WO2023131318A1 - Anticorps dirigé contre la covid-19, réactif et kit de détection de la covid-19 - Google Patents

Anticorps dirigé contre la covid-19, réactif et kit de détection de la covid-19 Download PDF

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Publication number
WO2023131318A1
WO2023131318A1 PCT/CN2023/071104 CN2023071104W WO2023131318A1 WO 2023131318 A1 WO2023131318 A1 WO 2023131318A1 CN 2023071104 W CN2023071104 W CN 2023071104W WO 2023131318 A1 WO2023131318 A1 WO 2023131318A1
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Prior art keywords
antibody
seq
optionally
functional fragment
amino acid
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PCT/CN2023/071104
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English (en)
Chinese (zh)
Inventor
孟媛
李蔚芝
钟冬梅
游辉
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东莞市朋志生物科技有限公司
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Publication of WO2023131318A1 publication Critical patent/WO2023131318A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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]
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the technical field of antibodies, in particular, to an anti-new coronavirus antibody, a reagent and a kit for detecting the new coronavirus.
  • the present disclosure provides an anti-new coronavirus or its N protein antibody or functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include the duplicates shown in SEQ ID NO: 15
  • amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 include or are shown in sequence as SEQ ID NO: 1-6.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • Embodiments of the present disclosure also provide vectors comprising the nucleic acid molecules described above.
  • Embodiments of the present disclosure also provide recombinant cells containing the above vectors.
  • Embodiments of the present disclosure also provide a method for detecting novel coronavirus or its N protein in a test sample, comprising:
  • the immune complex further includes a second antibody that binds to the novel coronavirus or its N protein antigen.
  • the disclosure provides an anti-new coronavirus antibody, a reagent and a kit for detecting the new coronavirus, the antibody can specifically bind to the N protein of the new coronavirus, and has a high affinity for it, and the antibody can be used to detect the new coronavirus It has good sensitivity and specificity.
  • the present disclosure provides a richer selection of antibodies for the detection of novel coronaviruses.
  • 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.
  • the pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • the mRNA was extracted from the hybridoma cell line that secretes the antibody against the N protein of the new coronavirus prepared in our laboratory, and the DNA product was obtained by RT-PCR method, and the product was inserted into pMD-18T after adding A reaction with rTaq DNA polymerase
  • the vector was transformed into DH5 ⁇ competent cells, and after the colonies grew out, heavy chain (Heavy Chain) and light chain (Light Chain) gene clones were taken respectively, and each 4 clones were sent to a gene sequencing company for sequencing.
  • the gene sequence obtained by the above sequencing into the kabat antibody database for analysis, and use VNTI11.5 software to analyze and confirm that the genes amplified by the heavy chain and light chain primer pairs are correct, and the genes amplified by the light chain
  • the gene sequence of VL (variable region of light chain) is 321bp, and there is a leader peptide sequence of 57bp in front of it
  • the gene sequence of VH (variable region of heavy chain) is 369bp, belonging to the VH1 gene family, with a 57bp leader peptide sequence in front of it (the sequences of the heavy and light chains of 9B54 are shown in SEQ ID NO: 19, 20, respectively).
  • pcDNA TM 3.4 Vector is a recombinant antibody eukaryotic expression vector constructed.
  • the expression vector has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and named it pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector in the future; according to the above pMD-18T
  • the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, and a 0.72kb light chain was amplified by PCR amplification Gene fragment and 1.40kb heavy chain gene fragment.
  • the cells were first cultured in a 125mL shake flask with an inoculation volume of 30mL, the medium was 100% Dynamis medium, and placed in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and 8% carbon dioxide. Cultivate for 72 hours, inoculate expansion culture at a seeding density of 500,000 cells/mL, the expansion volume is calculated according to production requirements, and the medium is 100% Dynamis medium. Then every 72h expansion once. When the amount of cells meets the production requirements, strictly control the inoculation density to about 500,000 cells/mL for production.
  • Shake flask parameters rotational speed 120r/min, temperature 37°C, carbon dioxide 8%.
  • Fed-batch feeding Feed feeding starts every day when the shake flask is cultured for 72 hours.
  • HyCloneTM Cell BoostTM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day. Replenish until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3g/L on the sixth day. Receive samples on the 13th day.
  • Affinity purification was performed using a protein A affinity column. Take 2 ⁇ g of the purified antibody for reducing SDS-PAGE, and 2 ⁇ g of foreign control antibody as a control.
  • the electropherogram is shown in Figure 1 below. After reducing SDS-PAGE, two bands are displayed, one Mr is 50KD, and the other Mr is 28KD .
  • serial number sequence fragment SEQ ID NO: 1 DYNMG SEQ ID NO: 2 AIIPNNGGTIYNQKFKG SEQ ID NO: 3 EAYRDYDVKTWF SEQ ID NO: 4 SASQGIRNYLN SEQ ID NO: 5 YTSTLHS SEQ ID NO: 6 QQYSKFP SEQ ID NO: 7 EVLLQQSGPELVNPGASVKIPCKASGYTFT SEQ ID NO: 8 WVKQSHGKSLEWIG SEQ ID NO: 9 KATLTVDESSSTAYMELRRSLTSEDTAVYYCAR SEQ ID NO: 10 AYWGQGTLVTVSA SEQ ID NO: 11 DIQMTQTTSSLSASLGDRVTISC SEQ ID NO: 12 WYQQKPDGTVKLLIY SEQ ID NO: 13 GVPSRFSGSGSGTDYSLTISNLEPEDIATYFC SEQ ID NO: 14 YTFGGGTKLEIK

