WO2022036788A1 - 新冠病毒rbd特异性单克隆抗体及其线性抗原表位和应用 - Google Patents

新冠病毒rbd特异性单克隆抗体及其线性抗原表位和应用 Download PDF

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WO2022036788A1
WO2022036788A1 PCT/CN2020/115480 CN2020115480W WO2022036788A1 WO 2022036788 A1 WO2022036788 A1 WO 2022036788A1 CN 2020115480 W CN2020115480 W CN 2020115480W WO 2022036788 A1 WO2022036788 A1 WO 2022036788A1
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rbd
monoclonal antibody
protein
specific monoclonal
novel coronavirus
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PCT/CN2020/115480
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English (en)
French (fr)
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金艾顺
李婷婷
韩晓建
王应明
胡超
李胜龙
王建为
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重庆医科大学
冯玉林
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Priority claimed from CN202010839226.6A external-priority patent/CN111909260B/zh
Priority claimed from CN202010839857.8A external-priority patent/CN111944026B/zh
Application filed by 重庆医科大学, 冯玉林 filed Critical 重庆医科大学
Publication of WO2022036788A1 publication Critical patent/WO2022036788A1/zh
Priority to US18/111,434 priority Critical patent/US20230374115A1/en

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • G01N33/577—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the invention belongs to the field of immunization technology, and particularly relates to a novel coronavirus RBD-specific monoclonal antibody and its linear epitope and application.
  • SARS-CoV-2 belongs to the genus Coronaviridae of the Coronaviridae family of the order Nidovirales, and it is the virus with the largest genome among the RNA viruses known to humans, with a length of 27 to 32 kb and a 4 major structural proteins, namely spike protein (S protein), membrane protein (M protein), envelope protein (E protein) and nucleocapsid protein (N protein), virus entry into cells depends on S protein and S protein
  • S protein spike protein
  • M protein membrane protein
  • E protein envelope protein
  • N protein nucleocapsid protein
  • the receptor binding domain (RBD) of the S protein has two subunits, S1 and S2.
  • the receptor binding site (RBD) is located on the S1 subunit. Its main function is to recognize the host cell surface receptor and mediate the interaction with the host cell.
  • N protein is a basic phosphoprotein, and its central region is combined with the viral genome RNA to form a coiled nucleocapsid helix, which is the core structure of the viral genetic material in the bag, and is the most expressed in infected cells. one of the viral proteins.
  • 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-mentioned patent has limited blocking effect on the virus, and the above-mentioned patent obtains the antibody cDNA by labeling plasma cells. The elicited humoral immune response is limited.
  • the Chinese invention patent application with application publication number CN111440229A discloses a new coronavirus T cell epitope.
  • the patent uses the IEDB resource Class I Immunogenicity tool to predict the T cell epitope of the new coronavirus N protein, and analyzes 600 immunogenic , 181 non-immunogenic 9mer peptides, but no linear epitopes recognized by B cells have been reported in the literature.
  • the purpose of the present invention is to provide a novel coronavirus RBD-specific monoclonal antibody that can be specifically recognized by B cells and its linear epitope and application.
  • the present invention provides a novel coronavirus RBD-specific monoclonal antibody, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1; the amino acid sequence of the light chain is shown in SEQ ID NO: 2 (mAb 3-CQTS126) .
  • the above-mentioned novel coronavirus RBD-specific monoclonal antibody is obtained by sorting RBD-specific memory B cells, and then obtaining antibody variable region cDNA from the mRNA of RBD-specific memory B cells.
  • the present invention also provides the application of the above-mentioned novel coronavirus RBD-specific monoclonal antibody in the preparation of reagents, vaccines or drugs for detecting or diagnosing SARS-CoV-2, wherein the drugs include novel coronavirus RBD-specific monoclonal antibodies and pharmaceutically acceptable
  • novel coronavirus RBD-specific monoclonal antibodies and pharmaceutically acceptable
  • the excipients, diluents or carriers for The application of the above expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines in the preparation of products is described.
