WO2023125520A1 - CAMEL-DERIVED NANOBODY WITH HIGH-AFFINITY FOR α, β, γ AND δ MUTANT STRAINS OF SARS-COV-2 - Google Patents

CAMEL-DERIVED NANOBODY WITH HIGH-AFFINITY FOR α, β, γ AND δ MUTANT STRAINS OF SARS-COV-2 Download PDF

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WO2023125520A1
WO2023125520A1 PCT/CN2022/142281 CN2022142281W WO2023125520A1 WO 2023125520 A1 WO2023125520 A1 WO 2023125520A1 CN 2022142281 W CN2022142281 W CN 2022142281W WO 2023125520 A1 WO2023125520 A1 WO 2023125520A1
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antibody
antigen
cov
sars
protein
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PCT/CN2022/142281
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杨鹏远
王楷
刘兰兰
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中国科学院生物物理研究所
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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/569—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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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

Definitions

  • the invention belongs to the fields of biotechnology, immunoassay and biomedicine, and in particular relates to broad-spectrum high-affinity antibodies or antigen-binding fragments and their applications in the detection, diagnosis, prevention and treatment of coronaviruses, especially SARS-CoV-2 Use in detection, diagnosis, prevention and treatment of Alpha mutant strain, Beta mutant strain, Gamma mutant strain and/or Delta mutant strain.
  • the novel coronavirus SARS-CoV-2 is an RNA virus of the genus Betacoronavirus.
  • the virus has the characteristics of strong transmissibility, high lethality and rapid mutation.
  • SARS-CoV-2 causes respiratory infections that lead to viral pneumonia and acute respiratory distress syndrome (ARDS) in some patients. At the same time, it can also trigger a cytokine storm and cause multiple organ damage.
  • ARDS acute respiratory distress syndrome
  • the original strain of the new coronavirus has been isolated so far, and new mutant viruses such as D614G mutant, B.1.1.7 mutant, B.1.351 mutant, B.1.429 mutant, and P.1 mutant have emerged continuously during the global spread.
  • strain, B.1.617.2 mutant strain, etc. not only greatly enhanced the transmissibility and lethality of the virus, caused repeated outbreaks of the epidemic in most countries around the world, but also caused the continuous reduction of vaccine protection.
  • Nanobody is a single-domain antibody that only contains the heavy chain antibody antigen-binding domain VHH. Compared with traditional polyclonal antibodies, monoclonal antibodies and single-chain antibodies, it has many obvious advantages, such as small size, and can pass through conventional antibodies. Into the tissues and organs (such as the sheath, spinal cord, brain, etc.); strong stability, no need for cold chain transportation and cold storage; low immunogenicity, easy to carry out humanization transformation.
  • the present invention takes SARS-CoV-2 virus surface spike protein (Spike protein, i.e.
  • S protein as the target, and develops a variety of SARS-CoV-2 virus mutations that can simultaneously recognize multiple SARS-CoV-2 virus mutations by constructing a phage display nanobody immune library and biological panning
  • the camel-derived high-affinity nanobody of the strain is of great significance for laying the foundation for the mechanism research, clinical diagnosis and treatment of new coronary pneumonia, as well as the strategic reserve for possible outbreaks of new coronaviruses in the future.
  • the technical problem to be solved by the present invention is to provide a broad-spectrum, high-affinity antibody against coronavirus, which can effectively detect, block, and treat coronavirus, especially the original strain of SARS-CoV-2 virus and its mutants .
  • anti-SARS-CoV-2 Nanobodies are provided, which can be combined with British mutant strain (Alpha; B.1.1.7), South African mutant strain (Beta; B.1.351), Brazilian mutant strain (Gamma; P.1) binds to the S1 subunit (also known as S1 protein) of the Indian mutant (Delta; B.1.617.2) S protein, and the affinity reaches nanomolar levels.
  • nanobodies against coronaviruses such as SARS-CoV-2 are provided, which can effectively block the infection of SARS-CoV-2 pseudoviruses to hACE2 overexpressed 293T cells, and neutralize them half effectively.
  • the concentration reaches nanomolar level.
  • the establishment of various ELISA detection methods based on antigen/antibody reactions and the development of detection products can be carried out.
  • multivalent isogenetic engineering based on the same or multiple Nanobodies can be performed.
  • Nanobody against a coronavirus such as SARS-CoV-2
  • said Nanobody comprising the following amino acid sequence and functional properties:
  • the antibody can have the amino acid sequence of the hypervariable region CDR1 shown in any of SEQ ID NO: 9-15; any of SEQ ID NO: 16-22 The amino acid sequence of the hypervariable region CDR2 shown; and the amino acid sequence of the hypervariable region CDR3 shown in any one of SEQ ID NO:23-30;
  • Nanobodies have nanomolar affinity to coronaviruses such as SARS-CoV-2 virus Alpha mutants, Beta mutants, Gamma mutants and Delta mutants;
  • Nanobodies effectively block the infection of SARS-CoV-2 pseudoviruses to hACE2 overexpressed 293T cells.
  • the present invention also provides a biological material containing the nucleic acid molecule encoding the antibody, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, virus vector or engineering bacteria.
  • the present invention also provides any of the following applications of the antibody:
  • the enzyme label plate of the coronavirus such as the SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain antigen.
  • the amount of the added enzyme-labeled secondary antibody bound to the bound nanobody is small, and finally the substrate is added Liquid and chromogenic solution, the color reaction is shallow, and the OD value detected by the microplate reader is low; on the contrary, when the nanobody is combined with the solid-phase antigen, the measured OD value is high, according to the amount of nanobody added and The binding curves of Nanobodies and SARS-CoV-2 were drawn corresponding to the OD values of the wells.
  • the present invention provides the following technical solutions:
  • An antibody or antigen-binding fragment thereof the amino acid sequence of which comprises CDR1 shown in any of SEQ ID NO: 9-15, CDR2 shown in any of SEQ ID NO: 16-22, and CDR2 shown in SEQ ID NO: CDR3 shown in any one of 23-30;
  • the antigen-binding fragment is, for example, Fv, Fab, Fab', scFv, F(ab') 2 , multivalent or multispecific fragments.
  • the antibody or antigen-binding fragment is an antibody comprising a sequence obtained by truncating amino acids from the 1st to the 130th amino acid from the N-terminal of any of the sequences shown in SEQ ID NO: 1-8, or SEQ ID NO: Antibodies or antigen-binding fragments with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to any of the sequences shown in 1-8.
  • a genetically engineered antibody comprising the antibody or antigen-binding fragment described in item 1 or 2; preferably, the genetically engineered antibody is a humanized antibody, a chimeric antibody, a multivalent or multispecific antibody.
  • a fusion protein comprising the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3; preferably, the fusion protein further comprises a tag polypeptide, a detection protein or an auxiliary protein.
  • a conjugate comprising the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4; preferably, the conjugate further comprises a detectable Labels, contrast agents, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, liposomes, viral coat proteins or VLPs, or combinations thereof.
  • a nucleic acid molecule encoding the antibody or antigen-binding fragment as described in item 1-2, the genetically engineered antibody as described in item 3, the fusion protein as described in item 4, or the conjugated protein as described in item 5 A substance, wherein the nucleic acid molecule is RNA, DNA or cDNA.
  • An expression vector comprising the nucleic acid molecule described in item 6;
  • the expression vector may be a DNA, RNA, viral vector, plasmid, expression cassette, transposon, other gene transfer system, or a combination thereof;
  • the expression vectors include viral vectors, such as phage vectors, lentiviruses, adenoviruses, AAV viruses, retroviruses, other protein expression systems, or combinations thereof.
  • viral vectors such as phage vectors, lentiviruses, adenoviruses, AAV viruses, retroviruses, other protein expression systems, or combinations thereof.
  • a host cell comprising the expression vector described in item 7; wherein, the host cell is a host cell for expressing a foreign protein, such as a prokaryotic expression cell, a eukaryotic expression cell, or a transgenic cell line; preferably, the The host cells include prokaryotic cells, yeast cells, insect cells, plant cells, and animal cells.
  • a method for preparing the antibody or antigen-binding fragment as described in item 1-2, the genetically engineered antibody as described in item 3, the fusion protein as described in item 4, or the conjugate as described in item 5, comprising Separating/recovering the target protein or polypeptide from the tissue sample or culture described in 9.
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4 or the conjugate described in item 5 as an active ingredient;
  • the pharmaceutical composition is an inhaled atomized drug, a drug for mucosal or epidermal application, a drug for subcutaneous injection, a drug for blood vessel transfusion, or a combination thereof; preferably, the drug also includes a pharmaceutical excipient or carrier .
  • the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4 or the conjugate described in item 5 is used in the preparation of prevention, treatment and/or Use in products or medicines for the diagnosis of coronavirus infection.
  • the coronavirus includes HCoV-NL63, SARS-CoV-1, SARS-CoV-2, HCoV-229E, MERS-CoV, HCoV-OC43, HCoV-HKU1 or other viruses with similar surface Coronavirus with S protein structure.
  • the mutant strains of SARS-CoV-2 virus include D614G mutant strain, B.1.1.7 mutant strain, B.1.351 mutant strain, B.1.429 mutant strain, P.1 mutant strain, B .1.617.2 mutant strains, etc.
  • functional polypeptides such as purification tags, detection tags, identification tags, conjugation tags, and functional verification tags included in the tag polypeptides, such as His tag, HA tag, Flag tag, c-Myc tag , Avi tags, etc.
  • the detection protein contained in the fusion protein includes functional proteins such as fluorescent protein, fluorescein-labeled protein, and peroxidase, such as FPs protein, HRP protein, Alexa Fluor-labeled protein, FITC-labeled protein, etc. protein etc.
  • functional proteins such as fluorescent protein, fluorescein-labeled protein, and peroxidase, such as FPs protein, HRP protein, Alexa Fluor-labeled protein, FITC-labeled protein, etc. protein etc.
  • the auxiliary protein contained in the fusion protein is a protein used for functions such as assisting folding, assisting expression, assisting dissolution, and shielding toxic proteins, such as GST protein, MBP protein, SUMO protein, NusA protein, etc. protein.
  • the antibody provided by the invention for anti-coronavirus such as SARS-CoV-2 effectively overcomes the shortcomings of current coronavirus such as SARS-CoV-2 recovered patients with less serum sources, high cost and unstable structure, and has high affinity and high sensitivity , high and high capacity, high output, high stability, low cost and capable of rapid mass production.
  • the antibody provided by the present invention can be used not only for initial infection blocking, early infection diagnosis, and middle and late infection treatment, but also for scientific research tools and in vitro rapid detection, such as the production of ELISA detection/diagnostic kits, colloidal gold detection/diagnostic reagents box.
  • the ELISA detection method established by the antibody of the present invention can accurately and sensitively detect whether a sample contains coronavirus such as SARS-CoV-2 virus.
  • the sample pretreatment process is simple, less time-consuming, and can detect a large number of samples at the same time, and the cost of sample detection is much lower than traditional nucleic acid detection methods.
  • the application of the antibody of the present invention to colloidal gold detection/diagnostic kits can quickly and accurately detect whether a sample contains coronaviruses such as SARS-CoV-2 virus, and is useful for solving large-scale crowd infections and environmental and cargo sample pollution screening and identification important practical significance.
  • Fig. 1 is the binding curve of Nanobody of the present invention and SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain S1 protein;
  • Fig. 2 is the affinity curve (taking antibody A1 as example) of Nanobody of the present invention and SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain S1 protein;
  • Fig. 3 is the neutralization inhibition curve of Nanobody of the present invention to SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain pseudovirus.
  • Figure 4 shows the sequence of the antibody of the present invention and its CDR region
  • FIG. 5 is a plasmid map of pComb3Xss used in Example 1.
  • FIG. 5 is a plasmid map of pComb3Xss used in Example 1.
  • the nanobody can be prepared as follows: the original strain SARS-CoV-2 protein is used as an immunogen to immunize camels of experimental animals, the total RNA of peripheral blood lymphocytes is extracted, and after inversion The nanobody heavy chain (VHH) gene fragment was cloned by recording and nested PCR, and the gene fragment was cloned into a phagemid vector by restriction enzyme digestion, and then transformed into Escherichia coli with high-efficiency electrotransformation, and the phage nanobody was constructed by assisting phage rescue.
  • VHH nanobody heavy chain
  • the prepared nanobody molecule is small, highly soluble, high temperature resistant, easy to purify, and easy to express.
  • the SARS-CoV-2 virus wild type original strain S protein and RBD protein are used as immunogens
  • the SARS-CoV-2 virus wild type original strain S1 protein, SARS-CoV-2 virus Alpha Mutant strain S1 protein, Beta mutant strain S1 protein, Gamma mutant strain S1 protein and Delta mutant strain S1 protein were used as coated antigens, all of which were purchased from Beijing Sino Biological Co., Ltd.
  • the microtiter plate is a 96-well microtiter plate, and the coating concentration of the coated antigen is 1 ug/mL.
  • the enzyme-labeled secondary antibody is an anti-HA tag antibody labeled with horseradish peroxidase, and the concentration is 0.1 ⁇ g/mL.
  • Abcam Company item number: ab1265.
  • the chromogenic solution A is prepared from 1 g of carbamide peroxide, 10.3 g of citric acid, 35.8 g of Na 2 HPO 4 ⁇ 12H 2 O, 100 ⁇ L of Tween-20 and 1000 mL of distilled water, with a pH value of 5.
  • chromogenic liquid B liquid is formulated by tetramethylbenzidine 700mg, DMSO 40mL, citric acid 10.3g and distilled water 1000mL, pH value 2.4.
  • the reaction termination solution is 2M sulfuric acid solution.
  • Leukocytes were isolated from peripheral blood after the fifth immunization, total RNA was extracted, and the VHH gene was cloned by reverse transcription PCR and nested PCR (the systems and parameters of reverse transcription PCR and nested PCR were as follows)
  • the fragment was modified with the restriction endonuclease SfiI, and the VHH gene fragment was connected to the phagemid pComb3Xss by T4 ligase (a gift from the laboratory of Professor Bruce D Hammock of UC Davis), and efficiently transformed into Escherichia coli ER2738 (laboratory Preservation, also commercially available, such as purchased from British NEB company), constructing a phage nanobody library of SARS-CoV-2.
  • the reverse transcription kit uses PrimeScript TM RT-PCR Kit, purchased from Takara Company, product number: AK2701.
  • the reverse transcription system is as follows:
  • Nested PCR (purchased from TAKATA company, item number: 6210A)
  • the reaction system is as follows:
  • the reaction system is as follows:
  • GSP-RT CGCCATCAATRTACCAGTTGA (SEQ ID NO: 31)
  • R represents base A/G
  • W represents base A/T
  • K represents base G/T
  • the amplified product is subjected to the next round of screening, ensuring that the addition amount of each round of screening is the same, the antigen coating concentration and the S protein competition elution concentration are reduced by 2 times, the titer of each round is calculated, and a single clone is selected for amplification and ELISA identification. Positive single clones were obtained after 3 rounds of panning.
  • Embodiment 3 the expression of SARS-CoV-2 Nanobody
  • the positive monoclonal plasmid was extracted, transformed into Escherichia coli TOP10F' competent cells (purchased from Thermo Fisher), and spread on solid medium after recovery for overnight culture. The next day, a single clone was picked and cultured in SB-carboxybenzyl medium, and IPTG was added to induce overnight expression; the next day, the cells were lysed with a high-pressure homogenizer, filtered with a filter membrane, and purified with a nickel column, that is, using a histidine tag and The affinity chromatography of nickel chloride in the nickel column is used to separate and purify the nanobodies to obtain high-purity anti-SARS-CoV-2 nanobodies, namely antibodies A1-A8. After amino acid sequencing analysis, the amino acid sequence of the obtained nanobodies is shown in SEQ ID NO:1-8.
  • Embodiment 4 the binding curve of nanobody and SARS-CoV-2 virus S1 protein
  • the SARS-CoV-2 virus Alpha mutant strain S1 protein, Beta mutant strain S1 protein, Gamma mutant strain S1 protein and Delta mutant strain S1 protein (Beijing Yiqiao Shenzhou Biological Co., Ltd.) were respectively coated on 96-well microtiter plates, The coating concentration of each well is 1ug/mL, and react overnight at 4°C; the next day, shake off the liquid in the well, wash 3 times with PBST containing 0.05% Tween, and invert the microplate on absorbent paper to pat dry; Add blocking solution, incubate at 37°C for 30 minutes, shake off the liquid in the well, wash 3 times with 0.05% PBST, invert the plate on absorbent paper and pat dry; Antibody solution, incubate at 37°C for 30 minutes; shake off the liquid in the well, wash 3 times with PBST, invert the plate on absorbent paper and pat dry; add enzyme-labeled secondary antibody (horseradish peroxidase-labeled anti-HA
  • Embodiment 5 the affinity curve of Nanobody and SARS-CoV-2 virus S1 protein
  • Affinity detection uses an avidin probe, which is detected using an Octecred 96 instrument.
  • the affinity detection method is a routine technical operation in the art, and the specific operations are as follows. Add 0.02% Tween-20 PBST to the 8 wells of the first column of a black non-binding 96-well plate; then add a concentration of 15ug/ml biotin-labeled SARS-CoV- 2 Virus Alpha mutant strain S1 protein, Beta mutant strain S1 protein, Gamma mutant strain S1 protein and Delta mutant strain S1 protein.
  • the balanced probe is immersed in the fourth row of nanobody dilution solution to carry out specific binding of antigen and antibody for 3 minutes;
  • the results are shown in Figure 2 and Table 1.
  • the results show that the eight Nanobodies have an affinity range of 0.28-0.82nM for the S1 protein of the Alpha mutant strain of SARS-CoV-2; and a range of 0.25-0.68nM for the S1 protein affinity of the Beta mutant strain;
  • the range of affinity for Gamma mutant S1 protein is: 0.27-0.95nM
  • the range of affinity for Delta mutant S1 protein is: 0.47-0.99nM.
  • Embodiment 6 the neutralization ability detection of nanobody to SARS-CoV-2 pseudovirus infection
  • the eight kinds of nanobodies described in the present invention are diluted to 10 concentration gradients with DMEM medium, and the final volume of each concentration is 50ul, wherein, only DMEM medium is contained in the 10th gradient and the nanobody concentration is 0 , and it was used as a control group, and then 3 ul of SARS-CoV-2 Alpha mutant strain pseudovirus capable of producing about 1x10 5 RLUs (related luciferase activity) (gifted by researcher Wang Haikun from Shanghai Pasteur Institute, Chinese Academy of Sciences, or also Can be purchased from Beijing Yunling Biotechnology Co., Ltd.) added to the nanoantibody diluent, mixed and incubated at 37 degrees for 60 minutes, and then 50ul containing 10,000 HEK293T-hACE2 cells (gifted by Wang Haikun Research Institute, Shanghai Pasteur Institute, Chinese Academy of Sciences, Alternatively, it can also be purchased from Nanjing Novizan Biotechnology Co., Ltd.) and added to the virus-antibody complex, mixed thoroughly and then added to
  • the neutralization EC 50 range for the Alpha mutant pseudovirus is 0.22-4.46nM
  • the neutralization EC 50 range for the Beta mutant pseudovirus is 0.81-10.33nM
  • the neutralizing EC 50 range of Delta mutant pseudovirus is 1.49-11.54nM.

Abstract

The present invention relates to a camel-derived nanobody specifically binding to a SARS-CoV-2 S protein and an antigen-binding fragment thereof, and specifically relates to a camel-derived nanobody or an antigen-binding fragment thereof capable of binding to surface S proteins of coronavirus mutant strains such as SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) with a high affinity. The nanobody or the antigen-binding fragment thereof can be used for preventing, detecting, diagnosing or treating infections caused by coronavirus, especially the SARS-CoV-2 virus.

Description

SARS-CoV-2α、β、γ和δ突变株骆驼源高亲和力纳米抗体SARS-CoV-2α, β, γ and δ mutant strain camel-derived high-affinity nanobodies 技术领域technical field
本发明属于生物技术、免疫检测和生物医药领域，具体涉及广谱型高亲和力抗体或抗原结合片段及其在冠状病毒的检测、诊断、预防、治疗中的用途，尤其涉及在SARS-CoV-2 Alpha突变株，Beta突变株，Gamma突变株和/或Delta突变株的检测、诊断、预防、治疗中的用途。The invention belongs to the fields of biotechnology, immunoassay and biomedicine, and in particular relates to broad-spectrum high-affinity antibodies or antigen-binding fragments and their applications in the detection, diagnosis, prevention and treatment of coronaviruses, especially SARS-CoV-2 Use in detection, diagnosis, prevention and treatment of Alpha mutant strain, Beta mutant strain, Gamma mutant strain and/or Delta mutant strain.
背景技术Background technique
新型冠状病毒SARS-CoV-2是一种β冠状病毒属RNA病毒。该病毒具有传播性强、致死率高和突变速度快等特点。SARS-CoV-2会引起呼吸道感染，从而导致一些患者出现病毒性肺炎和急性呼吸窘迫综合征(ARDS)。同时还会引发细胞因子风暴，引起多器官损伤。新冠病毒原始毒株被分离至今，在全球传播过程中不断出现的新型突变株病毒如D614G突变株、B.1.1.7突变株、B.1.351突变株、B.1.429突变株、P.1突变株、B.1.617.2突变株等，不仅大大增强了病毒的传播性和致死率，造成了全球大多数国家的疫情反复爆发，同时也造成了疫苗保护力的不断降低。The novel coronavirus SARS-CoV-2 is an RNA virus of the genus Betacoronavirus. The virus has the characteristics of strong transmissibility, high lethality and rapid mutation. SARS-CoV-2 causes respiratory infections that lead to viral pneumonia and acute respiratory distress syndrome (ARDS) in some patients. At the same time, it can also trigger a cytokine storm and cause multiple organ damage. The original strain of the new coronavirus has been isolated so far, and new mutant viruses such as D614G mutant, B.1.1.7 mutant, B.1.351 mutant, B.1.429 mutant, and P.1 mutant have emerged continuously during the global spread. strain, B.1.617.2 mutant strain, etc., not only greatly enhanced the transmissibility and lethality of the virus, caused repeated outbreaks of the epidemic in most countries around the world, but also caused the continuous reduction of vaccine protection.
在COVID-19患者治疗中使用一些小分子药物和干扰素进行抗病毒治疗，然而临床结果均显示无效或仅能在病毒感染的早期能起到有限的治疗效果,同时还会伴随一系列严重的药物副作用。已有研究证明抗体治疗策略是治疗冠状病毒患者，尤其是中晚期患者的最佳解决方案。利用含有大量中和抗体的COVID-19愈后患者血清治疗新冠病毒患者是行之有效的治疗策略。但患者血清疗法的局限性在于康复者血浆较难获得，数量较少，不能满足庞大患者群体需求，所以需要替代性的工程抗体进行治疗。In the treatment of COVID-19 patients, some small molecule drugs and interferon are used for antiviral therapy, but the clinical results show that they are ineffective or can only have a limited therapeutic effect in the early stage of viral infection, and it will also be accompanied by a series of serious Drug side effects. Studies have proven that antibody therapy strategies are the best solution for treating coronavirus patients, especially those in the middle and late stages. It is an effective therapeutic strategy to treat COVID-19 patients with the serum of post-COVID-19 patients containing a large amount of neutralizing antibodies. However, the limitation of patient serum therapy is that it is difficult to obtain the plasma of recovered patients, and the amount is small, which cannot meet the needs of a large patient population. Therefore, alternative engineered antibodies are needed for treatment.
纳米抗体(Nanobody)是只含有重链抗体抗原结合域VHH的单域抗体，与传统多克隆抗体、单克隆抗体和单链抗体相比具有诸多明显优势，比如体积小，可以穿过常规抗体无法进入的组织和器官(如鞘膜、 脊髓、大脑等)中；稳定性强，无需冷链运输和冷藏保存；免疫原性低，易于进行人源化改造。本发明将SARS-CoV-2病毒表面突刺蛋白(Spike蛋白，即S蛋白)做为靶标，通过构建噬菌体展示纳米抗体免疫文库、生物淘选研制出可同时识别多种SARS-CoV-2病毒突变株的骆驼源高亲和力纳米抗体，为新冠肺炎的机理研究、临床诊断和治疗打下基础，以及将来有可能出现的新型冠状病毒再次爆发的进行战略储备都具有重要意义。Nanobody (Nanobody) is a single-domain antibody that only contains the heavy chain antibody antigen-binding domain VHH. Compared with traditional polyclonal antibodies, monoclonal antibodies and single-chain antibodies, it has many obvious advantages, such as small size, and can pass through conventional antibodies. Into the tissues and organs (such as the sheath, spinal cord, brain, etc.); strong stability, no need for cold chain transportation and cold storage; low immunogenicity, easy to carry out humanization transformation. The present invention takes SARS-CoV-2 virus surface spike protein (Spike protein, i.e. S protein) as the target, and develops a variety of SARS-CoV-2 virus mutations that can simultaneously recognize multiple SARS-CoV-2 virus mutations by constructing a phage display nanobody immune library and biological panning The camel-derived high-affinity nanobody of the strain is of great significance for laying the foundation for the mechanism research, clinical diagnosis and treatment of new coronary pneumonia, as well as the strategic reserve for possible outbreaks of new coronaviruses in the future.
发明内容Contents of the invention
本发明所要解决的技术问题是提供一种针对冠状病毒的广谱型、高亲和力抗体，该抗体可有效检测、阻断、和治疗冠状病毒尤其是SARS-CoV-2病毒原始株及其突变株。The technical problem to be solved by the present invention is to provide a broad-spectrum, high-affinity antibody against coronavirus, which can effectively detect, block, and treat coronavirus, especially the original strain of SARS-CoV-2 virus and its mutants .
在本发明的具体实施方案中，提供了抗SARS-CoV-2纳米抗体，其可以和英国突变株(Alpha；B.1.1.7)、南非突变株(Beta；B.1.351)，巴西突变株(Gamma；P.1)和印度突变株(Delta；B.1.617.2)S蛋白的S1亚基(也称为S1蛋白)结合，亲和力均达到纳摩尔级别。In a specific embodiment of the present invention, anti-SARS-CoV-2 Nanobodies are provided, which can be combined with British mutant strain (Alpha; B.1.1.7), South African mutant strain (Beta; B.1.351), Brazilian mutant strain (Gamma; P.1) binds to the S1 subunit (also known as S1 protein) of the Indian mutant (Delta; B.1.617.2) S protein, and the affinity reaches nanomolar levels.
在本发明的具体实施方案中，提供了抗冠状病毒例如SARS-CoV-2的纳米抗体，其能够有效阻断SARS-CoV-2假病毒对hACE2过表达的293T细胞的感染，半有效中和浓度达到纳摩尔级别。In a specific embodiment of the present invention, nanobodies against coronaviruses such as SARS-CoV-2 are provided, which can effectively block the infection of SARS-CoV-2 pseudoviruses to hACE2 overexpressed 293T cells, and neutralize them half effectively. The concentration reaches nanomolar level.
在本发明的具体实施方案中，可以进行多种基于抗原/抗体反应的酶联免疫分析检测方法的建立和检测产品的开发。In a specific embodiment of the present invention, the establishment of various ELISA detection methods based on antigen/antibody reactions and the development of detection products can be carried out.
在本发明的具体实施方案中，可以进行同种或多种基于纳米抗体的多价化等基因工程改造。In a particular embodiment of the invention, multivalent isogenetic engineering based on the same or multiple Nanobodies can be performed.
在本发明的具体实施方案中，提供了针对冠状病毒例如SARS-CoV-2的纳米抗体，所述纳米抗体包含如下的氨基酸序列和功能特性：In a particular embodiment of the invention there is provided a Nanobody against a coronavirus such as SARS-CoV-2, said Nanobody comprising the following amino acid sequence and functional properties:
i)SEQ ID NO:1-8所示的氨基酸序列；或者所述抗体可具有SEQ ID NO:9-15任一所示的高度可变区CDR1氨基酸序列；SEQ ID NO:16-22 任一所示的高度可变区CDR2氨基酸序列；和SEQ ID NO:23-30任一所示的高度可变区CDR3氨基酸序列；i) the amino acid sequence shown in SEQ ID NO: 1-8; or the antibody can have the amino acid sequence of the hypervariable region CDR1 shown in any of SEQ ID NO: 9-15; any of SEQ ID NO: 16-22 The amino acid sequence of the hypervariable region CDR2 shown; and the amino acid sequence of the hypervariable region CDR3 shown in any one of SEQ ID NO:23-30;
ii)所述纳米抗体与冠状病毒例如SARS-CoV-2病毒Alpha突变株、Beta突变株，Gamma突变株和Delta突变株具有纳摩尔级别的亲和力；ii) the Nanobodies have nanomolar affinity to coronaviruses such as SARS-CoV-2 virus Alpha mutants, Beta mutants, Gamma mutants and Delta mutants;
iii)所述纳米抗体有效阻断SARS-CoV-2假病毒对hACE2过表达的293T细胞的感染。iii) The Nanobodies effectively block the infection of SARS-CoV-2 pseudoviruses to hACE2 overexpressed 293T cells.
本发明还提供一种含有所述编码所述抗体的核酸分子的生物材料，所述生物材料为重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体或工程菌。The present invention also provides a biological material containing the nucleic acid molecule encoding the antibody, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, virus vector or engineering bacteria.
本发明还提供所述抗体的以下任一应用：The present invention also provides any of the following applications of the antibody:
1)用于冠状病毒例如SARS-CoV-2病毒原始株及其突变株相关的科学研究；1) For scientific research related to coronaviruses such as the original strain of SARS-CoV-2 virus and its mutant strains;
2)用于冠状病毒例如SARS-CoV-2病毒原始株及其突变株表面S蛋白的检测；2) For the detection of S protein on the surface of coronaviruses such as the original strain of SARS-CoV-2 virus and its mutant strains;
3)用于研制冠状病毒例如SARS-CoV-2病毒原始株及其突变株检测试剂或ELISA检测试剂。3) It is used to develop detection reagents or ELISA detection reagents for coronaviruses such as the original strain of SARS-CoV-2 virus and its mutant strains.
本发明中，分析检测时，向包被有冠状病毒例如所述SARS-CoV-2病毒Alpha突变株、Beta突变株，Gamma突变株和Delta突变株抗原的酶标板的各孔中加入不同浓度所述纳米抗体，由于每个孔中的固相抗原含量均一致，因此当结合在固相抗原上的抗体少，加入的酶标二抗与被结合的纳米抗体结合量少，最后加入底物液和显色液，显色反应浅，用酶标仪检测的OD值低；反之，当所述纳米抗体和固相抗原结合多时，则所测的OD值高，根据加入的纳米抗体量和对应孔的OD值绘制纳米抗体和SARS-CoV-2的结合曲线。In the present invention, during analysis and detection, different concentrations are added to each well of the enzyme label plate of the coronavirus such as the SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain antigen. For the nanobody, since the solid-phase antigen content in each well is the same, when there are few antibodies bound to the solid-phase antigen, the amount of the added enzyme-labeled secondary antibody bound to the bound nanobody is small, and finally the substrate is added Liquid and chromogenic solution, the color reaction is shallow, and the OD value detected by the microplate reader is low; on the contrary, when the nanobody is combined with the solid-phase antigen, the measured OD value is high, according to the amount of nanobody added and The binding curves of Nanobodies and SARS-CoV-2 were drawn corresponding to the OD values of the wells.
具体地，本发明提供了以下技术方案：Specifically, the present invention provides the following technical solutions:
1、一种抗体或其抗原结合片段，其氨基酸序列包含由SEQ ID NO:9-15任一所示的CDR1、由SEQ ID NO:16-22任一所示的CDR2、由SEQ ID NO:23-30任一所示的CDR3；1. An antibody or antigen-binding fragment thereof, the amino acid sequence of which comprises CDR1 shown in any of SEQ ID NO: 9-15, CDR2 shown in any of SEQ ID NO: 16-22, and CDR2 shown in SEQ ID NO: CDR3 shown in any one of 23-30;
优选地，所述抗原结合片段例如为Fv、Fab、Fab'、scFv、F(ab') 2、多价化或多特异片段。 Preferably, the antigen-binding fragment is, for example, Fv, Fab, Fab', scFv, F(ab') 2 , multivalent or multispecific fragments.
2、根据项目1所述的抗体或抗原结合片段，其氨基酸序列如SEQ ID NO:1-8任一所示；2. The antibody or antigen-binding fragment according to item 1, whose amino acid sequence is as shown in any one of SEQ ID NO: 1-8;
或者所述抗体或抗原结合片段是包含将SEQ ID NO:1-8任一所示序列自N末端起第1位至130位氨基酸进行截短所获得的序列的抗体，或者是将SEQ ID NO:1-8任一所示序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的抗体或抗原结合片段。Or the antibody or antigen-binding fragment is an antibody comprising a sequence obtained by truncating amino acids from the 1st to the 130th amino acid from the N-terminal of any of the sequences shown in SEQ ID NO: 1-8, or SEQ ID NO: Antibodies or antigen-binding fragments with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to any of the sequences shown in 1-8.
3、基因工程抗体，其包含项目1或2所述的抗体或抗原结合片段；优选地，所述基因工程抗体为人源化抗体、嵌合抗体、多价化或多特异性抗体。3. A genetically engineered antibody comprising the antibody or antigen-binding fragment described in item 1 or 2; preferably, the genetically engineered antibody is a humanized antibody, a chimeric antibody, a multivalent or multispecific antibody.
4、融合蛋白，其包含项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体；优选地，融合蛋白还包含标签多肽、检测蛋白或辅助蛋白。4. A fusion protein comprising the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3; preferably, the fusion protein further comprises a tag polypeptide, a detection protein or an auxiliary protein.
5、偶联物，其包含项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白；优选地，所述偶联物还包含可检测标记物、造影剂、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、脂质体、病毒外壳蛋白或VLP，或其组合。5. A conjugate comprising the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4; preferably, the conjugate further comprises a detectable Labels, contrast agents, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, liposomes, viral coat proteins or VLPs, or combinations thereof.
6、一种核酸分子，其编码如项目1-2所述的抗体或抗原结合片段、如项目3所述的基因工程抗体、如项目4所述的融合蛋白或如项目5所述的偶联物，其中所述核酸分子为RNA、DNA或cDNA。6. A nucleic acid molecule encoding the antibody or antigen-binding fragment as described in item 1-2, the genetically engineered antibody as described in item 3, the fusion protein as described in item 4, or the conjugated protein as described in item 5 A substance, wherein the nucleic acid molecule is RNA, DNA or cDNA.
7、一种表达载体，其包含项目6所述的核酸分子；7. An expression vector comprising the nucleic acid molecule described in item 6;
任选地，所述表达载体可以是DNA、RNA、病毒载体、质粒、表达盒、转座子、其他基因转移系统、或其组合；Optionally, the expression vector may be a DNA, RNA, viral vector, plasmid, expression cassette, transposon, other gene transfer system, or a combination thereof;
优选地，所述表达载体包括病毒载体，如噬菌体载体、慢病毒、腺病毒、AAV病毒、逆转录病毒、其他蛋白表达系统、或其组合。Preferably, the expression vectors include viral vectors, such as phage vectors, lentiviruses, adenoviruses, AAV viruses, retroviruses, other protein expression systems, or combinations thereof.
8、宿主细胞，其包含项目7所述的表达载体；其中，所述宿主细胞是用于表达外源蛋白的宿主细胞，例如原核表达细胞、真核表达细胞、 转基因细胞系；优选地，所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞、植物细胞、动物细胞。8. A host cell comprising the expression vector described in item 7; wherein, the host cell is a host cell for expressing a foreign protein, such as a prokaryotic expression cell, a eukaryotic expression cell, or a transgenic cell line; preferably, the The host cells include prokaryotic cells, yeast cells, insect cells, plant cells, and animal cells.
9、组织样本或培养物，其通过培养项目8所述的宿主细胞获得。9. A tissue sample or culture obtained by culturing the host cell described in item 8.
10、蛋白或抗原结合片段，其从项目9所述的组织样本或培养物中分离获得。10. A protein or antigen-binding fragment isolated from the tissue sample or culture described in item 9.
11、制备项目1-2所述的抗体或抗原结合片段、如项目3所述的基因工程抗体、如项目4所述的融合蛋白或如项目5所述的偶联物的方法，包括从项目9所述的组织样本或培养物中分离/回收目的蛋白或多肽。11. A method for preparing the antibody or antigen-binding fragment as described in item 1-2, the genetically engineered antibody as described in item 3, the fusion protein as described in item 4, or the conjugate as described in item 5, comprising Separating/recovering the target protein or polypeptide from the tissue sample or culture described in 9.
12、药物组合物，其包含项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白或项目5所述的偶联物作为活性成分；例如，所述药物组合物为吸入式雾化药物、粘膜或表皮外用型药物、皮下注射型药物、血管输入型药物、或其组合；优选地，所述药物还包括药用赋形剂或载体。12. A pharmaceutical composition comprising the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4 or the conjugate described in item 5 as an active ingredient; For example, the pharmaceutical composition is an inhaled atomized drug, a drug for mucosal or epidermal application, a drug for subcutaneous injection, a drug for blood vessel transfusion, or a combination thereof; preferably, the drug also includes a pharmaceutical excipient or carrier .
13、含有项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白或项目5所述的偶联物的产品；例如，所述产品为口罩或空气净化器滤芯，环境、物体或人体表面消毒剂，或其组合；优选地，所述产品涂布在净化器滤芯中或溶解于消毒剂中用于雾化喷洒或表面擦拭。13. Products containing the antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4 or the conjugate described in item 5; for example, the product is A mask or an air purifier filter element, an environment, object or human surface disinfectant, or a combination thereof; preferably, the product is coated in a purifier filter element or dissolved in a disinfectant for atomized spraying or surface wiping.
14、项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白或项目5所述的偶联物在制备用于预防、治疗和/或诊断冠状病毒感染的产品或药物中的用途。14. The antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4 or the conjugate described in item 5 is used in the preparation of prevention, treatment and/or Use in products or medicines for the diagnosis of coronavirus infection.
15、项目1或2所述的抗体或抗原结合片段或项目3所述的基因工程抗体或项目4所述的融合蛋白或项目5所述的偶联物在制备用于以下功能的产品中的应用：15. The antibody or antigen-binding fragment described in item 1 or 2 or the genetically engineered antibody described in item 3 or the fusion protein described in item 4 or the conjugate described in item 5 in the preparation of products for the following functions application:
1)检测冠状病毒抗原，尤其是SARS-CoV-2病毒原始株及其突变株；1) Detection of coronavirus antigens, especially the original strain of SARS-CoV-2 virus and its mutant strains;
2)阻断冠状病毒感染，尤其是SARS-CoV-2病毒原始株及其突变株；2) Block coronavirus infection, especially the original strain of SARS-CoV-2 virus and its mutant strains;
3)消杀冠状病毒颗粒，尤其是SARS-CoV-2病毒原始株及其突变株；3) Eliminate coronavirus particles, especially the original strain of SARS-CoV-2 virus and its mutant strains;
4)诊断冠状病毒引起的相关疾病，尤其是SARS-CoV-2病毒原始株及其突变株；4) Diagnosis of related diseases caused by coronavirus, especially the original strain of SARS-CoV-2 virus and its mutant strains;
5)治疗冠状病毒引起的相关疾病，尤其是SARS-CoV-2病毒原始株及其突变株；5) Treatment of related diseases caused by coronaviruses, especially the original strain of SARS-CoV-2 virus and its mutant strains;
6)进行冠状病毒相关的基础科学研究，尤其是SARS-CoV-2病毒原始株及其突变株。6) Carry out basic scientific research related to coronavirus, especially the original strain of SARS-CoV-2 virus and its mutant strains.
在本发明的具体实施方案中，所述冠状病毒包括HCoV-NL63、SARS-CoV-1、SARS-CoV-2、HCoV-229E、MERS-CoV、HCoV-OC43、HCoV-HKU1或其他具有相似表面S蛋白结构的冠状病毒。In specific embodiments of the present invention, the coronavirus includes HCoV-NL63, SARS-CoV-1, SARS-CoV-2, HCoV-229E, MERS-CoV, HCoV-OC43, HCoV-HKU1 or other viruses with similar surface Coronavirus with S protein structure.
在本发明的具体实施方案中，SARS-CoV-2病毒的突变株包括D614G突变株、B.1.1.7突变株、B.1.351突变株、B.1.429突变株、P.1突变株、B.1.617.2突变株等。In specific embodiments of the present invention, the mutant strains of SARS-CoV-2 virus include D614G mutant strain, B.1.1.7 mutant strain, B.1.351 mutant strain, B.1.429 mutant strain, P.1 mutant strain, B .1.617.2 mutant strains, etc.
在本发明的具体实施方案中，所述标签多肽中所包含纯化标签、检测标签、鉴定标签、偶联标签、功能验证标签等功能多肽，例如His标签、HA标签、Flag标签、c-Myc标签、Avi标签等。In a specific embodiment of the present invention, functional polypeptides such as purification tags, detection tags, identification tags, conjugation tags, and functional verification tags included in the tag polypeptides, such as His tag, HA tag, Flag tag, c-Myc tag , Avi tags, etc.
在本发明的具体实施方案中，所述融合蛋白中所包含的检测蛋白包括荧光蛋白、荧光素标记蛋白、过氧化物酶等功能蛋白，例如FPs蛋白、HRP蛋白、Alexa Fluor标记蛋白、FITC标记蛋白等。In a specific embodiment of the present invention, the detection protein contained in the fusion protein includes functional proteins such as fluorescent protein, fluorescein-labeled protein, and peroxidase, such as FPs protein, HRP protein, Alexa Fluor-labeled protein, FITC-labeled protein, etc. protein etc.
在本发明的具体实施方案中，所述融合蛋白中所包含的辅助蛋白为用于辅助折叠、辅助表达、辅助溶解、屏蔽毒性蛋白等功能的蛋白，例如GST蛋白、MBP蛋白、SUMO蛋白、NusA蛋白。In a specific embodiment of the present invention, the auxiliary protein contained in the fusion protein is a protein used for functions such as assisting folding, assisting expression, assisting dissolution, and shielding toxic proteins, such as GST protein, MBP protein, SUMO protein, NusA protein, etc. protein.
技术效果technical effect
本发明提供的用于抗冠状病毒例如SARS-CoV-2的抗体，有效克服目前冠状病毒例如SARS-CoV-2康复患者血清来源少，成本高，结构不稳定等缺点，具有高亲和力、高灵敏度、高中和能力、高产量、高稳定性，低成本并能进行大批量快速生产。本发明提供的抗体除了可以用于 初期感染阻断、早期感染诊断、中晚期感染治疗外，还可以用于科研工具和体外快速检测，例如生产ELISA检测/诊断试剂盒、胶体金检测/诊断试剂盒。The antibody provided by the invention for anti-coronavirus such as SARS-CoV-2 effectively overcomes the shortcomings of current coronavirus such as SARS-CoV-2 recovered patients with less serum sources, high cost and unstable structure, and has high affinity and high sensitivity , high and high capacity, high output, high stability, low cost and capable of rapid mass production. The antibody provided by the present invention can be used not only for initial infection blocking, early infection diagnosis, and middle and late infection treatment, but also for scientific research tools and in vitro rapid detection, such as the production of ELISA detection/diagnostic kits, colloidal gold detection/diagnostic reagents box.
本发明的所述抗体建立的ELISA检测方法能准确灵敏地检测样品中是否含有冠状病毒例如SARS-CoV-2病毒。样品的前处理过程简单，耗时少，能同时检测大量的样品，样品检测成本远低于传统的核酸检测方法。本发明所述的抗体应用于胶体金检测/诊断试剂盒可以迅速且准确检测样品中是否含有冠状病毒例如SARS-CoV-2病毒，对解决大规模人群感染和环境、货物样品污染筛查鉴定具有重要的现实意义。The ELISA detection method established by the antibody of the present invention can accurately and sensitively detect whether a sample contains coronavirus such as SARS-CoV-2 virus. The sample pretreatment process is simple, less time-consuming, and can detect a large number of samples at the same time, and the cost of sample detection is much lower than traditional nucleic acid detection methods. The application of the antibody of the present invention to colloidal gold detection/diagnostic kits can quickly and accurately detect whether a sample contains coronaviruses such as SARS-CoV-2 virus, and is useful for solving large-scale crowd infections and environmental and cargo sample pollution screening and identification important practical significance.
附图说明Description of drawings
为了更清楚地说明本发明或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the present invention or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings in the following description are the For some embodiments of the present invention, those of ordinary skill in the art can also obtain other drawings based on these drawings on the premise of not paying creative efforts.
图1为本发明所述纳米抗体与SARS-CoV-2病毒Alpha突变株、Beta突变株，Gamma突变株和Delta突变株S1蛋白的结合曲线；Fig. 1 is the binding curve of Nanobody of the present invention and SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain S1 protein;
图2为本发明所述纳米抗体与SARS-CoV-2病毒Alpha突变株、Beta突变株，Gamma突变株和Delta突变株S1蛋白的亲和力曲线(以抗体A1为例)；Fig. 2 is the affinity curve (taking antibody A1 as example) of Nanobody of the present invention and SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain S1 protein;
图3为本发明所述纳米抗体对SARS-CoV-2病毒Alpha突变株、Beta突变株，Gamma突变株和Delta突变株假病毒的中和抑制曲线。Fig. 3 is the neutralization inhibition curve of Nanobody of the present invention to SARS-CoV-2 virus Alpha mutant strain, Beta mutant strain, Gamma mutant strain and Delta mutant strain pseudovirus.
图4显示了本发明所述抗体的序列及其CDR区；Figure 4 shows the sequence of the antibody of the present invention and its CDR region;
图5为实施例1中所使用的pComb3Xss的质粒图谱。FIG. 5 is a plasmid map of pComb3Xss used in Example 1. FIG.
具体实施方式Detailed ways
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明中的附图，对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明 中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention , but not all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用仪器等未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。所述方法如无特别说明均为常规方法，所述原材料如无特别说明均能从公开商业途径而得。If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The instruments used and those whose manufacturers are not indicated are all conventional products that can be purchased through formal channels. The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from open commercial channels unless otherwise specified.
若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.
下述实施例中的实验方法，如无特殊说明，均为常规方法。The experimental methods in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.
根据本发明的一些优选实施例，所述纳米抗体可以按如下方法进行制备：将原始毒株SARS-CoV-2蛋白作为免疫原免疫实验动物骆驼，提取外周血淋巴细胞的总RNA，经反转录及巢式PCR，克隆出纳米抗体重链(VHH)基因片段，通过酶切连接，将基因片段克隆至噬菌粒载体，高效电转化至大肠杆菌，经辅助噬菌体拯救，构建得到噬菌体纳米抗体库，筛选出SARS-CoV-2纳米抗体，将其进行表达纯化，得到灵敏度高的SARS-CoV-2纳米抗体，并且与流行的突变毒株有较高的交叉反应。制备的纳米抗体分子小，可溶性强，耐高温，易纯化，易表达。According to some preferred embodiments of the present invention, the nanobody can be prepared as follows: the original strain SARS-CoV-2 protein is used as an immunogen to immunize camels of experimental animals, the total RNA of peripheral blood lymphocytes is extracted, and after inversion The nanobody heavy chain (VHH) gene fragment was cloned by recording and nested PCR, and the gene fragment was cloned into a phagemid vector by restriction enzyme digestion, and then transformed into Escherichia coli with high-efficiency electrotransformation, and the phage nanobody was constructed by assisting phage rescue. library, screen out SARS-CoV-2 Nanobodies, express and purify them, and obtain SARS-CoV-2 Nanobodies with high sensitivity, and have high cross-reactivity with popular mutant strains. The prepared nanobody molecule is small, highly soluble, high temperature resistant, easy to purify, and easy to express.
根据本发明的一些优选实施例，所述SARS-CoV-2病毒wild type原始株S蛋白和RBD蛋白作为免疫原，SARS-CoV-2病毒wild type原始株S1蛋白、SARS-CoV-2病毒Alpha突变株S1蛋白、Beta突变株S1蛋白，Gamma突变株S1蛋白和Delta突变株S1蛋白作为包被的抗原，均购买于北京义翘神州生物有限公司。According to some preferred embodiments of the present invention, the SARS-CoV-2 virus wild type original strain S protein and RBD protein are used as immunogens, the SARS-CoV-2 virus wild type original strain S1 protein, SARS-CoV-2 virus Alpha Mutant strain S1 protein, Beta mutant strain S1 protein, Gamma mutant strain S1 protein and Delta mutant strain S1 protein were used as coated antigens, all of which were purchased from Beijing Sino Biological Co., Ltd.
所述酶标板为96孔酶标板，包被抗原的包被浓度为1ug/mL。The microtiter plate is a 96-well microtiter plate, and the coating concentration of the coated antigen is 1 ug/mL.
所述酶标记的二抗为辣根过氧化物酶标记的抗HA标签抗体，浓度为0.1μg/mL。购自Abcam公司，商品编号:ab1265。The enzyme-labeled secondary antibody is an anti-HA tag antibody labeled with horseradish peroxidase, and the concentration is 0.1 μg/mL. Purchased from Abcam Company, item number: ab1265.
所述显色液A液由过氧化脲1g、柠檬酸10.3g、Na 2HPO 4·12H 2O35.8g、吐温-20 100μL和蒸馏水1000mL配制而成，pH值5。 The chromogenic solution A is prepared from 1 g of carbamide peroxide, 10.3 g of citric acid, 35.8 g of Na 2 HPO 4 ·12H 2 O, 100 μL of Tween-20 and 1000 mL of distilled water, with a pH value of 5.
所述显色液B液由四甲基联苯胺700mg、DMSO 40mL、柠檬酸10.3g和蒸馏水1000mL配制而成，pH值2.4。Described chromogenic liquid B liquid is formulated by tetramethylbenzidine 700mg, DMSO 40mL, citric acid 10.3g and distilled water 1000mL, pH value 2.4.
所述反应终止液为2M的硫酸液。The reaction termination solution is 2M sulfuric acid solution.
实施例1、SARS-CoV-2纳米抗体库的构建Example 1, Construction of SARS-CoV-2 Nanobody Library
取200ug SARS-CoV-2病毒原始株S蛋白和RBD蛋白(北京义翘神州生物有限公司)与等体积完全弗氏佐剂混合，充分乳化后注射至骆驼，以后每隔两周加强免疫一次，其中在加强免疫中使用不完全弗氏佐剂与免疫原的混合液，颈背部皮下多点免疫，共免疫5次。从第三次免疫开始，每次免疫后一周从颈锁静脉采血并检测血清效价。Take 200ug of the original strain of SARS-CoV-2 virus S protein and RBD protein (Beijing Yiqiao Shenzhou Biological Co., Ltd.) and mix it with an equal volume of complete Freund's adjuvant, fully emulsify it and inject it into camels, and boost the immunization once every two weeks thereafter. Among them, the mixture of incomplete Freund's adjuvant and immunogen was used in the booster immunization, and the neck and back were subcutaneously immunized for 5 times in total. From the third immunization, blood was collected from the jugular vein one week after each immunization and the serum titer was detected.
从第5次免疫后的外周血中分离白细胞，提取总RNA，经反转录PCR及巢式PCR(其中，反转录PCR和巢式PCR的体系和参数如下所述)，克隆出VHH基因片段，用限制性内切酶SfiI修饰粘性末端，通过T4连接酶将VHH基因片段连接至噬菌粒pComb3Xss(UC Davis的Bruce D Hammock教授实验室惠赠)，高效电转化至大肠杆菌ER2738(实验室保存，也可商购获得，例如购自英国NEB公司)，构建SARS-CoV-2的噬菌体纳米抗体库。经测定，初级库容量达10 9cfu，加入辅助噬菌体(感染复数为20:1)M13KO7(购自NEB公司，货号：N0315S)进行拯救，得到噬菌体纳米抗体库，库容量为10 12pfu/mL，库的多样性较好。 Leukocytes were isolated from peripheral blood after the fifth immunization, total RNA was extracted, and the VHH gene was cloned by reverse transcription PCR and nested PCR (the systems and parameters of reverse transcription PCR and nested PCR were as follows) The fragment was modified with the restriction endonuclease SfiI, and the VHH gene fragment was connected to the phagemid pComb3Xss by T4 ligase (a gift from the laboratory of Professor Bruce D Hammock of UC Davis), and efficiently transformed into Escherichia coli ER2738 (laboratory Preservation, also commercially available, such as purchased from British NEB company), constructing a phage nanobody library of SARS-CoV-2. It was determined that the capacity of the primary library reached 10 9 cfu, and the helper phage (multiplicity of infection: 20:1) M13KO7 (purchased from NEB Company, item number: N0315S) was added for rescue to obtain a phage nanobody library with a library capacity of 10 12 pfu/mL , the diversity of the library is better.
反转录PCR：Reverse transcription PCR:
反转录试剂盒采用PrimeScript TM RT-PCR Kit，购自Takara公司，商品编号：AK2701。 The reverse transcription kit uses PrimeScript TM RT-PCR Kit, purchased from Takara Company, product number: AK2701.
反转录体系如下：The reverse transcription system is as follows:
Figure PCTCN2022142281-appb-000001
Figure PCTCN2022142281-appb-000001
65℃，反应5min。取出置于冰上，按以下体系加样，进行cDNA第一链合成。65°C, react for 5 minutes. Take it out and put it on ice, add sample according to the following system, and carry out cDNA first-strand synthesis.
Figure PCTCN2022142281-appb-000002
Figure PCTCN2022142281-appb-000002
30℃10min；42℃1h；72℃5min。10min at 30°C; 1h at 42°C; 5min at 72°C.
巢式PCR：(购自TAKATA公司，货号：6210A)Nested PCR: (purchased from TAKATA company, item number: 6210A)
第一轮PCR：The first round of PCR:
反应体系如下：The reaction system is as follows:
Figure PCTCN2022142281-appb-000003
Figure PCTCN2022142281-appb-000003
反应程序如下：The reaction procedure is as follows:
94℃预变性3min；Pre-denaturation at 94°C for 3 minutes;
Figure PCTCN2022142281-appb-000004
Figure PCTCN2022142281-appb-000004
72℃5min。72°C for 5min.
第二轮PCR：The second round of PCR:
反应体系如下：The reaction system is as follows:
Figure PCTCN2022142281-appb-000005
Figure PCTCN2022142281-appb-000005
Figure PCTCN2022142281-appb-000006
Figure PCTCN2022142281-appb-000006
反应程序如下：The reaction procedure is as follows:
94℃预变性3min；Pre-denaturation at 94°C for 3 minutes;
Figure PCTCN2022142281-appb-000007
Figure PCTCN2022142281-appb-000007
72℃延伸5min。Extend at 72°C for 5 min.
巢式PCR引物序列如下(5′-3′)：The sequence of the nested PCR primers is as follows (5'-3'):
GSP-RT：CGCCATCAATRTACCAGTTGA(SEQ ID NO:31)GSP-RT: CGCCATCAATRTACCAGTTGA (SEQ ID NO: 31)
LP-leader：GTGGTCCTGGCTGCTCTW(SEQ ID NO:32)LP-leader: GTGGTCCTGGCTGCTCTW (SEQ ID NO: 32)
R：CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG(SEQ ID NO:33)R: CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG (SEQ ID NO: 33)
F：CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC(SEQ ID NO:34)F: CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC (SEQ ID NO: 34)
其中，R表示碱基A/G，W表示碱基A/T，K表示碱基G/T。Wherein, R represents base A/G, W represents base A/T, and K represents base G/T.
实施例2、SARS-CoV-2纳米抗体的筛选Example 2, Screening of SARS-CoV-2 Nanobodies
在96孔酶标板的第1个孔包被SARS-CoV-2原始株病毒S蛋白抗原，包被浓度为1ug/mL，4℃过夜；次日，倒出包被液，用PBST洗涤3次，将酶标板第1、2两个孔用BSA封闭，室温孵育2h；倒出封闭液，用PBST洗涤3次；将实施例1获得的噬菌体纳米抗体库加入第1个孔，反应2h；倒出液体，在洁净的吸水纸上拍干，用PBST洗涤5次；将100μL SARS-CoV-2病毒Wild type原始株S1蛋白添加到第1个孔中，反应1h；吸出第1个孔中的液体，加入第2个孔，反应1h，除去与BSA结合的噬菌体；收集洗脱液，取5μL用于滴度测定，其余用于扩增。Coat the first well of a 96-well ELISA plate with the S protein antigen of the original strain of SARS-CoV-2 at a coating concentration of 1 ug/mL, overnight at 4°C; the next day, pour out the coating solution and wash with PBST for 3 First, block the first and second wells of the ELISA plate with BSA, and incubate at room temperature for 2 hours; pour out the blocking solution, and wash with PBST for 3 times; add the phage nanobody library obtained in Example 1 to the first well, and react for 2 hours ; Pour out the liquid, pat dry on clean absorbent paper, and wash 5 times with PBST; add 100 μL SARS-CoV-2 virus Wild type original strain S1 protein to the first well, react for 1 hour; suck out the first well Add the liquid in the second well, react for 1 h, and remove the phages bound to BSA; collect the eluate, take 5 μL for titer determination, and the rest for amplification.
将噬菌体洗脱液加入新鲜的大肠杆菌ER2738菌液(实验室保存，也可商购获得，例如购自NEB公司)，37℃，静置15min；加入羧苄青霉素和SB培养基，37℃，220rpm，培养2h；加入辅助噬菌体M13KO7(感染复数MOI＝20:1)(购自NEB公司，货号：N0315S)和卡那霉素，培养过夜；次日，离心取上清，加入PEG-NaCl溶液沉淀纯化噬菌体。Add the phage eluate to fresh Escherichia coli ER2738 bacterial liquid (preserved in the laboratory, and can also be obtained commercially, such as purchased from NEB Company), at 37°C, let stand for 15min; add carbenicillin and SB medium, at 37°C, Cultivate at 220rpm for 2 hours; add helper phage M13KO7 (MOI=20:1) (purchased from NEB Company, product number: N0315S) and kanamycin, and cultivate overnight; the next day, centrifuge to take the supernatant, and add PEG-NaCl solution Purify phage by precipitation.
将扩增产物进行下一轮筛选，保证每轮筛选的加入量相同，抗原包被浓度及S蛋白竞争洗脱浓度按2倍递减，计算每轮的滴度，挑取单克隆进行扩增及ELISA鉴定。经3轮淘选得到阳性单克隆。The amplified product is subjected to the next round of screening, ensuring that the addition amount of each round of screening is the same, the antigen coating concentration and the S protein competition elution concentration are reduced by 2 times, the titer of each round is calculated, and a single clone is selected for amplification and ELISA identification. Positive single clones were obtained after 3 rounds of panning.
实施例3、SARS-CoV-2纳米抗体的表达Embodiment 3, the expression of SARS-CoV-2 Nanobody
提取阳性单克隆质粒，转化至大肠杆菌TOP10F’感受态细胞(购自Thermo Fishier)，复苏后涂布于固体培养基过夜培养。次日，挑取单个克隆于SB-羧苄培养基中培养，加入IPTG诱导过夜表达；次日，用高压均浆仪裂解细胞，滤膜过滤后用镍柱纯化，即利用组氨酸标签与镍柱中氯化镍的亲和层析对纳米抗体进行分离纯化，得到高纯度的抗SARS-CoV-2纳米抗体，即抗体A1-A8，经氨基酸测序分析，所得纳米抗体的氨基酸序列如SEQ ID NO:1-8所示。The positive monoclonal plasmid was extracted, transformed into Escherichia coli TOP10F' competent cells (purchased from Thermo Fisher), and spread on solid medium after recovery for overnight culture. The next day, a single clone was picked and cultured in SB-carboxybenzyl medium, and IPTG was added to induce overnight expression; the next day, the cells were lysed with a high-pressure homogenizer, filtered with a filter membrane, and purified with a nickel column, that is, using a histidine tag and The affinity chromatography of nickel chloride in the nickel column is used to separate and purify the nanobodies to obtain high-purity anti-SARS-CoV-2 nanobodies, namely antibodies A1-A8. After amino acid sequencing analysis, the amino acid sequence of the obtained nanobodies is shown in SEQ ID NO:1-8.
实施例4、纳米抗体与SARS-CoV-2病毒S1蛋白的结合曲线Embodiment 4, the binding curve of nanobody and SARS-CoV-2 virus S1 protein
将SARS-CoV-2病毒Alpha突变株S1蛋白、Beta突变株S1蛋白，Gamma突变株S1蛋白和Delta突变株S1蛋白(北京义翘神州生物有限公司)分别包被于96孔酶标板上，每个孔包被浓度为1ug/mL，4℃过夜反应；次日，甩出孔中的液体，用含0.05％吐温的PBST洗3次，将酶标板倒置在吸水纸上拍干；加入封闭液，37℃孵育30分钟，甩出孔中的液体，用0.05％PBST洗3次，将酶标板倒置在吸水纸上拍干；分别加入100μL不同稀释倍数的实施例3所得的纳米抗体液，37℃孵育30分钟；甩出孔中的液体，用PBST洗3次，将酶标板倒置在吸水纸上拍干；加入酶标二抗(辣根过氧化物酶标记的抗HA标签抗体，购自Roche公司)，37℃孵育30分钟；甩出孔中的液体，用PBST洗板3次，拍干；取A液和B液等体积混匀，每孔加100μL，避光显色10～15分钟，加入终止液终止反应，酶标仪上测定各孔在波长为450nm处的OD值。 根据抗体浓度和对应孔中的OD值绘制纳米抗体和SARS-CoV-2病毒Alpha突变株S1蛋白、Beta突变株S1蛋白，Gamma突变株S1蛋白和Delta突变株S1蛋白的结合曲线(参见图1)。实验结果表明这8个纳米抗体与Alpha突变株S1蛋白、Beta突变株S1蛋白，Gamma突变株S1蛋白和Delta突变株S1蛋白均有较强的亲和力，说明所述纳米抗体具有一定的广谱性。The SARS-CoV-2 virus Alpha mutant strain S1 protein, Beta mutant strain S1 protein, Gamma mutant strain S1 protein and Delta mutant strain S1 protein (Beijing Yiqiao Shenzhou Biological Co., Ltd.) were respectively coated on 96-well microtiter plates, The coating concentration of each well is 1ug/mL, and react overnight at 4°C; the next day, shake off the liquid in the well, wash 3 times with PBST containing 0.05% Tween, and invert the microplate on absorbent paper to pat dry; Add blocking solution, incubate at 37°C for 30 minutes, shake off the liquid in the well, wash 3 times with 0.05% PBST, invert the plate on absorbent paper and pat dry; Antibody solution, incubate at 37°C for 30 minutes; shake off the liquid in the well, wash 3 times with PBST, invert the plate on absorbent paper and pat dry; add enzyme-labeled secondary antibody (horseradish peroxidase-labeled anti-HA Tag antibody (purchased from Roche Company), incubate at 37°C for 30 minutes; shake off the liquid in the well, wash the plate 3 times with PBST, and pat dry; take equal volumes of liquid A and liquid B and mix, add 100 μL to each well, and keep away from light After developing the color for 10-15 minutes, stop the reaction by adding a stop solution, and measure the OD value of each well at a wavelength of 450 nm on a microplate reader. Draw nanobody and SARS-CoV-2 virus Alpha mutant strain S1 protein, Beta mutant strain S1 protein, the binding curve of Gamma mutant strain S1 protein and Delta mutant strain S1 protein according to the OD value in antibody concentration and corresponding hole (see Fig. 1 ). The experimental results show that these 8 nanobodies have strong affinity with the Alpha mutant S1 protein, the Beta mutant S1 protein, the Gamma mutant S1 protein and the Delta mutant S1 protein, indicating that the nanobodies have certain broad-spectrum properties. .
实施例5、纳米抗体与SARS-CoV-2病毒S1蛋白的亲和力曲线Embodiment 5, the affinity curve of Nanobody and SARS-CoV-2 virus S1 protein
亲和力检测使用的是亲和素探针，利用Octec red 96仪器进行检测，所述亲和力检测方法是本领域常规技术操作，具体操作如下。向黑色无结合力的96孔板第一列的8个孔中加入0.02％吐温-20的PBST；再向第二列8个孔中加入浓度为15ug/ml生物素标记的SARS-CoV-2病毒Alpha突变株S1蛋白、Beta突变株S1蛋白，Gamma突变株S1蛋白和Delta突变株S1蛋白。第三、五、七、九，十一列中加入PBST,第四、六、八，十列中加入倍比稀释的本发明所述的纳米抗体，其中每列的第8个孔加入PBST，第十二列中加入甘氨酸2.0，所述上述液体均200ul每孔。大致程序如下：Affinity detection uses an avidin probe, which is detected using an Octecred 96 instrument. The affinity detection method is a routine technical operation in the art, and the specific operations are as follows. Add 0.02% Tween-20 PBST to the 8 wells of the first column of a black non-binding 96-well plate; then add a concentration of 15ug/ml biotin-labeled SARS-CoV- 2 Virus Alpha mutant strain S1 protein, Beta mutant strain S1 protein, Gamma mutant strain S1 protein and Delta mutant strain S1 protein. Add PBST to the third, fifth, seventh, ninth, and eleventh columns, and add the Nanobody of the present invention in the fourth, sixth, eighth, and tenth columns, wherein the eighth hole of each column is added with PBST, Glycine 2.0 was added to the twelfth column, and the above-mentioned liquids were 200ul per well. The general procedure is as follows:
1)先将8根亲和素探针(streptavidin-sensor，购自FORTEBIO,货号：18-5019)浸入到第一列PBST中进行平衡60s；1) First immerse 8 streptavidin-sensors (purchased from FORTEBIO, catalog number: 18-5019) into the first column of PBST for 60 seconds;
2)再将亲和素探针浸入到SARS-CoV-2S蛋白稀释液中结合3min；2) Then immerse the avidin probe into the SARS-CoV-2S protein dilution solution for binding for 3 minutes;
3)再回到第一，三列PBST中进行两次平衡；3) Go back to the first and perform two balances in the three columns of PBST;
4)平衡后的探针浸入到第四列纳米抗体稀释液中，进行抗原抗体的特异性结合，结合3min；4) The balanced probe is immersed in the fourth row of nanobody dilution solution to carry out specific binding of antigen and antibody for 3 minutes;
5)再回到第三列PBST中进行解离，解离10min。5) Return to the third column of PBST for dissociation for 10 minutes.
6)解离后探针在第十二列甘氨酸2.0中再生5s，将结合的纳米抗体完全洗脱下来；6) After dissociation, the probe is regenerated in the twelfth row of glycine 2.0 for 5 seconds, and the bound Nanobody is completely eluted;
7)再回到第十一列PBST中进行中和5s；7) Return to the eleventh column of PBST for neutralization for 5s;
8)重复6)7)两步；8) Repeat 6) and 7) two steps;
9)再将探针浸入第五列PBST中进行平衡；重复4)5)6)7)8)步骤依次检测其他纳米抗体与SARS-CoV-2S蛋白结合能力；9) Dip the probe into the fifth row of PBST for balance; repeat 4) 5) 6) 7) 8) steps to detect the binding ability of other nanobodies to SARS-CoV-2S protein in turn;
10)最后将实验数据导入到excel表中；10) Import the experimental data into the excel table at last;
结果参见图2和表1，结果显示八个纳米抗体对SARS-CoV-2病毒Alpha突变株S1蛋白亲和力范围为：0.28-0.82nM；对Beta突变株S1蛋白亲和力范围为：0.25-0.68nM；对Gamma突变株S1蛋白亲和力范围为：0.27-0.95nM，对Delta突变株S1蛋白亲和力范围为:0.47-0.99nM。The results are shown in Figure 2 and Table 1. The results show that the eight Nanobodies have an affinity range of 0.28-0.82nM for the S1 protein of the Alpha mutant strain of SARS-CoV-2; and a range of 0.25-0.68nM for the S1 protein affinity of the Beta mutant strain; The range of affinity for Gamma mutant S1 protein is: 0.27-0.95nM, and the range of affinity for Delta mutant S1 protein is: 0.47-0.99nM.
表1纳米抗体与SARS-CoV-2病毒Alpha突变株S1蛋白、Beta突变株S1蛋白，Gamma突变株S1蛋白和Delta突变株S1蛋白的亲和力常数K D(M) Table 1 Nanobody and SARS-CoV-2 virus Alpha mutant strain S1 protein, Beta mutant strain S1 protein, the affinity constant K D (M) of Gamma mutant strain S1 protein and Delta mutant strain S1 protein
Figure PCTCN2022142281-appb-000008
Figure PCTCN2022142281-appb-000008
实施例6、纳米抗体对SARS-CoV-2假病毒感的中和能力检测Embodiment 6, the neutralization ability detection of nanobody to SARS-CoV-2 pseudovirus infection
首先用DMEM培养基将本发明所述的八种纳米抗体倍比稀释10个浓度梯度，每个浓度的终体积为50ul，其中，第10个梯度中只含有DMEM培养基且纳米抗体浓度为0，并将其作为对照组，再将3ul能够产生约1x10 5RLUs(相关荧光素酶活性)的SARS-CoV-2 Alpha突变株假病毒(中国科学院上海巴斯德研究所王海坤研究员惠赠，或者也可购自北京云菱生物技术有限公司)加入纳米抗体稀释液中，混匀后37度孵育60min，再将50ul含有10000个HEK293T-hACE2细胞(中国科学院上海巴斯德研究所王海坤研究院惠赠，或者也可购自南京诺唯赞生物科技股份有限公司)加入到病毒-抗体复合物中，充分混匀再加入到96孔细胞培养板中，每个浓度的抗体设置3个重复孔。将细胞培养板放置于37℃培养 箱中，培养48h后，将细胞上清弃去，每孔加入100ul Bright-Glo(Promega)，反应2min，转移到白色96孔板中使用Varioskan Flash多功能读数仪检测萤火虫荧光素酶活性值(SARS-CoV-2病毒Beta突变株，Gamma突变株和Delta突变株假病毒中和实验与Alpha突变株假病毒中和实验步骤一致，仅加入的假病毒不同；假病毒和HEK293T-hACE2细胞均由上海巴斯德所王海坤研究员惠赠，或者也可购自北京云菱生物技术有限公司或南京诺唯赞生物科技股份有限公司)。以加入抗体后SARS-CoV-2 S假病毒对HEK293T-hACE2细胞感染率和纳米抗体浓度为横纵坐标绘制中和抑制曲线，最后根据曲线计算EC 50值。感染率＝(实验孔RLUs值-本底值)/(对照孔RLUs值-本底值)x100％，本底值为只加100ul Bright-Glo读取的值。结果参见图3，实验结果表明八种纳米抗体都能特异性中和SARS-CoV-2 S蛋白假病毒。对Alpha突变株假病毒的中和EC 50范围为：0.22-4.46nM,对Beta突变株假病毒的中和EC 50范围为：0.81-10.33nM，对Gamma突变株假病毒的中和EC 50范围为：1.65-10.31nM,对Delta突变株假病毒的中和EC 50范围为：1.49-11.54nM。 First, the eight kinds of nanobodies described in the present invention are diluted to 10 concentration gradients with DMEM medium, and the final volume of each concentration is 50ul, wherein, only DMEM medium is contained in the 10th gradient and the nanobody concentration is 0 , and it was used as a control group, and then 3 ul of SARS-CoV-2 Alpha mutant strain pseudovirus capable of producing about 1x10 5 RLUs (related luciferase activity) (gifted by researcher Wang Haikun from Shanghai Pasteur Institute, Chinese Academy of Sciences, or also Can be purchased from Beijing Yunling Biotechnology Co., Ltd.) added to the nanoantibody diluent, mixed and incubated at 37 degrees for 60 minutes, and then 50ul containing 10,000 HEK293T-hACE2 cells (gifted by Wang Haikun Research Institute, Shanghai Pasteur Institute, Chinese Academy of Sciences, Alternatively, it can also be purchased from Nanjing Novizan Biotechnology Co., Ltd.) and added to the virus-antibody complex, mixed thoroughly and then added to a 96-well cell culture plate. Three replicate wells are set for each concentration of antibody. Place the cell culture plate in a 37°C incubator, after culturing for 48 hours, discard the cell supernatant, add 100ul Bright-Glo (Promega) to each well, react for 2min, transfer to a white 96-well plate and use Varioskan Flash multifunctional reading The instrument detects firefly luciferase activity value (SARS-CoV-2 virus Beta mutant strain, Gamma mutant strain and Delta mutant strain pseudovirus neutralization experiment are consistent with Alpha mutant strain pseudovirus neutralization experiment steps, only the pseudovirus added is different; Pseudoviruses and HEK293T-hACE2 cells were donated by researcher Wang Haikun from the Shanghai Pasteur Institute, or purchased from Beijing Yunling Biotechnology Co., Ltd. or Nanjing Novizyme Biotechnology Co., Ltd.). Taking the infection rate of SARS-CoV-2 S pseudovirus on HEK293T-hACE2 cells after adding the antibody and the nanobody concentration as the horizontal and vertical coordinates, the neutralization inhibition curve was drawn, and finally the EC 50 value was calculated according to the curve. Infection rate=(experimental well RLUs value-background value)/(control well RLUs value-background value)×100%, the background value is the value read only by adding 100ul Bright-Glo. The results are shown in Figure 3. The experimental results show that the eight nanobodies can specifically neutralize the SARS-CoV-2 spike protein pseudovirus. The neutralization EC 50 range for the Alpha mutant pseudovirus is 0.22-4.46nM, the neutralization EC 50 range for the Beta mutant pseudovirus is 0.81-10.33nM, and the neutralization EC 50 range for the Gamma mutant pseudovirus 1.65-10.31nM, and the neutralizing EC 50 range of Delta mutant pseudovirus is 1.49-11.54nM.
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still be Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.
Figure PCTCN2022142281-appb-000009
Figure PCTCN2022142281-appb-000009
Figure PCTCN2022142281-appb-000010
Figure PCTCN2022142281-appb-000010

Claims (15)

  1. 一种抗体或其抗原结合片段，其氨基酸序列包含由SEQ ID NO:9-15任一所示的CDR1、由SEQ ID NO:16-22任一所示的CDR2、由SEQ ID NO:23-30任一所示的CDR3；An antibody or antigen-binding fragment thereof, the amino acid sequence of which comprises CDR1 shown in any of SEQ ID NO: 9-15, CDR2 shown in any of SEQ ID NO: 16-22, and CDR2 shown in any of SEQ ID NO: 23- 30 any of the indicated CDR3s;
    优选地，所述抗原结合片段例如为Fv、Fab、Fab'、scFv、F(ab') 2、多价化或多特异片段。 Preferably, the antigen-binding fragment is, for example, Fv, Fab, Fab', scFv, F(ab') 2 , multivalent or multispecific fragments.
  2. 根据权利要求1所述的抗体或抗原结合片段，其氨基酸序列如SEQ ID NO:1-8任一所示；The antibody or antigen-binding fragment according to claim 1, whose amino acid sequence is as shown in any one of SEQ ID NO: 1-8;
    或者所述抗体或抗原结合片段是包含将SEQ ID NO:1-8任一所示序列自N末端起第1位至130位氨基酸进行截短所获得的序列的抗体，或者是将SEQ ID NO:1-8任一所示序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的抗体或抗原结合片段。Or the antibody or antigen-binding fragment is an antibody comprising a sequence obtained by truncating amino acids from the 1st to the 130th amino acid from the N-terminal of any of the sequences shown in SEQ ID NO: 1-8, or SEQ ID NO: Antibodies or antigen-binding fragments with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to any of the sequences shown in 1-8.
  3. 基因工程抗体，其包含权利要求1或2所述的抗体或抗原结合片段；优选地，所述基因工程抗体为人源化抗体、嵌合抗体、多价化或多特异性抗体。A genetically engineered antibody comprising the antibody or antigen-binding fragment of claim 1 or 2; preferably, the genetically engineered antibody is a humanized antibody, a chimeric antibody, a multivalent or multispecific antibody.
  4. 融合蛋白，其包含权利要求1或2所述的抗体或抗原结合片段或权利要求3所述的基因工程抗体；优选地，融合蛋白还包含标签多肽、检测蛋白或辅助蛋白。A fusion protein comprising the antibody or antigen-binding fragment of claim 1 or 2 or the genetically engineered antibody of claim 3; preferably, the fusion protein further comprises a tag polypeptide, a detection protein or an auxiliary protein.
  5. 偶联物，其包含权利要求1或2所述的抗体或抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白；优选地，所述偶联物还包含可检测标记物、造影剂、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、脂质体、病毒外壳蛋白或VLP，或其组合。A conjugate, which comprises the antibody or antigen-binding fragment described in claim 1 or 2 or the genetically engineered antibody described in claim 3 or the fusion protein described in claim 4; preferably, the conjugate also comprises Detection of markers, contrast agents, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, liposomes, viral coat proteins or VLPs, or combinations thereof.
  6. 一种核酸分子，其编码如权利要求1-2所述的抗体或抗原结合片段、如权利要求3所述的基因工程抗体、如权利要求4所述的融合蛋白或如权利要求5所述的偶联物，其中所述核酸分子为RNA、DNA或cDNA。A nucleic acid molecule encoding the antibody or antigen-binding fragment as claimed in claim 1-2, the genetically engineered antibody as claimed in claim 3, the fusion protein as claimed in claim 4 or the antibody as claimed in claim 5 Conjugates, wherein the nucleic acid molecule is RNA, DNA or cDNA.
  7. 一种表达载体，其包含权利要求6所述的核酸分子；An expression vector comprising the nucleic acid molecule of claim 6;
    任选地，所述表达载体可以是DNA、RNA、病毒载体、质粒、表达盒、转座子、其他基因转移系统、或其组合；Optionally, the expression vector may be a DNA, RNA, viral vector, plasmid, expression cassette, transposon, other gene transfer system, or a combination thereof;
    优选地，所述表达载体包括病毒载体，如噬菌体载体、慢病毒、腺病毒、AAV病毒、逆转录病毒、其他蛋白表达系统、或其组合。Preferably, the expression vectors include viral vectors, such as phage vectors, lentiviruses, adenoviruses, AAV viruses, retroviruses, other protein expression systems, or combinations thereof.
  8. 宿主细胞，其包含权利要求7所述的表达载体；其中，所述宿主细胞是用于表达外源蛋白的宿主细胞，例如原核表达细胞、真核表达细胞、转基因细胞系；优选地，所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞、植物细胞、动物细胞。A host cell comprising the expression vector of claim 7; wherein, the host cell is a host cell for expressing a foreign protein, such as a prokaryotic expression cell, a eukaryotic expression cell, a transgenic cell line; preferably, the Host cells include prokaryotic cells, yeast cells, insect cells, plant cells, animal cells.
  9. 组织样本或培养物，其通过培养权利要求8所述的宿主细胞获得。A tissue sample or culture obtained by culturing the host cell of claim 8.
  10. 蛋白或抗原结合片段，其从权利要求9所述的组织样本或培养物中分离获得。The protein or antigen-binding fragment, which is isolated from the tissue sample or culture according to claim 9.
  11. 制备权利要求1-2所述的抗体或抗原结合片段、如权利要求3所述的基因工程抗体、如权利要求4所述的融合蛋白或如权利要求5所述的偶联物的方法，包括从权利要求9所述的组织样本或培养物中分离/回收目的蛋白或多肽。The method for preparing the antibody or antigen-binding fragment as claimed in claims 1-2, the genetically engineered antibody as claimed in claim 3, the fusion protein as claimed in claim 4 or the conjugate as claimed in claim 5, comprising Separating/recovering the target protein or polypeptide from the tissue sample or culture according to claim 9.
  12. 药物组合物，其包含权利要求1或2所述的抗体或抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或权利要求5所述的偶联物作为活性成分；例如，所述药物组合物为吸入式雾化药物、粘膜或表皮外用型药物、皮下注射型药物、血管输入型药物、或其组合；优选地，所述药物还包括药用赋形剂或载体。A pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 1 or 2 or the genetically engineered antibody of claim 3 or the fusion protein of claim 4 or the conjugate of claim 5 as an active Ingredient; for example, the pharmaceutical composition is an inhaled nebulized drug, a drug for mucosal or epidermal application, a drug for subcutaneous injection, a drug for blood vessel transfusion, or a combination thereof; preferably, the drug also includes a pharmaceutical excipient or carrier.
  13. 含有权利要求1或2所述的抗体或抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或权利要求5所述的偶联物的产品；例如，所述产品为口罩或空气净化器滤芯，环境、物体或人体表面消毒剂，或其组合；优选地，所述产品涂布在净化器滤芯中或溶解于消毒剂中用于雾化喷洒或表面擦拭。Products containing the antibody or antigen-binding fragment described in claim 1 or 2 or the genetically engineered antibody described in claim 3 or the fusion protein described in claim 4 or the conjugate described in claim 5; for example, the The product is a mask or an air purifier filter element, an environment, object or human surface disinfectant, or a combination thereof; preferably, the product is coated in a purifier filter element or dissolved in a disinfectant for atomized spraying or surface wiping.
  14. 权利要求1或2所述的抗体或抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或权利要求5所述的偶联物在制备用于预防、治疗和/或诊断冠状病毒感染的产品或药物中的用途。The antibody or antigen-binding fragment described in claim 1 or 2 or the genetically engineered antibody described in claim 3 or the fusion protein described in claim 4 or the conjugate described in claim 5 is used in the preparation of prevention, treatment and /or use in products or medicines for the diagnosis of coronavirus infection.
  15. 权利要求1或2所述的抗体或抗原结合片段或权利要求3所述的基因工程抗体或权利要求4所述的融合蛋白或权利要求5所述的偶联物在制备用于以下功能的产品中的应用：The antibody or antigen-binding fragment described in claim 1 or 2 or the genetically engineered antibody described in claim 3 or the fusion protein described in claim 4 or the conjugate described in claim 5 are used in the preparation of products for the following functions Apps in:
    1)检测冠状病毒抗原，尤其是SARS-CoV-2病毒原始株及其突变株；1) Detection of coronavirus antigens, especially the original strain of SARS-CoV-2 virus and its mutant strains;
    2)阻断冠状病毒感染，尤其是SARS-CoV-2病毒原始株及其突变株；2) Block coronavirus infection, especially the original strain of SARS-CoV-2 virus and its mutant strains;
    3)消杀冠状病毒颗粒，尤其是SARS-CoV-2病毒原始株及其突变株；3) Eliminate coronavirus particles, especially the original strain of SARS-CoV-2 virus and its mutant strains;
    4)诊断冠状病毒引起的相关疾病，尤其是SARS-CoV-2病毒原始株及其突变株；4) Diagnosis of related diseases caused by coronavirus, especially the original strain of SARS-CoV-2 virus and its mutant strains;
    5)治疗冠状病毒引起的相关疾病，尤其是SARS-CoV-2病毒原始株及其突变株；5) Treatment of related diseases caused by coronaviruses, especially the original strain of SARS-CoV-2 virus and its mutant strains;
    6)进行冠状病毒相关的基础科学研究，尤其是SARS-CoV-2病毒原始株及其突变株。6) Carry out basic scientific research related to coronavirus, especially the original strain of SARS-CoV-2 virus and its mutant strains.
PCT/CN2022/142281 2021-12-31 2022-12-27 CAMEL-DERIVED NANOBODY WITH HIGH-AFFINITY FOR α, β, γ AND δ MUTANT STRAINS OF SARS-COV-2 WO2023125520A1 (en)

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CN114409768A (en) * 2021-12-31 2022-04-29 中国科学院生物物理研究所 Camel source high affinity nano antibody of SARS-CoV-2alpha, beta, gamma and delta mutant strain
CN114805563B (en) * 2022-05-13 2024-04-02 中国科学院生物物理研究所 SARS-CoV-2alpha, gamma, delta and omicron mutant camel-derived high affinity nanobody

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN112094343A (en) * 2020-09-25 2020-12-18 中国科学技术大学 Alpaca source nano antibody combined with SARS-CoV-2RBD
CN112724248A (en) * 2021-01-28 2021-04-30 南京拓峰生物科技有限公司 Nano antibody capable of combining SARS-CoV-2 and application thereof
CN113563463A (en) * 2021-06-11 2021-10-29 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
CN114409768A (en) * 2021-12-31 2022-04-29 中国科学院生物物理研究所 Camel source high affinity nano antibody of SARS-CoV-2alpha, beta, gamma and delta mutant strain

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN112094343A (en) * 2020-09-25 2020-12-18 中国科学技术大学 Alpaca source nano antibody combined with SARS-CoV-2RBD
CN112724248A (en) * 2021-01-28 2021-04-30 南京拓峰生物科技有限公司 Nano antibody capable of combining SARS-CoV-2 and application thereof
CN113563463A (en) * 2021-06-11 2021-10-29 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
CN114409768A (en) * 2021-12-31 2022-04-29 中国科学院生物物理研究所 Camel source high affinity nano antibody of SARS-CoV-2alpha, beta, gamma and delta mutant strain

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