WO2022068895A1 - 抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体及其应用 - Google Patents

抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体及其应用 Download PDF

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WO2022068895A1
WO2022068895A1 PCT/CN2021/121803 CN2021121803W WO2022068895A1 WO 2022068895 A1 WO2022068895 A1 WO 2022068895A1 CN 2021121803 W CN2021121803 W CN 2021121803W WO 2022068895 A1 WO2022068895 A1 WO 2022068895A1
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amino acid
seq
variable region
acid sequences
chain variable
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PCT/CN2021/121803
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French (fr)
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丁莉丹
刘培
陈晖�
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南京金斯瑞生物科技有限公司
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Priority to CN202180067438.XA priority Critical patent/CN116419971A/zh
Publication of WO2022068895A1 publication Critical patent/WO2022068895A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the invention belongs to the field of virus detection and diagnosis, and relates to a monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane.
  • the present invention also relates to the application of the monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane.
  • Severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) belongs to the beta genus of coronavirus, with a diameter of 60nm to 140nm, with a capsule, and the particles are round or oval, often polymorphic Its genetic characteristics are significantly different from SARSr-CoV and MERSrCoV. SARS-CoV-2 is the seventh coronavirus discovered so far that can infect humans. The disease caused by the virus is called novel coronavirus disease 2019 (COVID-19), and as of August 13, 2020, more than 20.52 million cases of COVID-19 have been reported to WHO, according to Worldometer and nearly 740,000 deaths.
  • COVID-19 novel coronavirus disease 2019
  • SARS-CoV-2 also utilizes its highly glycosylated spike protein (Spike protein, S protein, S protein) to complete host cell receptor binding and virus infection in the form of trimers.
  • ECD extra cellular domain
  • the RBD (receptor-binding domain) region of the S1 subunit can recognize and bind to the angiotensin-converting enzyme 2 (human angiotensin-converting enzyme 2, hACE2) of the host cell, and the S2 subunit mediates the membrane fusion between the virus and the host cell.
  • angiotensin-converting enzyme 2 human angiotensin-converting enzyme 2, hACE2
  • nucleic acid testing requires relatively high throat or nasopharyngeal swab sampling. Improper sample collection techniques, storage conditions, and PCR operations may result in false negative and false positive results, resulting in significant delays in early diagnosis and follow-up management. Prompt life support treatment and preventive quarantine pose serious challenges.
  • the present invention provides a monoclonal antibody against the outer membrane region of the SARS-CoV-2 spike protein or a functional fragment thereof, the antibody or functional fragment thereof comprising a heavy chain variable region and a light chain variable region ,in
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3,
  • the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118 or 124 or the same as shown in Variants with up to three (eg, one, two, or three) amino acid mutations in the amino acid sequence;
  • the HCDR2 comprises a variant selected from the group consisting of SEQ ID NOs: 35, 41, 47, 53, 59, 65, 71, 77, 83 , 89, 95, 101, 107, 113, 119, or 125, or a variant with up to three amino acid mutations to the amino acid sequence shown;
  • the HCDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 36, 42, 48 , 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120 or 126 or variants with up to three amino acid mutation
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3,
  • the LCDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, or 127 or the amino acid sequence shown in Variants with up to three amino acid mutations are shown in the amino acid sequence;
  • the LCDR2 sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110,
  • the LCDR3 sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 39, 45, 51, 57, 63, 69, 75 , 81, 87, 93, 99, 105, 111, 117, 123 or 129 or variants with up to
  • the HCDR1 sequence comprises a sequence selected from SEQ ID NO: 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, or 124
  • the amino acid sequence of the HCDR2 sequence comprising amino acids selected from the group consisting of SEQ ID NOs: 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119 or 125
  • the HCDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120 or 126
  • the LCDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the sequence:
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 34, 35 and 36 or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 37, 38 and 39 or variants having up to three amino acid mutations respectively from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 40, 41 and 42 or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 43, 44 and 45 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 49, 50 and 51 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 52, 53 and 54, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 55, 56 and 57 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 58, 59 and 60, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 61, 62, and 63 or variants with up to three amino acid mutations, respectively, from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 64, 65 and 66 or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 67, 68 and 69 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 70, 71 and 72, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 73, 74 and 75 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 76, 77 and 78 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 79, 80 and 81 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 82, 83 and 84, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 comprise, respectively, The amino acid sequences shown in SEQ ID NOs: 85, 86 and 87 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 88, 89 and 90, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 91, 92 and 93 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 94, 95 and 96, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 97, 98 and 99 or variants having up to three amino acid mutations respectively with the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 100, 101 and 102 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 103, 104 and 105 or variants with up to three amino acid mutations respectively from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 106, 107 and 108 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 109, 110, and 111 or variants with up to three amino acid mutations, respectively, from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 112, 113 and 114 or variants having up to three amino acid mutations respectively with the shown amino acid sequences; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 115, 116 and 117 or variants with up to three amino acid mutations respectively from the amino acid sequences shown;
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 118, 119 and 120, respectively, or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences set forth in SEQ ID NOs: 121, 122 and 123 or variants with up to three amino acid mutations, respectively, from the amino acid sequences set forth; or
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 124, 125 and 126 or variants having up to three amino acid mutations with the shown amino acid sequences, respectively; and LCDR1, LCDR2 and LCDR3 respectively comprise The amino acid sequences shown in SEQ ID NOs: 127, 128 and 129 or variants with up to three amino acid mutations respectively from the amino acid sequences shown.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the sequence:
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 34, 35 and 36 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 37, 38 and 39;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 40, 41 and 42 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 43, 44 and 45;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 49, 50 and 51;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 52, 53 and 54 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 55, 56 and 57;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 58, 59 and 60 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 61, 62 and 63;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 64, 65 and 66 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 67, 68 and 69;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 70, 71 and 72 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 73, 74 and 75:
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 76, 77 and 78 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 79, 80 and 81;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 82, 83 and 84 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 85, 86 and 87;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 88, 89 and 90 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 91, 92 and 93;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 94, 95 and 96 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 97, 98 and 99;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 100, 101 and 102 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 103, 104 and 105;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 106, 107 and 108 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 109, 110 and 111;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 112, 113 and 114 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 115, 116 and 117;
  • HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 118, 119 and 120, respectively, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 121, 122 and 123, respectively; or
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 124, 125 and 126, respectively; and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 127, 128 and 129, respectively.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the sequence:
  • amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NOs: 100, 101 and 102 and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 103, 104 and 105;
  • amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NOs: 112, 113 and 114 and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NOs: 115, 116 and 117;
  • the heavy chain variable region sequence comprises and SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or
  • the amino acid sequence shown in 32 has an amino acid sequence with at least 80% identity;
  • the amino acid sequences shown in 23, 25, 27, 29, 31 or 33 have amino acid sequences that are at least 80% identical.
  • the heavy chain variable region sequence comprises and SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or
  • the amino acid sequence shown in 32 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98% or 99% identical amino acid sequence;
  • the light chain variable region sequence comprises the same as SEQ ID NO: 3, 5, 7, 9, 11, 13, 15,
  • the amino acid sequence shown in 17, 19, 21, 23, 25, 27, 29, 31 or 33 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, Amino acid sequences that are 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.
  • the heavy chain variable region sequence comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 The amino acid sequence shown; the light chain variable region sequence comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 show the amino acid sequence.
  • the heavy chain variable region and light chain variable region are selected from the following sequences:
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 2 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 3
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 4 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 5
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 6 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 7
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 8 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 9
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 10 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 11
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 12 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 13
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 14 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 15
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 16 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 17
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 18 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 19
  • the sequence shown has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 20 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 21
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 22 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 23
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • variable region of the heavy chain comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 24 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 25
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 26 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 27
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 28 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 29
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 30 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 31
  • the sequence shown has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences; or
  • the heavy chain variable region comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of the sequence shown in SEQ ID NO: 32 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, said light chain variable region comprising the same as SEQ ID NO: 33
  • the sequence shown has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical amino acid sequences.
  • the heavy chain variable region and light chain variable region are selected from the following sequences:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:5;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 20
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29;
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:30, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:31;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33.
  • the heavy chain variable region and light chain variable region are selected from the following sequences:
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 25;
  • the present invention provides isolated polynucleotides encoding the above-described monoclonal antibodies against the extramembrane region of the SARS-CoV-2 spike protein or functional fragments thereof.
  • the polynucleotide comprises a nucleotide sequence encoding the heavy chain variable region of the above-mentioned anti-SARS-CoV-2 spike protein outer-membrane monoclonal antibody or a functional fragment thereof, and encoding the The nucleotide sequence of the light chain variable region of a monoclonal antibody against the extramembrane region of the SARS-CoV-2 spike protein or a functional fragment thereof.
  • the present invention provides an expression vector comprising the polynucleotide.
  • the present invention provides a host cell or cell-free expression system comprising the expression vector.
  • the present invention provides a pharmaceutical composition comprising the anti-SARS-CoV-2 spike protein monoclonal antibody or its functional fragment and pharmaceutically acceptable vector.
  • the present invention provides the application of the monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane or its functional fragment in the preparation of a drug for the treatment of coronavirus.
  • the present invention provides a kit for detecting coronavirus, which comprises the monoclonal antibody or its functional fragment.
  • the coronavirus is selected from SARS-CoV, MERS-Cov or SARS-Cov-2, preferably SARS-Cov-2. In other embodiments, the coronavirus is SARS-Cov-2.