  • the present invention also provides a product, including the above-mentioned new coronavirus RBD-specific monoclonal antibody; the use of the product is any of the following (b1)-(b4): (b1) combined with the new coronavirus SARS-CoV-2; (b2) Detection of binding to the new coronavirus SARS-CoV-2; (b3) binding to the S protein of the new coronavirus SARS-CoV-2; (b4) detection of the S protein of the new coronavirus SARS-CoV-2.
  • the present invention also discloses the linear antigenic epitope of the above-mentioned novel coronavirus RBD-specific monoclonal antibody, and the amino acid sequence is SEQ ID NO: 3.
  • the above-mentioned linear antigenic epitope is obtained by the following steps: first, after denaturing the S protein of the new coronavirus or the RBD protein of the new coronavirus, and then using the new coronavirus-specific monoclonal antibody to react with the denatured S protein or RBD protein Binding test is carried out, and then S protein or RBD protein is used for the synthesis of antigenic linear epitope segment to obtain linear antigenic epitope.
  • the present invention also provides the above-mentioned nucleic acid encoding a linear antigenic epitope, and a recombinant vector comprising the nucleic acid.
  • the monoclonal antibody provided by the present invention is combined with RBD, which provides a wider application value for antibody drug screening, diagnosis, prevention and treatment of new coronary pneumonia.
  • the monoclonal antibody provided by the present invention is obtained by sorting RBD-specific memory B cells. Compared with the prior art by sorting plasma cells, the monoclonal antibody prepared by the present invention can induce stronger humoral immunity The reason is that, compared to plasma cells, memory B cells can trigger a faster and stronger humoral immune response than the initial response.
  • 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.
  • the linear antigenic epitope obtained by the present invention has broad application prospects for the detection, diagnosis, vaccine development and therapeutic drug development of the new coronavirus. It can detect the state of humoral immune response after vaccination with the new crown vaccine, which can be used for small molecule drug development, vaccine research and development, etc.
  • Figure 1 is a cell sorting diagram of RBD-specific 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;
  • Figure 5 is a graph of the RBD-specific detection results of CQTS126
  • Figure 6 is a graph of the ELISA results of the binding of the new coronavirus RBD-specific monoclonal antibody to SEQ ID NO: 3;
  • Fig. 7 is the experimental result diagram that SEQ ID NO:3 is combined with patient plasma in experiment one, and is not combined with healthy person;
  • Figure 8 is a graph of the experimental results of the binding of SEQ ID NO: 3 to the RBD receptor ACE2 in experiment two.
  • This example provides a novel coronavirus RBD-specific monoclonal antibody (mAb 1-CQTS126), the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1; the amino acid sequence of the light chain is shown in SEQ ID NO: 2.
  • This embodiment also provides the application of the above-mentioned novel coronavirus RBD-specific monoclonal antibody in the preparation of reagents or medicines for detecting or diagnosing SARS-CoV-2.
  • the present embodiment can be used to prepare nucleic acid molecules using novel coronavirus RBD-specific monoclonal antibodies, 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 includes the above-mentioned novel coronavirus RBD-specific monoclonal antibody and a pharmaceutically acceptable excipient, diluent or carrier.
  • the new coronavirus RBD-specific monoclonal antibody obtained in this example can be used to prepare a product, and the product can be used for any one of the following (b1)-(b4): (b1) in combination with the new coronavirus SARS -CoV-2; (b2) Detects binding to novel coronavirus SARS-CoV-2; (b3) Binds S protein of novel coronavirus SARS-CoV-2; (b4) detects S protein of novel coronavirus SARS-CoV-2 .
  • This example also provides a screening method for the above-mentioned novel coronavirus RBD-specific monoclonal antibody.
  • a single RBD-specific memory B cell is obtained from the peripheral blood of a recovered patient with novel coronavirus pneumonia, and then the mRNA of the RBD-specific memory B cell is obtained. , and then construct the antibody variable region gene expression cassette by RT-PCR and nested PCR, and then transfer the antibody variable region gene expression cassette into 293T cells to express the antibody and collect the supernatant.
