WO2022037616A1 - 新冠病毒rbd特异性单克隆抗体cqts004及其应用 - Google Patents

新冠病毒rbd特异性单克隆抗体cqts004及其应用 Download PDF

Info

Publication number
WO2022037616A1
WO2022037616A1 PCT/CN2021/113261 CN2021113261W WO2022037616A1 WO 2022037616 A1 WO2022037616 A1 WO 2022037616A1 CN 2021113261 W CN2021113261 W CN 2021113261W WO 2022037616 A1 WO2022037616 A1 WO 2022037616A1
Authority
WO
WIPO (PCT)
Prior art keywords
rbd
novel coronavirus
cqts004
monoclonal antibody
specific monoclonal
Prior art date
Application number
PCT/CN2021/113261
Other languages
English (en)
French (fr)
Inventor
金艾顺
李婷婷
韩晓建
王应明
胡超
李胜龙
Original Assignee
重庆医科大学
冯玉林
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 重庆医科大学, 冯玉林 filed Critical 重庆医科大学
Publication of WO2022037616A1 publication Critical patent/WO2022037616A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • 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 invention belongs to the technical field of monoclonal antibodies, and in particular relates to a novel coronavirus RBD-specific monoclonal antibody CQTS004 and applications thereof.
  • Antibodies are immunoglobulin molecules composed of four polypeptide chains, including two heavy chains (H chains) and two light chains (L chains).
  • the H chain consists of a heavy chain variable region (VH) and a heavy chain constant region, and the heavy chain constant region consists of three regions CH1, CH2 and CH3.
  • the L chain is composed of the L chain variable region (VL) and the light chain constant region, and the light chain constant region is composed of the CL region.
  • VH and VL can be further divided into hypervariable regions called complementarity determining regions (CDRs) and alternately conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • SARS-CoV-2 coronavirus
  • N protein spike protein
  • M protein membrane protein
  • E protein receptor binding site
  • the plasma of patients contains high concentrations of specific antigen-neutralizing antibodies, which can neutralize the pathogen of the new coronavirus and mediate an effective immune response after infusion into the patient. Therefore, the use of convalescent plasma is expected to provide an effective treatment for patients infected with the new coronavirus. , reduce the mortality rate and protect the life safety of patients.
  • the Chinese invention patent application with the application publication number CN111303280A discloses a fully human monoclonal antibody against SARS-CoV-2 with high neutralization activity.
  • the above patent provides a fully human monoclonal antibody whose recognition region is the S1 non-RBD region,
  • the fully human monoclonal antibody obtained in the above patent has limited blocking effect on the virus, and the above patent obtains the antibody cDNA by labeling plasma cells, but Compared with plasma cells, memory B cells respond quickly after activation, so memory B cells can trigger a faster and stronger humoral immune response than the initial response, while plasma cells trigger a limited humoral immune response.
  • the purpose of the present invention is to provide a novel coronavirus RBD-specific monoclonal antibody CQTS004 that is directed against RBD and can trigger a stronger humoral immune response and its application.
  • the new coronavirus RBD-specific monoclonal antibody CQTS004 the heavy chain amino acid sequence is shown in SEQ ID NO: 1; the light chain amino acid sequence is shown in SEQ ID NO: 2.
  • the present invention also provides the application of the above-mentioned novel coronavirus RBD-specific monoclonal antibody CQTS004 in the preparation of reagents or vaccines or medicines for detecting or diagnosing SARS-CoV-2, wherein the medicines include the novel coronavirus described in claim 1 RBD-specific monoclonal antibody CQTS004 and pharmaceutical excipients, diluents or carriers; nucleic acid molecules encoding the above-mentioned new coronavirus RBD-specific monoclonal antibody CQTS004 are also provided; expression cassettes, recombinants containing the above-mentioned nucleic acid molecules are also provided Vectors, recombinant bacteria or transgenic cell lines; applications of the above expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines in preparing products are also provided.
  • the present invention also provides a product, including the above-mentioned new coronavirus RBD-specific monoclonal antibody CQTS004; the use of the product is any of the following (b1)-(b4): (b1) combined with the new coronavirus SARS-CoV-2; (b2) ) to detect the binding to the new coronavirus SARS-CoV-2; (b3) to detect the S protein of the new coronavirus SARS-CoV-2; (b4) to detect the S protein of the new coronavirus SARS-CoV-2.
  • the above-mentioned novel coronavirus RBD-specific monoclonal antibody CQTS004 is obtained by sorting RBD-specific memory B cells, and then obtaining antibody variable region cDNA through the mRNA of RBD-specific memory B cells.
  • the monoclonal antibody CQTS004 provided by the present invention has RBD specificity. Compared with the monoclonal antibody against the non-RBD region of S1, the monoclonal antibody CQTS004 provided by the present invention is combined with RBD, which is used for antibody drug screening, diagnosis and prevention. And the treatment of new coronary pneumonia provides a wider range of application value.
  • the monoclonal antibody CQTS004 provided by the present invention is obtained by sorting RBD-specific memory B cells. Compared with the prior art of sorting plasma cells, the monoclonal antibody CQTS004 prepared by the present invention can induce more intense Humoral immune response. In addition, the present invention only performs subsequent RT-PCR, nested PCR and antibody function analysis for RBD-specific memory B cells, which greatly improves the specific binding ability of the monoclonal antibody to RBD.
  • Figure 1 is a cell sorting diagram of memory B cells analyzed by flow cytometry
  • Figure 2 is a cell sorting diagram of RBD-specific memory B cells analyzed by flow cytometry
  • Figure 3 is a gel electrophoresis image of a single-cell antibody gene PCR product
  • Figure 4 is an agarose gel electrophoresis image after PCR amplification of an antibody gene expression cassette comprising CMV promoter, WPRE- ⁇ or WPRE- ⁇ element;
  • FIG. 5 is a graph showing experimental results of RBD specificity.
  • the embodiment provides a novel coronavirus RBD-specific monoclonal antibody CQTS004, the heavy chain amino acid sequence is shown in SEQ ID NO:1; the light chain amino acid sequence is shown in SEQ ID NO:2.
  • This example also provides the application of the above-mentioned novel coronavirus RBD-specific monoclonal antibody CQTS004 in the preparation of reagents or drugs for detecting or diagnosing SARS-CoV-2.
  • the RBD-specific monoclonal antibodies obtained in this example can be used to prepare nucleic acid molecules, or to prepare expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules, or to prepare pharmaceutical compositions.
  • the pharmaceutical composition comprises the above-mentioned novel coronavirus RBD-specific monoclonal antibody CQTS004 and a pharmaceutically acceptable excipient, diluent or carrier.