  • the present invention provides a method for preparing a monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane or a functional fragment thereof, comprising:
  • PBMC peripheral blood mononuclear Cell
  • variable region coding sequence for recombinant antibody production to obtain a functional monoclonal antibody against the outer region of the SARS-CoV-2 spike protein membrane.
  • the monoclonal antibody is a rabbit-derived, chimeric, humanized or human antibody. In some preferred embodiments, the monoclonal antibody is of rabbit origin. In other preferred embodiments, the monoclonal antibody is humanized.
  • the monoclonal antibody against the membrane outer region of the SARS-CoV-2 spike protein developed by the present invention can specifically bind to the membrane outer region of the S protein, and can specifically recognize S1 or S2.
  • the monoclonal antibodies provided can recognize 11 epitopes of antigens, and the diversity of antibodies facilitates the development of detection kits.
  • Fig. 1 is the result figure of the rabbit serum titer detection result after immunization
  • Fig. 2 shows the result of SDS-PAGE identification after purification of some monoclonal antibodies
  • Figure 3 is a graph showing the results of affinity detection between monoclonal antibody and S-ECD recombinant protein
  • Fig. 4 is a paired detection diagram of monoclonal antibody 3C4 and antibody 39G6 on recombinant protein S-ECD;
  • Figure 5 is a graph showing the paired detection of recombinant protein S-ECD by monoclonal antibody 2F8 and antibody 39G6.
  • the present invention relates to a functional monoclonal antibody of the S protein membrane outer region of the virus SARS-CoV-2.
  • the embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • novel coronavirus SARS-CoV-2
  • 2019-nCoV refers to a new type of viral infection that began to appear and spread in 2019. It belongs to the genus beta coronavirus, with an envelope and round or Oval, often pleomorphic, 60-140 nm in diameter. Its genetic characteristics are significantly different from SARSr-Cov and MERSr-CoV. Studies have shown that it shares more than 85% homology with bat SARS-like coronavirus (bat-SL-CoVZC45). When isolated and cultured in vitro, 2019-nCov can be found in human respiratory epithelial cells in about 96 hours, while it takes about 6 days to isolate and culture in Vero E6 and Huh-7 cell lines.
  • antibody is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains (wherein two heavy (H) and two light (L) chains are connected to each other by disulfide bonds (ie, "complete antibody molecule")) , and multimers thereof (eg, IgM) or antigen-binding fragments thereof.
  • Each heavy chain consists of a heavy chain variable region ("HCVR” or “VH”) and a heavy chain constant region (consisting of the domains CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (“LCVR or "VL”) and a light chain constant region (CL).
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), Intervening are more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to hydroxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3 , CDR3, FR4.
  • the FRs of the antibody may be identical to human germline sequences or may be naturally or artificially modified.
  • the term "monoclonal antibody” refers to a homogeneous antibody directed against only a particular epitope. Each monoclonal antibody is directed against a single epitope on an antigen, in contrast to typical polyclonal antibody preparations that include different antibodies directed against different antigenic determinants (epitopes).
  • the modifier "monoclonal” denotes a uniform characteristic of an antibody and is not to be construed as requiring that the antibody be produced by any particular method.
  • the monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods, or obtained by screening methods as described elsewhere herein.
  • mutation refers to a monoclonal antibody or functional fragment thereof comprising an alteration of one or more (several) amino acid residues at one or more (several) positions, ie, a substitution, insertion and/or deletion of a polypeptide.
  • isolated polynucleotide refers to a polynucleotide that is not naturally found in nature, including polynucleotides isolated from nature (including in vivo) by biological techniques, as well as artificially synthesized polynucleotides.
  • An isolated polynucleotide can be genomic DNA, cDNA, mRNA, or other synthetic RNA, or a combination thereof. It should be pointed out that those skilled in the art can design the provided nucleotide sequences with different nucleotide sequences according to the amino acid sequences of the heavy chain variable region and light chain variable region provided herein and based on the degeneracy of codons. nucleotide sequences, but both encode the same amino acid sequence. These altered nucleotide sequences are also included within the scope of the present invention.
  • vector when referring to a polynucleotide refers to any molecule (eg, nucleic acid, plasmid or virus, etc.) used to transfer information encoded by a nucleotide into a host cell.
  • expression vector or “expression cassette” refers to a vector suitable for expressing a gene of interest (nucleotide sequence to be expressed) in a host cell, usually including parts of the gene of interest, promoter, terminator, marker gene and the like.
  • host cell refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a selected gene of interest.
  • the term includes progeny of a parent cell, whether or not the progeny is identical in morphology or genetic composition to the original parent cell, so long as the progeny has the selected gene of interest.
  • Commonly used host cells include bacteria, yeast, mammalian cells, and the like.
  • antibody functional fragment means antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable regions (eg, one or more CDRs) of the parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody.
  • antibody fragments capable of binding the coronavirus spike (S) protein or a portion thereof include, but are not limited to, sdAb (single domain antibodies), Fab (eg, antibodies obtained by papain digestion), F(ab')2 ( For example, by pepsin digestion), Fv or scFv (eg by molecular biology techniques).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and sustained release agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the Standard References in the latest edition of Remington's Pharmaceutical Sciences, which is hereby incorporated by reference in its entirety. Examples of suitable carriers or diluents include, but are not limited to, water, saline solution, ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and hydrophobic-hydrophobic vehicles such as fixed oils can also be used.
  • the use of pharmaceutically active media and agents is well known in the art. Except for those conventional media or agents that are incompatible with the active ingredient, its use in the ingredients can achieve the desired effect.
  • amino acid substitution refers to the replacement of existing amino acid residues with different amino acid residues in a predetermined (original) amino acid sequence.
  • amino acid substitutions are preferably made in accordance with the substitutions shown below:
  • Percent (%) amino acid sequence identity with respect to a peptide or polypeptide sequence is defined as after comparing the sequences and introducing gaps where necessary to obtain maximum percent sequence identity, and without considering any conservative substitutions as part of sequence identity, a candidate The percentage of amino acid residues in a sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence. Alignment of sequences to determine percent amino acid sequence identity can be performed in a variety of ways that are within the skill in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
  • administer and “treat” in reference to an animal, human, subject, cell, tissue, organ or biological fluid, it refers to combining an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, or biological fluid. Person, cell, tissue, organ or biological fluid contact.
  • administering and “treatment” can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cells includes contacting the agent with the cells and contacting the agent with a fluid, wherein the fluid is in contact with the cells.
  • administering and “treating” also mean in vitro and ex vivo treatment of cells, eg, by agents, diagnostic agents, binding compositions, or by other cells.
  • subject refers to an animal, preferably a mammal, more preferably a human, in need of alleviation, prevention and/or treatment of a disease or disorder such as a viral infection.
  • the term includes human subjects with or at risk of infection with a coronavirus such as SARS-CoV-2.
  • the term “effective amount” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • “Pharmaceutically acceptable carrier” refers to a carrier for administration, including various excipients, diluents and buffers, etc., which are suitable for human and/or animal administration without excessive adverse side effects, and at the same time Suitable for maintaining the viability of a drug or active agent located therein.
  • S-ECD spike protein extramembrane domain
  • the animal immunization antigen adopts recombinant protein S-ECD-His (the sequence is shown in SEQ ID NO: 1).
  • New Zealand white rabbits were immunized subcutaneously with 200 ⁇ g of S-ECD fusion protein. Subsequently, the immunization was repeated every other week, so that the experimental rabbits were boosted 3 times in total.
  • the serum titers of the two rabbits reached more than 10 5 after three immunizations (Fig. 1).
  • the experimental rabbit numbered 7967 was selected to collect sterile blood 7 days after the last immunization for subsequent antibody discovery.
  • the enriched cells were plated into a 96-well cell culture plate plated with feeder cells in advance at a density of 10 cells per well. Incubate the plate at 37 °C in 5% CO . After 6 days of incubation, fresh medium was exchanged, and on day 7 the overnight culture supernatants were collected and tested for the presence of antibodies against the extramembrane region of the S protein using ELISA binding as described below.
  • Indirect ELISA was used to assess the binding ability of the antibodies in the supernatant to the S-ECD protein.
  • ELISA plates were coated with 100 ⁇ l/well of recombinant S-ECD protein at 1 ⁇ g/ml in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 150 ⁇ l/well of PBST containing 1% BSA for 14 hours at 37°C. The blocking solution was then discarded and 100 ⁇ l of B cell culture supernatant was added to each plate, followed by incubation at 37°C for 1 hour.
  • RNA from total cells in wells with an OD value greater than 1.0 was extracted using TRIzol (Life Technology, 15596-026) and reverse transcribed into cDNA using universal primers (Prime Script TM 1 st Strand cDNA Synthesis Kit, Takara).
  • the rabbit immunoglobulin heavy and light chain V-region fragments were subsequently amplified by antibody signal peptide and constant region-specific primers, and the resulting PCR fragments were homologously recombined into the pCDNA3.4 vector using vector-specific primer pairs Inserts were sequenced.
  • strains of rabbit IgG antibodies were obtained, which are the unique V-region protein amino acids of 3B4, 2F6, 5D10, 3D5, 3C4, 2F8, 2G4, 5F7, 5E4, 5H7, 2F12, 3F4, 5A10, 3E5, 5C9, and 2E12 clones. sequences and plasmids.