  • the RBD specificity of the supernatant was detected by ELISA. Screening to obtain RBD-specific monoclonal antibodies specifically includes the following steps:
  • 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 primers designed in this example are designed with a general leader in the front section of the primers (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 six parts in total: (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 3min; 295°C denaturation for 15sec, 60°C annealing for 5sec, 72°C extension for 1min, 30-35cycles, in this example, 30cycles is preferred; 372°Cexternal extension for 5min, Store at 4°C.
  • the experimental reaction conditions of 2nd PCR are: 1 95°C pre-denaturation for 3min; 295°C denaturation for 15sec, 60°C annealing for 5s, 72°C extension for 1min, 30-35cycles, in this example, 35cycles is preferred; Store at 4°C.
  • BCR-ORF PCR amplification of promoter region (CMV promoter), WPRE- ⁇ (antibody gamma chain) and WPRE- ⁇ (antibody kappa chain), PCR amplification
  • CMV promoter promoter region
  • 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 3min; denaturation at 95°C for 15sec, annealing at 58°C for 15sec, extension at 72°C for 1.5min, 30 cycles; external extension at 72°C for 5min, 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
  • This example provides the linear epitope of the new coronavirus RBD-specific monoclonal antibody, and the amino acid sequence is shown in SEQ ID NO: 3.
  • This example also provides applications of the linear epitopes of the above-mentioned novel coronavirus RBD-specific monoclonal antibodies in the preparation of nucleic acids, recombinant vectors, host cells, compositions, vaccines, test strips, detection reagents or monoclonal antibodies.
  • This example also provides a screening method for the linear epitope of the above-mentioned novel coronavirus RBD-specific monoclonal antibody.
  • RBD protein or its antibody and its receptor ACE2
  • linear epitope of the new coronavirus antibody such as Mining of epitopes on spike protein of SARS-CoV-2 from COVID-19 patients (Cell Research(2020) 0:1–3; https://doi.org/10.1038/s41422-020-0366-x) is the prediction of S protein by software
  • the epitopes of SARS-CoV-2 were confirmed by blood analysis of patients with new coronary pneumonia during convalescence, but not by monoclonal antibodies.
  • the method of designing the epitope is different, and the linear epitope found is also different.
  • the S protein of the new coronavirus or the RBD protein of the new coronavirus is first denatured, and then the new coronavirus-specific monoclonal is used.
  • the antibody is subjected to a binding test with the denatured S protein or RBD protein, and then the S protein or RBD protein is used to synthesize the linear antigenic epitope segment of the antigen to obtain a linear antigenic epitope, which specifically includes the following steps:
  • ELISA method detects the antibody binding ability of the linear antigenic epitope of the antigen, and screens the linear antigenic epitope.
  • the specific principle is: if the antibody epitope is a spatial epitope, after being treated by SDS, mercaptoethanol, DTT, etc., the spatial conformation is destroyed. Antibodies are not recognized at this time. If it is a linear epitope, the antibody can still bind.
  • the purchase information for the coated board is CORNING, High Binding, Lot#20519008.
  • PBST machine-washed plate (Thermo Scientific wellwash versa) or hand-washed (the plate after machine washing still needs to be clapped manually/centrifuged with a microplate centrifuge (MPC-P25) for 1min, so that no water and air bubbles can be seen on the plate);
  • MPC-P25 microplate centrifuge
  • the purchase information for the coated board is CORNING, High Binding, Lot#20519008.
  • PBST machine-washed plate (Thermo Scientific wellwash versa) or hand-washed (the plate after machine washing still needs to be clapped manually/centrifuged with a microplate centrifuge (MPC-P25) for 1min, so that no water and air bubbles can be seen on the plate);
  • MPC-P25 microplate centrifuge
  • ACE2 protein purchased from Biyuntian
  • PBS final concentration 2 ⁇ g/ml
  • 10 ⁇ l/well coated on 384-well ELISA plate overnight at 4°C or coated at 37°C for 2h ( This example is preferably at 4°C overnight).
  • PBST machine-washed plate (Thermo Scientific wellwash versa) or hand-washed (the plate after machine washing still needs to be clapped manually/centrifuged with a microplate centrifuge (MPC-P25) for 1min, so that no water and air bubbles can be seen on the plate);
  • MPC-P25 microplate centrifuge
  • the RBD antigen peptide SEQ ID NO: 3 binds to ACRE2 of the RBD receptor.