  • this example obtains related products prepared by RBD-specific monoclonal antibodies, which can be used in any of the following (b1)-(b4): (b1) binding to the new coronavirus SARS-CoV-2; (b2) Detection of binding to the new coronavirus SARS-CoV-2; (b3) detection of the S protein of the new coronavirus SARS-CoV-2; (b4) detection of the S protein of the new coronavirus SARS-CoV-2.
  • the novel coronavirus RBD-specific monoclonal antibody CQTS004 was obtained by the following methods: first, a single RBD-specific memory B cell was obtained from the peripheral blood of recovered patients with novel coronavirus pneumonia, and then the mRNA of the RBD-specific memory B cell was obtained, and then RT- The antibody variable region gene expression cassette was constructed by PCR and nested PCR, and then the antibody variable region gene expression cassette was transfected into 293T cells to express the antibody and the supernatant was collected. The RBD specificity of the supernatant was detected by ELISA, and the RBD specificity was obtained by screening. Monoclonal antibodies.
  • Dead cell dye (Dead Dye) to remove the dead cells of PBMCs obtained by S1, and then use CD19, mIg-G, mIg-D and S-RBD to memory specific and high binding capacity of live RBDs in PBMCs B cells are stained and marked to screen out RBD-specific memory B cells; use flow cytometry to sort specific memory B cells into 96-well plates, each well has one specific memory B cell, and it is in- Store in a freezer at 80°C for later use.
  • Dead Dye dead cell dye
  • the preferred concentration range of Dead Dye staining in this embodiment is 1-2 ⁇ g/mL, and the preferred concentration of Dead Dye staining in this embodiment is 1.5 ⁇ g/mL;
  • CD19 is a B cell marker produced by Biolegend.
  • the concentration range of CD19 is 1-2 ⁇ g/mL, and the preferred concentration of CD19 in this embodiment is 1.5 ⁇ g/mL.
  • mIg-G is a B cell labeled surface receptor produced by Biolegend, and the concentration range during staining is 1-2 ⁇ g/mL.
  • the preferred concentration of mIg-G staining is 1.5 ⁇ g/mL; mIg-D is produced by Biolegend.
  • the concentration range of staining is 1-2 ⁇ g/mL, in this embodiment, the preferred concentration of mIg-D staining is 1.5 ⁇ g/mL;
  • S-RBD is the new coronavirus produced by sinobiological, which is a protein receptor domain , the concentration range during staining is 1-2 ⁇ g/mL, and in this embodiment, the preferred concentration of S-RBD during staining is 1.5 ⁇ g/mL.
  • the principle of using CD19, mIg-G, mIg-D and S-RBD to screen out RBD-specific memory B cells is as follows: PBMCs are treated with dead cell dye (Dead Dye), B cell marker CD19, memory B cells The cell markers mIg-G positive and mIg-D negative and memory B cells expressing RBD-specific IgG were stained, and then the CD19 cell population in the cell population was divided by flow cytometry, and then the CD19 cell population was separated from the CD19 positive cell population.
  • Dead Dye dead cell dye
  • the mRNA of a single RBD-specific memory B cell is obtained by sorting, and the variable region cDNA of the antibody is obtained by RT-PCR amplification.
  • the primer front section of the primer designed in this example is designed with a general leader (see Primer Sequence Table 1 and Primer Sequence Table 2), which effectively improves the amplification of antibody genes.
  • the experimental results are shown in Figure 3.
  • S3 and S4 were performed in a total of six parts: (1) extraction of mRNA from RBD-specific memory B cells; (2) single cell mRNA reverse transcription (RT); (3) G-tailing (TDT); (4) first Round PCR (1st PCR); (5) Second round PCR (2nd PCR); (6) BCR-ORF PCR amplification to construct gene expression cassette; (7) CMV, WPRE- ⁇ / ⁇ /l fragment amplification and CMV , BCR-V ⁇ / ⁇ /l (product of (6)), WPRE- ⁇ / ⁇ /l overlapping PCR (Overlap PCR) pre-ligation; (8) BCR- ⁇ ORF, BCR- ⁇ ORF, BCR-1 PCR amplification.
  • 2Washing Take out the Dynabeads oligo(dT) 25 microsphere bottle, vortex and mix well, suck up enough microspheres according to 2 ⁇ l/well, place it on the magnet block, let it stand for 30s, discard the supernatant, reconstitute it with 500 ⁇ l Lysis Buffer hanging;
  • Dispense Dispense the microspheres with an eight-connected tube, and then add 9 ⁇ l/well of the microspheres to the cell plate using a discharge gun;
  • Washing with Wash B Add Washing Buffer B at 8 ⁇ l/well, go back and forth 7-8 times to wash the microspheres thoroughly, discard the supernatant, and then add the pre-prepared reverse transcription (RT) reaction solution at 10 ⁇ l/well. Reagent preparation and reaction conditions are described in (2) below.
  • the 96-well plate was centrifuged at 600 ⁇ g for a short time, and then the 96-well plate was placed on the DynaMag TM -96side Magnet magnetic plate.
  • the formulation and reaction conditions are as described in (3) below.
  • the 96-well plate was centrifuged at 600 ⁇ g for a short time, and then placed on a DynaMag TM -96side Magnet magnetic plate. The supernatant was discarded with a row gun, and then 10 ⁇ l/well was added to the pre-prepared first-round PCR (1st PCR)
  • the reaction solution, reagent preparation and reaction conditions are described in (4) below.
  • the experimental reaction conditions of 1st PCR are: 1 95°C pre-denaturation for 3 min; 2 95°C denaturation for 15sec, 60°C annealing for 5sec, 72°C extension for 1min, 30-35 cycles, preferably 30 cycles in this example; 3 72°C external extension for 5min, Store at 4°C.
  • the experimental reaction conditions of 2nd PCR are as follows: (1) Pre-denaturation at 95°C for 3 min; (2) Denaturation at 95°C for 15sec, annealing at 60°C for 5s, extension at 72°C for 1min, 30-35cycles, 35cycles is preferred in this example; Store at 4°C.
  • CMV promoter CMV promoter
  • WPRE- ⁇ antibody gamma chain
  • WPRE- ⁇ antibody kappa chain
  • PCR amplification conditions were: 1 pre-denaturation at 95°C for 3 min; 2 denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, extension at 72°C for 1 min, 30 cycles; 3 external extension at 72°C for 5 min, and storage at 12°C.
  • PCR amplification conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 50°C for 15 sec, extension at 72°C for 1.5 min, 10 cycles; external extension at 72°C for 5 min, and storage at 12°C.
  • PCR amplification program pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 1.5 min, 30 cycles; extension at 72°C for 5 min, and storage at 12°C.
  • BCR- ⁇ ORF and BCR- ⁇ /ORF ethanol precipitation Take 30 ⁇ l of PCR products of BCR- ⁇ ORF and BCR- ⁇ ORF and put them in 8-connected tubes, then add 120 ⁇ l of absolute ethanol and 6 ⁇ l of sodium acetate solution, mix well, -80 °C stand for 30 min; 10000 rpm, centrifuge for 20 min, discard the supernatant, rinse with 200 ⁇ l of 70% ethanol and anhydrous ethanol in turn, fully volatilize the ethanol at 56 °C, add 40 ⁇ l of sterile water, shake to fully dissolve the precipitate, and detect The concentration of antibody variable region genes.
  • the Leader primers used in S3 and S4 are shown in the following primer sequence table 1:
  • the J-region primers used in S3 and S4 are shown in the following primer sequence table 2:
  • Blocking 80 ⁇ l of 5% BSA (BioFroxx, Cat. NO: 4240GR100) (prepared with PBST) was added to the above washed plate and placed in a 37° C. incubator for 1 h. Machine wash plates in PBST or hand wash.
  • BSA BioFroxx, Cat. NO: 4240GR100
  • PNPP diethanol aminesubstrate Buffer