  • 2F6 heavy chain variable region amino acid sequence (SEQ ID NO: 4):
  • 5D10 heavy chain variable region amino acid sequence (SEQ ID NO: 6):
  • 5D10 light chain variable region amino acid sequence (SEQ ID NO: 7):
  • 3D5 heavy chain variable region amino acid sequence (SEQ ID NO: 8):
  • 3C4 heavy chain variable region amino acid sequence (SEQ ID NO: 10):
  • Plasmids containing antibody heavy chain and light chain, respectively, were co-transfected into HEK293-6E (NRC, 11565) cells, and after culturing in shake flasks at 37°C for 6 days, the supernatant was collected for antibody purification.
  • the Protein A column was equilibrated with buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted 1:1 with 2x the above buffer and filter sterilized. The filtered supernatant was incubated with the protein A column for 2 hours at room temperature.
  • the IgG was eluted with sterile 0.1 M sodium citrate (pH 3.5), and the eluate was collected and washed with 1/9. Neutralize with one volume of sterile 1M Tris-HCl (pH 9.0). Under sterile conditions, the product was buffer exchanged to PBS (pH 7.4) to remove residual elution buffer, and antibodies were quantified by OD280nm using an extinction coefficient Ec (0.1%) of 1.43.
  • Purified antibodies were analyzed by SDS-PAGE using a BioRad electrophoresis system using 10% precast gels (GenScript, M42012C). The gel was stained with Estain2.0 (GenScript, L00687R) and the molecular size and purity were estimated by comparing the stained bands with Protein Ladder (Takara, 3452), as shown in Figure 2 for the detection results of 3B4, 3C4, and 5B10 antibodies .
  • Example 5 Binding of monoclonal antibody recombinant supernatant to viral S-ECD protein, S-ECD trimer protein, S-RBD protein, S1 protein and S2 protein
  • Indirect ELISA was used to evaluate antibodies in the supernatant after reconstitution against S-ECD protein (Genscript, Z03481), S-ECD trimer protein (produced by Genscript), S-RBD protein (Genscript, T80302), S1 protein (Genscript, T80302) , Z03485) and S2 protein (produced by Genscript).
  • ELISA plates were coated with 100 ⁇ l/well of 1 ⁇ g/ml of recombinant protein to be tested in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 150 ⁇ l/well of PBST containing 1% BSA for 1 hour at 37°C.
  • the negative judgment standard is that the OD value is less than 2.1 times of the negative control well, and the positive judgment standard is that the OD value is greater than 1.0.
  • the test results are shown in Table 2 ("+” means positive; "-” means negative).
  • S-RBD (SEQ ID NO: 130, NCBI Accession No: QNA38155.1):
  • Example 6 EC50 test of monoclonal antibody binding to viral S-ECD protein
  • Indirect ELISA was used to assess the binding ability of purified antibodies to S-ECD protein.
  • ELISA plates were coated with 100 ⁇ l/well of recombinant S-ECD protein at 0.5 ⁇ g/ml in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 250 ⁇ l/well of PBST containing 1% BSA for 2 hours at 37°C. Subsequently, the blocking solution was discarded, and 100 ⁇ l of 1 ⁇ g/ml purified antibody was added to the first well, and diluted in a 3-fold gradient, for a total of 11 test concentration gradients plus a blank well. It was then incubated at 37°C for 1 hour.
  • Antibody EC50 (ng/ml)
  • Antibody EC50 (ng/ml) 3B4 1.145 5E4 6.144 2F6 1.354 5H7 6.727 5D10 2.198 2F12 7.182 3D5 2.402 3F4 7.723 3C4 2.796 5A10 8.130 2F8 3.029 3E5 8.327 2G4 4.523 5C9 11.873 5F7 4.630 2E12 12.253
  • ELISA Competition ELISA was used to assess epitopes of cell culture supernatants.
  • ELISA plates were coated with 100 ⁇ l/well of recombinant S-ECD protein at 1 ⁇ g/ml in PBS for 2 hours at 37°C. Plates were washed with PBS-T (0.05% Tween) and blocked with 250 ⁇ l/well of PBST containing 1% BSA for 1 hour at 37°C. The blocking solution was then discarded, and 50 ⁇ l of each cell supernatant premixed with horseradish peroxidase-conjugated goat anti-rabbit IgG (Fc-specific) (GenScript, A01856) secondary antibody was added to each well and each cell 50 ⁇ l of supernatant stock solution.
  • Fc-specific horseradish peroxidase-conjugated goat anti-rabbit IgG
  • the difference of OD value is close to 1.0, and there is obvious competition effect.
  • the difference between the OD values of the supernatant B and the supernatant A-HRP mixture was about 0.9, there was an obvious competitive effect. Therefore, it can be determined that supernatant A and supernatant B recognize the same antigenic determinant.
  • the difference between the OD values of the supernatant C and the supernatant A-HRP mixture was not significantly different from the value of well 0, and there was no competition effect. Therefore, it can be determined and analyzed that supernatant C and supernatant A recognize different antigenic determinants of the antigen.
  • ELISA plates were coated with 100 ⁇ l/well of 2.5 ⁇ g/ml of unlabeled purified antibody (eg 3C4 or 2F8) in PBS overnight at 4°C. Plates were washed with PBST (0.05% Tween) and blocked with 250 ⁇ l/well of PBST containing 1% BSA for 2 hours at 37°C. Subsequently, the blocking solution was discarded, and 100 ⁇ l of recombinant S-ECD protein with a concentration of 20 ng/ml, 2-fold dilution and 0 ng/ml in the first well was added, and incubated at 37° C. for 1 hour.
  • PBST 0.05% Tween
  • the plate was washed 4 times with PBST, and a biotin-labeled antibody (such as 39G6-biotin) was added to the wells of the plate, 100 ⁇ l per well, and incubated at 37° C. for 1 hour. Plates were washed 4 times with PBST and incubated with 100 ⁇ l/well of streptavidin HRP (SA-HRP, GenScript) for 15 minutes at 37°C. Plates were finally washed 4 times with PBST, then TMB developer solution (GenScript) was added and incubated at 25°C for 15 minutes in the dark. The reaction was stopped by adding 50 ⁇ l of 1 M HCl stop solution (GenScript).
  • a biotin-labeled antibody such as 39G6-biotin
  • antibodies 3C4 and 39G6 are relatively good paired antibodies, and their sensitivity can reach 0-40pg/ml, which can be used as the development of ELISA kits for double-antibody sandwich detection of antigens.
  • antibodies 2F8 and 39G6 are also good paired antibodies, and their sensitivity can reach 0-40pg/ml.
  • the antibody 39G6 is derived from the rabbit monoclonal antibody specific for RBD in the PCT patent (PCT/CN2021/095228), and the variable region amino acid sequence of the antibody is shown in SEQ ID NO: 135 and SEQ ID NO: 136.

Abstract

属于病毒检测诊断领域，提供抗SARS-CoV-2刺突蛋白膜外区的单克隆杭体、其重链可变区和轻链可变区的氛基酸序列。提供的抗SARS-CoV-2刺突蛋白膜外区的单克隆杭体能够特异性地与S1或S2蛋白膜外区结合，可以用于SARS-CoV-2病毒杭原的检测，为SARS-CoV-2病毒的检测提供了可能和便利。

Description

抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体及其应用 技术领域
本发明属于病毒检测诊断领域，涉及一种抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体。本发明还涉及该抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体的应用。
背景技术
严重急性呼吸***综合征冠状病毒(Severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)属于冠状病毒的β属，直径60nm～140nm，有囊膜，颗粒呈圆形或椭圆形，常为多形性，其基因特征与SARSr-CoV和MERSrCoV有明显区别。SARS-CoV-2是目前发现的第7种可以感染人的冠状病毒。由该病毒引起的疾病称为新型冠状病毒疾病2019(coronavirus disease 2019，COVID-19)，据Worldometer报道，截止2020年8月13日，已向世卫组织报告了超过2052万例COVID-19病例和近74万例死亡。
与SARS-CoV相似，SARS-CoV-2也是利用其高度糖基化的刺突蛋白(Spike protein，S protein，S蛋白)，以三聚体形式完成宿主细胞受体结合和病毒侵染。ECD(extra cellular domain)是S蛋白的膜外区，S蛋白有两个亚基——S1和S2。S1亚基的RBD(receptor-binding domain)区可以识别并结合宿主细胞的血管紧张素转化酶2(human angiotensin-converting enzyme 2，hACE2)，S2亚基介导病毒与宿主细胞的膜融合。早期筛查、早诊断能够及时有效的筛选出感染患者，对感染患者进行有效的隔离和治疗，可以遏制病毒传染进一步扩展。但是，目前仅美国FDA和日本批准了少量可用于SARS-CoV-2病毒抗原检测的试剂，FDA认为，抗原检测将在对抗COVID-19的过程中发挥关键作用。目前国内临床还未批准针对SARS-CoV-2病毒抗原检测的试剂。
