WO2022110962A1 - 抗严重急性呼吸系统综合征ii型冠状病毒sars-cov-2的中和抗体 - Google Patents

抗严重急性呼吸系统综合征ii型冠状病毒sars-cov-2的中和抗体 Download PDF

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WO2022110962A1
WO2022110962A1 PCT/CN2021/116808 CN2021116808W WO2022110962A1 WO 2022110962 A1 WO2022110962 A1 WO 2022110962A1 CN 2021116808 W CN2021116808 W CN 2021116808W WO 2022110962 A1 WO2022110962 A1 WO 2022110962A1
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antibody
neutralizing antibody
cells
variable region
neutralizing
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刘密
王义鹏
刁璐
马琳
章铭辉
镇学初
龙亚秋
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Suzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • 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 relates to a neutralizing antibody against severe acute respiratory syndrome type II coronavirus SARS-COV-2, belonging to the technical field of biomedicine.
  • Pneumonia caused by infection with severe acute respiratory syndrome type II coronavirus (SARS-CoV 2) is a serious infectious disease with severe impact worldwide. Finding effective treatments against the virus is an urgent need.
  • Neutralizing antibody refers to an antibody that can eliminate the ability of virus infection after binding to a virus. It is a corresponding antibody produced by B lymphocytes when pathogenic microorganisms invade the body. It is a soluble protein secreted by adaptive immune response cells. When pathogenic microorganisms invade cells, they need to rely on specific molecules expressed by the pathogens themselves to bind to receptors on the cells, in order to infect cells and further expand. Neutralizing antibodies can bind to antigens on the surface of pathogenic microorganisms, thereby preventing the pathogenic microorganisms from adhering to target cell receptors and preventing cell invasion.
  • B cells secrete neutralizing antibodies into the blood, and the antibodies combine with the virus particles in the blood to prevent the virus from infecting cells and destroy the virus particles, thus "neutralizing” the virus. It can be seen that neutralizing antibodies play a major role in killing the free virus outside the cell.
  • Certain specific neutralizing antibodies from the blood of cured patients with viral infection have the effect of neutralizing the virus, so they can be used for the treatment of infectious diseases and make the virus lose its pathogenicity.
  • therapeutic antibodies have gradually played a good role in the treatment of various diseases.
  • Existing vaccines such as measles vaccine, polio vaccine, hepatitis B vaccine, and hepatitis A vaccine, all make vaccinated people produce neutralizing antibodies to prevent virus infection. Since neutralizing antibodies can destroy viruses before they enter cells and can remove free viruses outside cells in the body, neutralizing antibodies in the blood of recovered patients based on viral infections can be used for the treatment of viral infections.
  • the present invention extracts peripheral immune cells from the blood of recovered patients with COVID-19, and selects B cells that can bind to the novel coronavirus antigen protein-spike protein from the peripheral immune cells.
  • the analysis at the cellular level yielded the gene sequences encoding the heavy and light chains of the variable region of neutralizing antibodies in B cells. These sequences can be used to reconstruct and express neutralizing antibodies that can neutralize the new coronavirus in vitro, and are expected to be used for the treatment and prevention of diseases such as pneumonia caused by the new coronavirus.
  • the first object of the present invention is to provide a neutralizing antibody against severe acute respiratory syndrome type II coronavirus SARS-COV-2, including: a light chain variable region DNA sequence, and a heavy chain variable region DNA sequence ;
  • the nucleotide sequence of the light chain variable region DNA sequence is one or more combinations in SEQ ID NO.1-5;
  • the nucleotide sequence of the heavy chain variable region DNA sequence is one or more of SEQ ID NO.6-8.
  • the neutralizing antibody is a complete antibody comprising a constant region and a variable region, a partial antibody comprising only the variable region or a chimeric antibody comprising only the variable region.
  • the second object of the present invention is to provide a gene encoding the neutralizing antibody.
  • the third object of the present invention is to provide an expression vector carrying the gene.
  • the fourth object of the present invention is to provide a recombinant cell expressing the neutralizing antibody.
