CN118388640A - Fully human anti-novel coronavirus SARS-CoV-2 broad-spectrum antibody and its application - Google Patents

Abstract

Translated fromChinese

本发明涉及一种抗新型冠状病毒SARS‑CoV‑2的广谱抗体及其应用，所述广谱抗体包括重链和轻链，所述广谱抗体至少具有如下技术特征之一：(1)所述重链包括重链CDR1，其氨基酸序列为：GFTVSSNY；(2)所述重链包括重链CDR2，其氨基酸序列为：IYSGGST；(3)所述重链包括重链CDR3，其氨基酸序列为：ARDNPRVGSNY；(4)所述轻链包括轻链CDR1，其氨基酸序列为：QDINNY；(5)所述轻链包括轻链CDR2，其氨基酸序列为：DAS；(6)所述轻链包括轻链CDR3，其氨基酸序列为：QQCDNVPLT。本发明的抗新型冠状病毒SARS‑CoV‑2的广谱抗体能够有效阻断新型冠状病毒RBD与宿主受体蛋白ACE2的结合，且对多种新型冠状病毒具有强效的结合能力。

The present invention relates to a broad-spectrum antibody against the novel coronavirus SARS-CoV-2 and its application, wherein the broad-spectrum antibody comprises a heavy chain and a light chain, and the broad-spectrum antibody has at least one of the following technical features: (1) the heavy chain comprises a heavy chain CDR1, whose amino acid sequence is: GFTVSSNY; (2) the heavy chain comprises a heavy chain CDR2, whose amino acid sequence is: IYSGGST; (3) the heavy chain comprises a heavy chain CDR3, whose amino acid sequence is: ARDNPRVGSNY; (4) the light chain comprises a light chain CDR1, whose amino acid sequence is: QDINNY; (5) the light chain comprises a light chain CDR2, whose amino acid sequence is: DAS; (6) the light chain comprises a light chain CDR3, whose amino acid sequence is: QQCDNVPLT. The broad-spectrum antibody against the novel coronavirus SARS-CoV-2 of the present invention can effectively block the binding of the novel coronavirus RBD to the host receptor protein ACE2, and has a strong binding ability to a variety of novel coronaviruses.

Description

Translated fromChinese

一种全人源抗新型冠状病毒SARS-CoV-2的广谱抗体及其应用A fully human broad-spectrum antibody against the new coronavirus SARS-CoV-2 and its application

技术领域Technical Field

本发明涉及一种全人源抗新型冠状病毒SARS-CoV-2的广谱抗体及其应用，属于生物医药领域。The present invention relates to a fully human broad-spectrum antibody against the novel coronavirus SARS-CoV-2 and applications thereof, belonging to the field of biomedicine.

背景技术Background technique

SARS-CoV-2属β型冠状病毒，与SARS-CoV的核酸同源性为79.6％，其天然宿主尚不清楚。SARS-CoV-2可通过病毒外壳上的Spike糖蛋白S1亚基的受体识别结构域(receptorbinding domain，RBD)与ACE2结合，从而入侵宿主细胞。该病毒传染性强，传播途径广，能迅速适应人体环境，感染后在潜伏期即具有传播能力，同时还有一些无症状感染者报道，甚至在多种动物体内也检测到病毒核酸。SARS-CoV-2 is a beta coronavirus, with a 79.6% homology to SARS-CoV in nucleic acid. Its natural host is still unknown. SARS-CoV-2 can bind to ACE2 through the receptor binding domain (RBD) of the Spike glycoprotein S1 subunit on the viral shell, thereby invading host cells. The virus is highly contagious, has a wide range of transmission routes, can quickly adapt to the human environment, and has the ability to spread during the incubation period after infection. At the same time, there are also reports of asymptomatic infections, and viral nucleic acids have even been detected in a variety of animals.