基于PCR方法的病毒核酸检测是目前进行COVID-19诊断的金标准，也是无症状感染人群的必要筛查手段。但是，核酸检测对咽喉或鼻咽拭子采样要求比较高，样本采集手法、保存条件、PCR操作不当等均可能造成假阴性和假阳性的结果，导致早期诊断和后续管理的显著延迟，为提供及时的生命支持治疗和预防性检疫提出了严峻挑战。因此，制备抗SARS-CoV-2刺突蛋白ECD的单克隆抗体、开发抗原检测方法进行辅助诊断，与核酸检测相互补充，可弥补核酸检测时出现的假阴性和假阳性，提高疑似病例检测的准确率。中华 人民共和国国家卫生健康委员会《新型冠状病毒肺炎诊疗方案(第7版)》中增加了通过检测新冠病毒蛋白特异性抗体检测来辅助新冠肺炎的临床诊断。
发明内容
在一方面，本发明提供了一种抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体或其功能片段，所述抗体或其功能片段包含重链可变区和轻链可变区，其中
(a)所述重链可变区包含HCDR1、HCDR2和HCDR3，
所述HCDR1包含选自SEQ ID NO：34、40、46、52、58、64、70、76、82、88、94、100、106、112、118或124所示的氨基酸序列或与所示氨基酸序列具有至多三个(例如，一个、二个或三个)氨基酸突变的变体；所述HCDR2包含选自SEQ ID NO：35、41、47、53、59、65、71、77、83、89、95、101、107、113、119或125所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述HCDR3包含选自SEQ ID NO：36、42、48、54、60、66、72、78、84、90、96、102、108、114、120或126所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；以及
(b)所述轻链可变区包含LCDR1、LCDR2和LCDR3，
所述LCDR1序列包含选自SEQ ID NO：37、43、49、55、61、67、73、79、85、91、97、103、109、115、121或127所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述LCDR2序列包含选自SEQ ID NO：38、44、50、56、62、68、74、80、86、92、98、104、110、116、122或128所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述LCDR3序列包含选自SEQ ID NO：39、45、51、57、63、69、75、81、87、93、99、105、111、117、123或129所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体。
在一些实施方案中，所述HCDR1序列包含选自SEQ ID NO：34、40、46、52、58、64、70、76、82、88、94、100、106、112、118或124所示的氨基酸序列，所述HCDR2序列包含选自SEQ ID NO：35、41、47、53、59、65、71、77、83、89、95、101、107、113、119或125所示的氨基酸序列，所述HCDR3序列包含选自SEQ ID NO：36、42、48、54、60、66、72、78、84、90、96、102、108、114、120或126所示的氨基酸序列；以及所述LCDR1序列包含选自SEQ ID NO：37、43、49、55、61、67、73、79、85、91、97、103、109、115、121或127所示的氨基酸序列，所述LCDR2序列包含选自SEQ ID NO：38、44、 50、56、62、68、74、80、86、92、98、104、110、116、122或128所示的氨基酸序列，所述LCDR3序列包含选自SEQ ID NO：39、45、51、57、63、69、75、81、87、93、99、105、111、117、123或129所示的氨基酸序列。
在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下序列：
(a)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、35和36所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：37、38和39所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(b)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：40、41和42所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：43、44和45所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(c)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：46、47和48所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：49、50和51所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(d)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：52、53和54所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：55、56和57所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(e)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：58、59和60所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：61、62和63所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(f)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：64、65和66所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：67、68和69所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(g)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：70、71和72所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：73、74和75所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(h)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：76、77和78所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：79、80和81所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(i)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：82、83和84所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：85、86和87所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(j)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：88、89和90所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：91、92和93所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(k)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：94、95和96所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：97、98和99所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(l)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：100、101和102所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：103、104和105所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(m)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：106、107和108所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：109、110和111所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(n)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：112、113和114所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2 和LCDR3分别包含SEQ ID NO：115、116和117所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
(o)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：118、119和120所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：121、122和123所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；或
(p)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：124、125和126所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：127、128和129所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体。
在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下序列：
(a)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、35和36所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：37、38和39所示的氨基酸序列；
(b)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：40、41和42所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：43、44和45所示的氨基酸序列；
(c)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：46、47和48所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：49、50和51所示的氨基酸序列；
(d)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：52、53和54所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：55、56和57所示的氨基酸序列；
(e)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：58、59和60所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：61、62和63所示的氨基酸序列；
(f)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：64、65和66所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：67、68和69所示的氨基酸序列；
(g)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：70、71和72所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：73、74和75所示的氨基酸序列：
(h)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：76、77和78所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：79、80和81所示的氨基酸序列；
(i)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：82、83和84所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：85、86和87所示的氨基酸序列；
(j)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：88、89和90所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：91、92和93所示的氨基酸序列；
(k)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：94、95和96所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：97、98和99所示的氨基酸序列；
(l)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：100、101和102所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：103、104和105所示的氨基酸序列；
(m)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：106、107和108所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：109、110和111所示的氨基酸序列；
(n)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：112、113和114所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：115、116和117所示的氨基酸序列；
(o)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：118、119和120所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：121、122和123所示的氨基酸序列；或
(p)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：124、125和126所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：127、128和129所示的氨基酸序列。
在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下序列：
(a)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：34、35和36所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：37、38和39所示；
(b)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：40、41和42所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：43、44和45所示；
(c)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：46、47和48所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：49、50和51所示；
(d)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：52、53和54所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：55、56和57所示；