  • the fifth object of the present invention is to provide the application of the neutralizing antibody in the preparation of a medicine for treating pneumonia COVID-19.
  • the sixth object of the present invention is to provide a kit for treating pneumonia COVID-19, which contains the neutralizing antibody.
  • the invention extracts peripheral immune cells from the blood of recovered patients with COVID-19, screens B cells that can bind to the novel coronavirus antigen protein-spike protein, and then analyzes the single B cells that produce antibodies at the single cell level, The gene sequences encoding the heavy and light chains of neutralizing antibody variable regions in B cells were obtained. These sequences can be used to reconstruct and express neutralizing antibodies that can neutralize the new coronavirus in vitro, and are expected to be used for the treatment and prevention of diseases such as pneumonia caused by the new coronavirus.
  • Figure 1 is a schematic diagram of the process of screening neutralizing antibodies using single B cells in the blood of patients who have recovered from COVID-19;
  • Figure 2 is the experimental result of using SDS-PAGE to analyze the antibody after expression and purification
  • Figure 3 shows the pseudovirus neutralization test results of negative control antibody Pmab (no neutralizing effect) and neutralizing antibody HC5K VH/B5K VL (IC 50 ⁇ 0.1273 ⁇ g/mL);
  • Figure 4 shows the results of the neutralization experiment of the neutralizing antibody D5K VH/B5K VL pseudovirus (IC 50 ⁇ 0.00033 ⁇ g/mL);
  • Figure 5 shows the pseudovirus neutralization test results of neutralizing antibody HC5K VH/D4K VL (IC 50 ⁇ 0.00084 ⁇ g/mL) and neutralizing antibody HF4L VH/D5K VL (IC 50 ⁇ 0.0046 ⁇ g/mL);
  • Figure 6 shows the pseudovirus neutralization test results of neutralizing antibody HC5K VH/D5K VL (IC 50 ⁇ 0.0066 ⁇ g/mL) and neutralizing antibody HD5K VH/A5K VL (IC 50 ⁇ 0.0017 ⁇ g/mL);
  • Figure 7 shows the pseudovirus neutralization test results of neutralizing antibody HF4L VH/A5K VL (IC 50 ⁇ 0.6049 ⁇ g/mL) and neutralizing antibody HF4L VH/B5K VL (IC 50 ⁇ 0.6077 ⁇ g/mL);
  • Fig. 8 is the result of pseudovirus neutralization experiment of neutralizing antibody HD5K VH/D4K VL (IC 50 ⁇ 0.1996 ⁇ g/mL).
  • Example 1 Isolation and extraction of single B cells that can be combined with SARS-COV-2
  • PBMCs peripheral blood mononuclear cells
  • Fc block was added to each PBMC sample first, and after 15 minutes of action, APC-H7-labeled anti-human CD3 antibody, BV421-labeled anti-human CD19 antibody, BB700-labeled anti-human CD27 antibody, and Biotin-labeled SARS-COV- 2 Spike protein, Biotin-tagged SARS-COV-2 Spike RBD. Streptavidin-APC was then added and allowed to act for 30 minutes. Afterwards, FACS AriaTMIII flow cytometer was used to load and analyze the antibody-labeled PBMC samples.
  • CD3 is a specific surface marker for T cells
  • CD19 is a specific surface marker for B cells
  • CD27 is a specific surface marker for memory B cells.
  • APC-positive cells are cells that can specifically bind to SARS-COV-2.
  • the cells selected from CD3 - CD19 + CD27 + APC + are the memory B cells we are looking for that can specifically bind to SARS-COV-2.
  • the B cells of this type were isolated from single B cells by flow cytometry and directly injected into a 96-well plate pre-loaded with cell lysate, sealed immediately, and quickly frozen on dry ice, and then stored at -80 °C until later. use. In this way, single B cells that can specifically bind to SARS-COV-2 can be isolated.
  • Example 2 Amplification and sequencing of variable region light and heavy chains in single B cells
  • B cells are cells that secrete antibodies
  • sequence information of antibodies is stored in B cells that can bind to SARS-COV-2.