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Communicable Diseases (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Oncology (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明属于单克隆抗体技术领域，具体公开了新冠病毒RBD特异性单克隆抗体CQTS004以及上述新冠病毒RBD特异性单克隆抗体CQTS004的应用。本发明对于新型冠状病毒SARS-CoV-2引起疾病的预防、临床治疗和诊断试剂的研发均具有重要的科学意义和应用前景。

Description

新冠病毒RBD特异性单克隆抗体CQTS004及其应用 技术领域
本发明属于单克隆抗体技术领域，尤其涉及一种新冠病毒RBD特异性单克隆抗体CQTS004及其应用。
背景技术
抗体是由四条多肽链组成的免疫球蛋白分子，四条多肽链包括两条重链(H链)和两条轻链(L链)。H链由重链可变区(VH)和重链恒定区组成，重链恒定区由三个区CH1、CH2和CH3组成。L链由L链可变区(VL)和轻链恒定区组成，轻链恒定区由CL区组成。VH和VL可进一步分成称为互补决定区(CDRs)的高变区和称为构架区(FR)交替分布的保守区。
目前研究发现：新冠病毒(SARS-CoV-2)具有四种主要的结构蛋白，分别为刺突蛋白(S蛋白)、核衣壳蛋白(N蛋白)、膜蛋白(M蛋白)和包膜蛋白(E蛋白)，其中S蛋白有两个亚基：S1和S2，受体结合位点(RBD)位于S1亚基上，其主要功能是识别宿主细胞表面受体，介导与宿主细胞的融合。
患者血浆中含有高浓度特异性的抗原中和抗体，输入患者体内后，可以中和新冠病原体，介导有效的免疫反应，因此利用恢复期血浆有望成为救治感染新冠病毒的患者提供有效的治疗手段，降低死亡率，保障患者生命安全。
申请公开号为CN111303280A的中国发明专利申请公开了一种高中和活性抗SARS-CoV-2全人源单克隆抗体，上述专利提供的是识别区域为S1非RBD区的全人源单克隆抗体，但是由于新冠病毒入侵宿主细胞是通过RBD与宿主细胞的ACE2结合，所以上述专利获得的全人源单克隆抗体对病毒的阻断效果有限，并且上述专利是通过标记浆细胞而获得抗体cDNA，但是与浆细胞相比，是记忆B细胞活化后迅速做出反应，所以记忆B细胞能够引发比初次反应更快，也更强烈的体液免疫反应，而浆细胞所引发的体液免疫反应有限。
发明内容
本发明的目的在于提供一种针对RBD，并且能够引发更强烈体液免疫反应的新冠病毒RBD特异性单克隆抗体CQTS004和应用。
为了达到上述目的，本发明的技术方案如下：
新冠病毒RBD特异性单克隆抗体CQTS004，重链氨基酸序列如SEQ ID NO:1所示；轻链氨基酸 序列如SEQ ID NO:2所示。
本发明还提供了上述新冠病毒RBD特异性单克隆抗体CQTS004，在制备检测或诊断SARS-CoV-2试剂或疫苗或药物中的应用，其中药物包括权利要求1所述权利要求所述的新冠病毒RBD特异性单克隆抗体CQTS004和药学上的赋形剂、稀释剂或载体；还提供了编码上述新冠病毒RBD特异性单克隆抗体CQTS004的核酸分子；还提供了含有上述核酸分子的表达盒、重组载体、重组菌或转基因细胞系；还提供了上述表达盒、重组载体、重组菌或转基因细胞系在制备产品中的应用。
本发明还提供了产品，包括上述新冠病毒RBD特异性单克隆抗体CQTS004；产品用途如下(b1)-(b4)中的任一种：(b1)结合新型冠状病毒SARS-CoV-2；(b2)检测结合新型冠状病毒SARS-CoV-2；(b3)结合新型冠状病毒SARS-CoV-2的S蛋白；(b4)检测新型冠状病毒SARS-CoV-2的S蛋白。
优选的，上述新冠病毒RBD特异性单克隆抗体CQTS004均通过分选RBD特异性记忆B细胞，再通过RBD特异性记忆B细胞的mRNA获得抗体可变区cDNA。
本发明的原理和有益效果在于：
(1)本发明提供的单克隆抗体CQTS004具有RBD特异性，与针对S1非RBD区的单可隆抗体相比，本发明提供的单克隆抗体CQTS004与RBD结合，为抗体药物筛选，诊断、预防和治疗新冠肺炎提供了更加广泛的应用价值。
(2)本发明提供的单克隆抗体CQTS004是通过分选RBD特异性记忆B细胞而得到，与通过分选浆细胞的现有技术相比，本发明制备的单克隆抗体CQTS004能够引发更强烈的体液免疫反应。另外，本发明只针对RBD特异性记忆B细胞进行后续RT-PCR、巢式PCR和抗体功能分析，大大提高了单克隆抗体与RBD特异性结合能力。
附图说明
图1为采用流式细胞仪分析记忆B细胞的细胞分选图；
图2为采用流式细胞仪分析RBD特异性记忆B细胞的细胞分选图；
图3为单细胞抗体基因PCR产物凝胶电泳图；
图4为PCR扩增包含CMV启动子、WPRE-γ或WPRE-κ元件抗体基因表达盒后的琼脂糖凝胶电泳图；
图5为RBD特异性的实验结果图。
具体实施方式
下面将结合本发明实施例的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领 域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
实施例提供一种新冠病毒RBD特异性单克隆抗体CQTS004，重链氨基酸序列如SEQ ID NO:1所示；轻链氨基酸序列如SEQ ID NO:2所示。
本实施例还提供了上述新冠病毒RBD特异性单克隆抗体CQTS004，在制备检测或诊断SARS-CoV-2试剂或药物中的应用。
在实际生产时，可以采用本实施例得到RBD特异性单克隆抗体制备核酸分子，或者制备包含该核酸分子的表达盒、重组载体、重组菌或转基因细胞系，或者制备药物组合物。该药物组合物包括上述新冠病毒RBD特异性单克隆抗体CQTS004和药学上可接受的赋形剂、稀释剂或载体。
在应用时，本实施例得到RBD特异性单克隆抗体制备的相关产品，可具有如下(b1)-(b4)中的任一种的用途：(b1)结合新型冠状病毒SARS-CoV-2；(b2)检测结合新型冠状病毒SARS-CoV-2；(b3)结合新型冠状病毒SARS-CoV-2的S蛋白；(b4)检测新型冠状病毒SARS-CoV-2的S蛋白。
新冠病毒RBD特异性单克隆抗体CQTS004通过以下方法得到：首先从新冠肺炎康复患者的外周血中分选得到单个RBD特异性记忆B细胞，然后获得RBD特异性记忆B细胞的mRNA，再通过RT-PCR和巢式PCR构建抗体可变区基因表达盒，再将抗体可变区基因表达盒转导入293T细胞表达抗体并收集上清，用ELISA法检测上清的RBD特异性，筛选得到RBD特异性单克隆抗体。
具体包括以下步骤：
S1、采集若干名新冠肺炎康复患者外周血，分离得到PBMC，在-80℃的冰箱中冻存备用。
S2、首先采用去死细胞染料(Dead Dye)去除S1得到的PBMC的死细胞，然后采用CD19、mIg-G、mIg-D和S-RBD对PBMC中活的RBD特异性并且结合能力高的记忆B细胞染色标记，筛选出针对RBD特异性记忆B细胞；使用流式细胞分选仪将特异性记忆B细胞分选到96孔板上，每个孔内有一个特异性记忆B细胞，在-80℃的冰箱中冻存备用。
具体地，本实施例优选的Dead Dye染色时的浓度范围为1-2μg/mL，本实施例优选Dead Dye染色时的浓度为1.5μg/mL；CD19为Biolegend生产的B细胞标记物，染色时的浓度范围为1-2μg/mL，本实施例优选CD19染色时的浓度为1.5μg/mL。mIg-G为Biolegend生产的B细胞标表面受体，染色时的浓度范围为1-2μg/mL，本实施例优选mIg-G染色时的浓度为1.5μg/mL；mIg-D为Biolegend生产的B细胞表面受体，染色时的浓度范围为1-2μg/mL，本实施例优选mIg-D染色时的浓度为1.5μg/mL；S-RBD为sinobiological生产的新冠病毒是蛋白受体结构域，染色时的浓度范围为1-2μg/mL，本实施例优选S-RBD染色时的浓度为1.5μg/mL。