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Pulmonology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente divulgation relève du domaine technique des anticorps et concerne un anticorps dirigé contre la COVID-19, un réactif et un kit pour détecter la COVID-19. L'anticorps dirigé contre la COVID-19 selon la présente divulgation comprend une région déterminant la complémentarité de chaîne lourde et une région déterminant la complémentarité de chaîne légère. L'anticorps a une bonne affinité pour la protéine N de la COVID-19, et l'anticorps est hautement sensible et spécifique pour la détection de la COVID-19. La présente divulgation concerne une sélection d'anticorps supérieure pour la détection de la COVID-19.
PCT/CN2023/071104 2022-01-10 2023-01-06 Anticorps dirigé contre la covid-19, réactif et kit de détection de la covid-19 WO2023131318A1 (fr)

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CN202210022971.0A CN116444657B (zh) 2022-01-10 2022-01-10 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒
CN202210022971.0 2022-01-10

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Cited By (1)

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CN117447602A (zh) * 2023-12-22 2024-01-26 北京索莱宝科技有限公司 猪IgM的抗体及其应用

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CN112239501A (zh) * 2020-10-29 2021-01-19 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒
CN112239500A (zh) * 2020-10-29 2021-01-19 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体和检测新型冠状病毒的试剂盒
CN115197316A (zh) * 2021-11-08 2022-10-18 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒

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CN113150136B (zh) * 2021-04-30 2022-04-01 杭州贤至生物科技有限公司 新型冠状病毒n蛋白单克隆抗体的制备
CN113249337B (zh) * 2021-07-15 2021-10-08 天津一瑞生物科技股份有限公司 鼠抗新型冠状病毒n蛋白杂交瘤细胞株，单克隆抗体及应用

Patent Citations (4)

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CN112239501A (zh) * 2020-10-29 2021-01-19 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒
CN112239500A (zh) * 2020-10-29 2021-01-19 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体和检测新型冠状病毒的试剂盒
CN114426575A (zh) * 2020-10-29 2022-05-03 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体和检测新型冠状病毒的试剂盒
CN115197316A (zh) * 2021-11-08 2022-10-18 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117447602A (zh) * 2023-12-22 2024-01-26 北京索莱宝科技有限公司 猪IgM的抗体及其应用
CN117447602B (zh) * 2023-12-22 2024-03-19 北京索莱宝科技有限公司 猪IgM的抗体及其应用

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