(e)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别与SEQ ID NO：58、59和60所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：61、62和63所示；
(f)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：64、65和66所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：67、68和69所示；
(g)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：70、71和72所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：73、74和75所示；
(h)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：76、77和78所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：79、80和81所示；
(i)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：82、83和84所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：85、86和87所示；
(j)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：88、89和90所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：91、92和83所示；
(k)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别与SEQ ID NO：94、95和96所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：97、98和99所示；
(l)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：100、101和102所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：103、104和105所示；
(m)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别与SEQ ID NO：106、107和108所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：109、110和111所示；
(n)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：112、113和114所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：115、116和117所示；
(o)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：118、119和120所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：121、122和123所示；或
(p)所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：124、125和126所示以及LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：127、128和129所示。
在一些实施方案中，所述重链可变区序列包含与SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30或32所示氨基酸序列具有至少80％一致性的氨基酸序列；以及所述轻链可变区序列包含与SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31或33所示氨基酸序列具有至少80％一致性的氨基酸序列。在一些实施方案中，所述重链可变区序列包含与SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30或32所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；所述轻链可变区序列包含与SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31或33所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列。
在一些实施方案中，所述重链可变区序列包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30或32所示氨基酸序列；所述轻链可变区序列包含SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31或33所示氨基酸序列。
在一些实施方案中，所述重链可变区和轻链可变区选自如下序列：
(a)所述重链可变区包含与SEQ ID NO：2所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：3所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(b)所述重链可变区包含与SEQ ID NO：4所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：5所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(c)所述重链可变区包含与SEQ ID NO：6所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、 98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：7所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(d)所述重链可变区包含与SEQ ID NO：8所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：9所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(e)所述重链可变区包含与SEQ ID NO：10所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：11所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(f)所述重链可变区包含与SEQ ID NO：12所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：13所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(g)所述重链可变区包含与SEQ ID NO：14所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：15所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(h)所述重链可变区包含与SEQ ID NO：16所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：17所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(i)所述重链可变区包含与SEQ ID NO：18所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、 98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：19所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(j)所述重链可变区包含与SEQ ID NO：20所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：21所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(k)所述重链可变区包含与SEQ ID NO：22所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：23所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(l)所述重链可变区包含与SEQ ID NO：24所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：25所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(m)所述重链可变区包含与SEQ ID NO：26所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：27所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(n)所述重链可变区包含与SEQ ID NO：28所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：29所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；
(o)所述重链可变区包含与SEQ ID NO：30所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、 98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：31所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列；或
(p)所述重链可变区包含与SEQ ID NO：32所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：33所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％一致性的氨基酸序列。
在一些实施方案中，所述重链可变区和轻链可变区选自如下序列：
(a)所述重链可变区包含SEQ ID NO：2所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：3所示的氨基酸序列；
(b)所述重链可变区包含如SEQ ID NO：4所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：5所示的氨基酸序列；
(c)所述重链可变区包含如SEQ ID NO：6所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列；
(d)所述重链可变区包含如SEQ ID NO：8所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列；
(e)所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列；
(f)所述重链可变区包含如SEQ ID NO：12所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列；
(g)所述重链可变区包含SEQ ID NO：14所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：15所示的氨基酸序列；
(h)所述重链可变区包含如SEQ ID NO：16所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：17所示的氨基酸序列；
(i)所述重链可变区包含如SEQ ID NO：18所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：19所示的氨基酸序列；
(j)所述重链可变区包含如SEQ ID NO：20所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：21所示的氨基酸序列；
(k)所述重链可变区包含如SEQ ID NO：22所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：23所示的氨基酸序列；
(l)所述重链可变区包含如SEQ ID NO：24所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：25所示的氨基酸序列；
(m)所述重链可变区包含SEQ ID NO：26所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：27所示的氨基酸序列；
(n)所述重链可变区包含如SEQ ID NO：28所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：29所示的氨基酸序列；
(o)所述重链可变区包含如SEQ ID NO：30所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：31所示的氨基酸序列；或
(p)所述重链可变区包含如SEQ ID NO：32所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：33所示的氨基酸序列。
在一个具体实施方案中，所述重链可变区和轻链可变区选自如下序列：
(a)所述重链可变区的氨基酸序列如SEQ ID NO：2所示，所述轻链可变区的氨基酸序列如SEQ ID NO：3所示；
(b)所述重链可变区的氨基酸序列如SEQ ID NO：4所示，所述轻链可变区的氨基酸序列如SEQ ID NO：5所示；
(c)所述重链可变区的氨基酸序列如SEQ ID NO：6所示，所述轻链可变区的氨基酸序列如SEQ ID NO：7所示；
(d)所述重链可变区的氨基酸序列如SEQ ID NO：8所示，所述轻链可变区的氨基酸序列如SEQ ID NO：9所示；
(e)所述重链可变区的氨基酸序列如SEQ ID NO：10所示，所述轻链可变区的氨基酸序列如SEQ ID NO：11所示；
(f)所述重链可变区的氨基酸序列如SEQ ID NO：12所示，所述轻链可变区的氨基酸序列如SEQ ID NO：13所示；
(g)所述重链可变区的氨基酸序列如SEQ ID NO：14所示，所述轻链可变区的氨基酸序列如SEQ ID NO：15所示；
(h)所述重链可变区的氨基酸序列如SEQ ID NO：16所示，所述轻链可变区的氨基酸序列如SEQ ID NO：17所示；
(i)所述重链可变区的氨基酸序列如SEQ ID NO：18所示，所述轻链可变区的氨基酸序列如SEQ ID NO：19所示；
(j)所述重链可变区的氨基酸序列如SEQ ID NO：20所示，所述轻链可变区的氨基酸序列如SEQ ID NO：21所示；
(k)所述重链可变区的氨基酸序列如SEQ ID NO：22所示，所述轻链可变区的氨基酸序列如SEQ ID NO：23所示；
(l)所述重链可变区的氨基酸序列如SEQ ID NO：24所示，所述轻链可变区的氨基酸序列如SEQ ID NO：25所示；
(m)所述重链可变区的氨基酸序列如SEQ ID NO：26所示，所述轻链可变区的氨基酸序列如SEQ ID NO：27所示；
(n)所述重链可变区的氨基酸序列如SEQ ID NO：28所示，所述轻链可变区的氨基酸序列如SEQ ID NO：29所示；
(o)所述重链可变区的氨基酸序列如SEQ ID NO：30所示，所述轻链可变区的氨基酸序列如SEQ ID NO：31所示；或
(p)所述重链可变区的氨基酸序列如SEQ ID NO：32所示，所述轻链可变区的氨基酸序列如SEQ ID NO：33所示。
在另一方面，本发明提供了编码上述的抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体或其功能片段的分离的多核苷酸。
在一些实施方案中，所述多核苷酸包含编码上述抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体或其功能片段的重链可变区的核苷酸序列，和编码所述抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体或其功能片段的轻链可变区的核苷酸序列。
在另一方面，本发明提供了包含所述多核苷酸的表达载体。
在另一方面，本发明提供了包含所述表达载体的宿主细胞或无细胞表达***。
在另一方面，本发明提供了一种药物组合物，所述药物组合物包含所述的抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体或其功能片段和药学上可接受的载体。
在另一方面，本发明提供了所述的抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体或其功能片段在制备治疗冠状病毒药物中的应用。
在另一方面，本发明提供了一种检测冠状病毒的试剂盒，所述试剂盒中包含所述的单克隆抗体或其功能片段。