Abstract

一种新冠病毒RBD特异性单克隆抗体，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。新冠病毒RBD特异性单克隆抗体的线性抗原表位，氨基酸序列为SEQ ID NO:3。

Description

新冠病毒RBD特异性单克隆抗体及其线性抗原表位和应用 技术领域
本发明属于免疫技术领域，尤其涉及新冠病毒RBD特异性单克隆抗体及其线性抗原表位和应用。
背景技术
SARS-CoV-2属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)，是目前人类已知的RNA病毒中基因组最大的病毒，其长度为27至32kb，并具有至少4个主要结构蛋白，分别为刺突蛋白(S蛋白)、膜蛋白(M蛋白)、包膜蛋白(E蛋白)和核衣壳蛋白(N蛋白)，病毒进入细胞取决于S蛋白和S蛋白的受体结合域(RBD)，S蛋白有S1和S2两个亚基，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；N蛋白是一种碱性磷蛋白，其中央区与病毒基因组RNA结合，形成卷曲的核衣壳螺旋，是包裏病毒遗传物质的核心结构，是在感染细胞中是表达量最高的病毒蛋白之一。
目前针对新发病原体COVID-19尚无特效药物针对性治疗，疫苗研发尚需时日。近期治愈出院的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，所引发的体液免疫反应有限。
另外，新冠病毒抗原表位的研究对于新冠病毒的预防、检测、诊断和治疗均具有重要意义。申请公开号为CN111440229A的中国发明专利申请公开了一种新冠病毒T细胞表位，该专利利用IEDB资源Class I Immunogenicity工具预测了新冠病毒N蛋白的T细胞表位，分析了600个具有免疫原性、181个不具有免疫原性的9mer肽，但是目前还未有文献报道B细胞识别的线性抗原表位。
发明内容
本发明的目的在于提供一种能够特异性被B细胞识别的新冠病毒RBD特异性单克隆抗体及其线性抗原表位和应用。
为了达到上述目的，本发明的技术方案如下：
为了达到上述目的，本发明提供了新冠病毒RBD特异性单克隆抗体，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(单抗3-CQTS126)。
优选地，上述新冠病毒RBD特异性单克隆抗体通过分选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而得到。
本发明还提供了上述新冠病毒RBD特异性单克隆抗体，在制备检测或诊断SARS-CoV-2试剂、疫苗或药物中的应用，其中药物包括新冠病毒RBD特异性单克隆抗体和药学上可接受的赋形剂、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；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。
本发明还公开了上述新冠病毒RBD特异性单克隆抗体的线性抗原表位，氨基酸序列为SEQ ID NO:3。
优选地，上述线性抗原表位通过以下步骤得到：首先对新冠病毒的S蛋白或新冠病毒的RBD蛋白进行变性反应后，再用新冠病毒特异性单克隆抗体与变性反应后的S蛋白或RBD蛋白进行结合试验，再将S蛋白或RBD蛋白进行抗原线性抗原表位段的合成而获得线性抗原表位。
本发明还提供了上述编码线性抗原表位的核酸，以及包含该核酸的重组载体。
本发明的原理和有益效果在于：
(1)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能够引发更强烈的体液免疫反应，原因在于：与浆细胞相比，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。
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。
(3)本发明获得的线性抗原表位对于新冠病毒检测、诊断、疫苗研发和治疗药物的研发均具有广泛的应用前景，例如，使用本发明所提供的线性抗原表位检测患者体内的抗体滴度，检测接种新冠疫苗后的体液免疫反应状态，可以用于小分子药物开发、疫苗研发等。
附图说明
图1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CQTS126的RBD特异性检测结果图；
图6为新冠病毒RBD特异性单克隆抗体与SEQ ID NO:3结合的ELISA结果图；
图7为实验一中SEQ ID NO:3与患者血浆结合，而与健康人不结合的实验结果图；
图8为实验二中SEQ ID NO:3与RBD受体ACE2结合的实验结果图。
具体实施方式
下面通过具体实施方式进一步详细说明：
实施例1
本实施例提供新冠病毒RBD特异性单克隆抗体(单抗1-CQTS126)，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，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采用新冠病毒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特异性单克隆抗体和药学上可接受的赋形剂、稀释剂或载体。