  • RT-PCR was used to reverse transcription to obtain a cDNA library, and then nested PCR technology was used to amplify the DNA sequence in a single cell. Since the primers used in the experiment are specific primers for the variable region of the light chain or the variable region of the heavy chain, the obtained sequences are the sequences of the light chain and heavy chain portions of the antibody variable region in the single B cell. The amplified sequences are subjected to DNA sequencing analysis to obtain the sequence information of the variable region light chain and heavy chain of the antibody contained in a single B cell.
  • Single B cell RT-PCR was performed using the sorted single B cells as templates.
  • the PCR system configuration and sample addition were completed in a biological safety cabinet and placed on ice for operation.
  • the RT-PCR reaction system and reaction conditions were as follows:
  • This application obtains a total of 5 light chain variable region sequences and 3 heavy chain variable region sequences from B cells of recovered patients that can bind to the new coronavirus spike protein.
  • the five light chain variable region sequences and the three heavy chain variable region sequences can be freely combined to form an antibody, which is a neutralizing antibody of the new coronavirus SARS-COV-2.
  • the 5 light chain variable regions are named A5K VL (SEQ ID NO.1), B5K VL (SEQ ID NO.2), D4K VL (SEQ ID NO.3), D5K VL (SEQ ID NO.4) ) and D7K VL (SEQ ID NO.5)
  • the three heavy chain variable regions were named as HD5K VH (SEQ ID NO.6), HF4L VH (SEQ ID NO.7) and HC5K VH (SEQ ID NO.8 ).
  • Antibody expression and purification were performed as follows:
  • 9 antibodies were constructed, namely: HC5K VH/B5K VL, HD5K VH/D4K VL, HF4L VH/A5K VL, HF4L VH/B5K VL, HD5K VH/B5K VL, HC5K VH/D4K VL, HF4L VH /D5K VL, HC5K VH/D5K VL and HD5K VH/A5K VL.
  • Primers were designed according to the corresponding antibody light and heavy chains to amplify the corresponding VH and VL sequences. Using recombinase (ClonExpress II One Step Cloning Kit, C112-01/02, Vazyme), the amplified VH and VL sequences were recombined into the antibody expression vector backbone plasmid, and the bacterial solution was sent to sequencing for verification.
  • the cloned bacteria that were verified to be correct were inoculated into LB (Amp 100ug/ml) medium, cultured at 37°C overnight, and the plasmid was extracted with a plasmid extraction kit the next day, and the concentration was determined.
  • the plasmid/PEI mixture was added to the cell suspension, and the flask was shaken gently during the addition, and placed at 37° C., 8% CO 2 , and cultured at 125 rpm.
  • the supernatant was harvested by centrifugation.
  • the supernatant was filtered with a 0.4um membrane filter and docked to a purification column.
  • the Protein A affinity filler Mabselect SuReTM from GE was selected to remove the medium components and enrich the target protein.
  • the purified protein was analyzed by SDS-PAGE
  • the purified antibody was analyzed by SDS-PAGE electrophoresis.
  • the separating gel concentration used during the analysis was 4-20%.
  • the sample was first reduced, then the reduced sample was added to the Loading Buffer, boiled at 70°C for 10 min, and then loaded for analysis. The results are shown in Figure 2.
  • the pseudovirus carrying the 2019-nCoV spike protein was used to evaluate the neutralizing antibody of 2019-nCoV.
  • the virus system uses HIV-1 carrying a luciferase reporter gene as the viral backbone, and at the same time expresses the new coronavirus Spike protein on the virus shell, and the formed pseudovirion infects exogenous cell lines with high expression of ACE2, which highly simulates the passage of the new coronavirus through the virus.
  • the degree of infection of target cells by the pseudovirus particles was positively correlated with the luminescence value of luciferase, and negatively correlated with the neutralizing activity of antibodies.
  • the neutralizing antibody binds to the S protein of the virus coat in vitro, blocking the site on the S protein that binds to ACE2, so that the S protein cannot bind to ACE2, and the virus cannot invade cells; on the contrary, antibodies without neutralizing activity cannot interfere with the S protein. Binding to ACE2 on the cell surface, the virus entering the cell will express the Fluc protein, and after reacting with the luminescent substrate, its luminescence value is detected by a microplate reader.