在一些实施方案中，所述冠状病毒选自SARS-CoV、MERS-Cov或SARS-Cov-2，优选为SARS-Cov-2。在另一些实施方案中，所述冠状病毒为SARS-Cov-2。
在另一方面，本发明提供了一种制备抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体或其功能片段的方法，包括
1)以SARS-CoV-2刺突蛋白膜外区免疫动物，在所述动物中产生针对SARS-CoV-2刺突蛋白膜外区的免疫反应；
2)分离所述动物的外周血单核细胞(PBMC，Peripheral Blood Mononuclear Cell)，富集抗原阳性B细胞，并进行筛选，得到特异性识别SARS-CoV-2刺突蛋白膜外区的阳性克隆：
3)对所述阳性克隆进行基因测序，获得抗SARS-CoV-2刺突蛋白膜外区抗体的重链和轻链的可变区编码序列；
4)用所述可变区编码序列进行重组抗体生产，获得功能性抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体。
在一些实施方案中，所述的单克隆抗体是兔源、嵌合的、人源化的或人的抗体。在一些优选实施方案中，所述的单克隆抗体是兔源的。在另一些优选实施方案中，所述的单克隆抗体是人源化的。
有益效果
与PCR检测相比，血清学检测具有检测周期短、通量高和工作量少的优势。本发明开发的抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体能够特异性地与S蛋白膜外区结合，并且能够特异性的识别S1或者S2。提供的单克隆抗体可以识别抗原的11种表位，抗体的多样性为检测试剂盒的开发提供了便利。
附图说明
图1为免疫之后的兔血清效价检测结果图；
图2为部分单克隆抗体纯化后SDS-PAGE鉴定结果图；
图3为单克隆抗体与S-ECD重组蛋白亲和力检测结果图；
图4为单克隆抗体3C4与抗体39G6对重组蛋白S-ECD配对检测图；
图5为单克隆抗体2F8与抗体39G6对重组蛋白S-ECD配对检测图。
具体实施方式
本发明涉及一种具有功能性的病毒SARS-CoV-2 S蛋白膜外区的单克隆抗体，下面将结合实施例对本发明的实施方案进行详细描述。除非另有说明，本发明所用的技术和科学术语与本发明所属领域的普通技术员通常所理解的含义相同。
术语“新型冠状病毒”(SARS-CoV-2)，亦称为2019-nCoV，是指2019年开始出现并蔓延的新型病毒感染，其属于β属冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm。其基因特征与SARSr-Cov和MERSr-CoV有明显区别。研究显示，其与蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)同源性达85％以上。体外分离培养时，2019-nCov 96个小时左右即可在人呼吸道上皮细胞内发现，而在Vero E6和Huh-7细胞系中分离培养需约6天。
术语″抗体″意在指由四条多肽链组成的免疫球蛋白分子(其中两条重链(H)和两条轻链(L)通过二硫键相互连接(即″完整的抗体分子″))，以及其多聚体(例如IgM)或其抗原结合片段。每条重链由重链可变区(“HCVR”或“VH”)和重链恒定区(由结构域CH1、CH2和CH3组成)组成。每条轻链由轻链可变区(“LCVR或“VL”)和轻链恒定区(CL)组成。VH和VL区可进一步细分为称为互补决定区(CDR)的高变区，其间插有更保守的区称为框架区(FR)。每个VH和VL由三个CDR和四个FR组成，以下列顺序从氨基末端至羟基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。在本发明的一些实施方案中，抗体(或其抗原结合片段)的FR可与人种系序列相同或可经天然或人工修饰。
术语“单克隆抗体”指均一的仅针对某一特定抗原表位的抗体。与典型的包括针对不同抗原决定簇(表位)的不同抗体的普通多克隆抗体制剂相比，每种单克隆抗体针对抗原上的单个抗原决定簇。修饰语“单克隆”表示抗体的均一特征，不解释为需要通过任何特定方法产生的抗体。本发明的单克隆抗体优选通过重组DNA方法产生，或通过本发明其他地方描述的筛选方法获得。
术语“突变”是指单克隆抗体或其功能片段包含一个或多个(数个)位置的一个或多个(数个)氨基酸残基的变更，即取代、***和/或缺失的多肽。取代是指用不同的氨基酸替代占据某位置的氨基酸；缺失是指除去占据某位置的氨基酸；而***是指在占据某位置的氨基酸邻接处且在之后添加1-3个氨基酸。
术语“分离的多核苷酸”指非自然界中天然存在状态的多核苷酸，包括通过生物学技术从自然界(包括生物体内)分离出的多核苷酸，也包括人工合成的多核苷酸。分离的多核苷酸可以是基因组DNA、cDNA、mRNA或合成的其他RNA，或者它们的组合。需要指出的是，本领域技术人员可以根据本文所提供的重链可变区和轻链可变区的氨基酸序列，基于密码子简并性，设计出提供的核苷酸序列不完全相同的核苷酸序列，但都编码相同的氨基酸序列。这些经改动的核苷酸序列也包括在本发明的范围内。
当涉及多核苷酸时，术语“载体”指用于将核苷酸编码信息转移到宿主细胞内的任一种分子(例如核酸、质粒或病毒等)。术语“表达载体”或“表达盒”指适于在宿主细胞内表达目的基因(待表达核苷酸序列)的载体，通常包括目的基因、启动子、终止子、标记基因等部分。
术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等。
术语“抗体功能片段”意即抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。例如，能够结合冠状病刺突(S)蛋白或其部分的抗体片段，包括但不限于sdAb(单域抗体)、Fab(例如，抗体经木瓜蛋白酶消化而得到)、F(ab’)2(例如，通过胃蛋白酶消化得到)、Fv或scFv(例如通过分子生物学技术得到)。
术语“药学上可接受的载体”包括与药物给药相容的任何和所有溶剂，分散剂，包被物，抗细菌和抗真菌药剂，等渗和缓释剂，及其类似物。合适的载体在Remington’s Pharmaceutical Sciences最新版中的标准参考文件中有所叙述，其通过在此引述而全部合并于本文。合适的载体或稀释液例子包括，但不局限于，水，盐溶液，ringer’s液，葡萄糖溶液，和5％人血清白蛋白。也可以使用脂质体和疏-水介质如不挥发油。药物活性物质的介质和药剂的使用在本领域中是熟知的。除了那些对于活性成分不相容的常规介质或试剂以外，其在成分中的使用都可以达到预期效果。
术语“氨基酸替换”，指在预先确定的(初始)氨基酸序列中，用不同的氨基酸残基代替现有的氨基酸残基。一般而言，本领域技术人员公认在多肽非必需区的单个氨基酸取代基本上不改变生物学活性(参见例如Watson等，Molecular Biology ofthe Gene(基因的分子生物学)，The Benjamin/Cummings Pub.Co.，第224页(第四版，1987))。这样的例示性取代优选依照以下所示的取代来进行：
示例性保守氨基酸取代
原残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His
Asp(D) Glu；Asn
Cys(C) Ser；Ala
Gln(Q) Asn
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
关于肽或多肽序列的“百分比(％)氨基酸序列一致性”定义为对比序列并在必要时引入缺口以获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分率。可以本领域技术范围内的多种方式进行序列对比以测定百分比氨基酸序列同一性，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定测量对比的适宜参数，包括对所比较的序列全长获得最大对比所需的任何算法。
当用“给予”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触，其中所述流液与细胞接触。“给予”和“治疗”还意味着例如通过试剂、诊断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
如本文使用的术语“受试者”指需要缓解、预防和/或治疗疾病或病症如病毒感染的动物，优选哺乳动物，更优选人。术语包括具有冠状病毒如SARS-CoV-2感染或处于具有冠状病毒如SARS-CoV-2感染风险的人受试者。
提及药物组合物时，本文所使用的术语“有效量的”指可对人和/或动物产生功能或活性且可被人和/或动物所接受的量。“药学上可接受的载体”指用于给药的载体，包括各种赋形剂、稀释剂和缓冲剂等，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。
除非另外特别说明，否则单数的使用包括复数。除非另外特别说明，否则词语“一个(a)”或“一个(an)”意指“至少一个”。除非另外说明，否则“或”的使用意指“和/或”。短语“至少一个”的含义等同于短语“一个或多个”的含义。此外，术语“包括(including)”以及其他形式诸如“包括(includes)”和“包括(included)”的使用不是限制性的。此外，除非另外特别说明，否则术语诸如“要素”或“组分”包括包含一个单元的元素或组分以及包含多于一个单元的元素和组分。
除非另有说明，下文描述的实施例的方法和材料均为可以通过市场购买获得的常规产品。本发明所属领域技术员将会理解，下文描述的方法和材料，仅是示例性的，而不应视为限定本发明的范围。
实施例1：SARS-CoV-2刺突蛋白膜外区(S-ECD)蛋白动物免疫
动物免疫抗原采用重组蛋白S-ECD-His(序列如SEQ ID NO：1所示)。用含200μg S-ECD融合蛋白皮下免疫新西兰白兔。随后，每隔一周重复免疫，从而对实验兔进行加强免疫，共3次。2只兔血清效价在3次免疫之后均达到10 5以上(图1)。选择编号为7967的实验兔在最后一次免疫后7天采集无菌血进行后续抗体发现工作。
S-ECD-His氨基酸序列(SEQ ID NO：1，NCBI登录号：QLB39105.1)：
Figure PCTCN2021121803-appb-000001
Figure PCTCN2021121803-appb-000002
实施例2：B细胞的获得以及单克隆抗体的筛选
1)抗原阳性B细胞的获得
采集兔无菌血15ml进行PBMC分离，然后采用抗原对抗原阳性细胞进行富集。富集后的细胞按照10个细胞每孔的密度铺到提前铺有饲养细胞的96孔细胞培养板中。将板在37℃下在5％CO 2中孵育。6天的孵育之后，更换新鲜的培养基，第7天收集培养过夜的上清，开始使用下文所述的ELISA结合来测试针对S蛋白膜外区的抗体的存在情况。
2)ELISA结合检测方法
间接ELISA用于评估上清液中抗体对于S-ECD蛋白的结合能力。将ELISA板用100μl/孔的PBS中1μg/ml的重组S-ECD蛋白在4℃下包被过夜。用PBS-T(0.05％吐温)洗涤板，并将其用150μl/孔的含1％BSA的PBST在37℃封闭14、时。随后弃去封闭液，向每个板加入100μlB细胞培养上清液，然后在37℃孵育1小时。将板用PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗兔IgG(Fc-特异性)二抗(GenScript，A01856)37℃孵育0.5小时。将板用PBST洗涤五次，然后加入TMB显色液并在室温下在黑暗中孵育13分钟。通过加入50μl的1M HCl终止液(国药，10011018)终止反应。使用酶标仪在450nm下读板。选择OD值大于1.0的阳性孔进行后续实验。
实施例3：单克隆抗体的可变区测序
使用TRIzol(Life Technology，15596-026)提取OD值大于1.0的孔内总细胞的RNA，并利用通用引物(Prime Script TM 1 st Strand cDNA Synthesis Kit，Takara)将其逆转录为cDNA。随后通过抗体信号肽和恒定区特异性引物扩增兔免疫球蛋白重链和轻链V-区域片段，并将所得的PCR片段通过同源重组至pCDNA3.4载体中，使用载体特异性引物对***片段进行测序。最终获取了16株兔IgG抗体，分别为3B4，2F6，5D10，3D5，3C4，2F8，2G4，5F7，5E4，5H7，2F12，3F4，5A10，3E5，5C9，2E12克隆的独特V-区域蛋白氨基酸序列及质粒。
3B4重链可变区氨基酸序列(SEQ ID NO：2)：
Figure PCTCN2021121803-appb-000003
3B4轻链可变区氨基酸序列(SEQ ID NO：3)：
Figure PCTCN2021121803-appb-000004
2F6重链可变区氨基酸序列(SEQ ID NO：4)：
Figure PCTCN2021121803-appb-000005
2F6轻链可变区氨基酸序列(SEQ ID NO：5)：
Figure PCTCN2021121803-appb-000006
5D10重链可变区氨基酸序列(SEQ ID NO：6)：
Figure PCTCN2021121803-appb-000007
5D10轻链可变区氨基酸序列(SEQ ID NO：7)：
Figure PCTCN2021121803-appb-000008
3D5重链可变区氨基酸序列(SEQ ID NO：8)：
Figure PCTCN2021121803-appb-000009
3D5轻链可变区氨基酸序列(SEQ ID NO：9)：
Figure PCTCN2021121803-appb-000010
3C4重链可变区氨基酸序列(SEQ ID NO：10)：
Figure PCTCN2021121803-appb-000011
3C4轻链可变区氨基酸序列(SEQ ID NO：11)：
Figure PCTCN2021121803-appb-000012
2F8重链可变区氨基酸序列(SEQ ID NO：12)：
Figure PCTCN2021121803-appb-000013
2F8轻链可变区氨基酸序列(SEQ ID NO：13)：
Figure PCTCN2021121803-appb-000014
2G4重链可变区氨基酸序列(SEQ ID NO：14)：
Figure PCTCN2021121803-appb-000015
2G4轻链可变区氨基酸序列(SEQ ID NO：15)：
Figure PCTCN2021121803-appb-000016
5F7重链可变区氨基酸序列(SEQ ID NO：16)：
Figure PCTCN2021121803-appb-000017
5F7轻链可变区氨基酸序列(SEQ ID NO：17)：
Figure PCTCN2021121803-appb-000018
5E4重链可变区氨基酸序列(SEQ ID NO：18)：
Figure PCTCN2021121803-appb-000019
5E4轻链可变区氨基酸序列(SEQ ID NO：19)：
Figure PCTCN2021121803-appb-000020
5H7重链可变区氨基酸序列(SEQ ID NO：20)：
Figure PCTCN2021121803-appb-000021
5H7轻链可变区氨基酸序列(SEQ ID NO：21)：
Figure PCTCN2021121803-appb-000022
2F12重链可变区氨基酸序列(SEQ ID NO：22)：
Figure PCTCN2021121803-appb-000023
2F12轻链可变区氨基酸序列(SEQ ID NO：23)：
Figure PCTCN2021121803-appb-000024
3F4重链可变区氨基酸序列(SEQ ID NO：24)：
Figure PCTCN2021121803-appb-000025
3F4轻链可变区氨基酸序列(SEQ ID NO：25)：
Figure PCTCN2021121803-appb-000026
5A10重链可变区氨基酸序列(SEQ ID NO：26)：
Figure PCTCN2021121803-appb-000027
5A10轻链可变区氨基酸序列(SEQ ID NO：27)：
Figure PCTCN2021121803-appb-000028
3E5重链可变区氨基酸序列(SEQ ID NO：28)：
Figure PCTCN2021121803-appb-000029
3E5轻链可变区氨基酸序列(SEQ ID NO：29)：
Figure PCTCN2021121803-appb-000030
5C9重链可变区氨基酸序列(SEQ ID NO：30)：
Figure PCTCN2021121803-appb-000031
5C9轻链可变区氨基酸序列(SEQ ID NO：31)：
Figure PCTCN2021121803-appb-000032
2E12重链可变区氨基酸序列(SEQ ID NO：32)：
Figure PCTCN2021121803-appb-000033
2E12轻链可变区氨基酸序列(SEQ ID NO：33)：
Figure PCTCN2021121803-appb-000034
兔IgG重链恒定区氨基酸序列(SEQ ID NO：133)：
Figure PCTCN2021121803-appb-000035
兔IgG轻链恒定区氨基酸序列(SEQ ID NO：134)：
Figure PCTCN2021121803-appb-000036
表1抗体的CDR区序列
Figure PCTCN2021121803-appb-000037
Figure PCTCN2021121803-appb-000038
Figure PCTCN2021121803-appb-000039