通过流式细胞仪分选RBD特异性记忆B细胞的，通过CD19、mIg-G、mIg-D和S-RBD对PBMC 的细胞分选得到对S-RBD具有特异性记忆B细胞的细胞分选图如图1和图2所示，其中图2中的Batch ID 0428、0505、0522、0528是筛选批次。本实施例采用CD19、mIg-G、mIg-D和S-RBD筛选出针对RBD特异性记忆B细胞的原理在于：将PBMC用去死细胞染料(Dead Dye)、B细胞标记物CD19、记忆B细胞标记物mIg-G阳性和mIg-D阴性以及RBD特异性IgG表达的记忆B细胞进行染色，然后使用流式细胞分析仪将细胞群中CD19细胞群划分出来，再通过从CD19阳性细胞群中划分mIg-G +mIg-D -细胞群，再从mIg-G +mIg-D -细胞群划分RBD阳性的记忆B细胞，再通过流式细胞分选仪将RBD阳性的记忆B细胞进行分选。
S3、分选得到单个RBD特异性记忆B细胞的mRNA，采用RT-PCR扩增获得抗体可变区cDNA。具体地，使用RT-PCR扩增抗体可变区cDNA时，本实施例所设计的引物的引物前段设计通用Leader(参见引物序列表一和引物序列表二)，有效提高了抗体基因的扩增率，实验结果如图3所示。
S4、采用巢式PCR扩增S1-S3得到的抗体可变区cDNA，构建抗体可变区基因表达盒。
S3和S4总共通过以下六部分进行：(1)提取RBD特异性记忆B细胞的mRNA；(2)单个细胞mRNA逆转录(RT)；(3)加G尾(TDT)；(4)第一轮PCR(1st PCR)；(5)第二轮PCR(2nd PCR)；(6)BCR-ORF PCR扩增构建基因表达盒；(7)CMV、WPRE-γ/κ/l片段扩增及CMV、BCR-Vγ/κ/l((6)的产物)、WPRE-γ/κ/l重叠PCR(Overlap PCR)预连接；(8)BCR-γORF、BCR-κORF、BCR-lPCR扩增。
各部分反应液配制及反应条件如下：
(1)采用Dynabeads TM mRNA DIRECT TM Purification Kit(Thermo Fisherscientific)进行单细胞mRNA提取，具体包括以下步骤：
①离心：从-80℃冰箱中取出分选有单个RBD特异性记忆B细胞的96孔板后，600×g离心30s，使细胞离心于孔底部；
②清洗：将Dynabeads oligo(dT)25微球瓶取出后涡旋混匀，按照2μl/孔吸取足量微球，放置于磁铁块上，静置30s，弃上清，用500μl的Lysis Buffer重悬；
③配制：按照9μl/孔Lysis Buffer加入到50mL的离心管中，将上述500μl微球悬液加入其中，用枪吹匀；
④分装：用八连管分装微球，随后采用排枪将其按照9μl/孔加入到细胞板中；
⑤润洗：96孔板贴膜，随后润洗管壁四周，共2个循环；
⑥孵育：室温静置5min，使RBD特异性记忆B细胞的mRNA充分释放并结合到微球上，孵育结束后，600×g瞬时离心，使微球离心于孔底部。将96孔板放置于DynaMag TM-96side Magnet磁板上，用排枪吸弃上清；
⑦Wash A清洗：按照8μl/孔加入Washing Buffer A，来回走板7-8次，使微球充分洗涤，弃上清；
⑧Wash B清洗：按照8μl/孔加入Washing Buffer B，来回走板7-8次，使微球充分洗涤，弃上清，随后按照10μl/孔加入预先配制的逆转录(RT)反应液。试剂配制及反应条件如下述(2)描述。
(2)逆转录(RT)(10μl体系)：所需配制的试剂如下表1所示。
试剂名称 体积
DEPC-H 2O 4.5μl
5×primerscript Buffer 2.0μl
2.5mM dNTP 2.0μl
RNase Inhibitor 1μl
Sample beads
PrimerScriptⅡRTase 0.5μl
总体积 10μl
反应条件：42℃for 60min(每20min混合一次)。
反应结束后，600×g瞬时离心96孔板，然后将96孔板放置于DynaMag TM-96side Magnet磁板上，用排枪吸弃上清，随后按照10μl/孔加入预先配制的TDT反应液，试剂配制及反应条件如下述(3)描述。
(3)加G尾(TDT)(10μl体系)：所需配制的试剂如下表2所示。
试剂名称 体积
H 2O 6.4μl
5×TdT buffer 2.0μl
10mM dGTP 0.5μl
0.1％BSA 1.0μl
Sample beads
TdT 0.1μl
总体积 10μl
反应条件：37℃for 40min(每20min混合一次)。
反应结束，600×g瞬时离心96孔板，然后将其放置于DynaMag TM-96side Magnet磁板上，用排枪吸弃上清，随后按照10μl/孔加入预先配制的第一轮PCR(1st PCR)反应液，试剂配制及反应条件如下述(4)描述。
(4)1st PCR(10μl体系)(引物序列参见引物序列表)：所需配制的试剂如下表3所示：
试剂名称 体积
H 2O 1.9μl
2×GC Buffer 5μl
2.5mM dNTP 1μl
FP:AP3-dC(10μM) 0.5μl
RP1:Cg-1st(10μM) 0.5μl
RP2:Ck-1st(10μM) 0.5μl
RP3:CI-RT(10μM) 0.5μl
PrimesTAR 0.1μl
sample beads
总体积 10μl
基于PCR原理，1st PCR的实验反应条件为：①95℃预变性3min；②95℃变性15sec，60℃退火5sec，72℃延伸1min，30-35cycles，本实施例优选30cycles；③72℃循环外延伸5min，4℃保存。
(5)第二轮PCR(2nd PCR)(10μl体系)(引物序列参见引物序列表一和引物序列表二)：所需配制的试剂如下表4所示：
试剂名称 体积
H 2O 1.5μl
2×GC Buffer 5μl
2.5mM dNTP 1μl
FP:MAC-AP3/AP3(10μM) 0.5μl
RP:Cg-nest/K20/CI-nest(10μM) 0.5μl
PrimesTAR 0.5μl
sample 1μl
总体积 10μl
基于PCR原理，2nd PCR的实验反应条件为：①95℃预变性3min；②95℃变性15sec，60℃退火5s，72℃延伸1min，30-35cycles，本实施例优选35cycles；72℃循环外延伸5min，4℃保存。
PCR结束后：每孔取4μl进行1.5％琼脂糖凝胶电泳。将Gamma链与Kappa链或Lamada链配对的细胞孔送测序。
(6)抗体表达盒(BCR-ORF)的扩增和构建
PCR扩增启动子区(CMV启动子)、WPRE-γ(抗体gamma链)和WPRE-κ(抗体kappa链)，PCR扩增体系如下表5所示：
Figure PCTCN2021113261-appb-000001
PCR扩增条件为：①95℃预变性3min；②95℃变性15sec，56℃退火15sec，72℃延伸1min，30cycles；③72℃循环外延伸5min，12℃保存。
(7)CMV、WPRE-γ/κ/l片段扩增及CMV、BCR-Vγ/κ/l、WPRE-γ/κ/l重叠PCR(Overlap PCR)预连接，实验体系如下表6所示：
Figure PCTCN2021113261-appb-000002
PCR扩增条件为：95℃预变性3min；95℃变性15sec，50℃退火15sec，72℃延伸1.5min，10cycles；72℃循环外延伸5min，12℃保存。
(8)BCR-γORF、BCR-κORF、BCR-lPCR扩增：实验体系如下表7所示：
Figure PCTCN2021113261-appb-000003
PCR扩增程序：95℃预变性3min；95℃变性15sec，58℃退火15sec，72℃延伸1.5min，30cycles； 72℃循环外延伸5min，12℃保存。
扩增后，采用琼脂糖凝胶电泳，凝胶成像分析得到的抗体可变区基因大小是否正确，实验结果如图4所示，Marker在中间位置，条带在5000bp处。
BCR-γORF和BCR-κ/ORF乙醇沉淀：BCR-γORF和BCR-κORF的PCR产物各取30μl置于8连管中，再加入120μl无水乙醇，6μl醋酸钠溶液，充分混匀，-80℃静置30min；10000rpm，离心20min，弃上清，依次用200μl的70％乙醇和无水乙醇各漂洗一次，于56℃乙醇充分挥发，加入40μl无菌水，振荡，使沉淀充分溶解，检测抗体可变区基因的浓度。