目前，全球各国已有多种疫苗经批准紧急投入使用中，然而根据前期冠状病毒感染研究经验，冠状病毒疫苗诱导的抗体可能难以提供长期的保护，对于迅速突变的新型冠状病毒感染，其保护范围及保护时间均相当有限。同时，在尚未有效治疗药物的流行早期，就有给予重症患者使用康复期患者的血浆治疗的案例，且具有明显的效果。恢复期血浆治疗由于缺乏随机对照临床试验，导致其疗效一直没有下定论。此外，为了确保血浆的安全性，血浆输注前必须进行肝炎病毒、艾滋病毒、梅毒和寄生虫等血液传播病原体的筛查，还需要考虑批次间的变异性和血型的匹配等，十分复杂。因此，在康复者外周血筛选具有中和作用的人源单克隆抗体，是预防和治疗COVID-19的另一切实可行的方法。其中从恢复期病人体内获得的全人源单克隆抗体更具有成药潜能。高亲和力和高中和活性的人源抗体对新型冠状病毒疫情控制和重症患者治疗方面都具有重大的应用价值。At present, many vaccines have been approved for emergency use in countries around the world. However, according to the experience of previous coronavirus infection research, antibodies induced by coronavirus vaccines may not provide long-term protection. For the rapidly mutating new coronavirus infection, the scope and duration of protection are quite limited. At the same time, in the early stage of the epidemic when there was no effective treatment, there were cases of using plasma from convalescent patients to treat severe patients, and it had obvious effects. Due to the lack of randomized controlled clinical trials, the efficacy of convalescent plasma therapy has not been determined. In addition, in order to ensure the safety of plasma, blood-borne pathogens such as hepatitis virus, HIV, syphilis and parasites must be screened before plasma transfusion, and the variability between batches and blood type matching must also be considered, which is very complicated. Therefore, screening human monoclonal antibodies with neutralizing effects in the peripheral blood of convalescents is another feasible method for preventing and treating COVID-19. Among them, fully human monoclonal antibodies obtained from convalescent patients have more potential for drug development. Human antibodies with high affinity and high neutralizing activity have great application value in the control of the new coronavirus epidemic and the treatment of critically ill patients.

SARS-CoV-2是一种正链单股RNA病毒，RNA病毒的特点是突变率高，并且随着时间的推移会积累突变，而这种突变累积是驱动病毒进化和基因组变异，从而逃避宿主免疫并产生耐药性的主要驱动力(Duffy S，PLoS Biol,2018 08；16(8))。最近研究表明SARS-CoV-2仍在全世界范围内进化，Korber B等研究发现了SARS-CoV-2 13个正在累积的S蛋白突变，其中614位的显性突变体(天冬氨酸(D)被甘氨酸(G)取代，D614G突变)远在流行初期就已经出现，该突变体已在世界许多地方占据主导地位。随后，Cassia W等发现相比D614，G614感染患者的临床样本具有更高水平的病毒RNA，并且感染患者年龄趋于年轻化。目前已经有五种新冠突变株被世卫组织列为值得关注的突变株(VOC)，除了正在流行的Omicron突变株外，还有Alpha、Beta、Gamma和Delta突变株。新冠突变株的最大特点是会对疫苗免疫和中和抗体产生逃逸，目前新冠Omicron突变株已经对多株上市以及处于临床实验的新冠中和抗体产生了抵抗。因此需要发掘能抵抗新冠突变株逃逸的强效广谱单克隆抗体抗体以应对当前流行的Omicron突变株。SARS-CoV-2 is a positive-stranded single-stranded RNA virus. RNA viruses are characterized by a high mutation rate and the accumulation of mutations over time. This accumulation of mutations is the main driving force for viral evolution and genomic variation, thereby evading host immunity and developing drug resistance (Duffy S, PLoS Biol, 2018 08; 16(8)). Recent studies have shown that SARS-CoV-2 is still evolving worldwide. Korber B et al. found 13 accumulating S protein mutations in SARS-CoV-2, among which the dominant mutant at position 614 (aspartic acid (D) replaced by glycine (G), D614G mutation) appeared long before the early stage of the epidemic and has become dominant in many parts of the world. Subsequently, Cassia W et al. found that clinical samples of G614-infected patients had higher levels of viral RNA than D614, and the age of infected patients tended to be younger. Currently, five new coronavirus mutants have been listed as mutants of concern (VOC) by the World Health Organization. In addition to the Omicron mutant, there are also Alpha, Beta, Gamma and Delta mutants. The biggest feature of the new coronavirus mutant strain is that it can escape vaccine immunity and neutralizing antibodies. At present, the new coronavirus Omicron mutant strain has become resistant to many new coronavirus neutralizing antibodies that are on the market and in clinical trials. Therefore, it is necessary to discover strong and broad-spectrum monoclonal antibodies that can resist the escape of new coronavirus mutant strains to deal with the currently popular Omicron mutant strain.