实施例4：基于重组表达的单克隆抗体的生产
将分别包含抗体重链和轻链的质粒共转染至HEK293-6E(NRC，11565)细胞，于37℃摇瓶中培养6天后，收取上清用于抗体纯化。将蛋白A柱用含有0.05M Tris和1.5M NaCl(pH8.0)的缓冲液平衡。随后将收获的细胞培养物上清液，使用2×上述缓冲液1：1稀释并过滤除菌。将过滤的上清液和蛋白A柱室温孵育2小时，使用1×上述缓冲液洗涤柱后，使用无菌0.1M柠檬酸钠(pH3.5)洗脱IgG，收集洗脱液并用九分之一体积的无菌1M Tris-HCl(pH9.0)中和。在无菌条件下，将所述产品缓冲液交换为PBS(pH7.4)以除去残余的洗脱缓冲液，使用1.43的消光系数Ec(0.1％)通过OD280nm对抗体进行定量。
纯化的抗体通过BioRad电泳***用10％预制胶(GenScript，M42012C)通过SDS-PAGE来分析。将所述凝胶用Estain2.0(GenScript，L00687R)染色并通过比较染色带与Protein Ladder(Takara，3452)来估计分子大小和纯度，如图2所示为3B4，3C4，5B10抗体检测结果图。
实施例5：单克隆抗体重组上清对病毒S-ECD蛋白、S-ECD三聚体蛋白、S-RBD蛋白、S1蛋白和S2蛋白的结合
间接ELISA用于评估重组后上清液中抗体对于S-ECD蛋白(Genscript，Z03481)、S-ECD三聚体蛋白(由Genscript生产)、S-RBD蛋白(Genscript，T80302)、S1蛋白(Genscript，Z03485)和S2蛋白(由Genscript生产)的结合能力。将ELISA板用100μl/孔的PBS中1μg/ml 的重组待检蛋白在4℃下包被过夜。用PBS-T(0.05％吐温)洗涤板，并将其用150μl/孔的含1％BSA的PBST在37℃封闭1小时。随后弃去封闭液，向每个板加入100μl重组表达上清液，然后在37℃孵育1小时。将板用PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗兔IgG(Fc-特异性)二抗(GenScript，A01856)37℃孵育0.5小时。将板用PBST洗涤五次，然后加入TMB显色液并在室温下在黑暗中孵育13分钟。通过加入50μl的1M HCl终止液(国药，10011018)终止反应。使用酶标仪在450nm下读板。选择S-RBD蛋白结合阴性、S-ECD三聚体蛋白或者S1蛋白或者S2蛋白结合阳性的克隆进行后续实验。检测原蛋白氨基酸序列如下。
阴性判断标准为OD值小于阴性对照孔2.1倍，阳性判断标准为OD值大于1.0。检测结果如表2所示(“+”表示阳性；“-”表示阴性)。
表2抗体与S-ECD、S-RBD、S-ECD三聚体、S1及S2蛋白结合检测
抗体 S-ECD结合 S-RBD结合 S-ECD三聚体结合 S1结合 S2结合
3B4 + - + - +
2F6 + - + - +
5D10 + - + - +
3D5 + - + + -
3C4 + - + - +
2F8 + - + + -
2G4 + - + - +
5F7 + - + - +
5E4 + - + + -
5H7 + - + - +
2F12 + - + - +
3F4 + - + - +
5A10 + - + + -
3E5 + - + + -
5C9 + - + - +
2E12 + - + - -
S-RBD(SEQ ID NO：130，NCBI登录号：QNA38155.1)：
Figure PCTCN2021121803-appb-000040
S1(SEQ ID NO：131，NCBI登录号：QKU53385.1)：
Figure PCTCN2021121803-appb-000041
S2(SEQ ID NO：132，NCBI登录号：QNA42621.1)：
Figure PCTCN2021121803-appb-000042
实施例6：单克隆抗体对病毒S-ECD蛋白结合的EC 50测试
间接ELISA用于评估纯化抗体对于S-ECD蛋白的结合能力。将ELISA板用100μl/孔的PBS中0.5μg/ml的重组S-ECD蛋白在4℃下包被过夜。用PBS-T(0.05％吐温)洗涤板，并将其用250μl/孔的含1％BSA的PBST在37℃封闭2小时。随后弃去封闭液，向首孔加入1μg/ml的纯化抗体100μl，并按照3倍梯度稀释，共计11个测试浓度梯度外加一个空白孔。然后在37℃下孵育1小时。将板用PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗兔IgG(Fc-特异性)二抗(GenScript，A01856)37℃孵育0.5小时。ELISA板用PBST洗涤四次，然后加入TMB显色液(GenScript)并在25℃下在黑暗中孵育15分钟。通过加入50μl的1M HCl终止液(国药，10011018)终止反应。使用酶标仪在450nm下读板，各克隆的EC 50检测曲线如图3，EC 50值如表3所示：
表3抗体对S-ECD蛋白结合的EC 50值
抗体 EC 50(ng/ml) 抗体 EC 50(ng/ml)
3B4 1.145 5E4 6.144
2F6 1.354 5H7 6.727
5D10 2.198 2F12 7.182
3D5 2.402 3F4 7.723
3C4 2.796 5A10 8.130
2F8 3.029 3E5 8.327
2G4 4.523 5C9 11.873
5F7 4.630 2E12 12.253
实施例7：单克隆抗体表位鉴定
竞争ELISA用于评估细胞培养上清的抗原表位。将ELISA板用100μl/孔的PBS中1μg/ml的重组S-ECD蛋白在37℃下包被2小时。用PBS-T(0.05％吐温)洗涤板，并将其用250μl/孔的含1％BSA的PBST在37℃封闭1小时。随后弃去封闭液，每孔分别加入每个预先与缀合辣根过氧化物酶的山羊抗兔IgG(Fc-特异性)(GenScript，A01856)二抗混合的细胞上清50μl和每个细胞上清原液50μl。然后在37℃下孵育1小时。将板用PBST洗涤4次，然后加入TMB显色液(GenScript)并在25℃下在黑暗中孵育15分钟。通过加入50μl的1M HCl终止液(GenScript)终止反应。使用酶标仪在450nm下读板。16种抗体共检测到 11种表位，汇总如表4。我们经过第一轮表位归类得到初步的归类结果，在此基础上每个表位选择一个抗体加上剩余的其他未归类抗体进行第二轮归类，表位归类的判断逻辑是：A、B、C代表不同的克隆，假如上清A的自身竞争孔的OD为0.545，与空白孔的值相比，OD值的差值接近1.0，有明显的竞争效果。当上清B和上清A-HRP混合液的OD值的差值大约为0.9，有明显的竞争效果。所以可判定分析出上清A和上清B识别的是相同的抗原决定簇。上清C和上清A-HRP混合液的OD值的差值与0孔的值相比，没有明显差异，没有竞争效果。所以可判定分析出上清C与上清A识别的是抗原的不同抗原决定簇。
表4抗体表位归类结果
Figure PCTCN2021121803-appb-000043
实施例8：单克隆抗体的配对检测
将ELISA板用100μl/孔的PBS中2.5μg/ml的未标记纯化抗体(如3C4或2F8)在4℃下包被过夜。用PBST(0.05％吐温)洗涤板，并将其用250μl/孔的含1％BSA的PBST在37℃封闭2小时。随后弃去封闭液，分别加入首孔为20ng/ml，2倍稀释浓度和0ng/ml重组S-ECD蛋白100μl，在37℃孵育1小时。将板用PBST洗涤4次，将生物素标记的抗体(如39G6-biotin)加入板孔中，每孔加入100μl，37℃孵育1小时。将板用PBST洗涤4次，并用100μl/孔的抗生物素蛋白链菌素HRP(SA-HRP，GenScript)37℃孵育15分钟。最后将板用PBST洗涤4次，然后加入TMB显色液(GenScript)并在25℃下在黑暗中孵育15分钟。通过加入50μl的1M HCl终止液(GenScript)终止反应。使用酶标仪在450nm下读板，具体OD值如表5所示。选择2.5ug/ml的抗体(3C4或2F8)包被板子，然后加入不同浓度的检测原进行反应，最后加入生物素标记的检测抗体(39G6-biotin)，使用SA-HRP进行反应显色。选择抗原为0的孔值低于0.1，检测抗体的值随抗原梯度降低有很好的线性关系(R值大于0.99)，配对结果如图4和图5所示。判定抗体3C4和39G6是一个比较好的配对抗体，其灵敏度可达0-40pg/ml，可以作为双抗体夹心检测抗原的ELISA试剂盒的开发。同时抗体2F8和39G6也是一个比较好的配对抗体，其灵敏度可达0-40pg/ml。其中，抗体39G6为来源于PCT专利(PCT/CN2021/095228)中特异性针对RBD的兔单克隆抗体，其抗体的可变区氨基酸序列如SEQ ID NO：135和SEQ ID NO：136所示。
39G6重链可变区氨基酸序列(SEQ ID NO：135)：
Figure PCTCN2021121803-appb-000044
39G6轻链可变区氨基酸序列(SEQ ID NO：136)：
Figure PCTCN2021121803-appb-000045
表5配对抗体数据
Figure PCTCN2021121803-appb-000046

Claims (18)

  1. 一种抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体或其功能片段，所述抗体或其功能片段包含重链可变区和轻链可变区，其中
    (a)所述重链可变区包含HCDR1、HCDR2和HCDR3，
    所述HCDR1包含选自SEQ ID NO：34、40、46、52、58、64、70、76、82、88、94、100、106、112、118或124所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述HCDR2包含选自SEQ ID NO：35、41、47、53、59、65、71、77、83、89、95、101、107、113、119或125所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述HCDR3包含选自SEQ ID NO：36、42、48、54、60、66、72、78、84、90、96、102、108、114、120或126所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；以及
    (b)所述轻链可变区包含LCDR1、LCDR2和LCDR3，
    所述LCDR1序列包含选自SEQ ID NO：37、43、49、55、61、67、73、79、85、91、97、103、109、115、121或127所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述LCDR2序列包含选自SEQ ID NO：38、44、50、56、62、68、74、80、86、92、98、104、110、116、122或128所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体；所述LCDR3序列包含选自SEQ ID NO：39、45、51、57、63、69、75、81、87、93、99、105、111、117、123或129所示的氨基酸序列或与所示氨基酸序列具有至多三个氨基酸突变的变体。
  2. 根据权利要求1所述的单克隆抗体或其功能片段，其中，
    所述HCDR1序列包含选自SEQ ID NO：34、40、46、52、58、64、70、76、82、88、94、100、106、112、118或124所示的氨基酸序列；所述HCDR2序列包含选自SEQ ID NO：35、41、47、53、59、65、71、77、83、89、95、101、107、113、119或125所示的氨基酸序列；所述HCDR3序列包含选自SEQ ID NO：36、42、48、54、60、66、72、78、84、90、96、102、108、114、120或126所示的氨基酸序列；以及
    所述LCDR1序列包含选自SEQ ID NO：37、43、49、55、61、67、73、79、85、91、97、103、109、115、121或127所示的氨基酸序列；所述LCDR2序列包含选自SEQ ID NO：38、44、50、56、62、68、74、80、86、92、98、104、110、116、122或128所示的氨基酸序列；所述LCDR3序列包含选自SEQ ID NO：39、45、51、57、63、69、75、81、87、93、99、105、111、117、123或129所示的氨基酸序列。
  3. 根据权利要求1或2所述的单克隆抗体或其功能片段，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下序列：
    (a)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、35和36所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：37、38和39所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (b)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：40、41和42所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：43、44和45所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (c)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：46、47和48所示氨基酸序列或与与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：49、50和51所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (d)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：52、53和54所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：55、56和57所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (e)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：58、59和60所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：61、62和63所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (f)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：64、65和66所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：67、68和69所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (g)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：70、71和72所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：73、74和75所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (h)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：76、77和78所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：79、80和81所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (i)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：82、83和84所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：85、86和87所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (j)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：88、89和90所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：91、92和93所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (k)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：94、95和96所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：97、98和99所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (l)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：100、101和102所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：103、104和105所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (m)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：106、107和108所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：109、110和111所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (n)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：112、113和114所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：115、116和117所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；