在应用时，可以采用本实施例得到的新冠病毒RBD特异性单克隆抗体制备产品，该产品可具有如下(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。
本实施例还提供了上述新冠病毒RBD特异性单克隆抗体的筛选方法，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。
具体的，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而得到。具体的，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有通用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
Primes TAR 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 PCTCN2020115480-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 PCTCN2020115480-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 PCTCN2020115480-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，CQTS126为所需要的单克隆抗体，OD值为0.1以上为阳性。
实施例2
本实施例提供新冠病毒RBD特异性单克隆抗体的线性抗原表位，氨基酸序列如SEQ ID NO:3所示。
本实施例还提供上述新冠病毒RBD特异性单克隆抗体的线性抗原表位在制备核酸、重组载体、宿主细胞、组合物、疫苗、检测试纸、检测试剂或者单克隆抗体方面的应用。
本实施例还提供了上述新冠病毒RBD特异性单克隆抗体的线性抗原表位的筛选方法。目前，有多篇文献报道通过结构分析新冠病毒S蛋白、RBD蛋白或其抗体与其受体ACE2的空间结构的关键氨基酸或氨基酸位点，但是有关新冠抗体线性抗原表位的报道很少，例如Mining of epitopes on spike protein of SARS-CoV-2from COVID-19patients(Cell Research(2020)0:1–3；https://doi.org/10.1038/s41422-020-0366-x)是通过软件预测S蛋白的抗原表位，通过新冠肺炎患者恢复期血液分析，但不是通过单克隆抗体验证。本实施例从设计抗原表位方法不同，同时发现的线性抗 原表位也不一样，本实验首先对新冠病毒的S蛋白或新冠病毒的RBD蛋白进行变性反应后，再用新冠病毒特异性单克隆抗体与变性反应后的S蛋白或RBD蛋白进行结合试验，再将S蛋白或RBD蛋白进行抗原线性抗原表位段的合成而获得线性抗原表位，具体包括以下步骤：
1.设计并合成线性抗原表位；
2.ELISA法检测抗原线性抗原表位的抗体结合能力，筛选线性抗原表位，具体原理在于：若抗体表位为空间表位，经SDS，巯基乙醇、DTT等处理后，空间构象被破坏，此时抗体不能识别。若为线性抗原表位，则抗体依然能结合。
(1)耗材和试剂：链霉亲和素涂层板(Thermo Fisher，P#15504)；Wash Buffer(使用
Figure PCTCN2020115480-appb-000004
Protein-Free Blocking Buffers配制，P#37573)，在上述Buffer的基础上加入0.1％BSA(biofroxx,P#)；山羊F(ab')2抗人IgG(Fab')2的购买信息为Alkaline Phosphatase、Abcam,P#ab98532；PNPP Substrate的购买信息为Thermo Fisher、P#34045。
(2)第一天：RBD化学合成的抗原肽使用PBS稀释(终浓度20μg/ml)，10μl/孔，包被384孔ELISA板在4℃下过夜或37℃下包被2h(本实施例优选在4℃下过夜)。NOTE：加完后瞬时离心。
包被板的购买信息为CORNING，High Binding，Lot#20519008。
(3)第二天：
(a1)配制PBST(0.05％Tween 20，Cat#TB220)：1L的PBS中加入0.5ml的Tween 20；
PBST机洗板子(Thermo Scientific 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)；
(a2)封闭：80μl 5％BSA(BioFroxx,Cat.NO:4240GR100)(PBST配制)37℃下封闭1h；
(a3)加入新冠病毒RBD特异性单克隆抗体10μl/孔(20μg/ml)，室温1小时后洗板5次；
(a4)加入50ul山羊F(ab')2抗人IgG(Fab')2(Alkaline Phosphatase)(Abcam,P#ab98532)，室温30分钟；
(a5)100ul wash buffer清洗5次，加50ul反应底物PNPP；
(a6)检测吸光度值(OD405nm)。