发明内容Summary of the invention

针对现有技术的不足，本发明的目的在于提供一种抗新型冠状病毒SARS-CoV-2强效的广谱抗体及其应用。In view of the deficiencies in the prior art, the object of the present invention is to provide a potent broad-spectrum antibody against the new coronavirus SARS-CoV-2 and its application.

为了解决上述技术问题，本发明的技术方案如下：In order to solve the above technical problems, the technical solution of the present invention is as follows:

一种抗新型冠状病毒SARS-CoV-2的广谱抗体(VSM12-37)，所述广谱抗体包括重链和轻链，所述广谱抗体至少具有如下技术特征之一：A broad-spectrum antibody (VSM12-37) against the novel coronavirus SARS-CoV-2, the broad-spectrum antibody comprising a heavy chain and a light chain, the broad-spectrum antibody having at least one of the following technical features:

(1)所述重链包括重链CDR1，其氨基酸序列为：GFTVSSNY；(1) The heavy chain includes a heavy chain CDR1, whose amino acid sequence is: GFTVSSNY;

(2)所述重链包括重链CDR2，其氨基酸序列为：IYSGGST；(2) the heavy chain includes a heavy chain CDR2, the amino acid sequence of which is: IYSGGST;

(3)所述重链包括重链CDR3，其氨基酸序列为：ARDNPRVGSNY；(3) the heavy chain includes a heavy chain CDR3, the amino acid sequence of which is: ARDNPRVGSNY;

(4)所述轻链包括轻链CDR1，其氨基酸序列为：QDINNY；(4) the light chain includes a light chain CDR1, whose amino acid sequence is: QDINNY;

(5)所述轻链包括轻链CDR2，其氨基酸序列为：DAS；(5) The light chain includes a light chain CDR2, whose amino acid sequence is: DAS;

(6)所述轻链包括轻链CDR3，其氨基酸序列为：QQCDNVPLT。(6) The light chain includes a light chain CDR3, whose amino acid sequence is: QQCDNVPLT.

进一步地，所述广谱抗体至少具有如下技术特征之一：Furthermore, the broad-spectrum antibody has at least one of the following technical features:

(a)所述重链包括重链CDR1-3，重链CDR1的氨基酸序列为：GFTVSSNY，重链CDR2的氨基酸序列为：IYSGGST，重链CDR3的氨基酸序列为：ARDNPRVGSNY；(a) the heavy chain includes heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is: GFTVSSNY, the amino acid sequence of heavy chain CDR2 is: IYSGGST, and the amino acid sequence of heavy chain CDR3 is: ARDNPRVGSNY;

(b)所述轻链包括轻链CDR1-3，轻链CDR1的氨基酸序列为：QDINNY，轻链CDR2的氨基酸序列为：DAS，轻链CDR3的氨基酸序列为：QQCDNVPLT。(b) The light chain includes light chain CDR1-3, the amino acid sequence of light chain CDR1 is: QDINNY, the amino acid sequence of light chain CDR2 is: DAS, and the amino acid sequence of light chain CDR3 is: QQCDNVPLT.

进一步地，所述广谱抗体具有如下技术特征：所述重链包括重链CDR1，其氨基酸序列为：GFTVSSNY；所述重链包括重链CDR2，其氨基酸序列为：IYSGGST；所述重链包括重链CDR3，其氨基酸序列为：ARDNPRVGSNY；所述轻链包括轻链CDR1，其氨基酸序列为：QDINNY；所述轻链包括轻链CDR2，其氨基酸序列为：DAS；所述轻链包括轻链CDR3，其氨基酸序列为：QQCDNVPLT。Furthermore, the broad-spectrum antibody has the following technical characteristics: the heavy chain includes a heavy chain CDR1, whose amino acid sequence is: GFTVSSNY; the heavy chain includes a heavy chain CDR2, whose amino acid sequence is: IYSGGST; the heavy chain includes a heavy chain CDR3, whose amino acid sequence is: ARDNPRVGSNY; the light chain includes a light chain CDR1, whose amino acid sequence is: QDINNY; the light chain includes a light chain CDR2, whose amino acid sequence is: DAS; the light chain includes a light chain CDR3, whose amino acid sequence is: QQCDNVPLT.