S3和S4所用到的Leader引物参见如下的引物序列表一：
primer ID sequence
GV_01 CGGTACCGCGGGCCCGGGAatggactggacctggagcatccttttcttggtggc
GV_02 CGGTACCGCGGGCCCGGGAatggactggacctggaggatcctcttcttggtggc
GV_03 CGGTACCGCGGGCCCGGGAatggactgcacctggaggatcctcttcttggtggc
GV_04 CGGTACCGCGGGCCCGGGAatggactggaactggaggatcctttttttggtggt
GV_05 CGGTACCGCGGGCCCGGGAatggactggacctggagaatcctcttcttggtggc
GV_06 CGGTACCGCGGGCCCGGGAatggactggacctggagggtcttctgcttgctggc
GV_07 CGGTACCGCGGGCCCGGGAatggactggatttggaggatcctcttcttggtggg
GV_08 CGGTACCGCGGGCCCGGGAatggactggacctggaggttcctctttgtggtggc
GV_09 CGGTACCGCGGGCCCGGGAatggacacgttttgctccacactcctgctgctgac
GV_10 CGGTACCGCGGGCCCGGGAatggacacactttgctacacactcctgctgctgac
GV_11 CGGTACCGCGGGCCCGGGAatggacacactttgctccacgctcctgctgctgac
GV_12 CGGTACCGCGGGCCCGGGAatggacatactttgttccacgctcctgctactgac
GV_13 CGGTACCGCGGGCCCGGGAatggagtttgggctgagctgggttttccttgttgc
GV_14 CGGTACCGCGGGCCCGGGAatggagttggggctgagctgggttttccttgttgc
GV_15 CGGTACCGCGGGCCCGGGAatggaatttgggctgagctgggtttttcttgctgg
GV_16 CGGTACCGCGGGCCCGGGAatggaactggggctccgctgggttttccttgttgc
GV_17 CGGTACCGCGGGCCCGGGAatggagtttggactgagctgggttttccttgttgc
GV_18 CGGTACCGCGGGCCCGGGAatggagttggggctgtgctgggttttccttgttgc
GV_19 CGGTACCGCGGGCCCGGGAatggagtttgggcttagctgggttttccttgttgc
GV_20 CGGTACCGCGGGCCCGGGAatggagttttggctgagctgggttttccttgttgc
GV_21 CGGTACCGCGGGCCCGGGAatgacggagtttgggctgagctgggttttccttgt
GV_22 CGGTACCGCGGGCCCGGGAatggagttctggctgagctgggttctccttgttgc
GV_23 CGGTACCGCGGGCCCGGGAatggaattggggctgagctgggttttccttgttgc
GV_24 CGGTACCGCGGGCCCGGGAatggagttgggactgagctggattttccttttggc
GV_25 CGGTACCGCGGGCCCGGGAatgaaacacctgtggttcttcctcctgctggtggc
GV_26 CGGTACCGCGGGCCCGGGAatgaagcacctgtggtttttcctcctgctggtggc
GV_27 CGGTACCGCGGGCCCGGGAatgaagcacctgtggttcttcctcctgctggtggc
GV_28 CGGTACCGCGGGCCCGGGAatgaaacatctgtggttcttccttctcctggtggc
GV_29 CGGTACCGCGGGCCCGGGAatgcaagtgggggcctctccacttaaacccaggct
GV_30 CGGTACCGCGGGCCCGGGAatggggtcaaccgccatcctcgccctcctcctggc
GV_31 CGGTACCGCGGGCCCGGGAatgtctgtctccttcctcatcttcctgcccgtgct
GV_32 CGGTACCGCGGGCCCGGGAatggactggacctggagcatcctcttcttggtggc
GV_33 CGGTACCGCGGGCCCGGGAatgcttgtatgtgtgcttttgtattctttcagatt
KV_01 CGGTACCGCGGGCCCGGGAatgagggcccccactcagctcctggggctcctggt
KV_02 CGGTACCGCGGGCCCGGGAatggaaatgagggtccccgctcagctcctggggct
KV_03 CGGTACCGCGGGCCCGGGAatggacatgagagtcctcgctcagctcctggggct
KV_04 CGGTACCGCGGGCCCGGGAatggacatgagggtccctgctcagctcctgggact
KV_05 CGGTACCGCGGGCCCGGGAatggacatgagggtccccgctcagctcctggggct
KV_06 CGGTACCGCGGGCCCGGGAatgagggtccccgctcagctcctggggctcctgct
KV_07 CGGTACCGCGGGCCCGGGAatggacatgagggtcctcgctcagctcctggggct
KV_08 CGGTACCGCGGGCCCGGGAatggacatgagggtgcccgctcagcgcctggggct
KV_09 CGGTACCGCGGGCCCGGGAatgaggctccttgctcagcttctggggctgctaat
KV_10 CGGTACCGCGGGCCCGGGAatgaggctccctgctcagctcctggggctgctaat
KV_11 CGGTACCGCGGGCCCGGGAatggaagccccagcacagcttctcttcctcctgct
KV_12 CGGTACCGCGGGCCCGGGAatggaagccccagcgcagcttctcttcctcctgct
KV_13 CGGTACCGCGGGCCCGGGAatggaaaccccagcgcagcttctcttcctcctgct
KV_14 CGGTACCGCGGGCCCGGGAatggaagccccagctcagcttctcttcctcctgct
KV_15 CGGTACCGCGGGCCCGGGAatggaaccatggaagccccagcacagcttcttctt
KV_16 CGGTACCGCGGGCCCGGGAatggtgttgcagacccaggtcttcatttctctgtt
KV_17 CGGTACCGCGGGCCCGGGAatggggtcccaggttcacctcctcagcttcctcct
KV_18 CGGTACCGCGGGCCCGGGAatgttgccatcacaactcattgggtttctgctgct
KV_19 CGGTACCGCGGGCCCGGGAatggtgtccccgttgcaattcctgcggcttctgct
LV_01 CGGTACCGCGGGCCCGGGAatgccctgggctctgctcctcctgaccctcctcac
LV_02 CGGTACCGCGGGCCCGGGAatggccctgactcctctcctcctcctgctcctctc
LV_03 CGGTACCGCGGGCCCGGGAatggcctggtctcctctcctcctcactctcctcgc
LV_04 CGGTACCGCGGGCCCGGGAatgacctgctcccctctcctcctcacccttctcat
LV_05 CGGTACCGCGGGCCCGGGAatggccggcttccctctcctcctcaccctcctcac