  • Sample preparation transfer the pseudovirus from -80°C to 4°C refrigerator or thaw on ice in advance, dilute the virus to 1-2 ⁇ 10 4 TCID 50 /mL with DMEM medium containing 10% FBS before use If the transfected ACE2 overexpressing cell line was not purchased by our company, the optimal TCID50 needs to be explored by yourself).
  • sample-pseudovirus mixture in the 96-well cell culture plate and the two groups of controls were divided into three and transferred to a 96-well white plate, 50 ⁇ L/well (3 replicates).
  • the neutralizing antibodies of the present invention all have a certain ability to neutralize the new coronavirus.
  • the neutralizing ability of each antibody against the new coronavirus varies.
  • the reported IC50 values of neutralizing antibodies against 2019-nCoV in virus neutralization experiments are mostly in the range of 0.5 ⁇ g/mL to 0.0012 ⁇ g/mL.
  • some antibodies such as HD5K VH/B5K VL and HC5K VH/D4K VL have IC50s of 0.00033 ⁇ g/mL and 0.00084 ⁇ g/mL in the virus neutralization experiment, which are larger than Most SARS-CoV-2 neutralizing antibodies are stronger at neutralizing the virus.
  • Antibody Virus neutralization assay IC 50 ( ⁇ g/mL) HC5K VH/B5K VL 0.1273 HD5K VH/D4K VL 0.1996 HF4L VH/A5K VL 0.6049 HF4L VH/B5K VL 0.6077 HD5K VH/B5K VL 0.00033 HC5K VH/D4K VL 0.00084 HF4L VH/D5K VL 0.0046 HC5K VH/D5K VL 0.0066 HD5K VH/A5K VL 0.0017

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Abstract

公开了一种抗严重急性呼吸系统综合征II型冠状病毒SARS-COV-2的中和抗体，属于生物医药技术领域。从COVID-19康复患者血液中提取外周免疫细胞，并从中筛选可以和新冠病毒抗原蛋白-刺突蛋白结合的B细胞，然后对以生产抗体的单个B细胞在单细胞水平上进行分析，获得了B细胞内编码中和抗体可变区重链和轻链的基因序列。这些序列即可用来体外重新构建和表达可中和新冠病毒的中和抗体，有望用于新冠病毒所引起的肺炎等疾病的治疗和预防。

Description

抗严重急性呼吸系统综合征II型冠状病毒SARS-COV-2的中和抗体 技术领域
本发明涉及一种抗严重急性呼吸系统综合征II型冠状病毒SARS-COV-2的中和抗体，属于生物医药技术领域。