进一步地，所述重链还包括重链FR1-4，重链FR1的氨基酸序列为EVQLVESGGGLVQPGGSLRLSCAVS，重链FR2的氨基酸序列为MSWVRQAPGKGLEWVSL，重链FR3的氨基酸序列为YYADSVKGRFTVSRDRSKNTLYLQMNSLRAEDTALYYC，重链FR4的氨基酸序列为WGQGTLVTVSS。Furthermore, the heavy chain also includes heavy chain FR1-4, the amino acid sequence of heavy chain FR1 is EVQLVESGGGLVQPGGSLRLSCAVS, the amino acid sequence of heavy chain FR2 is MSWVRQAPGKGLEWVSL, the amino acid sequence of heavy chain FR3 is YYADSVKGRFTVSRDRSKNTLYLQMNSLRAEDTALYYC, and the amino acid sequence of heavy chain FR4 is WGQGTLVTVSS.

进一步地，所述轻链还包括轻链FR1-4，轻链FR1的氨基酸序列为DIQMTQSPSSLSASVGDRVTITCQAS，轻链FR2的氨基酸序列为LNWYQQKPGKAPKLLIY，轻链FR3的氨基酸序列为NLEAGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC，轻链FR4的氨基酸序列为FGGGTKVEIK。Furthermore, the light chain also includes light chain FR1-4, the amino acid sequence of light chain FR1 is DIQMTQSPSSLSASVGDRVTITCQAS, the amino acid sequence of light chain FR2 is LNWYQQKPGKAPKLLIY, the amino acid sequence of light chain FR3 is NLEAGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC, and the amino acid sequence of light chain FR4 is FGGGTKVEIK.

进一步地，所述广谱抗体的重链可变区的氨基酸序列如SEQ ID NO：1所示；所述广谱抗体的轻链可变区的氨基酸序列如SEQ ID NO：2所示。Furthermore, the amino acid sequence of the heavy chain variable region of the broad-spectrum antibody is shown as SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the broad-spectrum antibody is shown as SEQ ID NO: 2.

进一步地，编码广谱抗体的重链可变区的基因序列如SEQ ID NO：3所示；编码广谱抗体的轻链可变区的基因序列如SEQ ID NO：4所示。Furthermore, the gene sequence encoding the heavy chain variable region of the broad-spectrum antibody is shown in SEQ ID NO: 3; the gene sequence encoding the light chain variable region of the broad-spectrum antibody is shown in SEQ ID NO: 4.

编码如上所述的广谱抗体的核酸分子。Nucleic acid molecules encoding broad-spectrum antibodies as described above.

进一步地，所述核酸分子包括核苷酸序列SEQ ID NO：3和/或SEQ ID NO：4。Furthermore, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:3 and/or SEQ ID NO:4.

如上所述的广谱抗体在制备用于诊断试剂或诊断试剂盒或药物中的应用。Use of the broad-spectrum antibody as described above in the preparation of a diagnostic reagent or a diagnostic kit or a drug.

进一步地，所述药物具有针对新型冠状病毒SARS-CoV-2的中和作用；优选地，所述新型冠状病毒SARS-CoV-2包括新型冠状病毒SARS-CoV-2 Wuhan-Hu-1病毒株、新型冠状病毒Alpha突变株、新型冠状病毒Beta突变株、新型冠状病毒Gamma突变株、新型冠状病毒Delta突变株、新型冠状病毒Omicron突变株中的一种或几种。Furthermore, the drug has a neutralizing effect on the new coronavirus SARS-CoV-2; preferably, the new coronavirus SARS-CoV-2 includes one or more of the new coronavirus SARS-CoV-2 Wuhan-Hu-1 virus strain, the new coronavirus Alpha mutant strain, the new coronavirus Beta mutant strain, the new coronavirus Gamma mutant strain, the new coronavirus Delta mutant strain, and the new coronavirus Omicron mutant strain.