LV_06 CGGTACCGCGGGCCCGGGAatggcctgggctctgctgctcctcaccctcctcac
LV_07 CGGTACCGCGGGCCCGGGAatggcatggatccctctcttcctcggcgtccttgc
LV_08 CGGTACCGCGGGCCCGGGAatggcctggacccctctcctgctccccctcctcac
LV_09 CGGTACCGCGGGCCCGGGAatggcctggacccctctctggctcactctcctcac
LV_10 CGGTACCGCGGGCCCGGGAatggcctggaccgttctcctcctcggcctcctctc
LV_11 CGGTACCGCGGGCCCGGGAatggcctggatccctctacttctccccctcttcac
LV_12 CGGTACCGCGGGCCCGGGAatggcctggatccctctcctgctccccctcctcat
LV_13 CGGTACCGCGGGCCCGGGAatggcctggaccgctctccttctgagcctccttgc
LV_14 CGGTACCGCGGGCCCGGGAatggcctgggtctccttctacctactgcccttcat
LV_15 CGGTACCGCGGGCCCGGGAatggcctggaccccactcctcctcctcttccctct
LV_16 CGGTACCGCGGGCCCGGGAatggcctggactcctctcctcctcctgctcctctc
LV_17 CGGTACCGCGGGCCCGGGAatggcctgggctccactacttctcaccctcctcgc
LV_18 CGGTACCGCGGGCCCGGGAatggcctggactcctctctttctgttcctcctcac
LV_19 CGGTACCGCGGGCCCGGGAatggcctgcatgatgcttctccttgggctccttgc
LV_20 CGGTACCGCGGGCCCGGGAatggcctggatgatgcttctcctcggactccttgc
LV_21 CGGTACCGCGGGCCCGGGAatggcctgggctcctctgctcctcaccctcctcag
S3和S4所用到的所用到J-region引物参见如下的引物序列表二：
primer ID sequence
IGHJ_01 GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCAGGGTGCCCTGGCCCCAGT
IGHJ_02 GATGGGCCCTTGGTGGAGGGTGAGGAGACAGTGACCAGGGTGCCACGGCCCCAGA
IGHJ_03 GATGGGCCCTTGGTGGAGGGTGAAGAGACGGTGACCATTGTCCCTTGGCCCCAGA
IGHJ_04 GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCGTGGTCCCTTGCCCCCAGA
IGKJ_01 GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC
IGKJ_02 GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC
IGKJ_03 GATGGTGCAGCCACAGTTCGTTTGATATCCACTTTGGTCCCAGGGCCGAAAGTGA
IGKJ_04 GATGGTGCAGCCACAGTTCGTTTGATCTCCACCTTGGTCCCTCCGCCGAAAGTGA
IGKJ_05 GATGGTGCAGCCACAGTTCGTTTAATCTCCAGTCGTGTCCCTTGGCCGAAGGTGA
IGLJ_01 GGGGCAGCCTTGGGCTGACCTAGGACGGTGACCTTGGTCCCAGTTCCGAAGACAT
IGLJ_02 GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTTGGTCCCTCCGCCGAATACCA
IGLJ_03 GGGGCAGCCTTGGGCTGACCTAAAATGATCAGCTGGGTTCCTCCACCAAATACAA
IGLJ_04 GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCGGTCCCCTCACCAAACACCC
IGLJ_05 GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCCGTCCCCTCACCAAACACCC
IGLJ_06 GGGGCAGCCTTGGGCTGACCGAGGACGGTCACCTTGGTGCCACTGCCGAACACAT
IGLJ_07 GGGGCAGCCTTGGGCTGACCGAGGACGGTCAGCTGGGTGCCTCCTCCGAACACAG
IGLJ_08 GGGGCAGCCTTGGGCTGACCGAGGGCGGTCAGCTGGGTGCCTCCTCCGAACACAG
S5、将S4得到的抗体可变区基因表达盒转导入293T细胞48小时内表达抗体并收集上清，用ELISA法检测上清的RBD特异性，筛选RBD特异性全人源单克隆抗体。
(A)使用PBS稀释抗原(终浓度2μg/mL)，10μl/孔，包被384孔ELISA板4℃过夜或37℃包被2h(本实施例优选4℃过夜)。NOTE：加完后瞬时离心保证液体在底部。
实验体系如下表8所示：
试剂名称 货号 原浓度 终浓度 稀释比
SARS-COV-2RBD Cat:40592-V08H 200μg/mL 2μg/mL 1:100
Goat pab to Hu IgG－ALP Cat:ab97221 1mg/mL 2μg/mL 1:500
(B)配制PBST(0.05％Tween 20，Cat#TB220)：1L的PBS加入0.5mL的Tween 20；
PBST机洗板子(Thermoscientific wellwash versa)或者手洗(机洗完的板子依然要手动拍板/使用微孔板离心机(MPC-P25)离心1min，使板子看不见有水和气泡)。
封闭：80μl的5％BSA(BioFroxx，Cat.NO:4240GR100)(PBST配制)加入上述洗好的板子里，放置于37℃的孵育箱孵育1h。PBST机洗板子或者手洗。
(C)加样及标准品。其中，标准品：10μl/well原浓度1μg/mL，梯度稀释为250ng/mL、125ng/mL、62.5ng/mL、31.25ng/mL、15.63ng/mL、7.81ng/mL、3.9ng/mL和1.95ng/mL。(封闭液稀释)；样品： 转染抗体基因的细胞上清液。阴性对照/空白孔：封闭液10μl/well。
在37℃孵育30min。PBST机洗板子或者手洗。
(D)加二抗，加入的浓度为10μl/well，然后在37℃下孵育30min。
实验体系如下表9所示：
二抗名称 货号 原浓度 终浓度 稀释比
goat-anti-human IgG-ALP A18808 1.5mg/ml 0.3μg/ml 1:5000
Goat pab to Hu IgG-ALP Ab98532 0.5mg/ml 0.25μg/ml 1:2000
PBST机洗板子或者手洗。10μl/well的PNPP(对硝基苯磷酸二钠)，使用(Thermoscientific Muttiskan GO)检测5min、10min、15min、20min、25min、30min、35min、40min、45min、50min、55min和60min的OD(450mm)值。50mg的PNPP粉末(Thermo，Prod#34045)+40mL的ddH 2O+10mL的Diethanol aminesubstrate Buffer(5X)，PNPP避光4℃储存。
实验结果如图5所示，OD值大于0.1为阳性，图5中新冠病毒RBD特异性单克隆抗体CQTS004的OD值大于0.1。
以上所述仅为本发明的优选实施例，对本发明而言仅是说明性的，而非限制性的；本领域普通技术人员理解，在本发明权利要求所限定的精神和范围内可对其进行许多改变，修改，甚至等效变更，但都将落入本发明的保护范围内。