背景技术
抗严重急性呼吸系统综合征II型冠状病毒(SARS-CoV 2)感染引起的肺炎(COVID-19)是严重传染性疾病，并在全球范围内造成了严重影响。寻找针对该病毒的有效治疗方案是一种迫切需求。
中和抗体是指与病毒结合后能消除病毒感染能力的抗体，是当病原微生物侵入机体时由B淋巴细胞而产生的相应的抗体，是由适应性免疫应答细胞分泌的一种可溶性蛋白。病原微生物入侵细胞时需要依赖病原体自身表达的特定分子与细胞上的受体结合，才能感染细胞，并进一步扩增。中和抗体能够与病原微生物表面的抗原结合，从而阻止该病原微生物黏附靶细胞受体，防止侵入细胞。病毒侵入人体之后，B细胞把中和抗体分泌到血液里，抗体与血液里的病毒颗粒结合，阻止病毒感染细胞，破坏病毒颗粒，这样就把病毒“中和”掉了。由此可见，中和抗体在杀灭细胞外的游离病毒时起主要作用。
来自病毒感染治愈患者血液的某些特异性中和抗体具有中和病毒的作用，因而可用于感染性疾病的治疗，使病毒丧失致病力。随着抗体制备技术的进步，治疗性抗体已逐渐在多种疾病的治疗方面发挥了很好的效果。现有的疫苗如麻疹疫苗、脊髓灰质炎疫苗、乙肝疫苗、甲肝疫苗，都是使接种者产生中和抗体以预防病毒感染的。由于中和抗体可在病毒进入细胞之前破坏病毒并可清除体内细胞外的游离病毒，所以，基于病毒感染痊愈患者血液中的中和抗体可用于病毒感染的治疗。
发明内容
为解决上述技术问题，本发明从COVID-19康复患者血液中提取外周免疫细胞，并从中筛选可以和新冠病毒抗原蛋白-刺突蛋白结合的B细胞，然后对以生产抗体的单个B细胞在单细胞水平上进行分析，获得了B细胞内编码中和抗体可变区重链和轻链的基因序列。这些序列即可用来体外重新构建和表达可中和新冠病毒的中和抗体，有望用于新冠病毒所引起的肺炎等疾病的治疗和预防。
本发明的第一个目的是提供一种抗严重急性呼吸系统综合征II型冠状病毒SARS-COV-2的中和抗体，包括：轻链可变区DNA序列，以及重链可变区DNA序列；
所述轻链可变区DNA序列的核苷酸序列为SEQ ID NO.1-5中的一种或多种组合；
所述重链可变区DNA序列的核苷酸序列为SEQ ID NO.6-8中的一种或多种。
进一步地，所述的中和抗体为包含恒定区和可变区的完整抗体、只包含可变区的部分抗体或只包含可变区的嵌合抗体。
本发明的第二个目的是提供一种编码所述的中和抗体的基因。
本发明的第三个目的是提供一种携带所述的基因的表达载体。
本发明的第四个目的是提供一种表达所述的中和抗体的重组细胞。
本发明的第五个目的是提供所述的中和抗体在制备治疗肺炎COVID-19的药物中的应用。
本发明的第六个目的是提供一种治疗肺炎COVID-19的试剂盒，所述试剂盒内含有所述的中和抗体。
本发明的有益效果是：
本发明从COVID-19康复患者血液中提取外周免疫细胞，并从中筛选可以和新冠病毒抗原蛋白-刺突蛋白结合的B细胞，然后对以生产抗体的单个B细胞在单细胞水平上进行分析，获得了B细胞内编码中和抗体可变区重链和轻链的基因序列。这些序列即可用来体外重新构建和表达可中和新冠病毒的中和抗体，有望用于新冠病毒所引起的肺炎等疾病的治疗和预防。
附图说明
图1为利用COVID-19康复患者血液中的单个B细胞筛选中和抗体流程示意图；
图2为采用SDS-PAGE分析表达纯化后的抗体实验结果；
图3为阴性对照抗体Pmab(无中和效果)和中和抗体HC5K VH/B5K VL(IC 50～0.1273μg/mL)假病毒中和实验结果；
图4为中和抗体D5K VH/B5K VL假病毒中和实验结果(IC 50～0.00033μg/mL)；
图5为中和抗体HC5K VH/D4K VL(IC 50～0.00084μg/mL)和中和抗体HF4L VH/D5K VL(IC 50～0.0046μg/mL)假病毒中和实验结果；
图6为中和抗体HC5K VH/D5K VL(IC 50～0.0066μg/mL)和中和抗体HD5K VH/A5K VL(IC 50～0.0017μg/mL)假病毒中和实验结果；