    (o)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：118、119和120所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2 和LCDR3分别包含SEQ ID NO：121、122和123所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；或
    (p)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：124、125和126所示氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体；以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：127、128和129所示的氨基酸序列或与所示氨基酸序列分别具有至多三个氨基酸突变的变体。
  4. 根据权利要求3中所述的单克隆抗体或其功能片段，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下序列：
    (a)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、35和36所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：37、38和39所示的氨基酸序列；
    (b)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：40、41和42所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：43、44和45所示的氨基酸序列；
    (c)所述HCDR1、HCDR2和HCDR3分别包含SEQ IDNO：46、47和48所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：49、50和51所示的氨基酸序列；
    (d)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：52、53和54所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：55、56和57所示的氨基酸序列；
    (e)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：58、59和60所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：61、62和63所示的氨基酸序列；
    (f)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：64、65和66所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：67、68和69所示的氨基酸序列；
    (g)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：70、71和72所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：73、74和75所示的氨基酸序列；
    (h)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：76、77和78所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：79、80和81所示的氨基酸序列；
    (i)所述HCDR1、HCDR2和HCDR3分别包含SEQ IDNO：82、83和84所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：85、86和87所示的氨基酸序列；
    (j)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：88、89和90所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：91、92和93所示的氨基酸序列；
    (k)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：94、95和96所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：97、98和99所示的氨基酸序列；
    (l)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：100、101和102所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：103、104和105所示的氨基酸序列；
    (m)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：106、107和108所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：109、110和111所示的氨基酸序列；
    (n)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：112、113和114所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：115、116和117所示的氨基酸序列；
    (o)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：118、119和120所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：121、122和123所示的氨基酸序列；或
    (p)所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：124、125和126所示氨基酸序列以及LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：127、128和129所示的氨基酸序列。
  5. 根据权利要求1～4中任一项所述的单克隆抗体或其功能片段，其中，所述重链可变区序列包含与SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30或32所示氨基酸序列具有至少80％一致性的氨基酸序列；以及
    所述轻链可变区序列包含与SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31或33所示氨基酸序列具有至少80％一致性的氨基酸序列。
  6. 根据权利要求5中所述的单克隆抗体或其功能片段，其中，
    所述重链可变区序列包含SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30或32所示的氨基酸序列；以及所述轻链可变区序列包含SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、31或33所示的氨基酸序列。
  7. 根据权利要求1～6中任一项所述单克隆抗体或其功能片段，所述重链可变区和轻链可变区选自如下序列：
    (a)所述重链可变区包含与SEQ ID NO：2所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：3所示序列具有至少80％一致性的氨基酸序列；
    (b)所述重链可变区包含与SEQ ID NO：4所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：5所示序列具有至少80％一致性的氨基酸序列；
    (c)所述重链可变区包含与SEQ ID NO：6所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：7所示序列具有至少80％一致性的氨基酸序列；
    (d)所述重链可变区包含与SEQ ID NO：8所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：9所示序列具有至少80％一致性的氨基酸序列；
    (e)所述重链可变区包含与SEQ ID NO：10所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：11所示序列具有至少80％一致性的氨基酸序列；
    (f)所述重链可变区包含与SEQ ID NO：12所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：13所示序列具有至少80％一致性的氨基酸序列；
    (g)所述重链可变区包含与SEQ ID NO：14所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：15所示序列具有至少80％一致性的氨基酸序列；
    (h)所述重链可变区包含与SEQ ID NO：16所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：17所示序列具有至少80％一致性的氨基酸序列；
    (i)所述重链可变区包含与SEQ ID NO：18所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：19所示序列具有至少80％一致性的氨基酸序列；
    (j)所述重链可变区包含与SEQ ID NO：20所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：21所示序列具有至少80％一致性的氨基酸序列；
    (k)所述重链可变区包含与SEQ ID NO：22所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：23所示序列具有至少80％一致性的氨基酸序列；
    (l)所述重链可变区包含与SEQ ID NO：24所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：25所示序列具有至少80％一致性的氨基酸序列；
    (m)所述重链可变区包含与SEQ ID NO：26所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：27所示序列具有至少80％一致性的氨基酸序列；
    (n)所述重链可变区包含与SEQ ID NO：28所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：29所示序列具有至少80％一致性的氨基酸序列；
    (o)所述重链可变区包含与SEQ ID NO：30所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：31所示序列具有至少80％一致性的氨基酸序列；或
    (p)所述重链可变区包含与SEQ ID NO：32所示序列具有至少80％一致性的氨基酸序列，所述轻链可变区包含与SEQ ID NO：33所示序列具有至少80％一致性的氨基酸序列。
  8. 根据权利要求7所述单克隆抗体或其功能片段，所述重链可变区和轻链可变区选自如下序列：
    (a)所述重链可变区包含SEQ ID NO：2所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：3所示的氨基酸序列；
    (b)所述重链可变区包含如SEQ ID NO：4所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：5所示的氨基酸序列；
    (c)所述重链可变区包含如SEQ ID NO：6所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列；
    (d)所述重链可变区包含如SEQ ID NO：8所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列；
    (e)所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列；
    (f)所述重链可变区包含如SEQ ID NO：12所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列；
    (g)所述重链可变区包含SEQ ID NO：14所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：15所示的氨基酸序列；
    (h)所述重链可变区包含如SEQ ID NO：16所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：17所示的氨基酸序列；
    (i)所述重链可变区包含如SEQ ID NO：18所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：19所示的氨基酸序列；
    (j)所述重链可变区包含如SEQ ID NO：20所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：21所示的氨基酸序列；
    (k)所述重链可变区包含如SEQ ID NO：22所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：23所示的氨基酸序列；
    (l)所述重链可变区包含如SEQ ID NO：24所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：25所示的氨基酸序列；
    (m)所述重链可变区包含SEQ ID NO：26所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：27所示的氨基酸序列；
    (n)所述重链可变区包含如SEQ ID NO：28所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：29所示的氨基酸序列；
    (o)所述重链可变区包含如SEQ ID NO：30所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：31所示的氨基酸序列；或
    (p)所述重链可变区包含如SEQ ID NO：32所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO：33所示的氨基酸序列。
  9. 编码权利要求1～8中任一项所述的抗SARS-CoV-2刺突蛋白膜外区的单克隆抗体或其功能片段的分离的多核苷酸。
  10. 根据权利要求9所述的多核苷酸，其特征在于，所述多核苷酸包含编码所述单克隆抗体或其功能片段的重链可变区的核苷酸序列，和编码所述单克隆抗体或其功能片段的轻链可变区的核苷酸序列。
  11. 包含根据权利要求9或10所述的多核苷酸的表达载体。
  12. 包含根据权利要求11所述表达载体的宿主细胞或无细胞表达***。
  13. 一种药物组合物，所述药物组合物包含权利要求1～8中任一项所述的单克隆抗体或其功能片段和药学上可接受的载体。
  14. 权利要求1～8中任一项所述单克隆抗体或其功能片段在制备治疗冠状病毒药物中的应用。
  15. 根据权利要求14所述的应用，所述冠状病毒选自SARS-CoV、MERS-Cov或SARS-Cov-2，优选为SARS-Cov-2。
  16. 一种检测冠状病毒的试剂盒，所述试剂盒中包含权利要求1～8中任一项所述的单克隆抗体或其功能片段。
  17. 根据权利要求16所述的试剂盒，所述冠状病毒选自SARS-CoV、MERS-Cov或SARS-Cov-2，优选为SARS-Cov-2。
  18. 根据权利要求1～8中任一项所述的单克隆抗体或其功能片段，其特征在于，所述抗体是兔源的、嵌合的、人源化的或者人的。
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