Claims (6)

  1. 新冠病毒RBD特异性单克隆抗体CQTS004，其特征在于，重链氨基酸序列如SEQ ID NO:1所示；轻链氨基酸序列如SEQ ID NO:2所示。
  2. 根据权利要求1所述的新冠病毒RBD特异性单克隆抗体CQTS004，其特征在于，通过分选RBD特异性记忆B细胞，再通过RBD特异性记忆B细胞的mRNA获得抗体可变区cDNA。
  3. 根据权利要求1或2所述的新冠病毒RBD特异性单克隆抗体CQTS004，在制备检测或诊断SARS-CoV-2试剂或药物中的应用，其中药物包括权利要求1或2所述的新冠病毒RBD特异性单克隆抗体CQTS004和药学上的赋形剂、稀释剂或载体。
  4. 编码权利要求1或2所述的新冠病毒RBD特异性单克隆抗体CQTS004的核酸分子。
  5. 含有权利要求4所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
  6. 根据权利要求5所述的表达盒、重组载体、重组菌或转基因细胞系在制备产品中的应用，其特征在于，产品用途如下(b1)-(b4)中的任一种：(b1)结合新型冠状病毒SARS-CoV-2；(b2)检测结合新型冠状病毒SARS-CoV-2；(b3)结合新型冠状病毒SARS-CoV-2的S蛋白；(b4)检测新型冠状病毒SARS-CoV-2的S蛋白。
PCT/CN2021/113261 2020-08-19 2021-08-18 新冠病毒rbd特异性单克隆抗体cqts004及其应用 WO2022037616A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010839864.8 2020-08-19
CN202010839864.8A CN111909263B (zh) 2020-08-19 2020-08-19 新冠病毒rbd特异性单克隆抗体和应用

Publications (1)

Publication Number Publication Date
WO2022037616A1 true WO2022037616A1 (zh) 2022-02-24

Family

ID=73279100

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/113261 WO2022037616A1 (zh) 2020-08-19 2021-08-18 新冠病毒rbd特异性单克隆抗体cqts004及其应用

Country Status (2)

Country Link
CN (7) CN117003862A (zh)
WO (1) WO2022037616A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023155902A1 (en) 2022-02-18 2023-08-24 Chongqing Mingdao Haoyue Biotechnology Co., Ltd. Intranasal formulations and anti-sars-cov-2-spike protein antibodies

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7116256B1 (ja) 2020-04-02 2022-08-09 リジェネロン・ファーマシューティカルズ・インコーポレイテッド 抗ｓａｒｓ－ｃｏｖ－２－スパイク糖タンパク質抗体および抗原結合断片
CR20220660A (es) 2020-06-03 2023-02-17 Regeneron Pharma Métodos para tratar o prevenir infecciones por sars-cov2 y covid-19 con anticuerpos de glicoproteína espícular anti-sars-cov-2
CN117003862A (zh) * 2020-08-19 2023-11-07 重庆医科大学 新冠病毒rbd特异性单克隆抗体和应用
CN115340602A (zh) * 2020-08-19 2022-11-15 重庆医科大学 新冠病毒rbd特异性单克隆抗体和应用
WO2022102744A1 (ja) * 2020-11-13 2022-05-19 株式会社ハカレル ＳＡＲＳ－ＣｏＶ－２のスパイクタンパク質に対する抗体
WO2022112392A1 (en) * 2020-11-26 2022-06-02 Memo Therapeutics Ag Anti-sars-cov-2 antibody molecules
WO2022179561A1 (en) * 2021-02-24 2022-09-01 The University Of Hong Kong Neutralizing antibodies against covid-19 and methods of use thereof
CN113388031B (zh) * 2021-08-17 2021-11-09 上海浙江大学高等研究院 单克隆抗体35b5及其制备方法和用途
CN113388030B (zh) * 2021-08-17 2021-11-23 上海浙江大学高等研究院 单克隆抗体32c7及其制备方法和用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111153991A (zh) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 一种人SARS-CoV-2单克隆抗体及其制备方法和应用
CN111187354A (zh) * 2020-02-20 2020-05-22 北京新创生物工程有限公司 新型冠状病毒(SARS-CoV-2)IgM/IgG抗体检测试剂盒
CN111303280A (zh) * 2020-03-22 2020-06-19 中国人民解放军军事科学院军事医学研究院 高中和活性抗SARS-CoV-2全人源单克隆抗体及应用
CN111505285A (zh) * 2020-04-27 2020-08-07 东莞博识生物科技有限公司 SARS-CoV-2检测芯片及其应用
CN111909263A (zh) * 2020-08-19 2020-11-10 重庆医科大学 新冠病毒rbd特异性单克隆抗体和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB202003632D0 (en) * 2020-03-12 2020-04-29 Harbour Antibodies Bv SARS-Cov-2 (SARS2, COVID-19) antibodies
CN111303279B (zh) * 2020-03-17 2022-02-15 北京凯因科技股份有限公司 一种针对新型冠状病毒的单域抗体及其应用
CN111856027B (zh) * 2020-04-16 2022-05-10 中国科学院苏州纳米技术与纳米仿生研究所 适用于无明显症状者排查的新冠病毒抗体检测试剂盒
CN111995675B (zh) * 2020-05-15 2021-03-23 潍坊医学院 一种针对新冠病毒SARS-CoV-2棘突蛋白RBD区的单克隆抗体及其应用
RU2723008C9 (ru) * 2020-05-19 2021-02-09 федеральное государственное бюджетное учреждение «Национальный исследовательский центр эпидемиологии и микробиологии имени почетного академика Н.Ф. Гамалеи» Министерства здравоохранения Российской Федерации Способ получения штамма клеток яичника китайского хомячка, продуцента рекомбинантного белка RBD вируса SARS-CoV-2, штамм клеток яичника китайского хомячка, продуцент рекомбинантного белка RBD вируса SARS-CoV-2, способ получения рекомбинантного белка RBD вируса SARS-CoV-2, тест-система для иммуноферментного анализа сыворотки или плазмы крови человека и ее применение

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111187354A (zh) * 2020-02-20 2020-05-22 北京新创生物工程有限公司 新型冠状病毒(SARS-CoV-2)IgM/IgG抗体检测试剂盒
CN111153991A (zh) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 一种人SARS-CoV-2单克隆抗体及其制备方法和应用
CN111303280A (zh) * 2020-03-22 2020-06-19 中国人民解放军军事科学院军事医学研究院 高中和活性抗SARS-CoV-2全人源单克隆抗体及应用
CN111505285A (zh) * 2020-04-27 2020-08-07 东莞博识生物科技有限公司 SARS-CoV-2检测芯片及其应用
CN111909263A (zh) * 2020-08-19 2020-11-10 重庆医科大学 新冠病毒rbd特异性单克隆抗体和应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023155902A1 (en) 2022-02-18 2023-08-24 Chongqing Mingdao Haoyue Biotechnology Co., Ltd. Intranasal formulations and anti-sars-cov-2-spike protein antibodies

Also Published As

Publication number Publication date
CN114920832B (zh) 2023-10-13
CN116813760B (zh) 2024-06-25
CN117003862A (zh) 2023-11-07
CN111909263B (zh) 2022-10-11
CN117736313A (zh) 2024-03-22
CN111909263A (zh) 2020-11-10
CN116813760A (zh) 2023-09-29
CN118085068A (zh) 2024-05-28
CN114920832A (zh) 2022-08-19
CN115925896A (zh) 2023-04-07

Similar Documents

Publication Publication Date Title
WO2022037616A1 (zh) 新冠病毒rbd特异性单克隆抗体cqts004及其应用
WO2022037033A1 (zh) 新冠病毒rbd特异性单克隆抗体和应用
CN111909260B (zh) 新冠病毒rbd特异性单克隆抗体和应用
CN111909261B (zh) 新冠病毒rbd特异性单克隆抗体和应用
CN111925440B (zh) 新冠病毒rbd特异性单克隆抗体和应用
CN111925442B (zh) 新冠病毒rbd特异性单克隆抗体和应用
CN111925441B (zh) 新冠病毒rbd特异性单克隆抗体和应用
CN111925443B (zh) 新冠病毒rbd特异性单克隆抗体和应用
CN111909262B (zh) 新冠病毒rbd特异性单克隆抗体和应用
WO2022036788A1 (zh) 新冠病毒rbd特异性单克隆抗体及其线性抗原表位和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21857708

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21857708

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 21857708

Country of ref document: EP

Kind code of ref document: A1