图7为中和抗体HF4L VH/A5K VL(IC 50～0.6049μg/mL)和中和抗体HF4L VH/B5K VL(IC 50～0.6077μg/mL)假病毒中和实验结果；
图8为中和抗体HD5K VH/D4K VL(IC 50～0.1996μg/mL)假病毒中和实验结果。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：可和SARS-COV-2结合的单个B细胞的分离提取
抽取多个新冠病毒肺炎康复患者血液约15mL，采用Ficoll梯度法分离提取每个康复患者血液中的外周血单个核细胞(PBMC)，并将所得PBMC洗涤两遍后备用。
在每个PBMC样品中先加入Fc block，作用15分钟后依次加入APC-H7标记的抗人CD3抗体，BV421标记的抗人CD19抗体，BB700标记的抗人CD27抗体，Biotin标记的SARS-COV-2刺突蛋白，Biotin标记的SARS-COV-2 Spike RBD。尔后加入Streptavidin-APC并作用30分钟。尔后采用FACS AriaTMIII流式细胞仪上样分析收集抗体标记好的PBMC样品。CD3为T细胞的特异性表面标志物，CD19为B细胞的特异性表面标志物，CD27为记忆性B细胞的特异性表面标志物，Biotin标记的两种抗原可以和APC标记的Streptavidin特异性的结合，所以APC阳性的细胞即是可以和SARS-COV-2特异性结合的细胞。选取CD3 -CD19 +CD27 +APC +的细胞即是我们在寻找的可以和SARS-COV-2特异性结合的记忆性B细胞。将该类B细胞利用流式细胞仪进行单个B细胞分离并直接打入预先载有细胞裂解液的96孔板，立即封膜，并置于干冰上迅速冷冻，然后置于-80℃保存待用。这样即可以分离得到能和SARS-COV-2特异性结合的单个B细胞。
实施例2：单个B细胞中可变区轻链和重链的扩增和测序
因为B细胞为分泌抗体的细胞，所以能和SARS-COV-2结合的B细胞内储存有抗体的序列信息。将96孔板中已经裂解得到的单个B细胞裂解液分成3份。一份用于进行Kappa轻链序列分析，一份进行lamda轻链的序列分析，另一份进行重链序列的分析。
在该部分实验中，先用RT-PCR反转录得到cDNA文库，然后利用巢式PCR技术扩增单个细胞中的DNA序列。因为实验中所使用的的引物为轻链可变区或重链可变区特异性的引物，所以所得序列即为该单个B细胞中的抗体可变区轻链和重链部分的序列。将扩增所得序列进行DNA测序分析即得单个B细胞中所包含的抗体可变区轻链和重链的序列信息。
RT-PCR扩增单B细胞抗体轻重链可变区基因
设计RT-PCR引物(SEQ ID NO.9-25)，如表1所示：
表1
Figure PCTCN2021116808-appb-000001
以分选出的单个B细胞作为模板，进行单个B细胞RT-PCR。PCR体系配置及加样均在生物安全柜中完成并置于冰上操作，RT-PCR反应体系及反应条件如下：
PCR反应体系：
表2
成分 体系 终浓度
无RNA酶的水 - -
5×RT-PCR缓冲液 10.0μl 1×
dNTP混合物 2.0μl 每种dNTP 400μM
A引物 - 0.6μM
B引物 - 0.6μM
混合酶 2.0μl  
RNA酶抑制剂 - 5-10个单位
模板RNA - 1pg–2μg
总体系 50.0μl -
PCR反应条件：
Figure PCTCN2021116808-appb-000002
巢式PCR扩增单B细胞抗体轻重链可变区基因
设计巢式PCR引物(SEQ ID NO.26-38)，如表3所示：
表3
Figure PCTCN2021116808-appb-000003
以RT-PCR产物为模板，建立三个PCR反应体系，分别扩增抗体的H链、κ链、λ链的可变区基因，每个反应体系分别使用与其对应的混合引物，巢式PCR反应体系及反应条件如下：
PCR反应体系：
表4
PCR试剂 每个样品的体积(μl)(25μl体系) 最终浓度
Taq DNA聚合酶 0.25 50U ml -1
10×缓冲液 2.5 1×
dNTPs(10mM) 0.5 200μM
正向引物:VH3a和VH3b或PanVκ 0.5 1.2μM