结论：如图6所示，通过是否与新冠病毒RBD特异性单克隆抗体CQTS047、CQTS050和CQTS126结合的实验结果，筛选线性抗原表位SEQ ID NO:3。
实验一：线性抗原表位检测新冠肺炎患者恢复期血清抗体
(1)耗材和试剂：链霉亲和素涂层板(Thermo Fisher，P#15504)；Wash Buffer(使用
Figure PCTCN2020115480-appb-000005
Protein-Free Blocking Buffers配制，P#37573)，在上述Buffer的基础上加入0.1％BSA(biofroxx,P#)；山羊F(ab')2抗人IgG(Fab')2(Alkaline Phosphatase)(Abcam,P#ab98532)；PNPP Substrate(Thermo Fisher, P#34045)；
(2)第一天：RBD化学合成的抗原肽使用PBS稀释(终浓度20μg/ml)，10μl/孔，包被384孔ELISA板在4℃下过夜或37℃下包被2h(本实施例优选在4℃下过夜)。NOTE：加完后瞬时离心。
包被板的购买信息为CORNING，High Binding，Lot#20519008。
(3)第二天：
(a1)配制PBST(0.05％Tween 20，Cat#TB220)：1L的PBS中加入0.5ml的Tween 20；
PBST机洗板子(Thermo Scientific 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)；
(a2)封闭：80μl 5％BSA(BioFroxx,Cat.NO:4240GR100)(PBST配制)37℃下封闭1h；
(a3)加入新冠肺炎患者恢复期血浆和正常健康者(阴性对照)10μl/孔(原浓度)，室温1小时后洗板5次；
(a4)加入50ul山羊F(ab')2抗人IgG(Fab')2(Alkaline Phosphatase)(Abcam,P#ab98532)，室温30分钟；
(a5)100ul wash buffer清洗5次，加50ul反应底物PNPP；
(a6)检测吸光度值(OD405nm)。
结论：如图7所示，新冠病毒线性抗原表位SEQ ID NO:3与患者血浆结合，而与健康人不结合的实验结果图。
实验二：线性抗原表位与ACE2结合的实验
(1)第一天：ACE2蛋白(从碧云天购入)使用PBS稀释(终浓度2μg/ml)，10μl/孔，包被384孔ELISA板在4℃下过夜或37℃下包被2h(本实施例优选在4℃下过夜)。NOTE：加完后瞬时离心。
(2)第二天：
(a1)配制PBST(0.05％Tween 20，Cat#TB220)：1L的PBS中加入0.5ml的Tween 20；
PBST机洗板子(Thermo Scientific 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)；
(a2)封闭：80μl 5％BSA(BioFroxx,Cat.NO:4240GR100)(PBST配制)37℃下封闭1h；
(a3)加入实施实验三中合成的RBD抗原肽10μl/孔(20μg/ml)。室温1小时后洗板5次；
(a4)加入50ul亲和素Strepavidin-ALP抗体(3310-10)(1:1000)，室温30分钟；
(a5)100ul wash buffer清洗5次，加50ul反应底物PNPP；
(a6)检测吸光度值(OD405nm)。
结论：如图8所示，RBD抗原肽SEQ ID NO:3与RBD受体的ACRE2结合。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (10)

  1. 新冠病毒RBD特异性单克隆抗体，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权利要求所述的新冠病毒RBD特异性单克隆抗体，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而得到。
  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，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所述的新冠病毒RBD特异性单克隆抗体和药学上可接受的赋形剂、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。
  7. 根据权利要求1所述的新冠病毒RBD特异性单克隆抗体的线性抗原表位，其特征在于，氨基酸序列为SEQ ID NO:3。
  8. 根据权利要求6所述的新冠病毒RBD特异性单克隆抗体的线性抗原表位，其特征在于，通过以下步骤得到：首先对新冠病毒的S蛋白或新冠病毒的RBD蛋白进行变性反应后，再用新冠病毒特异性单克隆抗体与变性反应后的S蛋白或RBD蛋白进行结合试验，再将S蛋白或RBD蛋白进行抗原线性抗原表位段的合成而获得线性抗原表位。
  9. 一种编码权利要求7或8所述的线性抗原表位的核酸。
  10. 一种包括权利要求9所述的核酸的重组载体。
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