coronavirus S protein S1 subunit and application thereof
CN112094342B (en) * 2020-09-25 2022-05-13 中国科学技术大学 Alpaca source nano antibody combined with SARS-CoV-2RBD
CN113563463B (en) * 2021-06-11 2022-05-17 中国医学科学院病原生物学研究所 Neutralizing nano antibody for resisting novel coronavirus SARS-CoV-2 and application thereof
CN114478758B (en) * 2022-04-01 2022-06-21 深圳市人民医院 Nano antibody of targeting SARS-CoV-2, preparation method and application

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021224606A1 (en) * 2020-05-04 2021-11-11 The Rosalind Franklin Institute Single domain antibodies binding to sars-cov-2 spike protein
WO2022020668A1 (en) * 2020-07-23 2022-01-27 The United State Of America, As Represented By The Secretary, Department Of Health And Human Services Nanobodies directed to coronavirus spike protein receptor binding domain and uses thereof
WO2022040423A2 (en) * 2020-08-19 2022-02-24 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Coronavirus nanobodies and methods for their use and identification
CN114031685A (en) * 2022-01-10 2022-02-11 中国人民解放军军事科学院军事医学研究院 Fully human anti-new coronavirus broad-spectrum neutralizing antibody ZW2G10 and application thereof
CN114106166A (en) * 2022-01-29 2022-03-01 中国疾病预防控制中心病毒病预防控制所 Humanized anti-neocoronavirus neutralizing antibody D2 and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MITCHELL HO.: ""Perspectives on the development of neutralizing antibodies against SARS-CoV-2"" *
TIMOTHY F CZAJKA等: ""Slaying SARS-CoV-2 One (Single-domain) Antibody at a Time"" *

Also Published As

Publication number Publication date
CN115043933A (en) 2022-09-13
CN114478757A (en) 2022-05-13
CN114478757B (en) 2022-07-05
CN115043935A (en) 2022-09-13
CN115043936A (en) 2022-09-13
CN115043934B (en) 2023-06-27
CN115043937B (en) 2023-06-02
CN115043936B (en) 2023-06-27
CN115043934A (en) 2022-09-13
CN115043935B (en) 2023-06-27
CN115043933B (en) 2023-08-08

Similar Documents

Publication Publication Date Title
CN114163523B (en) Single-domain antibody for novel coronavirus and application thereof
CN115043937A (en) Nano antibody targeting new coronavirus, and preparation method and application thereof
CN114478758B (en) Nano antibody of targeting SARS-CoV-2, preparation method and application
CN112010965B (en) Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof
CN111269313B (en) Monoclonal antibody for detecting novel coronavirus and application of monoclonal antibody in preparation of kit
Walter et al. Sybodies targeting the SARS-CoV-2 receptor-binding domain
CN113912710B (en) Monoclonal antibody for resisting novel coronavirus N protein and application thereof
CN111505286A (en) Novel coronavirus specific antibody double-antigen sandwich E L ISA detection kit and preparation method thereof
CN110903394B (en) Polypeptide capable of binding CD4 and application thereof
CN116041498B (en) Single-domain antibody specifically binding SARS-CoV-2 spike protein and application thereof
CN113248579B (en) Novel coronavirus (2019-ncov) epitope, antibody and application thereof
CN111999497B (en) ELISA kit for detecting rabies virus glycoprotein antigen and application thereof
CN114057870B (en) Labeled antibody for detecting novel coronavirus Delta mutant strain and application thereof
KR101969696B1 (en) Monoclonal antibodies of specifically binding to spike S2 protein of MERS-CoV
KR20070103548A (en) Monoclonal antibody against nucleocapsid protein of sars coronavirus and the use thereof
CN116621974A (en) Novel coronavirus SARS-CoV-2 broad spectrum neutralization nano antibody and application thereof
CN114478755B (en) Fully human antibody against novel coronavirus, composition and application thereof
WO2019165019A1 (en) Antibodies to human respiratory syncytial virus protein f pre-fusion conformation and methods of use therefor
WO2021233433A1 (en) Anti-sars-cov-2 spike protein monoclonal antibody
KR20220055423A (en) Detection method of SARS-CoV-2 using novel SARS-CoV-2 specific antibody
KR100832867B1 (en) Monoclonal antibody against nucleocapsid protein of SARS coronavirus and the use thereof
CN114656554B (en) Nano antibody of chikungunya virus E2 protein and application thereof
CN117777280A (en) Novel coronavirus SARS-CoV-2 broad spectrum neutralization single domain antibody and application thereof
WO2022112392A1 (en) Anti-sars-cov-2 antibody molecules
CN116063474A (en) Shark single domain antibody targeting SARS-CoV-2-S1-RBD, application and kit thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant