WO2024055928A1 - 抗猴痘病毒抗体或其抗原结合片段、检测猴痘病毒的试剂和试剂盒 - Google Patents

抗猴痘病毒抗体或其抗原结合片段、检测猴痘病毒的试剂和试剂盒 Download PDF

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WO2024055928A1
WO2024055928A1 PCT/CN2023/117997 CN2023117997W WO2024055928A1 WO 2024055928 A1 WO2024055928 A1 WO 2024055928A1 CN 2023117997 W CN2023117997 W CN 2023117997W WO 2024055928 A1 WO2024055928 A1 WO 2024055928A1
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seq
antibody
antigen
binding fragment
variable region
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PCT/CN2023/117997
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English (en)
French (fr)
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孟媛
钟冬梅
唐丽娜
游辉
曹慧方
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菲鹏生物股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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

Definitions

  • the application number of this disclosure request submitted to the State Intellectual Property Office of China on September 16, 2022 is 202211130858.0, titled "Anti-monkeypox virus antibodies or functional fragments thereof, reagents and kits for detecting monkeypox virus", in 2022
  • the application number submitted to the State Intellectual Property Office of China on September 16 was 202211126049.2, titled “Anti-monkeypox virus antibodies or functional fragments thereof, reagents and kits for detecting monkeypox virus", and was submitted to China on September 16, 2022
  • the application number of the State Intellectual Property Office is 202211126051.
  • the application number is 202211370957.6, titled "Anti-monkeypox virus antibodies or functional fragments thereof, reagents and kits for detecting monkeypox virus”.
  • the application number submitted to the China State Intellectual Property Office on November 3, 2022 is 202211370953.8.
  • the name is "Anti-monkeypox virus antibodies or functional fragments thereof, reagents and kits for detecting monkeypox virus”
  • the application number submitted to the China State Intellectual Property Office on November 3, 2022 is 202211371076.6, titled “Anti-monkeypox virus” "Viral antibodies or functional fragments thereof, reagents and kits for detecting monkeypox virus", the entire content of which is incorporated into this disclosure by reference.
  • the present disclosure relates to the field of antibody technology, and specifically to anti-monkeypox virus antibodies or antigen-binding fragments thereof, reagents and kits for detecting monkeypox virus.
  • Monkeypox is caused by monkeypox virus (MPXV).
  • Monkeypox virus is a double-stranded DNA virus and a member of the genus Orthopoxvirus in the family Poxviridae.
  • Monkeypox is a viral zoonotic disease that occurs mainly in the tropical rainforest areas of Central and West Africa and is occasionally exported to other areas.
  • Current research has found that the virus invades the human body through mucous membranes and damaged skin. Humans are mainly infected through contact with the exudates, blood, and other body fluids of infected animals, or through bites or scratches from infected animals. It is mainly transmitted between people through close contact, but can also be transmitted through droplets. It is also possible to be infected by contact with items contaminated with the virus, and it can also be transmitted vertically through the placenta. Sexual transmission cannot be ruled out.
  • monkeypox In terms of clinical symptoms, the infection symptoms of monkeypox are similar to those of smallpox, but the clinical symptoms of monkeypox are milder, and the incubation period is 5-12 days, usually 6-13 days. Patients initially present with symptoms such as fever, headache, swollen lymph nodes, muscle aches, and severe fatigue. Later, they may cause rashes on the face and body. Monkeypox is a self-limiting disease, and people usually recover within two to three weeks; however, in children, pregnant women, or people who are immunosuppressed due to other health conditions, monkeypox may lead to other secondary infections. Serious illnesses such as pneumonia, sepsis and encephalitis.
  • PCR amplification method is the main detection method for detecting monkeypox virus at home and abroad, but it has high requirements on equipment, detection site and environmental conditions, and there are detection problems. Shortcomings such as long time and low throughput make it inconvenient for large-scale detection of people.
  • the immunoassay based on colloidal gold has the advantages of being fast, convenient, and easy to carry, so it has become a hot spot for research and development.
  • the detection performance of colloidal gold depends on the performance of antibodies against monkeypox virus.
  • the purpose of this disclosure is to provide anti-monkeypox virus antibodies or antigen-binding fragments thereof, reagents and kits for detecting monkeypox virus or monkeypox virus antigens.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, the HCDR1, HCDR2, HCDR3 comprising or being With SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:31, SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ The amino acid sequence consistent with HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in ID NO:80, SEQ ID NO:81 or SEQ ID NO:82; the LCDR1, LCDR2 and LCDR3 include or are identical to SEQ ID NO:8 , SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:50, SEQ ID NO:88, SEQ ID NO:89 or SEQ ID NO:90, the LCDR1, LCDR2
  • the CDRs are defined by Kabat, Chothia, AbM, Contact or IMGT systems.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, which includes HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3.
  • the HCDR1, HCDR2, HCDR3, and LCDR1 are selected from any combination of the following:
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes HCDR1 ⁇ described in any of the above embodiments.
  • HCDR3 and the light chain variable region include LCDR1 ⁇ LCDR3 described in any of the above embodiments; and the heavy chain variable region is as with SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:31, SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:80, SEQ ID NO:81 or SEQ ID NO:82 have at least 80 % identity of the amino acid sequence shown and/or the light chain variable region is as shown in SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:50, SEQ ID NO:88, SEQ ID NO:89 or SEQ ID NO:90 is shown as an amino acid sequence having at least
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, the heavy chain variable region such as SEQ ID NO: 7, SEQ ID NO: 19.
  • SEQ ID NO:31, SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:80, SEQ ID NO:81 or SEQ ID NO:82 is shown
  • the light chain variable region is as SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:50, SEQ ID NO:88, SEQ ID NO: 89 or SEQ ID NO:90.
  • the heavy chain variable region and light chain variable region in the above-mentioned first, second, third or fourth aspect are selected from any combination of the following:
  • the antibody or antigen-binding fragment thereof described in the first, second, third or fourth aspect above includes a heavy chain variable region and a light chain variable region in any combination of the following:
  • the antibody or antigen-binding fragment thereof described in the above first, second, third or fourth aspect further includes a constant region.
  • the constant region includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from the kappa or lambda type Light chain constant region.
  • the species of the constant region is derived from cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans. Any kind.
  • the heavy chain constant region is as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto.
  • the light chain constant region is as shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain and a light chain, and the amino acid sequence of the heavy chain is such as SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 35. SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96 shown; the amino acid sequence of the light chain is such as SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 57, SEQ ID NO: 97, SEQ ID NO: 98 or SEQ ID NO: 99 shown.
  • the heavy chain and light chain are selected from any combination of the following:
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, which binds to the amino acid sequence of monkeypox virus antigenic protein with the antibody or antigen-binding fragment thereof described in any of the above embodiments. the same epitope among them; or, the antibody or antigen-binding fragment thereof competitively binds to the monkeypox virus antigen with the antibody or antigen-binding fragment thereof described in any of the above embodiments.
  • the antigen-binding fragment includes a heavy chain variable region and a light chain variable region
  • the heavy chain variable region includes HCDR1 to HCDR3 described in any of the above embodiments
  • the light chain variable region includes LCDR1 to LCDR3 described in any of the above embodiments.
  • the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.
  • embodiments of the present disclosure provide an antibody conjugate, which includes the antibody or antigen-binding fragment thereof as described in the previous embodiments.
  • the antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.
  • the antibody conjugate further includes a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.
  • the antibody conjugate further includes a label coupled to the antibody or antigen-binding fragment thereof.
  • the label is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle labels.
  • the label is colloidal gold.
  • embodiments of the present disclosure provide a reagent or kit, which includes the antibody or antigen-binding fragment thereof as described in the preceding embodiments or the antibody conjugate as described in the preceding embodiments.
  • the reagent or kit further includes a second antibody that binds to the antibody or antigen-binding fragment thereof.
  • the reagent or kit further includes a second antibody that binds to monkeypox virus or monkeypox virus antigen.
  • embodiments of the present disclosure provide a method for detecting monkeypox virus or monkeypox virus antigen, which includes: using the antibody or antigen-binding fragment thereof, antibody conjugate or reagent or reagent as described in the previous embodiment.
  • the cartridge comes into contact with the monkeypox virus or monkeypox virus antigen in the sample to be tested to form an immune complex.
  • the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment thereof.
  • the immune complex further includes a second antibody that binds to monkeypox virus or monkeypox virus antigen.
  • embodiments of the present disclosure provide a nucleic acid encoding the antibody or antigen-binding fragment thereof described in the preceding embodiments.
  • embodiments of the present disclosure provide a vector containing the nucleic acid described in the previous embodiments.
  • embodiments of the present disclosure provide a cell containing the nucleic acid or vector described in the previous embodiments.
  • embodiments of the present disclosure provide a method of preparing the antibody or antigen-binding fragment thereof described in the preceding embodiment, which includes: culturing the cells described in the preceding embodiment.
  • embodiments of the present disclosure provide antibodies or antigen-binding fragments thereof, antibody conjugates, reagents or kits as described in the previous embodiments for detecting monkeypox virus or monkeypox virus antigens or for preparing and detecting monkeypox virus. or application of monkeypox virus antigens in products.
  • the anti-monkeypox virus antibody disclosed in the present disclosure includes the above-mentioned heavy chain complementarity-determining region and light chain complementarity-determining region.
  • the antibody provides an important source of raw materials for the detection of monkeypox virus or monkeypox virus antigen, and has improved affinity or activity.
  • Figure 1 shows the reducing SDS-PAGE results of Anti-MPXV 4G21.
  • Figure 2 shows the reducing SDS-PAGE results of Anti-MPXV 2F19.
  • Figure 3 shows the results of reducing SDS-PAGE of Anti-MPXV 17A16.
  • Figure 4 shows the results of reducing SDS-PAGE of Anti-MPXV 11D3RMb1 ⁇ 4.
  • Figure 5 shows the results of reducing SDS-PAGE of Anti-MPXV 6F13RMb1 ⁇ 3.
  • antibody is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.
  • CDR complementarity determining region
  • CDRs refers to the highly variable regions of the heavy and light chains of an immunoglobulin, including one or more, or even all, of the opposing antibodies or the region of major amino acid residues that are responsible for the binding of an antigen-binding fragment to the antigen or epitope it recognizes.
  • CDRs refer to the highly variable regions of the heavy and light chains of the antibody.
  • the heavy chain complementarity determining region is represented by HCDR, and the three CDRs contained in the heavy chain variable region include HCDR1, HCDR2, and HCDR3; the light chain complementarity determining region is represented by LCDR, and the three CDRs contained in the light chain variable region include The CDRs include LCDR1, LCDR2 and LCDR3.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, the HCDR1, HCDR2, HCDR3 comprising or being
  • the amino acid sequence consistent with HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO:7; the LCDR1, LCDR2 and LCDR3 include or are identical to the LCDR1, HCDR2 and HCDR3 of the light chain variable region shown in SEQ ID NO:8 The identical amino acid sequences of LCDR2 and LCDR3.
  • CDRs are well known in the art. Common ones include Kabat, Chothia, AbM, Contact or IMGT, etc. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. There are other methods of defining CDRs that may not strictly follow one of the above methods but will still overlap with at least part of the CDRs defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results for specific residues or groups of residues, e.g. The C-termini of HCDR3 and LCDR3 defined by Kabat are each shortened by 1 to 2 amino acids. As used herein, CDRs may refer to CDRs defined by any method known in the art, including combinations of methods.
  • the CDRs are defined by Kabat, Chothia, AbM, Contact or IMGT systems.
  • the CDRs are based on the CDRs defined by the Kabat system, with the C-terminus of HCDR3 shortened by 2 amino acids, and the C-terminus of LCDR3 shortened by 1 amino acid.
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 include SEQ ID NO: 1 to 3 in sequence or are shown in SEQ ID NO: 1 to 3 in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 include in sequence SEQ ID NO:4 ⁇ 6 or as shown in SEQ ID NO:4 ⁇ 6.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, the amino acid sequences of HCDR1, HCDR2 and HCDR3
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 include SEQ ID NO:1 ⁇ 3 or as shown in SEQ ID NO:1 ⁇ 3 in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 include SEQ ID NO:4 ⁇ 6 in sequence or as shown in SEQ ID NO:4 ⁇ 6 in sequence. Show.
  • the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively correspond to H31 ⁇ H35, H50 ⁇ H65, H95 ⁇ H100D, L24 ⁇ 34, L50 ⁇ 56, and L89 ⁇ 96 under the Kabat number The amino acid of the position segment.
  • the antibody or antigen-binding fragment thereof further includes a framework region.
  • the "framework region” or “FR” region includes the heavy chain framework region and the light chain framework region, and refers to the region in the antibody heavy chain variable region and light chain variable region other than the CDR; where, the heavy chain region
  • the chain framework region (HFR) can be further subdivided into contiguous regions separated by CDRs, including the HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region (LFR) can be further subdivided into contiguous regions separated by CDRs
  • the adjacent region includes the LFR1, LFR2, LFR3 and LFR4 framework regions.
  • the heavy chain variable region is obtained by HCDR and HFR arranged in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by LCDR and LFR arranged in the following combination: LFR1-LCDR1- LFR2-LCDR2-LFR3-LCDR3-LFR4.
  • "-" represents a peptide bond.
  • the combined fragments are arranged sequentially from the upstream (N-terminal) to the downstream (C-terminal) of the sequence.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes HCDR1 ⁇ described in any of the above embodiments.
  • HCDR3 and the light chain variable region include LCDR1 ⁇ LCDR3 described in any of the above embodiments;
  • the heavy chain variable region is as shown in an amino acid sequence with at least 80% identity to SEQ ID NO: 7 and/or
  • the light chain variable region is represented by an amino acid sequence having at least 80% identity with SEQ ID NO:8.
  • the heavy chain variable region is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% similar to SEQ ID NO:7 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
  • the identity of the amino acid sequence is shown; the light chain variable region has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with SEQ ID NO:8 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences are shown.
  • the antibody or antigen-binding fragment thereof described in the first, second or third aspect above includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region is such as SEQ ID NO:7 is shown; the light chain variable region is shown as SEQ ID NO:8.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, and the heavy chain variable region is as shown in SEQ ID NO: 7; The light chain variable region is shown in SEQ ID NO:8.
  • the antibody or antigen-binding fragment thereof further comprises a constant region.
  • the constant region includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from kappa Type or lambda light chain constant region.
  • the species source of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose Any kind of person.
  • the heavy chain constant region is as set forth in SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto.
  • the light chain constant region is as set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto.
  • "having at least 80% identity” may mean having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain and a light chain.
  • the heavy chain is as shown in SEQ ID NO: 11, and the light chain is as shown in SEQ ID NO: 12. Show.
  • embodiments of the present disclosure also provide an antibody or an antigen-binding fragment thereof, which binds to the amino acid of the monkeypox virus antigen protein with the antibody or antigen-binding fragment thereof described in any of the above embodiments.
  • the same epitope in the sequence; or, the antibody or antigen-binding fragment thereof competitively binds to the monkeypox virus antigen with the antibody or antigen-binding fragment thereof described in any of the above embodiments.
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 6.98 ⁇ 10 ⁇ 8 M.
  • the antibody or antigen-binding fragment thereof has a KD ⁇ 10 -08 M, a KD ⁇ 10 -09 M, a KD ⁇ 10 -10 M, a KD ⁇ 10 -11 M or a KD ⁇ 10 -12
  • the affinity of M binds monkeypox virus antigen.
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 2.23 ⁇ 10 ⁇ 10 M.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, the HCDR1, HCDR2, HCDR3 comprising or being
  • the amino acid sequence consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 19; the LCDR1, LCDR2, and LCDR3 include or are identical to the LCDR1, HCDR2, and HCDR3 of the light chain variable region shown in SEQ ID NO: 20 The identical amino acid sequences of LCDR2 and LCDR3.
  • the CDRs are defined by Kabat, Chothia, AbM, Contact or IMGT systems.
  • the CDRs are based on the CDRs defined in the Kabat system, with 2 amino acids each shortened at the C terminus of HCDR3 and LCDR3.
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 include SEQ ID NO: 13-15 in sequence or are shown in SEQ ID NO: 13-15 in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 include in sequence SEQ ID NO:16 ⁇ 18 or as shown in SEQ ID NO:16 ⁇ 18.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, the amino acid sequences of HCDR1, HCDR2 and HCDR3
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 include SEQ ID NO:13 ⁇ 15 or as shown in SEQ ID NO:13 ⁇ 15 in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 include SEQ ID NO:16 ⁇ 18 in sequence or as shown in SEQ ID NO:16 ⁇ 18 in sequence. Show.
  • the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively correspond to H31 ⁇ H35, H50 ⁇ H65, H95 ⁇ H100C, L24 ⁇ 34, L50 ⁇ 56, and L89 ⁇ 95 under the Kabat number The amino acid of the position segment.
  • the antibody or antigen-binding fragment thereof further includes a framework region.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes HCDR1 ⁇ described in any of the above embodiments.
  • HCDR3 and the light chain variable region include LCDR1 ⁇ LCDR3 described in any of the above embodiments;
  • the heavy chain variable region is as shown in an amino acid sequence with at least 80% identity to SEQ ID NO: 19 and/or
  • the light chain variable region is shown as an amino acid sequence having at least 80% identity with SEQ ID NO:20.
  • the heavy chain variable region is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% similar to SEQ ID NO: 19 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity of the amino acid sequence shown;
  • the light chain variable Areas such as SEQ ID NO:20 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 Amino acid sequences with %, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity are shown.
  • the antibody or antigen-binding fragment thereof described in the seventh, eighth or ninth aspect above includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region is such as SEQ ID NO:19 is shown; the light chain variable region is shown as SEQ ID NO:20.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, said The heavy chain variable region is shown in SEQ ID NO:19; the light chain variable region is shown in SEQ ID NO:20.
  • the antibody or antigen-binding fragment thereof further comprises a constant region.
  • the constant region includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from kappa Type or lambda light chain constant region.
  • the species source of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose Any kind of person.
  • the heavy chain constant region is as set forth in SEQ ID NO: 21 or an amino acid sequence having at least 80% identity thereto.
  • the light chain constant region is as set forth in SEQ ID NO: 22 or an amino acid sequence having at least 80% identity thereto.
  • "having at least 80% identity” may mean having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain and a light chain, the heavy chain is as shown in SEQ ID NO: 23, and the light chain is as shown in SEQ ID NO: 24 shown.
  • embodiments of the present disclosure also provide an antibody or an antigen-binding fragment thereof, which binds to the monkeypox virus antigen protein with the antibody or antigen-binding fragment thereof described in any of the above embodiments.
  • the same epitope in the amino acid sequence; or, the antibody or antigen-binding fragment thereof competitively binds to the monkeypox virus antigen with the antibody or antigen-binding fragment thereof described in any of the above embodiments.
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 6.34 ⁇ 10 ⁇ 8 M.
  • the antibody or antigen-binding fragment thereof has a KD ⁇ 10 -08 M, a KD ⁇ 10 -09 M, a KD ⁇ 10 -10 M, a KD ⁇ 10 -11 M or a KD ⁇ 10 -12
  • the affinity of M binds monkeypox virus antigen.
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 1.14 ⁇ 10 ⁇ 11 M.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1 to 3 include or are identical to SEQ ID NO: 31 or 38.
  • the amino acid sequence consistent with HCDR1 ⁇ 3 of the heavy chain variable region is shown; the LCDR1 ⁇ 3 includes or is an amino acid sequence consistent with LCDR1 ⁇ 3 of the light chain variable region shown in SEQ ID NO:32.
  • HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 in the same heavy chain variable region, and LCDR1, LCDR2 and LCDR3 are identical to LCDR1 in the same light chain variable region. , LCDR2, LCDR3 consistent amino acid sequence.
  • the HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 31; the LCDR1, LCDR2, and LCDR3 are the same amino acid sequences as those of the heavy chain variable region shown in SEQ ID NO: 32.
  • the consistent amino acid sequences of LCDR1, LCDR2, and LCDR3 in the chain variable region are the same amino acid sequences consistent with the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 31;
  • the LCDR1, LCDR2, and LCDR3 are the same amino acid sequences as those of the heavy chain variable region shown in SEQ ID NO: 32.
  • the consistent amino acid sequences of LCDR1, LCDR2, and LCDR3 in the chain variable region are consistent amino acid sequences consistent with the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 32.
  • the CDRs are defined by Kabat, Chothia, AbM, Contact or IMGT systems.
  • the CDRs are based on the CDRs defined by the Kabat system, with the C-terminus of HCDR3 shortened by 3 amino acids, and the C-terminus of LCDR3 shortened by 2 amino acids.
  • the amino acid sequence of HCDR1 includes SEQ ID NO:25 or is as shown in SEQ ID NO:25; the amino acid sequence of HCDR2 includes SEQ ID NO:26 or 37, or is as shown in SEQ ID NO : as shown in 26 or 37; the amino acid sequence of the HCDR3 includes SEQ ID NO:27 or as shown in SEQ ID NO:27; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 sequentially include SEQ ID NO:28 ⁇ 30 or As shown in sequence SEQ ID NO:28 ⁇ 30.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof.
  • the antibody or an antigen-binding fragment thereof includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3.
  • the amino acid sequence of HCDR1 includes SEQ ID NO: 25 or as shown in SEQ ID NO: 25;
  • the amino acid sequence of the HCDR2 includes SEQ ID NO: 26 or 37, or as shown in SEQ ID NO: 26 or 37;
  • the amino acid sequence of the HCDR3 includes SEQ ID NO :27 or as shown in SEQ ID NO:27;
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 sequentially include SEQ ID NO:28 ⁇ 30 or as shown in SEQ ID NO:28 ⁇ 30.
  • the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively correspond to H31 ⁇ H35, H50 ⁇ H65, H95 ⁇ H100A, L24 ⁇ 34, L50 ⁇ 56, and L89 ⁇ 95 under the Kabat number The amino acid of the position segment.
  • the antibody or antigen-binding fragment thereof further includes a framework region.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes the HCDR1 described in any of the above embodiments.
  • ⁇ HCDR3 and the light chain variable region include LCDR1 ⁇ LCDR3 described in any of the above embodiments;
  • the heavy chain variable region is as shown in an amino acid sequence having at least 80% identity with SEQ ID NO: 31 or 38 and/or the light chain variable region is represented by an amino acid sequence having at least 80% identity with SEQ ID NO:32.
  • the heavy chain variable region is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% similar to SEQ ID NO:31 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity as shown in the amino acid sequence, or as shown in SEQ ID NO.
  • :38 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or 100% identity of the amino acid sequence shown; the light chain variable region has at least 80%, 81%, 82%, 83% identity with SEQ ID NO:32 %,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical ammonia The amino acid sequence is shown.
  • the antibody or antigen-binding fragment thereof described in the thirteenth, fourteenth or fifteenth aspect above includes a heavy chain variable region and a light chain variable region, and the heavy chain can
  • the variable region is as shown in SEQ ID NO:31 or 38; the light chain variable region is as shown in SEQ ID NO:32.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, the heavy chain variable region being such as SEQ ID NO: 31 or SEQ ID NO SEQ ID NO: 32; the light chain variable region is shown in SEQ ID NO: 32.
  • the antibody or antigen-binding fragment thereof further comprises a constant region.
  • the constant region includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from kappa Type or lambda light chain constant region.
  • the species source of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose Any kind of person.
  • the heavy chain constant region is as set forth in SEQ ID NO: 33 or an amino acid sequence having at least 80% identity thereto.
  • the light chain constant region is as set forth in SEQ ID NO: 34 or an amino acid sequence having at least 80% identity thereto.
  • "having at least 80% identity” may mean having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, including a heavy chain and a light chain.
  • the heavy chain is as shown in SEQ ID NO: 35 or 39
  • the light chain is as shown in SEQ ID NO: 35 or 39. :36.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, which binds to the monkeypox virus antigen protein amino acid with the antibody or antigen-binding fragment thereof described in any of the above embodiments.
  • the same epitope in the sequence; or, the antibody or antigen-binding fragment thereof competitively binds to the monkeypox virus antigen with the antibody or antigen-binding fragment thereof described in any one of the above.
  • the same epitope is located in amino acid sequence 1 to 110 of monkeypox virus A29L protein; or, the antibody or antigen-binding fragment thereof is identical to the antibody or antigen thereof described in any of the above embodiments.
  • the binding fragment competitively binds to monkeypox virus A29L protein.
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 7.69 ⁇ 10 ⁇ 8 M.
  • the antibody or antigen-binding fragment thereof binds to a monkeypox virus antigen with an affinity of KD ⁇ 10-08M , KD ⁇ 10-09M , KD ⁇ 10-10M , KD ⁇ 10-11M or KD ⁇ 10-12M .
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 2.27 ⁇ 10 ⁇ 0 9 M.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1/HCDR2/HCDR3 combination is
  • the HCDR1/HCDR2/HCDR3 combination contained in the heavy chain variable region shown in any one of SEQ ID NO:46 to 49 is the same, and the LCDR1/LCDR2/LCDR3 combination is the same as the LCDR1 contained in the light chain variable region shown in SEQ ID NO:50 /LCDR2/LCDR3 combination is the same.
  • the CDRs may refer to CDRs defined by any method known in the art.
  • the CDRs are defined by Kabat, Chothia, IMGT, AbM, Contact or Lesk systems.
  • the antibody or antigen-binding fragment thereof includes:
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 include SEQ ID Nos: 40 ⁇ 42 or as shown in SEQ ID Nos: 40 ⁇ 42; the amino acid sequences of LCDR1, LCDR2 and LCDR3 include SEQ ID Nos: 43 ⁇ 45. Or as shown in SEQ ID No: 43 ⁇ 45.
  • the CDRs are based on the CDRs defined by the Kabat system, with the C-terminus of HCDR3 shortened by 3 amino acids and the C-terminus of LCDR3 shortened by 2 amino acids.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising: (a) the amino acid sequences of HCDR1, HCDR2 and HCDR3 sequentially comprise SEQ ID Nos: 40-42 Or as shown in SEQ ID No: 40 ⁇ 42 in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 include SEQ ID No: 43 ⁇ 45 in sequence or as shown in SEQ ID No: 43 ⁇ 45 in sequence.
  • the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively correspond to H31 ⁇ H35, H50 ⁇ H65, H95 ⁇ H100B, L24 ⁇ 34, L50 ⁇ 56, and L89 ⁇ 95 under the Kabat number The amino acid of the position segment.
  • the antibody or antigen-binding fragment thereof further includes a framework region.
  • the antibody or antigen-binding fragment thereof described in the nineteenth and twentieth aspects above includes:
  • Heavy chain variable region and light chain variable region includes the amino acid sequence shown in any one of SEQ ID NO: 46 to 49, or the amino acid sequence shown in any one of SEQ ID NO: 46 to 49 Amino acid sequence composition; the light chain variable region includes or consists of the amino acid sequence shown in SEQ ID NO:50.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising: (b) a heavy chain variable region and a light chain variable region; a heavy chain variable region; The region includes or consists of the amino acid sequence shown in any one of SEQ ID NO: 46 to 49; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 50 sequence, or consisting of the amino acid sequence shown in SEQ ID NO:50.
  • the antibody or functional fragment thereof includes:
  • a heavy chain variable region or/and light chain variable region whose amino acid sequence is at least 80% identical to the heavy chain variable region or/and light chain variable region sequence shown in (b), and includes (a) ) HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequences shown.
  • the antibody or antigen-binding fragment thereof includes the heavy chain framework region in the heavy chain variable region shown in any one of SEQ ID NO: 46 to 49, and the heavy chain framework region shown in SEQ ID NO: 50 The light chain framework region in the light chain variable region.
  • the amino acid sequence of the framework region of the antibody or antigen-binding fragment thereof may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% identical to the above-mentioned framework region. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the antibody or antigen-binding fragment thereof further comprises a constant region.
  • the constant region includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from kappa Type or lambda light chain constant region.
  • the species source of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose Any kind of person.
  • the heavy chain constant region includes SEQ ID NO: 51 or an amino acid sequence that is at least 80% identical thereto.
  • the heavy chain constant region consists of SEQ ID NO: 51 or an amino acid sequence having at least 80% identity thereto.
  • the light chain constant region includes SEQ ID NO: 52 or an amino acid sequence having at least 80% identity thereto.
  • the light chain constant region consists of SEQ ID NO: 52 or an amino acid sequence having at least 80% identity thereto.
  • the antibody or antigen-binding fragment thereof includes a heavy chain and a light chain;
  • the heavy chain includes an amino acid shown in any one of SEQ ID NO: 53 to 56 or having at least 80% identity with it. sequence; or, consisting of an amino acid sequence shown in any one of SEQ ID NO: 53 to 56 or having at least 80% identity with it;
  • the light chain includes SEQ ID NO: 57 or having at least 80% identity with it Amino acid sequence; or, consisting of SEQ ID NO: 57 or an amino acid sequence having at least 80% identity thereto.
  • the above-mentioned "at least 80% identity” can mean at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, and the variant site of identity is not in the CDRs region.
  • embodiments of the present disclosure also provide an antibody or an antigen-binding fragment thereof, the binding epitope of the antibody or an antigen-binding fragment thereof binds to any of the above-mentioned antibodies or an antigen-binding fragment thereof.
  • the epitopes are the same; or, the antibody or antigen-binding fragment thereof competitively binds to the same epitope as the antibody or antigen-binding fragment thereof described in any one of the above.
  • the same epitope is located in amino acid sequence 1 to 110 of monkeypox virus A29L protein; or, the antibody or antigen-binding fragment thereof is identical to any of the above-mentioned antibodies or antigens thereof.
  • the binding fragment competitively binds to monkeypox virus A29L protein.
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 7.36 ⁇ 10 ⁇ 8 M.
  • the antibody or antigen-binding fragment thereof has a KD ⁇ 10 -8 M, KD ⁇ 10 -9 M, KD ⁇ 10 -10 M, KD ⁇ 10 -11 M, and KD ⁇ 10 -12
  • the affinity of M binds monkeypox virus antigen.
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 1.78 ⁇ 10 ⁇ 9 M.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1/HCDR2/HCDR3 combination
  • the HCDR1/HCDR2/LCDR3 combination is the same as the heavy chain variable region shown in any one of SEQ ID NO:80 to SEQ ID NO:87
  • the LCDR1/LCDR2/LCDR3 combination is the same as SEQ ID NO:88 to SEQ ID NO: 91 Any of the light chain variable regions shown contains the same LCDR1/LCDR2/LCDR3 combination.
  • the CDRs may refer to CDRs defined by any method known in the art.
  • the CDRs are defined by Kabat, Chothia, IMGT, AbM, Contact or Lesk systems.
  • the CDRs are based on the CDRs defined by the Kabat system, with the N-terminus of HCDR3 extended by 1 amino acid and the C-terminus shortened by 2 amino acids; the C-terminus of LCDR3 is shortened by 2 amino acids.
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 58; the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 59 or 60; the HCDR3 includes the amino acid sequence shown in SEQ ID NO : the amino acid sequence shown in 104; the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 61; the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 62, 63, 64 or 65; the LCDR3 includes The amino acid sequence shown in SEQ ID NO:66.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, the HCDR1 comprising SEQ ID NO.
  • the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively correspond to Kabat The amino acids in the H31 ⁇ H35, H50 ⁇ H65, H94 ⁇ H96, L24 ⁇ 34, L50 ⁇ 56, L89 ⁇ 95 position segments under the numbering.
  • the amino acid sequence of HCDR1 in the body or antigen-binding fragment thereof in the twenty-third or twenty-fourth aspect is shown in SEQ ID NO: 58; the amino acid sequence of HCDR2 is shown in SEQ ID NO:59 or 60; the amino acid sequence of HCDR3 is shown in SEQ ID NO:104; the amino acid sequence of LCDR1 is shown in SEQ ID NO:61; the amino acid sequence of LCDR2 is shown in SEQ ID NO: 62, 63, 64 or 65; the amino acid sequence of LCDR3 is shown in SEQ ID NO: 66.
  • the amino acid sequence of HCDR1 in the body or antigen-binding fragment thereof in the twenty-third or twenty-fourth aspect is shown in SEQ ID NO: 58; the amino acid sequence of HCDR2 is shown in SEQ ID NO:59; the amino acid sequence of HCDR3 is shown in SEQ ID NO:104; the amino acid sequence of LCDR1 is shown in SEQ ID NO:61; the amino acid sequence of LCDR2 is shown in SEQ ID NO:62, 63, 64 or 65; the amino acid sequence of LCDR3 is shown in SEQ ID NO: 66.
  • the amino acid sequence of HCDR1 in the body or antigen-binding fragment thereof in the twenty-third or twenty-fourth aspect is shown in SEQ ID NO: 58; the amino acid sequence of HCDR2 is shown in SEQ ID NO:60; the amino acid sequence of HCDR3 is shown in SEQ ID NO:104; the amino acid sequence of LCDR1 is shown in SEQ ID NO:4; the amino acid sequence of LCDR2 is shown in SEQ ID NO:62, 63, 64 or 65; the amino acid sequence of LCDR3 is shown in SEQ ID NO: 66.
  • the antibody or antigen-binding fragment thereof also includes HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.
  • the HFR1 to HFR4 sequentially comprise SEQ ID NO: 67 to SEQ ID NO: 70 or an amino acid sequence having at least 80% identity thereto.
  • the HFR1 to HFR4 are shown in sequence as SEQ ID NO: 67 to SEQ ID NO: 70 or an amino acid sequence having at least 80% identity thereto.
  • the LFR1 to LFR4 sequentially comprise SEQ ID NO:76 to SEQ ID NO:79 or an amino acid sequence having at least 80% identity thereto.
  • the LFR1 to LFR4 are shown in sequence as SEQ ID NO: 76 to SEQ ID NO: 79 or an amino acid sequence having at least 80% identity thereto.
  • the amino acid sequence of the framework region of the antibody or antigen-binding fragment thereof can be the same as the above-mentioned framework region (SEQ ID NO: 67 to SEQ ID NO: 70 or SEQ ID NO: 76 to SEQ ID NO: 79 ) have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% identity.
  • amino acid sequence of HFR1 includes SEQ ID NO:71, or is as shown in SEQ ID NO:71.
  • the amino acid sequence of HFR2 includes SEQ ID NO: 72, 73 or 74, or is as shown in SEQ ID NO: 72, 73 or 74.
  • amino acid sequence of HFR3 includes SEQ ID NO:75, or is as shown in SEQ ID NO:75.
  • amino acid sequence of LFR1 includes SEQ ID NO:100, or is as shown in SEQ ID NO:100.
  • the amino acid sequence of LFR3 includes SEQ ID NO: 101, 102 or 103, or is as shown in SEQ ID NO: 101, 102 or 103.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising
  • the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 the light chain variable region contains the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, the HCDR1, HCDR2, HCDR3,
  • the amino acid sequences of LCDR1, LCDR2, and LCDR3 are the above-mentioned amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • amino acid sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are the above-mentioned amino acid sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.
  • the heavy chain variable region includes an amino acid sequence shown in any one of SEQ ID NOs: 80 to 87 or having at least 80% identity thereto.
  • the heavy chain variable region consists of an amino acid sequence shown in any one of SEQ ID NOs: 80 to 87 or having at least 80% identity thereto.
  • the light chain variable region includes an amino acid sequence shown in any one of SEQ ID NOs: 88 to 91 or having at least 80% identity thereto.
  • the light chain variable region consists of an amino acid sequence shown in any one of SEQ ID NOs: 88 to 91 or having at least 80% identity thereto.
  • variable region sequence of the antibody or antigen-binding fragment thereof may have at least 80%, 81% similarity with the above variable region (SEQ ID NO: 80 to 87 or SEQ ID NO: 88 to 91). %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the variant amino acid positions of the identity are not located in the CDRs region.
  • the heavy chain variable region is composed of the amino acid sequence shown in any one of SEQ ID NO: 80 to 87; the light chain variable region is composed of any one of SEQ ID NO: 88 to 91.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region consisting of It consists of the amino acid sequence shown in any one of SEQ ID NO: 80 to 87; the light chain variable region consists of the amino acid sequence shown in any one of SEQ ID NO: 88 to 91.
  • the heavy chain variable region in the antibody or antigen-binding fragment thereof described in the twenty-fifth or twenty-sixth aspect above consists of the amino acid sequence shown in any one of SEQ ID NO: 80 to 87 Composed; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:88.
  • the heavy chain variable region in the antibody or antigen-binding fragment thereof described in the twenty-fifth or twenty-sixth aspect above consists of the amino acid sequence shown in any one of SEQ ID NO: 80 to 87 Composed; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:89.
  • the heavy chain variable region in the antibody or antigen-binding fragment thereof described in the twenty-fifth or twenty-sixth aspect above consists of the amino acid sequence shown in any one of SEQ ID NO: 80 to 87 Composed; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:90.
  • the heavy chain variable region in the antibody or antigen-binding fragment thereof described in the twenty-fifth or twenty-sixth aspect above consists of the amino acid sequence shown in any one of SEQ ID NO: 80 to 87 Composed; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:91.
  • the antibody or antigen-binding fragment thereof further comprises a constant region.
  • the constant region includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from kappa Type or lambda light chain constant region.
  • the species source of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose Any kind of person.
  • the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 92 or has at least 80% identity thereto.
  • the heavy chain constant region consists of an amino acid sequence set forth in SEQ ID NO: 92 or having at least 80% identity thereto.
  • the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 93 or has at least 80% identity thereto.
  • the light chain constant region consists of an amino acid sequence set forth in SEQ ID NO: 93 or having at least 80% identity thereto.
  • the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% identical to the above constant region (SEQ ID NO: 92 or 93) , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the antibody or antigen-binding fragment thereof includes a heavy chain and a light chain.
  • the heavy chain includes an amino acid sequence shown in any one of SEQ ID NOs: 94 to 96 or having at least 80% identity thereto.
  • the heavy chain consists of an amino acid sequence shown in any one of SEQ ID NOs: 94 to 96 or having at least 80% identity thereto.
  • the light chain includes an amino acid sequence shown in any one of SEQ ID NOs: 97 to 99 or having at least 80% identity thereto.
  • the light chain consists of an amino acid sequence shown in any one of SEQ ID NOs: 97 to 99 or having at least 80% identity thereto.
  • the heavy chain or light chain sequence of the antibody or antigen-binding fragment thereof may have the same sequence as the heavy chain or light chain sequence described above (SEQ ID NOs: 94 to 96 or SEQ ID NOs: 97 to 99). At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identity.
  • the variant amino acid positions of the identity are not located in the CDRs region.
  • the heavy chain consists of the amino acid sequence shown in any one of SEQ ID NO: 94 to 96; the light chain consists of the amino acid sequence shown in any one of SEQ ID NO: 97 to 99.
  • embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof, the binding epitope of the antibody or the antigen-binding fragment thereof and the binding epitope of any of the above-mentioned antibodies or the antigen-binding fragment thereof.
  • the same epitope; or, the antibody or antigen-binding fragment thereof competitively binds to the same epitope as the antibody or antigen-binding fragment thereof described in any one of the above.
  • the same epitope is located in amino acid sequence 1 to 110 of monkeypox virus A29L protein; or, the antibody or antigen-binding fragment thereof is identical to the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.
  • the antibody or antigen-binding fragment thereof competitively binds to the monkeypox virus A29L protein.
  • the antibody or antigen-binding fragment thereof binds to a unique epitope of monkeypox virus A29L protein (MKPVA29L) but does not bind to monkeypox virus A29L protein, vaccinia virus 162 protein (CPXV162), and vaccinia virus A27L Common epitope of protein (VACA27L).
  • MKPVA29L monkeypox virus A29L protein
  • CPXV162 vaccinia virus 162 protein
  • VACA27L Common epitope of protein
  • the antibody or antigen-binding fragment thereof binds to the amino acid region within amino acid sequence 17 to 49 of the monkeypox virus A29L protein.
  • the antibody or antigen-binding fragment thereof specifically binds to monkeypox virus or monkeypox virus A29L protein (MKPVA29L) and is incompatible with vaccinia virus 162 protein (CPXV162) or vaccinia virus A27L protein (VACA27L). Cross-reactivity exists.
  • MKPVA29L monkeypox virus or monkeypox virus A29L protein
  • CPXV162 protein vaccinia virus 162 protein
  • VACA27L vaccinia virus A27L protein
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 7.91 ⁇ 10 ⁇ 8 M.
  • the antibody or antigen-binding fragment thereof has a KD ⁇ 10 -08 M, a KD ⁇ 10 -09 M, a KD ⁇ 10 -10 M, a KD ⁇ 10 -11 M or a KD ⁇ 10 -12
  • the affinity of M binds monkeypox virus antigen.
  • the antibody or antigen-binding fragment thereof binds monkeypox virus antigen with an affinity of KD ⁇ 1.47 ⁇ 10 -10 M or KD ⁇ 9.96 ⁇ 10 -10 M.
  • thermodynamic detection methods are common such as isothermal titration calorimetry (ITC); kinetic detection methods are common such as surface plasmon resonance (SPR) and biofilm optical interference (BLI); dynamic equilibrium detection methods are common such as enzyme-linked immunosorbent assay (ELISA) )wait.
  • ITC isothermal titration calorimetry
  • SPR surface plasmon resonance
  • BLI biofilm optical interference
  • ELISA enzyme-linked immunosorbent assay
  • KD is measured using a kinetic detection method; preferably, a surface plasmon resonance method, for example, by using a method such as Systematic biosensor system.
  • the antigen-binding fragment includes a heavy chain variable region and a light chain variable region
  • the heavy chain variable region includes HCDR1 to HCDR3 and the light chain described in any of the above embodiments.
  • the variable region includes LCDR1 to LCDR3 described in any of the above embodiments.
  • the antigen-binding fragment is selected from any one of F(ab') 2 , Fab', Fab, Fv and scFv of the antibody.
  • Antigen-binding fragments of the above antibodies generally have the same binding specificity as the antibody from which they are derived.
  • antigen-binding fragment and “functional fragment” having the same binding specificity as the antibody from which it is derived are used interchangeably.
  • the antigen-binding fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or methods of cleaving disulfide bonds through chemical reduction.
  • enzymatic digestion including pepsin or papain
  • cleaving disulfide bonds through chemical reduction.
  • those skilled in the art can easily obtain the above-mentioned antigen-binding fragments.
  • Antigen-binding fragments of the above-mentioned antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art, or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems.
  • embodiments of the present disclosure also provide an antibody conjugate, which includes the antibody or antigen-binding fragment thereof as described in the previous embodiment.
  • the antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.
  • the antibody conjugate further includes a label coupled to the antibody or antigen-binding fragment thereof.
  • the above-mentioned marker refers to a type of substance that has characteristics that can be directly observed by the naked eye or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which the corresponding target can be achieved. Qualitative or quantitative detection of substances.
  • the label is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.
  • the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, include but are not limited to fluorescein isothiocyanate (FITC), hydroxyl fluorescein (FAM), tetrachlorofluorescein). (TET), etc. or their analogs), rhodamine dyes and their derivatives (including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs ), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc.
  • fluorescein dyes and their derivatives for example, include but are not limited to fluorescein isothiocyanate (FITC), hydroxyl fluorescein (FAM), tetrachlorofluorescein). (TET), etc. or their analogs
  • Alexa series dyes and their derivatives for example, including But not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs
  • protein dyes and their derivatives for example, including but Not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinophycoxanthin-chlorophyll protein (preCP), etc.
  • the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate Deoxygenase.
  • the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.
  • the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, crustacean fluorescein and its derivatives, ruthenium bipyridyl and its derivatives, acridinium esters and its derivatives. Derivatives, dioxetane and its derivatives, lopranine and its derivatives and peroxyoxalate and its derivatives.
  • the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.
  • the colloids include, but are not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.
  • the colloidal metal includes, but is not limited to, colloidal gold or colloidal silver.
  • the colloidal metal is colloidal gold.
  • the antibody conjugate further includes a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.
  • the antibody is coupled to a solid support.
  • the solid support is selected from microspheres, plates and membranes.
  • the solid phase includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, wool Straw tubes, nylon and nitrocellulose membranes.
  • the solid phase carrier is a nitrocellulose membrane.
  • embodiments of the present disclosure also provide a reagent or kit, which includes the antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments or the antibody conjugate as described in any of the foregoing embodiments.
  • the reagent or kit further includes a second antibody that binds to the antibody or antigen-binding fragment thereof;
  • the reagent or kit further includes a second antibody that binds to monkeypox virus or monkeypox virus antigen.
  • the reagent or kit has improved detection sensitivity or specificity.
  • embodiments of the present disclosure also provide a method for detecting monkeypox virus or monkeypox virus antigen, which includes:
  • the antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments or the antibody conjugate as described in any of the foregoing embodiments or the reagent or kit as described in any of the foregoing embodiments is combined with the monkeypox virus in the sample to be detected or Monkeypox virus antigens come into contact and form immune complexes.
  • the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment thereof.
  • the immune complex further includes a second antibody that binds to monkeypox virus or monkeypox virus antigen.
  • the embodiments of the present disclosure also provide the antibody or antigen-binding fragment described in any of the preceding embodiments, the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in detecting monkeys.
  • the embodiments of the present disclosure also provide the antibodies or antigen-binding fragments described in any of the preceding embodiments, the antibody conjugates described in any of the preceding embodiments, or the reagents or kits described in any of the preceding embodiments.
  • the related diseases caused by monkeypox virus infection include at least one of fever, headache, lymphadenopathy, muscle aches, severe fatigue, pneumonia, sepsis and encephalitis.
  • the product includes a reagent or kit.
  • embodiments of the present disclosure also provide an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof described in any of the preceding embodiments.
  • embodiments of the present disclosure also provide a vector containing the isolated nucleic acid described in any of the preceding embodiments.
  • embodiments of the present disclosure also provide a cell containing the isolated nucleic acid described in any of the foregoing embodiments or the vector as described in any of the foregoing embodiments.
  • embodiments of the present disclosure also provide a method of preparing the antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, which includes: culturing the cells as described in any of the preceding embodiments.
  • the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by a gene sequencing company.
  • the hybridoma cell line secreting Anti-MPXV 4G21 monoclonal antibody is a hybridoma cell line prepared in our laboratory and is recovered and used for later use.
  • Extract mRNA from the hybridoma cell line secreting Anti-MPXV 4G21 monoclonal antibody obtain the DNA product by RT-PCR method, insert it into the pMD-18T vector, transform it into DH5 ⁇ competent cells, and grow the colony Take 4 positive clones of Heavy Chain and Light Chain genes and send them to a gene sequencing company for sequencing.
  • VNL gene sequence is 321bp, preceded by a 57bp leader peptide sequence
  • VH gene sequence is 363bp, belonging to the VH1 gene family, preceded by a 57bp leader peptide sequence.
  • the vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been modified to introduce a polyclonal enzyme cutting site, and is subsequently referred to as the 3.4A expression vector. Based on the above-mentioned sequencing results of the antibody variable region gene in pMD-18T, Anti-MPXV 4G21 was designed The VL and VH gene-specific primers of the antibody have restriction endonuclease cleavage sites and protective bases at both ends respectively. The 0.73KB Light Chain gene fragment and the 1.42KB Heavy Chain gene are amplified through PCR amplification method. fragment.
  • the Heavy Chain and Light Chain gene fragments were double digested with restriction endonucleases respectively, and the 3.4A vector was double digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain genes and the Light Chain genes were connected to 3.4 respectively.
  • expression vector A the recombinant expression plasmids of Heavy Chain and Light Chain were obtained respectively.
  • HEK293 cells Resuscitate HEK293 cells in advance, subculture to a 200ml system, so that the cell density reaches 3 to 5 ⁇ 10 6 cells/ml, and the cell viability is >95%; centrifuge and wash the cells, redissolve them in culture medium, and adjust the cell density to 2.9 ⁇ 10 6 cells/ml as cell diluent.
  • the cells were cultured in a 35°C constant-temperature incubator with a rotation speed of 120 rpm and a CO2 content of 8%. After 13 days, the samples were collected by centrifugation. The centrifugation supernatant was affinity purified using a protein A affinity chromatography column. Take 6 ⁇ g of the purified antibody and perform reducing SDS-PAGE. The electrophoresis pattern is shown in Figure 1. After reducing SDS-PAGE, two bands were displayed, one Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).
  • the Anti-MPXV 4G21 monoclonal antibody prepared above has the ability to bind to monkeypox virus antigens, its affinity and antibody activity are not ideal. Therefore, the applicant conducted directed mutations on the variable region of the antibody. That is, computer simulation of the antibody variable region structure, simulation of the structure of the antigen-antibody variable region interaction complex, analysis of the key amino acids of the antibody and mutation design were performed. Bidirectional primers covering the mutation site were designed and synthesized according to the mutation scheme, primers at both ends of the target DNA were synthesized, high-fidelity PCR reactions were performed, and the PCR products were cloned into the vector. The mutant antibody was then prepared according to the method of step 2 above. After screening, a monoclonal antibody with significantly improved affinity and antibody activity was obtained and named Anti-MPXV 4G21RMb1.
  • the amino acid sequences of its heavy chain and light chain are as follows.
  • the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by a gene sequencing company.
  • the hybridoma cell line secreting Anti-MPXV 2F19 monoclonal antibody is a hybridoma cell line prepared in our laboratory and is recovered and used for later use.
  • Extract mRNA from the hybridoma cell line secreting Anti-MPXV 2F19 monoclonal antibody obtain the DNA product through RT-PCR, insert it into the pMD-18T vector, transform it into DH5 ⁇ competent cells, and grow the colony Take 4 positive clones of Heavy Chain and Light Chain genes and send them to a gene sequencing company for sequencing.
  • VNL gene sequence 339bp, preceded by a 57bp leader peptide sequence
  • VH gene sequence 360bp, belonging to the VH1 gene family, preceded by a 57bp leader peptide sequence.
  • the vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been modified to introduce a polyclonal enzyme cutting site, and is subsequently referred to as the 3.4A expression vector. Based on the above-mentioned sequencing results of the antibody variable region gene in pMD-18T, Anti-MPXV 2F19 was designed The VL and VH gene-specific primers of the antibody have restriction enzyme cleavage sites and protective bases at both ends respectively. The 0.74KB Light Chain gene fragment and the 1.42KB Heavy Chain gene are amplified through PCR amplification method. fragment.
  • the Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was double-digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.
  • HEK293 cells Resuscitate HEK293 cells in advance, subculture to a 200ml system, so that the cell density reaches 3 to 5 ⁇ 10 6 cells/ml, and the cell viability is >95%; centrifuge and wash the cells, redissolve them in culture medium, and adjust the cell density to 2.9 ⁇ 10 6 cells/ml as cell diluent.
  • the cells were cultured in a 35°C constant-temperature incubator with a rotation speed of 120 rpm and a CO2 content of 8%. After 13 days, the samples were collected by centrifugation. The centrifugation supernatant was affinity purified using a protein A affinity chromatography column. Take 6 ⁇ g of the purified antibody and perform reducing SDS-PAGE. The electrophoresis pattern is shown in Figure 2. After reducing SDS-PAGE, two bands were shown, one Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).
  • the Anti-MPXV 2F19 monoclonal antibody prepared above has the ability to bind monkeypox virus antigen, its affinity and antibody activity are not ideal, so the applicant carried out directed mutation of the variable region of the antibody. That is, computers are used to simulate the structure of the antibody variable region, the structure of the interaction complex between the antigen and the antibody variable region, analysis of the key amino acids of the antibody, and mutation design. According to the mutation plan, bidirectional primers covering the mutation site are designed and synthesized, and primers at both ends of the target DNA are synthesized. , perform a high-fidelity PCR reaction, clone the PCR product into the vector, and then prepare the mutant antibody according to the method in step 2 above. After screening, a monoclonal antibody with significantly improved affinity and antibody activity was obtained and named Anti-MPXV 2F19RMb1. Its heavy chain and light chain amino acid sequences are as follows.
  • the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by a gene sequencing company.
  • the hybridoma cell line secreting Anti-MPXV 17A16 monoclonal antibody is a hybridoma cell line prepared in our laboratory, which is recovered and used for later use.
  • Extract mRNA from the hybridoma cell line secreting Anti-MPXV 17A16 monoclonal antibody obtain the DNA product by RT-PCR method, insert it into the pMD-18T vector, transform it into DH5 ⁇ competent cells, and grow the colony Take 4 positive clones of Heavy Chain and Light Chain genes and send them to a gene sequencing company for sequencing.
  • VNL gene sequence is 321bp, preceded by a 57bp leader peptide sequence
  • VH gene sequence is 357bp, belonging to the VH1 gene family, preceded by a 57bp leader peptide sequence.
  • pcDNA TM 3.4 vector is a constructed recombinant antibody eukaryotic expression vector that has been modified to introduce polyclonal enzyme cutting sites point, which will be referred to as the 3.4A expression vector in the future; based on the above sequencing results of the antibody variable region gene in pMD-18T, VL and VH gene-specific primers for the Anti-MPXV 17A16 antibody were designed, with restriction endonuclease digestion at both ends. Site and protective base, a 0.74KB Light Chain gene fragment and a 1.41kB Heavy Chain gene fragment were amplified through PCR amplification method.
  • the Heavy Chain and Light Chain gene fragments were double digested with restriction endonucleases respectively, and the 3.4A vector was double digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain genes and the Light Chain genes were connected to 3.4 respectively.
  • expression vector A the recombinant expression plasmids of Heavy Chain and Light Chain were obtained respectively.
  • HEK293 cells Resuscitate HEK293 cells in advance, subculture to a 200ml system, so that the cell density reaches 3 to 5 ⁇ 10 6 cells/ml, and the cell viability is >95%; centrifuge and wash the cells, redissolve them in culture medium, and adjust the cell density to 2.9 ⁇ 10 6 cells/ml as cell diluent.
  • the cells were cultured in a 35°C constant-temperature incubator with a rotation speed of 120 rpm and a CO2 content of 8%. After 13 days, the samples were collected by centrifugation. The centrifugation supernatant was affinity purified using a protein A affinity chromatography column. Take 6 ⁇ g of the purified antibody and perform reducing SDS-PAGE. The electrophoresis pattern is shown in Figure 3. After reducing SDS-PAGE, two bands were shown, one Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).
  • the Anti-MPXV 17A16 monoclonal antibody prepared above has the ability to bind to the monkeypox virus antigen, the affinity and antibody activity are not ideal, so the applicant carried out targeted mutation of the variable region of the antibody. That is, computers are used to simulate the structure of the antibody variable region, the structure of the interaction complex between the antigen and the antibody variable region, analysis of the key amino acids of the antibody, and mutation design. According to the mutation plan, bidirectional primers covering the mutation site are designed and synthesized, and primers at both ends of the target DNA are synthesized. , perform a high-fidelity PCR reaction, clone the PCR product into the vector, and then prepare the mutant antibody according to the method in step 2 above. After screening, monoclonal antibodies with significantly improved affinity and antibody activity were obtained and named Anti-MPXV 17A16RMb1 and Anti-MPXV 17A3RMb1. Their heavy chain and light chain amino acid sequences are as follows respectively.
  • the reactivity of the above-prepared antibody with monkeypox virus A29L protein was identified by ELISA.
  • the ELISA operation is as follows: coat the microtiter plate with 0.5 ⁇ g/ml MKPVA29L at 100 ul/well, and incubate at 37°C for 2 hours. BSA closed. Dilute the antibodies prepared above with PBS to 0.05ug/ml, 50ul/well, and incubate at 37°C for 30 minutes.
  • Negative control add PBS and incubate.
  • Color development Add 50ul of solution A and B each, and after 10 minutes, add 50ul of stop solution and read the value.
  • the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by a gene sequencing company.
  • the hybridoma cell line secreting Anti-MPXV 11D3 monoclonal antibody is a hybridoma cell line prepared in our laboratory, which is recovered and used for later use.
  • restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by a gene sequencing company.
  • Extract mRNA from the hybridoma cell line secreting Anti-MPXV 11D3 monoclonal antibody obtain the DNA product through RT-PCR, insert it into the pMD-18T vector, transform it into DH5 ⁇ competent cells, and grow the colony Take 4 positive clones of Heavy Chain and Light Chain genes and send them to a gene sequencing company for sequencing.
  • VNL gene sequence is 333bp, preceded by a 57bp leader peptide sequence
  • VH gene sequence is 366bp, belonging to the VH1 gene family, preceded by a 57bp leader peptide sequence.
  • the vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been modified to introduce a polyclonal enzyme cutting site, and is subsequently referred to as the 3.4A expression vector. Based on the above-mentioned sequencing results of the antibody variable region gene in pMD-18T, Anti-MPXV 11D3 was designed The VL and VH gene-specific primers of the antibody have restriction endonuclease cleavage sites and protective bases at both ends respectively. The 0.71KB Light Chain gene fragment and the 1.41KB Heavy Chain gene are amplified through PCR amplification method. fragment.
  • the Heavy Chain and Light Chain gene fragments were double digested with restriction endonucleases respectively, and the 3.4A vector was double digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain genes and the Light Chain genes were connected to 3.4 respectively.
  • expression vector A the recombinant expression plasmids of Heavy Chain and Light Chain were obtained respectively.
  • HEK293 cells Resuscitate HEK293 cells in advance, subculture to a 200ml system, so that the cell density reaches 3 to 5 ⁇ 10 6 cells/ml, and the cell viability is >95%; centrifuge and wash the cells, redissolve them in culture medium, and adjust the cell density to 2.9 ⁇ 10 6 cells/ml as cell diluent.
  • the cells were cultured in a 35°C constant-temperature incubator with a rotation speed of 120 rpm and a CO2 content of 8%. After 13 days, the samples were collected by centrifugation. The centrifugation supernatant was affinity purified using a protein A affinity chromatography column. Take 6 ⁇ g of the purified antibody and perform reducing SDS-PAGE. The electrophoresis pattern is shown in Figure 4. After reducing SDS-PAGE, two bands were shown, one Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).
  • the Anti-MPXV 11D3 monoclonal antibody prepared above has the ability to bind to the monkeypox virus antigen, its affinity and antibody activity are not ideal, so the applicant carried out targeted mutation of the variable region of the antibody. That is, computers are used to simulate the structure of the variable region of the antibody, the structure of the interaction complex between the antigen and the variable region of the antibody, analyze the key amino acids of the antibody, and design mutations. Create bidirectional primers covering the mutation site, synthesize primers at both ends of the target DNA, perform a high-fidelity PCR reaction, clone the PCR product into the vector, and then prepare the mutant antibody according to the method in 2 above. After screening, monoclonal antibodies with significantly improved affinity and antibody activity were obtained and named Anti-MPXV 11D3RMb1 to Anti-MPXV 11D3RMb4. Their heavy chain and light chain amino acid sequences are as follows.
  • the reactivity of the above-prepared antibody with monkeypox virus A29L protein was determined by ELISA.
  • the ELISA operation is as follows: coat the microtiter plate with 100ul/well of MKPVA29L at 0.5 ⁇ g/ml and 0.05 ⁇ g/ml respectively, and incubate at 37°C for 2 hours. BSA closed. Dilute the antibodies prepared above with PBS to 0.5ug/ml, 0.05ug/ml, 50ul/well, and incubate at 37°C for 30 minutes.
  • Secondary antibody 100ul/well, add goat anti-mouse IgG-HRP, and incubate at 37°C for 30 minutes.
  • the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by a gene sequencing company.
  • the hybridoma cell line secreting Anti-MPXV 6F13 monoclonal antibody is a hybridoma cell line prepared in our laboratory and is recovered and used for later use.
  • restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by a gene sequencing company.
  • Extract mRNA from the hybridoma cell line secreting Anti-MPXV 6F13 monoclonal antibody obtain the DNA product through RT-PCR, insert it into the pMD-18T vector, transform it into DH5 ⁇ competent cells, and grow the colony Take 4 positive clones of Heavy Chain and Light Chain genes and send them to a gene sequencing company for sequencing.
  • VNL gene sequence is 336bp, preceded by a 57bp leader peptide sequence
  • VH gene sequence is 339bp, belonging to the VH1 gene family, preceded by a 57bp leader peptide sequence.
  • the vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been modified to introduce a polyclonal enzyme cutting site, and is subsequently referred to as the 3.4A expression vector. Based on the above-mentioned sequencing results of the antibody variable region gene in pMD-18T, Anti-MPXV 6F13 was designed The VL and VH gene-specific primers of the antibody have restriction endonuclease cleavage sites and protective bases at both ends respectively. The 0.74KB Light Chain gene fragment and the 1.41KB Heavy Chain gene are amplified through PCR amplification method. fragment.
  • the Heavy Chain and Light Chain gene fragments were double digested with restriction endonucleases respectively, and the 3.4A vector was double digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain genes and the Light Chain genes were connected to 3.4 respectively.
  • expression vector A the recombinant expression plasmids of Heavy Chain and Light Chain were obtained respectively.
  • HEK293 cells Resuscitate HEK293 cells in advance, subculture to a 200ml system, so that the cell density reaches 3 to 5 ⁇ 10 6 cells/ml, and the cell viability is >95%; centrifuge and wash the cells, redissolve them in culture medium, and adjust the cell density to 2.9 ⁇ 10 6 cells/ml as cell diluent.
  • the cells were cultured in a 35°C constant-temperature incubator with a rotation speed of 120 rpm and a CO2 content of 8%. After 13 days, the samples were collected by centrifugation. The centrifugation supernatant was affinity purified using a protein A affinity chromatography column. Take 6 ⁇ g of the purified antibody and perform reducing SDS-PAGE. The electrophoresis pattern is shown in Figure 5. After reducing SDS-PAGE, two bands were shown, one Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).
  • the Anti-MPXV 6F13 monoclonal antibody prepared above has the ability to bind to the monkeypox virus antigen, the affinity and antibody activity are not ideal, so the applicant carried out targeted mutation of the variable region of the antibody. That is, computers are used to simulate the structure of the antibody variable region, the structure of the interaction complex between the antigen and the antibody variable region, analysis of the key amino acids of the antibody, and mutation design. According to the mutation plan, bidirectional primers covering the mutation site are designed and synthesized, and primers at both ends of the target DNA are synthesized. , perform a high-fidelity PCR reaction, clone the PCR product into the vector, and then prepare the mutant antibody according to the method in step 2 above. After screening, monoclonal antibodies with significantly improved affinity and antibody activity were obtained and named Anti-MPXV 6F13RMb1 to Anti-MPXV 6F13RMb3. Their heavy chain and light chain amino acid sequences are as follows.
  • Coating Coat the microtiter plate with 100ul/well of MKPVA29L, VCA27L, and CPXV162 at 0.5 ⁇ g/ml and 0.05 ⁇ g/ml respectively, and incubate at 37°C for 2 hours.
  • Blocking Take 120ul of BSA blocking solution and incubate at 37°C for 2 hours.
  • the above antibodies were diluted to 0.5ug/ml, 0.05ug/ml with PBS, 50ul/well, and incubated at 37°C for 30 minutes.
  • Secondary antibody 100ul/well, add goat anti-mouse IgG-HRP, and incubate at 37°C for 30 minutes.
  • amino acid sequence involved in this application is as follows:
  • the present disclosure provides anti-monkeypox virus antibodies or antigen-binding fragments thereof, reagents and kits for detecting monkeypox virus.
  • the anti-monkeypox virus antibody disclosed in the present disclosure includes a heavy chain complementarity-determining region and a light chain complementarity-determining region.
  • the antibody provides an important source of raw materials for the detection of monkeypox virus, has improved affinity or activity, and has excellent industrial practicability. .

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Abstract

一种抗猴痘病毒抗体或其抗原结合片段、检测猴痘病毒的试剂和试剂盒，涉及抗体领域。所述抗猴痘病毒抗体包括重链互补决定区和轻链互补决定区，该抗体为猴痘病毒的检测提供了重要的原料来源，具有改善的亲和力或活性。

Description

抗猴痘病毒抗体或其抗原结合片段、检测猴痘病毒的试剂和试剂盒
相关申请的交叉引用
本公开要求于2022年09月16日提交中国国家知识产权局的申请号为202211130858.0、名称为“抗猴痘病毒抗体或其功能性片段、检测猴痘病毒的试剂和试剂盒”，于2022年09月16日提交中国国家知识产权局的申请号为202211126049.2、名称为“抗猴痘病毒抗体或其功能性片段、检测猴痘病毒的试剂和试剂盒”，于2022年09月16日提交中国国家知识产权局的申请号为202211126051.X，名称为“抗猴痘病毒抗体或其功能性片段、检测猴痘病毒的试剂和试剂盒”，于2022年11月03日提交中国国家知识产权局的申请号为202211370957.6、名称为“抗猴痘病毒抗体或其功能性片段、检测猴痘病毒的试剂和试剂盒”，于2022年11月03日提交中国国家知识产权局的申请号为202211370953.8，名称为“抗猴痘病毒抗体或其功能性片段、检测猴痘病毒的试剂和试剂盒”和于2022年11月03日提交中国国家知识产权局的申请号为202211371076.6，名称为“抗猴痘病毒抗体或其功能性片段、检测猴痘病毒的试剂和试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及抗体技术领域，具体而言，涉及抗猴痘病毒抗体或其抗原结合片段、检测猴痘病毒的试剂和试剂盒。
背景技术
猴痘是由猴痘病毒(Monkeypox virus，MPXV)引起的。猴痘病毒是一种双链DNA病毒，属于痘病毒科正痘病毒属的一员。猴痘是一种病毒性人畜共患病，主要发生在中非和西非的热带雨林地区，偶尔输出到其它地区。目前研究发现，病毒经黏膜和破损的皮肤侵入人体。人主要通过接触感染动物病变渗出物、血液、其它体液，或被感染动物咬伤、抓伤而感染。人与人之间主要通过密切接触传播，也可通过飞沫传播，接触被病毒污染的物品也有可能感染，还可通过胎盘垂直传播。尚不能排除性传播。
在临床症状上，猴痘的感染症状与天花相似，但猴痘的临床症状较轻，潜伏期5-12天，多为6-13天。患者发病初期表现为发热、头痛、淋巴结肿大、肌肉酸痛、重度疲乏等症状，之后会引起面部和身体的皮疹。猴痘为自限性疾病，患者通常会在两到三个星期内自行康复；但对于儿童、孕妇或由于其他健康状况而免疫抑制的人来说，猴痘可能会导致其他继发性感染，如肺炎、败血症和脑炎等严重疾病。
目前，猴痘病毒的检测方法主要是PCR扩增法和免疫检测法，PCR扩增法是国内外检测猴痘病毒的主要检测方法，但对仪器设备、检测场地及环境条件要求高，存在检测时间长、通量低等缺点，不便于人群大规模检测。而基于胶体金的免疫检测法具有快速、方便、易携带等的优势，因此成为研发的热点，而胶体金的检测性能依赖于针对猴痘病毒的抗体的性能。
因此，本领域对于有效结合猴痘病毒并对其进行检测的抗体存在着强烈需求。
发明内容
本公开的目的在于提供抗猴痘病毒抗体或其抗原结合片段、检测猴痘病毒或猴痘病毒抗原的试剂和试剂盒。
第一方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1、HCDR2、HCDR3包括或为与SEQ ID NO:7、SEQ ID NO:19、SEQ ID NO:31、SEQ ID NO:38、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:80、SEQ ID NO:81或SEQ ID NO:82所示重链可变区的HCDR1、HCDR2、HCDR3一致的氨基酸序列；所述LCDR1、LCDR2、LCDR3包括或为与SEQ ID NO:8、SEQ ID NO:20、SEQ ID NO:32、SEQ ID NO:50、SEQ ID NO:88、SEQ ID NO:89或SEQ ID NO:90所示轻链可变区的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
可选地，所述CDRs由Kabat、Chothia、AbM、Contact或IMGT系统定义。
第二方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3的氨基酸序列选自以下任一组合：

第三方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区包括上述任一实施例所述的HCDR1～HCDR3和所述轻链可变区包括上述任一实施例所述的LCDR1～LCDR3；且所述重链可变区如与SEQ ID NO:7、SEQ ID NO:19、SEQ ID NO:31、SEQ ID NO:38、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:80、SEQ ID NO:81或SEQ ID NO:82具有至少80％同一性的氨基酸序列所示和/或所述轻链可变区如与SEQ ID NO:8、SEQ ID NO:20、SEQ ID NO:32、SEQ ID NO:50、SEQ ID NO:88、SEQ ID NO:89或SEQ ID NO:90具有至少80％同一性的氨基酸序列所示。
第四方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区如SEQ ID NO:7、SEQ ID NO:19、SEQ ID NO:31、SEQ ID NO:38、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:80、SEQ ID NO:81或SEQ ID NO:82所示，所述轻链可变区如SEQ ID NO:8、SEQ ID NO:20、SEQ ID NO:32、SEQ ID NO:50、SEQ ID NO:88、SEQ ID NO:89或SEQ ID NO:90所示。
可选地，上述第一方面、第二方面、第三方面或第四方面中的重链可变区和轻链可变区选自以下任一组合：
可选地，上述第一方面、第二方面、第三方面或第四方面所述抗体或其抗原结合片段包括以下任一组合的重链可变区和轻链可变区：
可选地，上述第一方面、第二方面、第三方面或第四方面所述抗体或其抗原结合片段还包含恒定区。
可选地，所述恒定区包括重链恒定区和/或轻链恒定区。
可选地，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中任一项的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区。
可选地，所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅和人中的任意一种。
可选地，所述重链恒定区如SEQ ID NO:9或与其具有至少80％同一性的氨基酸序列所示。
可选地，所述轻链恒定区如SEQ ID NO:10或与其具有至少80％同一性的氨基酸序列所示。
第五方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链和轻链，所述重链的氨基酸序列如SEQ ID NO:11、SEQ ID NO:23、SEQ ID NO:35、SEQ ID NO:39、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:94、SEQ ID NO:95或SEQ ID NO:96所示；所述轻链的氨基酸序列如SEQ ID NO:12、SEQ ID NO:24、SEQ ID NO:36、SEQ ID NO:57、SEQ ID NO:97、SEQ ID NO:98或SEQ ID NO:99所示。
可选地，所述重链和轻链选自以下任一组合：
第六方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段与上述任一实施例所述的抗体或其抗原结合片段结合猴痘病毒抗原蛋白氨基酸序列之中的同一表位；或，所述抗体或其抗原结合片段与上述任一实施例所述的抗体或其抗原结合片段竞争性结合猴痘病毒抗原。
可选地，所述抗原结合片段包括重链可变区和轻链可变区，所述重链可变区包括上述任一实施例所述的HCDR1～HCDR3和所述轻链可变区包括上述任一实施例所述的LCDR1～LCDR3。
可选地，所述抗原结合片段选自所述抗体的F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
第七方面，本公开实施例提供了一种抗体偶联物，其包括如前述实施例所述的抗体或其抗原结合片段。
可选地，所述抗体偶联物还包括与所述抗体或其抗原结合片段偶联的生物素或生物素衍生物。
可选地，所述抗体偶联物还包括与所述抗体或其抗原结合片段偶联的固相载体。
可选地，所述抗体偶联物还包括与所述抗体或其抗原结合片段偶联的标记物。
可选地，所述标记物选自荧光染料、酶、放射性同位素、化学发光试剂和纳米颗粒类标记物中的至少一种。
可选地，所述标记物为胶体金。
第八方面，本公开实施例提供了一种试剂或试剂盒，其包括如前述实施例所述的抗体或其抗原结合片段或如前述实施例所述的抗体偶联物。
可选地，所述试剂或试剂盒还包括第二抗体，所述第二抗体与所述抗体或其抗原结合片段结合。
可选地，所述试剂或试剂盒还包括第二抗体，所述第二抗体与猴痘病毒或猴痘病毒抗原结合。
第九方面，本公开实施例提供了一种检测猴痘病毒或猴痘病毒抗原的方法，其包括：将如前述实施例所述的抗体或其抗原结合片段、抗体偶联物或试剂或试剂盒与待测样本中的猴痘病毒或猴痘病毒抗原接触，形成免疫复合物。
可选地，所述免疫复合物还包括第二抗体，所述第二抗体与所述抗体或其抗原结合片段结合。
可选地，所述免疫复合物还包括第二抗体，所述第二抗体与猴痘病毒或猴痘病毒抗原结合。
第十方面，本公开实施例提供了一种核酸，其编码前述实施例所述的抗体或其抗原结合片段。
第十一方面，本公开实施例提供了一种载体，其含有前述实施例所述的核酸。
第十二方面，本公开实施例提供了一种细胞，其含有前述实施例所述的核酸或载体。
第十三方面，本公开实施例提供了一种制备前述实施例所述的抗体或其抗原结合片段的方法，其包括：培养前述实施例所述的细胞。
第十四方面，本公开实施例提供了如前述实施例所述的抗体或其抗原结合片段、抗体偶联物、试剂或试剂盒在检测猴痘病毒或猴痘病毒抗原或制备检测猴痘病毒或猴痘病毒抗原的产品中的应用。
本公开具有以下有益效果：
本公开公开的抗猴痘病毒抗体包括上述重链互补决定区和轻链互补决定区，该抗体为猴痘病毒或猴痘病毒抗原的检测提供了重要的原料来源，具有改善的亲和力或活性。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，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相关的附图。
图1为Anti-MPXV 4G21的还原性SDS-PAGE的结果。
图2为Anti-MPXV 2F19的还原性SDS-PAGE的结果。
图3为Anti-MPXV 17A16的还原性SDS-PAGE的结果。
图4为Anti-MPXV 11D3RMb1～4的还原性SDS-PAGE的结果。
图5为Anti-MPXV 6F13RMb1～3的还原性SDS-PAGE的结果。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
在本公开中，术语“抗体”在最广义上使用，其可以包括全长单克隆抗体，双特异性或多特异性抗体，以及嵌合抗体，只要它们展示所需的生物学活性。
在本公开中，术语“互补性决定区”、“CDR”或“CDRs”是指免疫球蛋白的重链和轻链的高度可变区，指包含一种或多种或者甚至全部的对抗体或抗原结合片段与其识别的抗原或表位的结合起作用的主要氨基酸残基的区域。在本公开具体实施方式中，CDRs是指所述抗体的重链和轻链的高度可变区。
在本公开中，重链互补决定区用HCDR表示，重链可变区中含有的3个CDR包括HCDR1、HCDR2和HCDR3；轻链互补决定区用LCDR表示，轻链可变区中含有的3个CDR包括LCDR1、LCDR2和LCDR3。
第一方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1、HCDR2、HCDR3包括或为与SEQ ID NO:7所示重链可变区的HCDR1、HCDR2、HCDR3一致的氨基酸序列；所述LCDR1、LCDR2、LCDR3包括或为与SEQ ID NO:8所示轻链可变区的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
CDRs的定义方法是本领域公知的。常见如Kabat、Chothia、AbM、Contact或IMGT等。Kabat等人是第一个为免疫球蛋白可变区提出标准化编号方案的人。在过去的几十年中，序列的积累导致了KABATMAN数据库的创建，Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。还有其他CDRs定义方法可能不严格遵循上述方法之一，但仍会与Kabat定义的CDRs的至少一部分重叠，尽管根据特定残基或残基组的预测或实验结果可能会缩短或延长它们，例如在Kabat定义的HCDR3和LCDR3的C端各缩短1～2个氨基酸。如本文所述，CDRs可以指由本领域已知的任何方法定义的CDRs，包括方法的组合。
在可选的实施方式中，所述CDRs由Kabat、Chothia、AbM、Contact或IMGT系统定义。
在可选的实施方式中，所述CDRs由在Kabat系统定义的CDRs基础上，在HCDR3的C端缩短2个氨基酸，和LCDR3的C端缩短1个氨基酸。
在可选的实施方式中，所述HCDR1、HCDR2和HCDR3的氨基酸序列依次包括SEQ ID NO:1～3或依次如SEQ ID NO:1～3所示；LCDR1、LCDR2和LCDR3的氨基酸序列依次包括SEQ ID NO:4～6或依次如SEQ ID NO:4～6所示。
第二方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1、HCDR2和HCDR3的氨基酸序列依次包括SEQ ID NO:1～3或依次如SEQ ID NO:1～3所示；LCDR1、LCDR2和LCDR3的氨基酸序列依次包括SEQ ID NO:4～6或依次如SEQ ID NO:4～6所示。
在可选的实施方式中，所述HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3分别对应Kabat编号下的H31～H35、H50～H65、H95～H100D、L24～34、L50～56、L89～96位置区段的氨基酸。
在可选的实施方式中，所述抗体或其抗原结合片段还包括框架区。
在本公开中，“框架区”或“FR”区包括重链框架区和轻链框架区，是指抗体重链可变区和轻链可变区中除CDR之外的区域；其中，重链框架区(HFR)可以被进一步细分成被CDR分隔开的毗邻区域，包含HFR1、HFR2、HFR3和HFR4框架区；轻链框架区(LFR)可以被进一步细分成被CDR分隔开的毗邻区域，包含LFR1、LFR2、LFR3和LFR4框架区。
重链可变区由HCDR与HFR按如下组合排列连接获得：HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4；轻链可变区由LCDR与LFR按如下组合排列连接获得：LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4。在本公开中，“-”表示肽键。组合片段从序列上游(N端)至下游(C端)依次排布。
第三方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区包括上述任一实施例所述的HCDR1～HCDR3和所述轻链可变区包括上述任一实施例所述的LCDR1～LCDR3；所述重链可变区如与SEQ ID NO:7具有至少80％同一性的氨基酸序列所示和/或所述轻链可变区如与SEQ ID NO:8具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述重链可变区如与SEQ ID NO:7具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％ 同一性的氨基酸序列所示；所述轻链可变区如与SEQ ID NO:8具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列所示。
在可选的实施方式中，上述第一方面、第二方面或第三方面所述抗体或其抗原结合片，包括重链可变区和轻链可变区，所述重链可变区如SEQ ID NO:7所示；所述轻链可变区如SEQ ID NO:8所示。
第四方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区如SEQ ID NO:7所示；所述轻链可变区如SEQ ID NO:8所示。
在可选的实施方式中，所述抗体或其抗原结合片段还包含恒定区。
在可选的实施方式中，所述恒定区包括重链恒定区和/或轻链恒定区。
在可选的实施方式中，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中任一项的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中，所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅和人中的任意一种。
在可选的实施方式中，所述重链恒定区如SEQ ID NO:9或与其具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述轻链恒定区如SEQ ID NO:10或与其具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述“具有至少80％的同一性”可以是具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性。
第五方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链和轻链，所述重链如SEQ ID NO:11所示，所述轻链如SEQ ID NO:12所示。
第六方面，本公开实施例还提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段与上述任一实施例所述的抗体或其抗原结合片段结合猴痘病毒抗原蛋白氨基酸序列之中的同一表位；或，所述抗体或其抗原结合片段与上述任一实施例所述的抗体或其抗原结合片段竞争性结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤6.98×10-8M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤10-08M、KD≤10-09M、KD≤10-10M、KD≤10-11M或KD≤10-12M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤2.23×10-10M的亲和力结合猴痘病毒抗原。
第七方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1、HCDR2、HCDR3包括或为与SEQ ID NO:19所示重链可变区的HCDR1、HCDR2、HCDR3一致的氨基酸序列；所述LCDR1、LCDR2、LCDR3包括或为与SEQ ID NO:20所示轻链可变区的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
在可选的实施方式中，所述CDRs由Kabat、Chothia、AbM、Contact或IMGT系统定义。
在可选的实施方式中，所述CDRs由在Kabat系统定义的CDRs基础上，在HCDR3和LCDR3的C端各缩短2个氨基酸。
在可选的实施方式中，所述HCDR1、HCDR2和HCDR3的氨基酸序列依次包括SEQ ID NO:13～15或依次如SEQ ID NO:13～15所示；LCDR1、LCDR2和LCDR3的氨基酸序列依次包括SEQ ID NO:16～18或依次如SEQ ID NO:16～18所示。
第八方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1、HCDR2和HCDR3的氨基酸序列依次包括SEQ ID NO:13～15或依次如SEQ ID NO:13～15所示；LCDR1、LCDR2和LCDR3的氨基酸序列依次包括SEQ ID NO:16～18或依次如SEQ ID NO:16～18所示。
在可选的实施方式中，所述HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3分别对应Kabat编号下的H31～H35、H50～H65、H95～H100C、L24～34、L50～56、L89～95位置区段的氨基酸。
在可选的实施方式中，所述抗体或其抗原结合片段还包括框架区。
第九方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区包括上述任一实施例所述的HCDR1～HCDR3和所述轻链可变区包括上述任一实施例所述的LCDR1～LCDR3；所述重链可变区如与SEQ ID NO:19具有至少80％同一性的氨基酸序列所示和/或所述轻链可变区如与SEQ ID NO:20具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述重链可变区如与SEQ ID NO:19具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列所示；所述轻链可变区如与SEQ ID NO:20具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列所示。
在可选的实施方式中，上述第七方面、第八方面或第九方面所述抗体或其抗原结合片，包括重链可变区和轻链可变区，所述重链可变区如SEQ ID NO:19所示；所述轻链可变区如SEQ ID NO:20所示。
第十方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述 重链可变区如SEQ ID NO:19所示；所述轻链可变区如SEQ ID NO:20所示。
在可选的实施方式中，所述抗体或其抗原结合片段还包含恒定区。
在可选的实施方式中，所述恒定区包括重链恒定区和/或轻链恒定区。
在可选的实施方式中，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中任一项的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中，所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅和人中的任意一种。
在可选的实施方式中，所述重链恒定区如SEQ ID NO:21或与其具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述轻链恒定区如SEQ ID NO:22或与其具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述“具有至少80％的同一性”可以是具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性。
第十一方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链和轻链，所述重链如SEQ ID NO:23所示，所述轻链如SEQ ID NO:24所示。
第十二方面，本公开实施例还提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段与上述任一实施例所述的抗体或其抗原结合片段结合猴痘病毒抗原蛋白氨基酸序列之中的同一表位；或，所述抗体或其抗原结合片段与上述任一实施例所述的抗体或其抗原结合片段竞争性结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤6.34×10-8M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤10-08M、KD≤10-09M、KD≤10-10M、KD≤10-11M或KD≤10-12M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤1.14×10-11M的亲和力结合猴痘病毒抗原。
第十三方面，本公开实施例提供了一种抗体或其抗原结合片段，包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1～3包括或为与SEQ ID NO:31或38所示重链可变区的HCDR1～3一致的氨基酸序列；所述LCDR1～3包括或为与SEQ ID NO:32所示轻链可变区的LCDR1～3一致的氨基酸序列。
需要说明的是，所述HCDR1、HCDR2和HCDR3为与同一条重链可变区中的HCDR1、HCDR2、HCDR3一致的氨基酸序列，LCDR1、LCDR2和LCDR3为与同一条轻链可变区中的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
例如所述HCDR1、HCDR2、HCDR3为与SEQ ID NO:31所示重链可变区的HCDR1、HCDR2、HCDR3一致的氨基酸序列；所述LCDR1、LCDR2、LCDR3为与SEQ ID NO:32所示轻链可变区的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
在可选的实施方式中，所述CDRs由Kabat、Chothia、AbM、Contact或IMGT系统定义。
在可选的实施方式中，所述CDRs由在Kabat系统定义的CDRs基础上，在HCDR3的C端缩短3个氨基酸，和LCDR3的C端缩短2个氨基酸。
在可选的实施方式中，所述HCDR1的氨基酸序列包括SEQ ID NO:25或如SEQ ID NO:25所示；所述HCDR2的氨基酸序列包括SEQ ID NO:26或37，或如SEQ ID NO:26或37所示；所述HCDR3的氨基酸序列包括SEQ ID NO:27或如SEQ ID NO:27所示；和所述LCDR1、LCDR2和LCDR3的氨基酸序列依次包括SEQ ID NO:28～30或依次如SEQ ID NO:28～30所示。
第十四方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1的氨基酸序列包括SEQ ID NO:25或如SEQ ID NO:25所示；所述HCDR2的氨基酸序列包括SEQ ID NO:26或37，或如SEQ ID NO:26或37所示；所述HCDR3的氨基酸序列包括SEQ ID NO:27或如SEQ ID NO:27所示；和所述LCDR1、LCDR2和LCDR3的氨基酸序列依次包括SEQ ID NO:28～30或依次如SEQ ID NO:28～30所示。
在可选的实施方式中，所述HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3分别对应Kabat编号下的H31～H35、H50～H65、H95～H100A、L24～34、L50～56、L89～95位置区段的氨基酸。
在可选的实施方式中，所述抗体或其抗原结合片段还包括框架区。
第十五方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区包括上述任一实施例所述的HCDR1～HCDR3和所述轻链可变区包括上述任一实施例所述的LCDR1～LCDR3；所述重链可变区如与SEQ ID NO:31或38具有至少80％同一性的氨基酸序列所示和/或所述轻链可变区如与SEQ ID NO:32具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述重链可变区如与SEQ ID NO:31具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列所示，或如与SEQ ID NO:38具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列所示；所述轻链可变区如与SEQ ID NO:32具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨 基酸序列所示。
在可选的实施方式中，上述第十三方面、第十四方面或第十五方面所述抗体或其抗原结合片，包括重链可变区和轻链可变区，所述重链可变区如SEQ ID NO:31或38所示；所述轻链可变区如SEQ ID NO:32所示。
第十六方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区如SEQ ID NO:31或SEQ ID NO:38所示；所述轻链可变区如SEQ ID NO:32所示。
在可选的实施方式中，所述抗体或其抗原结合片段还包含恒定区。
在可选的实施方式中，所述恒定区包括重链恒定区和/或轻链恒定区。
在可选的实施方式中，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中任一项的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中，所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅和人中的任意一种。
在可选的实施方式中，所述重链恒定区如SEQ ID NO:33或与其具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述轻链恒定区如SEQ ID NO:34或与其具有至少80％的同一性的氨基酸序列所示。
在可选的实施方式中，所述“具有至少80％的同一性”可以是具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性。
第十七方面，本公开实施例提供了一种抗体或其抗原结合片段，包括重链和轻链，所述重链如SEQ ID NO:35或39所示，所述轻链如SEQ ID NO:36所示。
第十八方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段与上述任一实施例所述的抗体或其抗原结合片段结合猴痘病毒抗原蛋白氨基酸序列之中的同一表位；或，所述抗体或其抗原结合片段与上述任一项所述的抗体或其抗原结合片段竞争性结合猴痘病毒抗原。
在可选的实施方式中，所述同一表位位于猴痘病毒A29L蛋白氨基酸序列1至110之中；或，所述抗体或其抗原结合片段与上述任一实施例所述的抗体或其抗原结合片段竞争性结合猴痘病毒A29L蛋白。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤7.69×10-8M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤10-08M、KD≤10-09M、KD≤10-10M、KD≤10-11M或KD≤10-12M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤2.27×10-09M的亲和力结合猴痘病毒抗原。
第十九方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1/HCDR2/HCDR3组合与SEQ ID NO:46～49任一所示重链可变区包含的HCDR1/HCDR2/HCDR3组合相同，所述LCDR1/LCDR2/LCDR3组合与SEQ ID NO:50所示轻链可变区包含的LCDR1/LCDR2/LCDR3组合相同。
在可选的实施方式中，所述CDRs可以指由本领域已知的任何方法定义的CDRs。
在可选的实施方式中，所述CDRs由Kabat、Chothia、IMGT、AbM、Contact或Lesk系统定义。
在可选的实施方式中，所述抗体或其抗原结合片段包括：
(a)HCDR1、HCDR2和HCDR3的氨基酸序列依次包括SEQ ID No:40～42或依次如SEQ ID No:40～42所示；LCDR1、LCDR2和LCDR3的氨基酸序列依次包括SEQ ID No:43～45或依次如SEQ ID No:43～45所示。
在可选的实施方式中，所述CDRs由在Kabat系统定义的CDRs基础上，在HCDR3的C端缩短3个氨基酸，LCDR3的C端缩短2个氨基酸。
第二十方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括：(a)HCDR1、HCDR2和HCDR3的氨基酸序列依次包括SEQ ID No:40～42或依次如SEQ ID No:40～42所示；LCDR1、LCDR2和LCDR3的氨基酸序列依次包括SEQ ID No:43～45或依次如SEQ ID No:43～45所示。
在可选的实施方式中，所述HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3分别对应Kabat编号下的H31～H35、H50～H65、H95～H100B、L24～34、L50～56、L89～95位置区段的氨基酸。
在可选的实施方式中，所述抗体或其抗原结合片段还包括框架区。
在可选的实施方式中，上述第十九方面、第二十方面所述的抗体或其抗原结合片段包括：
(b)重链可变区和轻链可变区；重链可变区包括SEQ ID NO:46～49任一所示的氨基酸序列，或由SEQ ID NO:46～49任一所示的氨基酸序列组成；轻链可变区包括SEQ ID NO:50所示的氨基酸序列，或由SEQ ID NO:50所示的氨基酸序列组成。
第二十一方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括：(b)重链可变区和轻链可变区；重链可变区包括SEQ ID NO:46～49任一所示的氨基酸序列，或由SEQ ID NO:46～49任一所示的氨基酸序列组成；轻链可变区包括SEQ ID NO:50所示的氨基酸序列，或由SEQ ID NO:50所示的氨基酸序列组成。
在可选的实施方式中，所述抗体或其功能性片段包括：
(c)氨基酸序列与(b)所示重链可变区或/和轻链可变区序列具有至少80％同一性的重链可变区或/和轻链可变区，且包括(a)所示序列的HCDR1～HCDR3和LCDR1～LCDR3。
在可选的实施方式中，所述抗体或其抗原结合片段包括SEQ ID NO:46～49任一所示的重链可变区中的重链框架区，和SEQ ID NO:50所示的轻链可变区中的轻链框架区。
在可选的实施方式中，所述抗体或其抗原结合片段的框架区氨基酸序列可以与上述框架区具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
在可选的实施方式中，所述抗体或其抗原结合片段还包含恒定区。
在可选的实施方式中，所述恒定区包括重链恒定区和/或轻链恒定区。
在可选的实施方式中，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中任一项的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中，所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅和人中的任意一种。
在可选的实施方式中，所述重链恒定区包括SEQ ID NO:51或与其具有至少80％的同一性的氨基酸序列。
在可选的实施方式中，所述重链恒定区由SEQ ID NO:51或与其具有至少80％同一性的氨基酸序列组成。
在可选的实施方式中，所述轻链恒定区包括SEQ ID NO:52或与其具有至少80％同一性的氨基酸序列。
在可选的实施方式中，所述轻链恒定区由SEQ ID NO:52或与其具有至少80％的同一性的氨基酸序列组成。
在可选的实施方式中，所述抗体或其抗原结合片段包括重链和轻链；所述重链包括SEQ ID NO:53～56任一所示或与其具有至少80％的同一性的氨基酸序列；或，由SEQ ID NO:53～56任一所示或与其具有至少80％的同一性的氨基酸序列组成；所述轻链包括SEQ ID NO:57或与其具有至少80％的同一性的氨基酸序列；或，由SEQ ID NO:57或与其具有至少80％的同一性的氨基酸序列组成。
具体地，上述“至少80％同一性”，可以是具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性，且同一性的变体位点不在CDRs区。
第二十二方面，本公开实施例还提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段的结合表位与上述任一项所述的抗体或其抗原结合片段的结合表位相同；或，所述抗体或其抗原结合片段与上述任一项所述的抗体或其抗原结合片段竞争性结合相同的表位。
在可选的实施方式中，所述相同的表位位于猴痘病毒A29L蛋白氨基酸序列1至110之中；或，所述抗体或其抗原结合片段与上述任一项所述的抗体或其抗原结合片段竞争性结合猴痘病毒A29L蛋白。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤7.36×10-8M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤10-8M、KD≤10-9M、KD≤10-10M、KD≤10-11M、KD≤10-12M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤1.78×10-9M的亲和力结合猴痘病毒抗原。
第二十三方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1/HCDR2/HCDR3组合与SEQ ID NO:80至SEQ ID NO:87任一所示重链可变区包含的HCDR1/HCDR2/HCDR3组合相同，所述LCDR1/LCDR2/LCDR3组合与SEQ ID NO:88至SEQ ID NO:91任一所示轻链可变区包含的LCDR1/LCDR2/LCDR3组合相同。
在可选的实施方式中，所述CDRs可以指由本领域已知的任何方法定义的CDRs。
在可选的实施方式中，所述CDRs由Kabat、Chothia、IMGT、AbM、Contact或Lesk系统定义。
在可选的实施方式中，所述CDRs由在Kabat系统定义的CDRs基础上，在HCDR3的N端延长1个氨基酸，在C端缩短2个氨基酸；LCDR3的C端缩短2个氨基酸。
在可选的实施方式中，所述HCDR1包括如SEQ ID NO:58所示的氨基酸序列；所述HCDR2包括如SEQ ID NO:59或60所示的氨基酸序列；所述HCDR3包括如SEQ ID NO:104所示的氨基酸序列；所述LCDR1包括如SEQ ID NO:61所示的氨基酸序列；所述LCDR2包括如SEQ ID NO:62、63、64或65所示的氨基酸序列；所述LCDR3包括如SEQ ID NO:66所示的氨基酸序列。
第二十四方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1包括如SEQ ID NO:58所示的氨基酸序列；所述HCDR2包括如SEQ ID NO:59或60所示的氨基酸序列；所述HCDR3包括如SEQ ID NO:104所示的氨基酸序列；所述LCDR1包括如SEQ ID NO:61所示的氨基酸序列；所述LCDR2包括如SEQ ID NO:62、63、64或65所示的氨基酸序列；所述LCDR3包括如SEQ ID NO:66所示的氨基酸序列。
在可选的实施方式中，所述HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3分别对应Kabat 编号下的H31～H35、H50～H65、H94～H96、L24～34、L50～56、L89～95位置区段的氨基酸。
在可选的实施方式中，上述第二十三方面或第二十四方面所述体或其抗原结合片段中HCDR1的氨基酸序列如SEQ ID NO:58所示；所述HCDR2的氨基酸序列如SEQ ID NO:59或60所示；所述HCDR3的氨基酸序列如SEQ ID NO:104所示；所述LCDR1的氨基酸序列如SEQ ID NO:61所示；所述LCDR2的氨基酸序列如SEQ ID NO:62、63、64或65所示；所述LCDR3的氨基酸序列如SEQ ID NO:66所示。
在可选的实施方式中，上述第二十三方面或第二十四方面所述体或其抗原结合片段中HCDR1的氨基酸序列如SEQ ID NO:58所示；所述HCDR2的氨基酸序列如SEQ ID NO:59所示；所述HCDR3的氨基酸序列如SEQ ID NO:104所示；所述LCDR1的氨基酸序列如SEQ ID NO:61所示；所述LCDR2的氨基酸序列如SEQ ID NO:62、63、64或65所示；所述LCDR3的氨基酸序列如SEQ ID NO:66所示。
在可选的实施方式中，上述第二十三方面或第二十四方面所述体或其抗原结合片段中HCDR1的氨基酸序列如SEQ ID NO:58所示；所述HCDR2的氨基酸序列如SEQ ID NO:60所示；所述HCDR3的氨基酸序列如SEQ ID NO:104所示；所述LCDR1的氨基酸序列如SEQ ID NO:4所示；所述LCDR2的氨基酸序列如SEQ ID NO:62、63、64或65所示；所述LCDR3的氨基酸序列如SEQ ID NO:66所示。
在可选的实施方式中，所述抗体或其抗原结合片段还包括HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3和LFR4。
在可选的实施方式中，所述HFR1至HFR4依次包括SEQ ID NO:67至SEQ ID NO:70或与其具有至少80％同一性的氨基酸序列。
在可选的实施方式中，所述HFR1至HFR4依次如SEQ ID NO:67至SEQ ID NO:70或与其具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述LFR1至LFR4依次包括SEQ ID NO:76至SEQ ID NO:79或与其具有至少80％同一性的氨基酸序列。
在可选的实施方式中，所述LFR1至LFR4依次如SEQ ID NO:76至SEQ ID NO:79或与其具有至少80％同一性的氨基酸序列所示。
在可选的实施方式中，所述抗体或其抗原结合片段的框架区氨基酸序列可以与上述框架区(SEQ ID NO:67至SEQ ID NO:70或SEQ ID NO:76至SEQ ID NO:79)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
在可选的实施方式中，所述HFR1的氨基酸序列包括SEQ ID NO:71，或如SEQ ID NO:71所示。
在可选的实施方式中，所述HFR2的氨基酸序列包括SEQ ID NO:72、73或74，或如SEQ ID NO:72、73或74所示。
在可选的实施方式中，所述HFR3的氨基酸序列包括SEQ ID NO:75，或如SEQ ID NO:75所示。
在可选的实施方式中，所述LFR1的氨基酸序列包括SEQ ID NO:100，或如SEQ ID NO:100所示。
在可选的实施方式中，所述LFR3的氨基酸序列包括SEQ ID NO:101、102或103，或如SEQ ID NO:101、102或103所示。
第二十五方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区含有HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4的序列结构，所述轻链可变区区含有LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4的序列结构，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3的氨基酸序列为上述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3的氨基酸序列。
在可选的实施方式中，所述HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3、LFR4的氨基酸序列为上述的HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3、LFR4的氨基酸序列。
在可选的实施方式中，所述重链可变区包括SEQ ID NO:80至87任一所示或与其具有至少80％同一性的氨基酸序列。
在可选的实施方式中，所述重链可变区由SEQ ID NO:80至87任一所示或与其具有至少80％同一性的氨基酸序列组成。
在可选的实施方式中，所述轻链可变区包括SEQ ID NO:88至91任一所示或与其具有至少80％同一性的氨基酸序列。
在可选的实施方式中，所述轻链可变区由SEQ ID NO:88至91任一所示或与其具有至少80％同一性的氨基酸序列组成。
在可选的实施方式中，所述抗体或其抗原结合片段的可变区序列可以与上述可变区(SEQ ID NO:80至87或SEQ ID NO:88至91)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
在可选的实施方式中，所述同一性的变体氨基酸位点不在CDRs区。
在可选的实施方式中，所述重链可变区由SEQ ID NO:80至87任一所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:88至91任一所示的氨基酸序列组成。
第二十六方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区由SEQ ID NO:80至87任一所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:88至91任一所示的氨基酸序列组成。
在可选的实施方式中，上述第二十五方面或第二十六方面所述抗体或其抗原结合片段中的重链可变区由SEQ ID NO:80至87任一所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:88所示的氨基酸序列组成。
在可选的实施方式中，上述第二十五方面或第二十六方面所述抗体或其抗原结合片段中的重链可变区由SEQ ID NO:80至87任一所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:89所示的氨基酸序列组成。
在可选的实施方式中，上述第二十五方面或第二十六方面所述抗体或其抗原结合片段中的重链可变区由SEQ ID NO:80至87任一所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:90所示的氨基酸序列组成。
在可选的实施方式中，上述第二十五方面或第二十六方面所述抗体或其抗原结合片段中的重链可变区由SEQ ID NO:80至87任一所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:91所示的氨基酸序列组成。
在可选的实施方式中，所述抗体或其抗原结合片段还包含恒定区。
在可选的实施方式中，所述恒定区包括重链恒定区和/或轻链恒定区。
在可选的实施方式中，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中任一项的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中，所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅和人中的任意一种。
在可选的实施方式中，所述重链恒定区包括SEQ ID NO:92所示或与其具有至少80％同一性的氨基酸序列。