反向引物:PW-Cgamma或CK494-516 0.5 1.2μM
无RNA酶的水 17.25-19.25(补齐体系至24μl) -
模板 1.0 -
PCR反应条件：
Figure PCTCN2021116808-appb-000004
实施例3：实验结果
本申请从康复患者可以结合新冠病毒刺突蛋白的B细胞中共得到5个轻链可变区序列，3个重链可变区序列。该5个轻链可变区序列和3个重链可变区序列可以自由组合成为抗体即为新冠病毒SARS-COV-2的中和抗体。
其中，5个轻链可变区分别命名为A5K VL(SEQ ID NO.1)、B5K VL(SEQ ID NO.2)、D4K VL(SEQ ID NO.3)、D5K VL(SEQ ID NO.4)和D7K VL(SEQ ID NO.5)，3个重链可变区分别命名为HD5K VH(SEQ ID NO.6)、HF4L VH(SEQ ID NO.7)和HC5K VH (SEQ ID NO.8)。
按照如下方法进行抗体的表达与纯化：
本实施例构建了9种抗体，分别为：HC5K VH/B5K VL、HD5K VH/D4K VL、HF4L VH/A5K VL、HF4L VH/B5K VL、HD5K VH/B5K VL、HC5K VH/D4K VL、HF4L VH/D5K VL、HC5K VH/D5K VL和HD5K VH/A5K VL。
1、载体构建
按照对应的抗体轻链和重链设计引物，扩增对应的VH和VL序列。利用重组酶(ClonExpress II One Step Cloning Kit，C112-01/02，Vazyme)，将扩增好的VH和VL序列重组至抗体表达载体骨架质粒上，菌液送测序验证。
2、质粒抽提
将验证正确的克隆菌接种到LB(Amp 100ug/ml)培养基中，37℃过夜培养，第二天用质粒大抽试剂盒抽提质粒，并测定浓度。
3、细胞瞬转表达
将抽提好的质粒瞬转293F细胞，具体如下：
1)计数处于对数生长期的293F，用新鲜的293培养基重新悬浮细胞，使其密度达到2*10 6/mL，总体积100mL。
2)用培养基稀释100ug质粒。
3)用培养基稀释PEI(1μg/μL)，将稀释后的PEI加到稀释后的质粒DNA中。旋转和/或颠倒试管或用移液器轻轻吹打2至3次进行混合。将复合物在室温下孵育约20分钟。
4)质粒/PEI混合物加入细胞悬液中，在添加过程中轻轻摇动摇瓶，放置在37℃，8％CO 2，125rpm中培养。
5)在转染后16-22小时向摇瓶中添加5％(v/v)补料，在添加过程中轻轻晃动摇瓶，将摇瓶放回37℃培养箱。
6)第6天收获上清。
4、抗体纯化
1)收获上清
利用离心机离心，收获上清。上清用0.4um滤膜过滤，对接纯化柱。
2)蛋白纯化
选择选用GE公司的Protein A亲和填料Mabselect SuReTM，去除培养基组分，富集目的蛋白。
5、采用SDS-PAGE分析纯化所得蛋白质
采用SDS-PAGE电泳分析纯化得到的抗体。在分析过程中所使用的分离胶浓度为4-20％。先将样品还原，然后还原样品加入Loading Buffer后70℃煮10min后上样分析。结果如图2所示。
实施例4：假病毒中和实验
本实验以携带新冠病毒刺突蛋白的假病毒来进行新冠中和抗体的评价。该病毒系统以携带荧光素酶报告基因的HIV-1为病毒骨架，同时在病毒外壳表达新冠病毒Spike蛋白，形成的假病毒颗粒感染外源性高表达ACE2的细胞系，高度模拟了新冠病毒通过Spike-ACE2对目的细胞的入侵过程，该假病毒颗粒感染目的细胞的程度与荧光素酶的发光值呈正相关，与抗体的中和活性呈负相关。
中和抗体在体外与病毒外壳的S蛋白结合，封闭了S蛋白上与ACE2结合的位点，导致S蛋白无法与ACE2结合，病毒无法入侵细胞；反之，没有中和活性的抗体无法干扰S蛋白与细胞表面ACE2的结合，进入细胞的病毒会表达Fluc蛋白，在与发光底物反应后通过酶标仪检测其发光值。
实验步骤：
1、样品准备：提前将假病毒从-80℃转至4℃冰箱或者冰上融化，使用前用含10％FBS的DMEM培养基稀释病毒至1-2×10 4TCID 50/mL(如侵染的ACE2过表达细胞株非我公司购买，则最佳TCID50需要自行摸索)。