在可选的实施方式中，所述重链恒定区由SEQ ID NO:92所示或与其具有至少80％同一性的氨基酸序列组成。
在可选的实施方式中，所述轻链恒定区包括SEQ ID NO:93所示或与其具有至少80％同一性的氨基酸序列。
在可选的实施方式中，所述轻链恒定区由SEQ ID NO:93所示或与其具有至少80％同一性的氨基酸序列组成。
具体地，所述恒定区序列可以与上述恒定区(SEQ ID NO:92或93)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
在可选的实施方式中，所述抗体或其抗原结合片段包括重链和轻链。
在可选的实施方式中，所述重链包括SEQ ID NO:94至96任一所示或与其具有至少80％同一性的氨基酸序列。
在可选的实施方式中，所述重链由SEQ ID NO:94至96任一所示或与其具有至少80％同一性的氨基酸序列组成。
在可选的实施方式中，所述轻链包括SEQ ID NO:97至99任一所示或与其具有至少80％同一性的氨基酸序列。
在可选的实施方式中，所述轻链由SEQ ID NO:97至99任一所示或与其具有至少80％同一性的氨基酸序列组成。
在可选的实施方式中，所述抗体或其抗原结合片段的重链或轻链序列可以与上述重链或轻链序列(SEQ ID NO:94至96或SEQ ID NO:97至99)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
在可选的实施方式中，所述同一性的变体氨基酸位点不在CDRs区。
在可选的实施方式中，所述重链由SEQ ID NO:94至96任一所示的氨基酸序列组成；所述轻链由SEQ ID NO:97至99任一所示的氨基酸序列组成。
第二十七方面，本公开实施例提供了一种抗体或其抗原结合片段，所述抗体或其抗原结合片段的结合表位与上述任一项所述的抗体或其抗原结合片段的结合表位相同；或，所述抗体或其抗原结合片段与上述任一项所述的抗体或其抗原结合片段竞争性结合相同的表位。
在可选的实施方式中，所述相同的表位位于猴痘病毒A29L蛋白氨基酸序列1至110之中；或，所述抗体或其抗原结合片段与权利要求1～5任一项所述的抗体或其抗原结合片段竞争性结合猴痘病毒A29L蛋白。
在可选的实施方式中，所述抗体或其抗原结合片段结合猴痘病毒A29L蛋白(MKPVA29L)的特有表位，而不结合猴痘病毒A29L蛋白、牛痘病毒162蛋白(CPXV162)和痘苗病毒A27L蛋白(VACA27L)的共同表位。
在可选的实施方式中，所述抗体或其抗原结合片段与猴痘病毒A29L蛋白氨基酸序列17至49之中的氨基酸区域结合。
在可选的实施方式中，所述抗体或其抗原结合片段特异性结合猴痘病毒或猴痘病毒A29L蛋白(MKPVA29L)，且与牛痘病毒162蛋白(CPXV162)或痘苗病毒A27L蛋白(VACA27L)不存在交叉反应性。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤7.91×10-8M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤10-08M、KD≤10-09M、KD≤10-10M、KD≤10-11M或KD≤10-12M的亲和力结合猴痘病毒抗原。
在可选的实施方式中，所述抗体或其抗原结合片段以KD≤1.47×10-09M或KD≤9.96×10-10M的亲和力结合猴痘病毒抗原。
抗体亲和力(KD)测定方法有很多种，根据检测原理可分为热力学检测方法、动力学检测方法和动态平衡检测方法。其中，热力学检测方法常见如等温滴定量热法(ITC)；动力学检测方法常见如表面等离子共振(SPR)及生物膜光干涉法(BLI)；动态平衡检测方法常见如酶联免疫吸附(ELISA)等。
在可选的实施方式中，KD的测定采用动力学检测方法；优选为，表面等离子共振方法，例如通过使用诸如系统的生物传感器系统。
在可选的实施方式中，所述抗原结合片段包括重链可变区和轻链可变区，所述重链可变区包括上述任一实施例所述的HCDR1～HCDR3和所述轻链可变区包括上述任一实施例所述的LCDR1～LCDR3。
在可选的实施方式中，所述抗原结合片段选自所述抗体的F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
上述抗体的抗原结合片段通常具有与其来源抗体相同的结合特异性。
在本公开中，术语“抗原结合片段”和具有与其来源抗体相同的结合特异性的“功能性性片段”可互换使用。
本领域技术人员根据本公开记载的内容容易理解到，上述抗体的抗原结合片段可以通过比如酶消化的方法(包括胃蛋白酶或木瓜蛋白酶)和/或通过化学还原分裂二硫键的方法获得。在本公开公开了完整抗体的结构基础上，本领域技术人员容易获得上述的抗原结合片段。
上述抗体的抗原结合片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪，比如Applied BioSystems等销售的自动肽合成仪合成获得。
另一方面，本公开实施例还提供了一种抗体偶联物，其包括如前述实施例所述的抗体或其抗原结合片段。
在可选的实施方式中，所述抗体偶联物还包括与所述抗体或其抗原结合片段偶联的生物素或生物素衍生物。
在可选的实施方式中，所述抗体偶联物还包括与所述抗体或其抗原结合片段偶联的标记物。
在可选的实施方式中，上述标记物是指具有能够被肉眼直接观察或被仪器检测或探测到的特性例如发光、显色、放射性等特性的一类物质，通过该特性可以实现对相应目标物的定性或定量检测。
在可选的实施方式中，所述标记物选自荧光染料、酶、放射性同位素、化学发光试剂和纳米颗粒类标记物中的至少一种。
在实际的使用过程中，本领域技术人员可以根据检测条件或实际需要选择合适的标记物，无论使用何种标记物，均属于本公开的保护范围。
在可选的实施方式中，所述荧光染料包括但不限于荧光素类染料及其衍生物(例如包括但不限于异硫氰酸荧光素(FITC)羟基光素(FAM)、四氯光素(TET)等或其类似物)、罗丹明类染料及其衍生物(例如包括但不限于红色罗丹明(RBITC)、四甲基罗丹明(TAMRA)、罗丹明B(TRITC)等或其类似物)、Cy系列染料及其衍生物(例如包括但不限于Cy2、Cy3、Cy3B、Cy3.5、Cy5、Cy5.5、Cy3等或其类似物)、Alexa系列染料及其衍生物(例如包括但不限于AlexaFluor350、405、430、488、532、546、555、568、594、610、33、647、680、700、750等或其类似物)和蛋白类染料及其衍生物(例如包括但不限于藻红蛋白(PE)、藻蓝蛋白(PC)、别藻蓝蛋白(APC)、多甲藻黄素-叶绿素蛋白(preCP)等)。
在可选的实施方式中，所述酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。
在可选的实施方式中，所述放射性同位素包括但不限于212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu和18F。
在可选的实施方式中，所述化学发光试剂包括但不限于鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物。
在可选的实施方式中，所述纳米颗粒类标记物包括但不限于纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒。
在可选的实施方式中，所述胶体包括但不限于胶体金属、胶体硒、分散型染料、染料标记的微球和乳胶。
在可选的实施方式中，所述胶体金属包括但不限于胶体金或胶体银。
在可选的实施方式中，所述胶体金属为胶体金。
在可选的实施方式中，所述抗体偶联物还包括与所述抗体或其抗原结合片段偶联的固相载体。抗体偶联物中，所述抗体偶联至固相载体。
在可选的实施方式中，所述固相载体选自微球、板和膜。
在可选的实施方式中，所述固相包括但不限于磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛 细管、尼龙和硝酸纤维素膜。
在可选的实施方式中，所述固相载体为硝酸纤维素膜。
另一方面，本公开实施例还提供了一种试剂或试剂盒，其包括如前述任意实施例所述的抗体或其抗原结合片段或如前述任意实施例所述的抗体偶联物。
在可选的实施方式中，所述试剂或试剂盒还包括第二抗体，所述第二抗体与所述抗体或其抗原结合片段结合；
在可选的实施方式中，所述试剂或试剂盒还包括第二抗体，所述第二抗体与猴痘病毒或猴痘病毒抗原结合。
在可选的实施方式中，所述试剂或试剂盒具有提高的检测灵敏度或特异性。
另一方面，本公开实施例还提供了一种检测猴痘病毒或猴痘病毒抗原的方法，其包括：
将如前述任意实施例所述的抗体或其抗原结合片段或如前述任意实施例所述的抗体偶联物或前述任意实施例所述的试剂或试剂盒与待检测样本中的猴痘病毒或猴痘病毒抗原接触，形成免疫复合物。
在优选的实施方式中，所述免疫复合物还包括第二抗体，所述第二抗体与所述抗体或其抗原结合片段结合。
在优选的实施方式中，所述免疫复合物还包括第二抗体，所述第二抗体与猴痘病毒或猴痘病毒抗原结合。
另一方面，本公开实施例还提供了前述任意实施例所述的抗体或抗原结合片段或前述任意实施例所述的抗体偶联物或前述任意实施例所述的试剂或试剂盒在检测猴痘病毒或猴痘病毒抗原或制备检测猴痘病毒或猴痘病毒抗原产品中的应用。
另一方面，本公开实施例还提供了前述任意实施例所述的抗体或抗原结合片段或前述任意实施例所述的抗体偶联物或前述任意实施例所述的试剂或试剂盒在制备具有以下至少一种用途的产品中的应用，所述用途包括：诊断或辅助诊断猴痘病毒感染引发的相关疾病，预测或辅助预测猴痘病毒感染引发相关疾病的预后疗效中的至少一种。
在可选的实施方式中，所述猴痘病毒感染引发的相关疾病包括发热、头痛、淋巴结肿大、肌肉酸痛、重度疲乏、肺炎、败血症和脑炎中的至少一种。
在可选的实施方式中，所述产品包括试剂或试剂盒。
另一方面，本公开实施例还提供了一种分离的核酸，其编码前述任意实施例所述的抗体或其抗原结合片段。
另一方面，本公开实施例还提供了一种载体，其含有前述任意实施例所述的分离的核酸。
另一方面，本公开实施例还提供了一种细胞，其含有前述任意实施例所述的分离的核酸或如前述任意实施例所述的载体。
另一方面，本公开实施例还提供了一种制备如前述任意实施例所述的抗体或其抗原结合片段的方法，其包括：培养如前述任意实施例所述的细胞。
在本公开公开了抗体或其抗原结合片段的氨基酸序列的基础上，本领域技术人员容易想到采用基因工程技术或其他技术(化学合成、重组表达)制备得到该抗体或其抗原结合片段，例如从能够重组表达如上任一项所述的抗体或其抗原结合片段的重组细胞的培养产物中分离纯化得到该抗体或其抗原结合片段，这对本领域技术人员来说是容易实现的，基于此，无论采用何种技术制备本公开的抗体或其抗原结合片段，其均属于本公开的保护范围。
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明，否则实践本公开将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编，1984)；《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编，1987)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《哺乳动物细胞用基因转移载体(Gene Transfer Vectors for Mammalian Cells)》(J.M.Miller和M.P.Calos编，1987)；《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编，1987)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编，1994)；以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编，2011)，所述文献中的每个文献均通过引用明确并入本文中。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实验1
实施例1-1 Anti-MPXV 4G21单克隆抗体的制备
1.表达质粒构建
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMARTTM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由基因测序公司完成。分泌Anti-MPXV 4G21单克隆抗体的杂交瘤细胞株为本实验室制备的杂交瘤细胞株，复苏备用。
1.1 Anti-MPXV 4G21抗体基因制备
从分泌Anti-MPXV 4G21单克隆抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，并将其插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取Heavy Chain及Light Chain基因的阳性克隆各4个克隆送基因测序公司进行测序。
1.2 Anti-MPXV 4G21抗体可变区基因的序列分析
将上述测序得到的基因序列放在Kabat抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中Light Chain扩增出的基因片段中，VL基因序列为321bp，其前方有57bp的前导肽序列；Heavy Chain引物对扩增出的基因片段中，VH基因序列为363bp，属于VH1基因家族，其前方有57bp的前导肽序列。
1.3重组抗体表达质粒的构建
pcDNATM 3.4vector为构建的重组抗体真核表达载体，该表达载体经改造引入多克隆酶切位点，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计Anti-MPXV 4G21抗体的VL和VH基因特异性引物，两端分别带有限制性内切酶酶切位点和保护碱基，通过PCR扩增方法扩出0.73KB的Light Chain基因片段和1.42kb的Heavy Chain基因片段。
Heavy Chain和Light Chain基因片段分别采用限制性内切酶进行双酶切，3.4A载体采用限制性内切酶进行双酶切，将片段和载体纯化回收后Heavy Chain基因和Light Chain基因分别连接3.4A表达载体中，分别得到Heavy Chain和Light Chain的重组表达质粒。
2.重组抗体的样品制备
提前复苏HEK293细胞，传代培养到200ml体系，使细胞密度达到3～5×106cells/ml，细胞活力>95％；离心清洗细胞，用培养基复溶，同时将细胞密度调整到2.9×106cells/ml，作为细胞稀释液。用培养基分别配制质粒DNA和转染试剂稀释液。将转染试剂稀释液加入到质粒DNA稀释液中，混匀后室温静置放置15min；将该混合物在1min内缓慢加入细胞稀释液中，混匀后取样计数，记录并观察细胞转染后的活力，并将其放置于35℃恒温培养箱中培养，转速120rmp，CO2含量8％，13天后离心收样。将离心上清用protein A亲和层析柱进行亲和纯化。取6μg纯化的抗体进行还原性SDS-PAGE，电泳图如图1所示。在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链)，另一条Mr为28KD(轻链)。
3.亲和力及活性优化
上述制备得到的Anti-MPXV 4G21单克隆抗体虽然具备结合猴痘病毒抗原的能力，但亲和力和抗体活性均不够理想，因而申请人通过对该抗体的可变区进行定向突变。即利用计算机进行抗体可变区结构模拟、抗原与抗体可变区作用复合物结构模拟、抗体关键氨基酸分析及突变设计，根据突变方案设计合成覆盖突变位点的双向引物，合成目的DNA两端引物，进行高保真PCR反应，将PCR产物克隆至载体，再按照上述步骤2的方法进行突变抗体的制备。经筛选，得到亲和力和抗体活性显著提升的单克隆抗体，并命名为Anti-MPXV 4G21RMb1，其重链和轻链氨基酸序列分别如下。
表1-1抗体序列
实施例1-2亲和力分析
提前稀释纯化抗体，同时对猴痘重组抗原(购自菲鹏生物)进行梯度稀释；利用已提前偶联羊抗小鼠IgG的CM5芯片，在Biacore 8K+设备上测试抗原抗体的结合解离曲线，仪器自动拟合获得亲和力常数、结合速率、解离速率。(KD表示平衡解离常数即亲和力常数；ka表示结合速率；kd表示解离速率)。
表1-2亲和力分析数据
实施例1-3活性鉴定
包被液(主要成分NaHCO3)稀释重组猴痘重组抗原(购自菲鹏生物)至1ug/ml，每孔100uL，4℃过夜；次日，洗涤液(主要成份Na2HPO4+Nacl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120uL，37℃，1h，拍干；加入稀释后的纯化抗体和对照抗体，100uL/孔，37℃，30min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100uL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50uL/孔)，加入显色液B液(50uL/孔)，10min；加入终止液，50uL/孔；酶标仪上450nm(参 考630nm)处读OD值。
表1-3活性数据
实施例1-4抗体稳定性考核
将上述抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降趋势，说明表达的抗体稳定。下表为考核21天的酶免活性检测OD结果。
表1-4稳定性数据
实验2
实施例2-1 Anti-MPXV 2F19单克隆抗体的制备
1.表达质粒构建
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMARTTM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由基因测序公司完成。分泌Anti-MPXV 2F19单克隆抗体的杂交瘤细胞株为本实验室制备的杂交瘤细胞株，复苏备用。
1.1 Anti-MPXV 2F19抗体基因制备
从分泌Anti-MPXV 2F19单克隆抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，并将其插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取Heavy Chain及Light Chain基因的阳性克隆各4个克隆送基因测序公司进行测序。
1.2 Anti-MPXV 2F19抗体可变区基因的序列分析
将上述测序得到的基因序列放在Kabat抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中Light Chain扩增出的基因片段中，VL基因序列为339bp，其前方有57bp的前导肽序列；Heavy Chain引物对扩增出的基因片段中，VH基因序列为360bp，属于VH1基因家族，其前方有57bp的前导肽序列。
1.3重组抗体表达质粒的构建
pcDNATM 3.4vector为构建的重组抗体真核表达载体，该表达载体经改造引入多克隆酶切位点，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计Anti-MPXV 2F19抗体的VL和VH基因特异性引物，两端分别带有限制性内切酶酶切位点和保护碱基，通过PCR扩增方法扩出0.74KB的Light Chain基因片段和1.42kb的Heavy Chain基因片段。
Heavy Chain和Light Chain基因片段分别采用限制性内切酶进行双酶切，3.4A载体采用限制性内切酶进行双酶切，将片段和载体纯化回收后Heavy Chain基因和Light Chain基因分别连接3.4A表达载体中，分别得到Heavy Chain和Light Chain的重组表达质粒。
2.重组抗体的样品制备
提前复苏HEK293细胞，传代培养到200ml体系，使细胞密度达到3～5×106cells/ml，细胞活力>95％；离心清洗细胞，用培养基复溶，同时将细胞密度调整到2.9×106cells/ml，作为细胞稀释液。用培养基分别配制质粒DNA和转染试剂稀释液。将转染试剂稀释液加入到质粒DNA稀释液中，混匀后室温静置放置15min；将该混合物在1min内缓慢加入细胞稀释液中，混匀后取样计数，记录并观察细胞转染后的活力，并将其放置于35℃恒温培养箱中培养，转速120rmp，CO2含量8％，13天后离心收样。将离心上清用protein A亲和层析柱进行亲和纯化。取6μg纯化的抗体进行还原性SDS-PAGE，电泳图如图2所示。在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链)，另一条Mr为28KD(轻链)。
3.亲和力及活性优化
上述制备得到的Anti-MPXV 2F19单克隆抗体虽然具备结合猴痘病毒抗原的能力，但亲和力和抗体活性均不够理想，因而申请人通过对该抗体的可变区进行定向突变。即利用计算机进行抗体可变区结构模拟、抗原与抗体可变区作用复合物结构模拟、抗体关键氨基酸分析及突变设计，根据突变方案设计合成覆盖突变位点的双向引物，合成目的DNA两端引物，进行高保真PCR反应，将PCR产物克隆至载体，再按照上述步骤2的方法进行突变抗体的制备。经筛选，得到亲和力和抗体活性显著提升的单克隆抗体，并命名为Anti-MPXV 2F19RMb1，其重链和轻链氨基酸序列分别如下。
表2-1抗体序列
实施例2-2亲和力分析
提前稀释纯化抗体，同时对猴痘重组抗原(购自菲鹏生物)进行梯度稀释；利用已提前偶联羊抗小鼠IgG的CM5芯片，在Biacore 8K+设备上测试抗原抗体的结合解离曲线，仪器自动拟合获得亲和力常数、结合速率、解离速率。(KD表示平衡解离常数即亲和力常数；ka表示结合速率；kd表示解离速率)。
表2-2亲和力分析数据
实施例2-3活性鉴定
包被液(主要成分NaHCO3)稀释重组猴痘重组抗原(购自菲鹏生物)至1ug/ml，每孔100uL，4℃过夜；次日，洗涤液(主要成份Na2HPO4+Nacl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120uL，37℃，1h，拍干；加入稀释后的纯化抗体和对照抗体，100uL/孔，37℃，30min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100uL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50uL/孔)，加入显色液B液(50uL/孔)，10min；加入终止液，50uL/孔；酶标仪上450nm(参考630nm)处读OD值。
表2-3活性数据
实施例2-4抗体稳定性考核
将上述抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降趋势，说明表达的抗体稳定。下表为考核21天的酶免活性检测OD结果。
表2-4稳定性数据
实验3
实施例3-1 Anti-MPXV 17A16单克隆抗体的制备
1.表达质粒构建
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMARTTM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由基因测序公司完成。分泌Anti-MPXV 17A16单克隆抗体的杂交瘤细胞株为本实验室制备的杂交瘤细胞株，复苏备用。
1.1 Anti-MPXV 17A16抗体基因制备
从分泌Anti-MPXV 17A16单克隆抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，并将其插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取Heavy Chain及Light Chain基因的阳性克隆各4个克隆送基因测序公司进行测序。
1.2 Anti-MPXV 17A16抗体可变区基因的序列分析
将上述测序得到的基因序列放在Kabat抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中Light Chain扩增出的基因片段中，VL基因序列为321bp，其前方有57bp的前导肽序列；Heavy Chain引物对扩增出的基因片段中，VH基因序列为357bp，属于VH1基因家族，其前方有57bp的前导肽序列。
1.3重组抗体表达质粒的构建
pcDNATM 3.4vector为构建的重组抗体真核表达载体，该表达载体经改造引入多克隆酶切位 点，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计Anti-MPXV 17A16抗体的VL和VH基因特异性引物，两端分别带有限制性内切酶酶切位点和保护碱基，通过PCR扩增方法扩出0.74KB的Light Chain基因片段和1.41kb的Heavy Chain基因片段。
Heavy Chain和Light Chain基因片段分别采用限制性内切酶进行双酶切，3.4A载体采用限制性内切酶进行双酶切，将片段和载体纯化回收后Heavy Chain基因和Light Chain基因分别连接3.4A表达载体中，分别得到Heavy Chain和Light Chain的重组表达质粒。
2.重组抗体的样品制备
提前复苏HEK293细胞，传代培养到200ml体系，使细胞密度达到3～5×106cells/ml，细胞活力>95％；离心清洗细胞，用培养基复溶，同时将细胞密度调整到2.9×106cells/ml，作为细胞稀释液。用培养基分别配制质粒DNA和转染试剂稀释液。将转染试剂稀释液加入到质粒DNA稀释液中，混匀后室温静置放置15min；将该混合物在1min内缓慢加入细胞稀释液中，混匀后取样计数，记录并观察细胞转染后的活力，并将其放置于35℃恒温培养箱中培养，转速120rmp，CO2含量8％，13天后离心收样。将离心上清用protein A亲和层析柱进行亲和纯化。取6μg纯化的抗体进行还原性SDS-PAGE，电泳图如图3所示。在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链)，另一条Mr为28KD(轻链)。
3.亲和力及活性优化
上述制备得到的Anti-MPXV 17A16单克隆抗体虽然具备结合猴痘病毒抗原的能力，但亲和力和抗体活性均不够理想，因而申请人通过对该抗体的可变区进行定向突变。即利用计算机进行抗体可变区结构模拟、抗原与抗体可变区作用复合物结构模拟、抗体关键氨基酸分析及突变设计，根据突变方案设计合成覆盖突变位点的双向引物，合成目的DNA两端引物，进行高保真PCR反应，将PCR产物克隆至载体，再按照上述步骤2的方法进行突变抗体的制备。经筛选，得到亲和力和抗体活性显著提升的单克隆抗体，并命名为Anti-MPXV 17A16RMb1和Anti-MPXV 17A3RMb1，其重链和轻链氨基酸序列分别如下。
表3-1抗体序列
实施例3-2亲和力分析
提前稀释纯化抗体，同时对猴痘重组抗原(购自菲鹏生物)进行梯度稀释；利用已提前偶联羊抗小鼠IgG的CM5芯片，在Biacore 8K+设备上测试抗原抗体的结合解离曲线，仪器自动拟合获得亲和力常数、结合速率、解离速率。(KD表示平衡解离常数即亲和力常数；ka表示结合速率；kd表示解离速率)。
表3-2亲和力分析数据
实施例3-3活性鉴定
包被液(主要成分NaHCO3)稀释重组猴痘重组抗原(购自菲鹏生物)至1ug/ml，每孔100uL，4℃过夜；次日，洗涤液(主要成份Na2HPO4+Nacl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120uL，37℃，1h，拍干；加入稀释后的纯化抗体和对照抗体，100uL/孔，37℃，30min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100uL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50uL/孔)，加入显色液B液(50uL/孔)，10min；加入终止液，50uL/孔；酶标仪上450nm(参考630nm)处读OD值。
表3-3活性数据
实施例3-4抗体稳定性考核
将上述抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降趋势，说明表达的抗体稳定。下表为考核21天的酶免活性检测OD结果。
表3-4稳定性数据
实施例3-5性能评估
通过ELISA，鉴定上述制备的抗体与猴痘病毒A29L蛋白的反应性。ELISA操作如下：用0.5μg/ml的MKPVA29L以100ul/孔包被微量滴定板，37℃孵育2h。BSA封闭。将上述制备的抗体均用PBS稀释至0.05ug/ml，50ul/孔，37℃孵育30min。
阴性对照：加入PBS孵育。
二抗：100ul/孔，羊抗鼠IgG-HRP 5K稀释in HIV-ED，37℃孵育30min。
显色：各加50ulA、B液，10min后加50ul终止液，读值。
结果显示上述制备的抗体与猴痘病毒A29L蛋白的反应性均优于对照。
表3-5性能评估数据
实验4
实施例4-1Anti-MPXV 11D3单克隆抗体的制备
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMARTTM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由基因测序公司完成。分泌Anti-MPXV 11D3单克隆抗体的杂交瘤细胞株为本实验室制备的杂交瘤细胞株，复苏备用。
1.表达质粒构建
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMARTTM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由基因测序公司完成。
1.1 Anti-MPXV 11D3抗体基因制备
从分泌Anti-MPXV 11D3单克隆抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，并将其插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取Heavy Chain及Light Chain基因的阳性克隆各4个克隆送基因测序公司进行测序。
1.2 Anti-MPXV 11D3抗体可变区基因的序列分析
将上述测序得到的基因序列放在Kabat抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中Light Chain扩增出的基因片段中，VL基因序列为333bp，其前方有57bp的前导肽序列；Heavy Chain引物对扩增出的基因片段中，VH基因序列为366bp，属于VH1基因家族，其前方有57bp的前导肽序列。
1.3重组抗体表达质粒的构建
pcDNATM 3.4vector为构建的重组抗体真核表达载体，该表达载体经改造引入多克隆酶切位点，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计Anti-MPXV 11D3抗体的VL和VH基因特异性引物，两端分别带有限制性内切酶酶切位点和保护碱基，通过PCR扩增方法扩出0.71KB的Light Chain基因片段和1.41kb的Heavy Chain基因片段。
Heavy Chain和Light Chain基因片段分别采用限制性内切酶进行双酶切，3.4A载体采用限制性内切酶进行双酶切，将片段和载体纯化回收后Heavy Chain基因和Light Chain基因分别连接3.4A表达载体中，分别得到Heavy Chain和Light Chain的重组表达质粒。
2.重组抗体的样品制备
提前复苏HEK293细胞，传代培养到200ml体系，使细胞密度达到3～5×106cells/ml，细胞活力>95％；离心清洗细胞，用培养基复溶，同时将细胞密度调整到2.9×106cells/ml，作为细胞稀释液。用培养基分别配制质粒DNA和转染试剂稀释液。将转染试剂稀释液加入到质粒DNA稀释液中，混匀后室温静置放置15min；将该混合物在1min内缓慢加入细胞稀释液中，混匀后取样计数，记录并观察细胞转染后的活力，并将其放置于35℃恒温培养箱中培养，转速120rmp，CO2含量8％，13天后离心收样。将离心上清用protein A亲和层析柱进行亲和纯化。取6μg纯化的抗体进行还原性SDS-PAGE，电泳图如图4所示。在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链)，另一条Mr为28KD(轻链)。
3.亲和力及活性优化
上述制备得到的Anti-MPXV 11D3单克隆抗体虽然具备结合猴痘病毒抗原的能力，但亲和力和抗体活性均不够理想，因而申请人通过对该抗体的可变区进行定向突变。即利用计算机进行抗体可变区结构模拟、抗原与抗体可变区作用复合物结构模拟、抗体关键氨基酸分析及突变设计，根据突变方案设计合 成覆盖突变位点的双向引物，合成目的DNA两端引物，进行高保真PCR反应，将PCR产物克隆至载体，再按照上述2的方法进行突变抗体的制备。经筛选，得到亲和力和抗体活性显著提升的单克隆抗体，并命名为Anti-MPXV 11D3RMb1至Anti-MPXV 11D3RMb4，其重链和轻链氨基酸序列分别如下。
表4-1抗体序列
实施例4-2亲和力分析
提前稀释纯化抗体，同时对猴痘重组抗原(购自菲鹏生物)进行梯度稀释；利用已提前偶联羊抗小鼠IgG的CM5芯片，在Biacore 8K+设备上测试抗原抗体的结合解离曲线，仪器自动拟合获得亲和力常数、结合速率、解离速率。(KD表示平衡解离常数即亲和力常数；ka表示结合速率；kd表示解离速率)。
表4-2亲和力分析数据
实施例4-3活性鉴定
包被液(主要成分NaHCO3)稀释重组猴痘重组抗原(购自菲鹏生物)至1ug/ml，每孔100uL，4℃过夜；次日，洗涤液(主要成份Na2HPO4+Nacl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120uL，37℃，1h，拍干；加入稀释后的纯化抗体和对照抗体，100uL/孔，37℃，30min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100uL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50uL/孔)，加入显色液B液(50uL/孔)，10min；加入终止液，50uL/孔；酶标仪上450nm(参考630nm)处读OD值。
表4-3活性数据
实施例4-4抗体稳定性考核
将上述抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降趋势，说明表达的抗体稳定。下表为考核21天的酶免活性检测OD结果。
表4-4稳定性数据
实施例4-5性能评估
通过ELISA，鉴定上述制备的抗体与猴痘病毒A29L蛋白的反应性。ELISA操作如下：分别用0.5μg/ml和0.05μg/ml的MKPVA29L以100ul/孔包被微量滴定板，37℃孵育2h。BSA封闭。将上述制备的抗体均用PBS稀释至0.5ug/ml、0.05ug/ml，50ul/孔，37℃孵育30min。
阴性对照：加入PBS孵育
二抗：100ul/孔，加入羊抗鼠IgG-HRP，37℃孵育30min。
显色：各加50ulA、B液，10min后加50ul终止液，读数。
结果显示上述制备的抗体与猴痘病毒A29L蛋白的反应性均优于对照。
表4-5性能评估数据
实验5
实施例5-1Anti-MPXV 6F13单克隆抗体的制备
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMARTTM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由基因测序公司完成。分泌Anti-MPXV 6F13单克隆抗体的杂交瘤细胞株为本实验室制备的杂交瘤细胞株，复苏备用。
1.表达质粒构建
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMARTTM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由基因测序公司完成。
1.1 Anti-MPXV 6F13抗体基因制备
从分泌Anti-MPXV 6F13单克隆抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，并将其插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取Heavy Chain及Light Chain基因的阳性克隆各4个克隆送基因测序公司进行测序。
1.2 Anti-MPXV 6F13抗体可变区基因的序列分析
将上述测序得到的基因序列放在Kabat抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中Light Chain扩增出的基因片段中，VL基因序列为336bp，其前方有57bp的前导肽序列；Heavy Chain引物对扩增出的基因片段中，VH基因序列为339bp，属于VH1基因家族，其前方有57bp的前导肽序列。
1.3重组抗体表达质粒的构建
pcDNATM 3.4vector为构建的重组抗体真核表达载体，该表达载体经改造引入多克隆酶切位点，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计Anti-MPXV 6F13抗体的VL和VH基因特异性引物，两端分别带有限制性内切酶酶切位点和保护碱基，通过PCR扩增方法扩出0.74KB的Light Chain基因片段和1.41kb的Heavy Chain基因片段。
Heavy Chain和Light Chain基因片段分别采用限制性内切酶进行双酶切，3.4A载体采用限制性内切酶进行双酶切，将片段和载体纯化回收后Heavy Chain基因和Light Chain基因分别连接3.4A表达载体中，分别得到Heavy Chain和Light Chain的重组表达质粒。
2.重组抗体的样品制备
提前复苏HEK293细胞，传代培养到200ml体系，使细胞密度达到3～5×106cells/ml，细胞活力>95％；离心清洗细胞，用培养基复溶，同时将细胞密度调整到2.9×106cells/ml，作为细胞稀释液。用培养基分别配制质粒DNA和转染试剂稀释液。将转染试剂稀释液加入到质粒DNA稀释液中，混匀后室温静置放置15min；将该混合物在1min内缓慢加入细胞稀释液中，混匀后取样计数，记录并观察细胞转染后的活力，并将其放置于35℃恒温培养箱中培养，转速120rmp，CO2含量8％，13天后离心收样。将离心上清用protein A亲和层析柱进行亲和纯化。取6μg纯化的抗体进行还原性SDS-PAGE，电泳图如图5所示。在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链)，另一条Mr为28KD(轻链)。
3.亲和力及活性优化
上述制备得到的Anti-MPXV 6F13单克隆抗体虽然具备结合猴痘病毒抗原的能力，但亲和力和抗体活性均不够理想，因而申请人通过对该抗体的可变区进行定向突变。即利用计算机进行抗体可变区结构模拟、抗原与抗体可变区作用复合物结构模拟、抗体关键氨基酸分析及突变设计，根据突变方案设计合成覆盖突变位点的双向引物，合成目的DNA两端引物，进行高保真PCR反应，将PCR产物克隆至载体，再按照上述步骤2的方法进行突变抗体的制备。经筛选，得到亲和力和抗体活性显著提升的单克隆抗体，并命名为Anti-MPXV 6F13RMb1至Anti-MPXV 6F13RMb3，其重链和轻链氨基酸序列分别如下。
表5-1抗体序列
实施例5-2亲和力分析
提前稀释纯化抗体，同时对猴痘重组抗原(购自菲鹏生物)进行梯度稀释；利用已提前偶联羊抗鼠IgG的CM5芯片，在Biacore 8K+设备上测试抗原抗体的结合解离曲线，仪器自动拟合获得亲和力常数、结合速率、解离速率。(KD表示平衡解离常数即亲和力常数；ka表示结合速率；kd表示解离速率)。
表5-2亲和力分析数据
实施例5-3活性鉴定
包被液(主要成分NaHCO3)稀释重组猴痘重组抗原(购自菲鹏生物)至1ug/ml，每孔100uL，4℃过夜；次日，洗涤液(主要成份Na2HPO4+Nacl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120uL，37℃，1h，拍干；加入稀释后的纯化抗体和对照抗体，100uL/孔，37℃，30min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100uL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50uL/孔)，加入显色液B液(50uL/孔)，10min；加入终止液，50uL/孔；酶标仪上450nm(参考630nm)处读OD值。
表5-3活性数据
实施例5-4抗体稳定性考核
将上述抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降趋势，说明表达的抗体稳定。下表为考核21天的酶免活性检测OD结果。
表5-4稳定性数据
实施例5-5抗体交叉反应性的鉴定
通过ELISA，鉴定上述制备的抗体与猴痘病毒A29L蛋白、牛痘病毒162蛋白和痘苗病毒A27L蛋白的交叉反应。ELISA操作如下：
包被：分别用0.5μg/ml和0.05μg/ml的MKPVA29L、VCA27L、CPXV162以100ul/孔包被微量滴定板，37℃孵育2h。
封闭：取120ul的BSA封闭液，在37℃孵育2h。
一抗/样品：上述抗体均用PBS稀释至0.5ug/ml、0.05ug/ml，50ul/孔，37℃孵育30min。
阴性对照：加入PBS孵育
二抗：100ul/孔，加入羊抗鼠IgG-HRP，37℃孵育30min。
显色：各加50ulA、B液，10min后加50ul终止液，读数。
结果如下表所示：
表5-5交叉反应性数据

以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
本申请涉及的氨基酸序列如下所示：




工业实用性
本公开提供了抗猴痘病毒抗体或其抗原结合片段、检测猴痘病毒的试剂和试剂盒。本公开公开的抗猴痘病毒抗体包括重链互补决定区和轻链互补决定区，该抗体为猴痘病毒的检测提供了重要的原料来源，具有改善的亲和力或活性，具备优异的工业实用性。

Claims (16)

  1. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，其特征在于，
    所述HCDR1～3包括与SEQ ID NO:7、SEQ ID NO:19、SEQ ID NO:31、SEQ ID NO:38、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:80、SEQ ID NO:81或SEQ ID NO:82所示重链可变区的HCDR1～3一致的氨基酸序列；
    所述LCDR1～3包括与SEQ ID NO:8、SEQ ID NO:20、SEQ ID NO:32、SEQ ID NO:50、SEQ ID NO:88、SEQ ID NO:89或SEQ ID NO:90所示轻链可变区的LCDR1～3一致的氨基酸序列。
  2. 根据权利要求1所述的抗体或其抗原结合片段，所述CDRs由Kabat、Chothia、AbM、Contact或IMGT系统定义。
  3. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，其特征在于，所述HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3的氨基酸序列选自以下任一组合：
  4. 一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，其特征在于，所述重链可变区包括权利要求1～3任一项所述的HCDR1～HCDR3和所述轻链可变区包括权利要求1～3任一项所述的LCDR1～LCDR3；且所述重链可变区如与SEQ ID NO:7、SEQ ID NO:19、SEQ ID NO:31、SEQ ID NO:38、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:80、SEQ ID NO:81或SEQ ID NO:82具有至少80％同一性的氨基酸序列所示和/或所述轻链可变区如与SEQ ID NO:8、SEQ ID NO:20、SEQ ID NO:32、SEQ ID NO:50、SEQ ID NO:88、SEQ ID NO:89或SEQ ID NO:90具有至少80％同一性的氨基酸序列所示。
  5. 一种抗体或其抗原结合片段，包括重链可变区和轻链可变区，其特征在于，所述重链可变区如SEQ ID NO:7、SEQ ID NO:19、SEQ ID NO:31、SEQ ID NO:38、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:80、SEQ ID NO:81或SEQ ID NO:82所示；所述轻链可变区如SEQ ID NO:8、SEQ ID NO:20、SEQ ID NO:32、SEQ ID NO:50、SEQ ID NO:88、SEQ ID NO:89或SEQ ID NO:90所示；
    可选地，所述重链可变区和轻链可变区选自以下任一组合：
  6. 根据权利要求1～5任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段还包含恒定区；
    可选地，所述恒定区包括重链恒定区和/或轻链恒定区；
    可选地，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中任一项的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区；
    可选地，所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅和人中的任意一种；
    可选地，所述重链恒定区如SEQ ID NO:9或与其具有至少80％同一性的氨基酸序列所示；
    可选地，所述轻链恒定区如SEQ ID NO:10或与其具有至少80％同一性的氨基酸序列所示。
  7. 一种抗体或其抗原结合片段，包括重链和轻链，所述重链的氨基酸序列如SEQ ID NO:11、SEQ ID NO:23、SEQ ID NO:35、SEQ ID NO:39、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:94、SEQ ID NO:95或SEQ ID NO:96所示，所述轻链的氨基酸序列如SEQ ID NO:12、SEQ ID  NO:24、SEQ ID NO:36、SEQ ID NO:57、SEQ ID NO:97、SEQ ID NO:98或SEQ ID NO:99所示；
    可选地，所述重链和轻链选自以下任一组合：
  8. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段与权利要求1～7任一项所述的抗体或其抗原结合片段结合猴痘病毒抗原蛋白氨基酸序列之中的同一表位；或，所述抗体或其抗原结合片段与权利要求1～7任一项所述的抗体或其抗原结合片段竞争性结合猴痘病毒抗原；
    可选地，所述抗原结合片段包括重链可变区和轻链可变区，所述重链可变区包括权利要求1～3任一项所述的HCDR1～HCDR3和所述轻链可变区包括权利要求1～3任一项所述的LCDR1～LCDR3；
    可选地，所述抗原结合片段选自所述抗体的F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
  9. 一种抗体偶联物，其特征在于，其包括如权利要求1～8任一项所述的抗体或其抗原结合片段；
    可选地，所述抗体偶联物还包括与所述抗体或其抗原结合片段偶联的生物素或生物素衍生物；
    可选地，所述抗体偶联物还包括与所述抗体或其抗原结合片段偶联的固相载体；
    可选地，所述抗体偶联物还包括与所述抗体或其抗原结合片段偶联的标记物；
    可选地，所述标记物选自荧光染料、酶、放射性同位素、化学发光试剂和纳米颗粒类标记物中的至少一种；
    可选地，所述标记物为胶体金。
  10. 一种试剂或试剂盒，其特征在于，其包括如权利要求1～8任一项所述的抗体或其抗原结合片段或如权利要求9所述的抗体偶联物；
    可选地，所述试剂或试剂盒还包括第二抗体，所述第二抗体与所述抗体或其抗原结合片段结合；
    可选地，所述试剂或试剂盒还包括第二抗体，所述第二抗体与猴痘病毒或猴痘病毒抗原结合。
  11. 一种检测猴痘病毒或猴痘病毒抗原的方法，其特征在于，其包括：
    将如权利要求1～8任一项所述的抗体或其抗原结合片段或如权利要求9所述的抗体偶联物或如权利要求10所述的试剂或试剂盒与待检测样本中的猴痘病毒或猴痘病毒抗原接触，形成免疫复合物；
    可选地，所述免疫复合物还包括第二抗体，所述第二抗体与所述抗体或其抗原结合片段结合；
    可选地，所述免疫复合物还包括第二抗体，所述第二抗体与猴痘病毒或猴痘病毒抗原结合。
  12. 一种核酸，其特征在于，其编码权利要求1～8任一项所述的抗体或其抗原结合片段。
  13. 一种载体，其特征在于，其含有权利要求12所述的核酸。
  14. 一种细胞，其特征在于，其含有权利要求12所述的核酸或权利要求13所述的载体。
  15. 一种制备权利要求1～8任一项所述的抗体或其抗原结合片段的方法，其特征在于，其包括：培养权利要求14所述的细胞。
  16. 如权利要求1～8任一项所述的抗体或其抗原结合片段、如权利要求9所述的抗体偶联物或如权利要求10所述的试剂或试剂盒在检测猴痘病毒或猴痘病毒抗原或制备检测猴痘病毒或猴痘病毒抗原的产品中的应用。
PCT/CN2023/117997 2022-09-16 2023-09-11 抗猴痘病毒抗体或其抗原结合片段、检测猴痘病毒的试剂和试剂盒 WO2024055928A1 (zh)

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