2、取新的96孔细胞培养板进行样本稀释。
3、在96孔细胞培养板第2列加入135μL待测样本。
4、第3-10列分别加入90μl的无血清DMEM，然后用排枪从第2列取45μl稀释液加入到第3列进行倍比稀释，直到稀释到第9列，最后一列多余的45μL的液体弃去。再向每孔加入90μl稀释后的假病毒溶液。病毒对照组(VC)加入90μL假病毒，细胞对照组(CC)只加入180μL含血清DMEM培养基，将96孔细胞培养板置于37℃培养箱孵育1h。
5、将96孔细胞培养板中的样本-假病毒混合液以及两组对照一分为三转至96孔白板中，50μL/well(3个重复)。
6、立即将50μl密度为0.4×10 6cells/ml(数量为2×10 4cells/well)的293-ACE2细胞铺入96孔白板中(不需要更换培养基)，置于37℃培养箱中孵育。
7、孵育20-24h后，向每孔中加入25μL，37℃预热的含有10％FBS的DMEM培养基。
8、继续培养至48h后，取出96孔白板，平衡至室温，每孔加入125μl室温平衡后的Bio-Lite报告基因检测试剂，震板2min，室温静置5min后用酶标仪检测化学发光值(RLU)。
9、数据处理：将抗体浓度的log值以及对应的RLU带入到graph pad软件中，计算并且比较IC50值。
实验结果如表5和图3～图8所示：本发明所述的中和抗体对新冠病毒均具有一定的中和能力。但是各个抗体对新冠病毒的中和能力强弱不一。病毒中和实验中的IC50值越低越好，说明只需要很少的抗体就能中和病毒。已经报道的新冠病毒的中和抗体在病毒中和实验中的IC50值多位于0.5μg/mL到0.0012μg/mL。而本发明所述的新冠病毒中和抗体中，有的抗体如HD5K VH/B5K VL和HC5K VH/D4K VL在病毒中和实验中的IC50达到了0.00033μg/mL和0.00084μg/mL，比大多数新冠病毒中和抗体对病毒的中和能力都要更强。
表5中和抗体病毒中和能力
抗体 病毒中和实验IC 50(μg/mL)
HC5K VH/B5K VL 0.1273
HD5K VH/D4K VL 0.1996
HF4L VH/A5K VL 0.6049
HF4L VH/B5K VL 0.6077
HD5K VH/B5K VL 0.00033
HC5K VH/D4K VL 0.00084
HF4L VH/D5K VL 0.0046
HC5K VH/D5K VL 0.0066
HD5K VH/A5K VL 0.0017
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，如对抗体可变区重链或轻链序列进行3％以内的碱基替换，或对抗体可变区重链或轻链序列所表达的抗体部分的氨基酸序列进行3％以内的氨基酸替换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (7)

  1. 一种抗严重急性呼吸系统综合征II型冠状病毒SARS-COV-2的中和抗体，其特征在于，包括：轻链可变区DNA序列，以及重链可变区DNA序列；
    所述轻链可变区DNA序列的核苷酸序列为SEQ ID NO.1-5中的一种或多种组合；
    所述重链可变区DNA序列的核苷酸序列为SEQ ID NO.6-8中的一种或多种。
  2. 根据权利要求1所述的中和抗体，其特征在于，所述的中和抗体为包含恒定区和可变区的完整抗体、只包含可变区的部分抗体或只包含可变区的嵌合抗体。
  3. 一种编码权利要求1或2所述的中和抗体的基因。
  4. 一种携带权利要求3所述的基因的表达载体。
  5. 一种表达权利要求1或2所述的中和抗体的重组细胞。
  6. 权利要求1或2所述的中和抗体在制备治疗肺炎COVID-19的药物中的应用。
  7. 一种治疗肺炎COVID-19的试剂盒，其特征在于，所述试剂盒内含有权利要求1或2所述的中和抗体。
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