如上所述的核酸分子在制备抗新型冠状病毒SARS-CoV-2的广谱抗体或药物中的应用。The use of the nucleic acid molecules as described above in the preparation of broad-spectrum antibodies or drugs against the new coronavirus SARS-CoV-2.

进一步地，所述药物具有针对新型冠状病毒SARS-CoV-2的中和作用；优选地，所述新型冠状病毒SARS-CoV-2包括新型冠状病毒SARS-CoV-2 Wuhan-Hu-1病毒株、新型冠状病毒Alpha突变株(B.1.1.7)、新型冠状病毒Beta突变株(B.1.351)、新型冠状病毒Gamma突变株(P1)、新型冠状病毒Delta突变株(B.1.617.2)、新型冠状病毒Omicron突变株(B.1.1.529)中的一种或几种。Furthermore, the drug has a neutralizing effect on the new coronavirus SARS-CoV-2; preferably, the new coronavirus SARS-CoV-2 includes one or more of the new coronavirus SARS-CoV-2 Wuhan-Hu-1 virus strain, the new coronavirus Alpha mutant strain (B.1.1.7), the new coronavirus Beta mutant strain (B.1.351), the new coronavirus Gamma mutant strain (P1), the new coronavirus Delta mutant strain (B.1.617.2), and the new coronavirus Omicron mutant strain (B.1.1.529).

含有如上所述的核酸分子的表达盒、重组载体、重组菌或转基因细胞系。An expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the nucleic acid molecule as described above.

可选地，本发明的抗新型冠状病毒SARS-CoV-2的广谱抗体的制备方法包括如下步骤：Optionally, the method for preparing a broad-spectrum antibody against the novel coronavirus SARS-CoV-2 of the present invention comprises the following steps:

S1、从新型冠状病毒SARS-CoV-2感染恢复患者外周血中分选获得SARS-CoV-2特异的Memory B细胞；S1. SARS-CoV-2-specific Memory B cells were isolated from the peripheral blood of patients who recovered from SARS-CoV-2 infection;

S2、扩增SARS-CoV-2特异的Memory B细胞的Ig可变序列，获得特异性扩增产物；S2, amplify the Ig variable sequence of SARS-CoV-2-specific memory B cells to obtain specific amplification products;

S3、构建表达质粒，体外转染、表达、纯化，获得抗新型冠状病毒SARS-CoV-2的广谱抗体。S3. Construct expression plasmids, perform in vitro transfection, expression, and purification to obtain broad-spectrum antibodies against the new coronavirus SARS-CoV-2.

本发明通过流式单细胞分析分选技术、单个B细胞PCR扩增抗体制备技术，从新冠康复患者PBMC中分选棘突蛋白(Spike)特异的单个记忆性B细胞，并直接克隆抗体重链以及轻链可变区序列，构建成表达质粒并表达纯化得到新型冠状病毒SARS-CoV-2的广谱抗体(VSM12-37)。The present invention uses flow cytometry single-cell analysis and sorting technology and single B cell PCR amplification antibody preparation technology to sort single memory B cells specific to the spike protein from PBMCs of recovered COVID-19 patients, directly clone the antibody heavy chain and light chain variable region sequences, construct an expression plasmid, and express and purify to obtain a broad-spectrum antibody (VSM12-37) against the new coronavirus SARS-CoV-2.

本发明的抗新型冠状病毒SARS-CoV-2的广谱抗体是特异性靶向RBD的广谱抗体，能够特异性结合多种新型冠状病毒突变株，具有强结合力，该特异性广谱抗体能够有效阻断多种新型冠状病毒突变株与受体蛋白的结合，是有效的广谱中和活性单抗。The broad-spectrum antibody against the new coronavirus SARS-CoV-2 of the present invention is a broad-spectrum antibody that specifically targets RBD, can specifically bind to a variety of new coronavirus mutant strains, and has strong binding force. This specific broad-spectrum antibody can effectively block the binding of a variety of new coronavirus mutant strains to receptor proteins and is an effective broad-spectrum neutralizing active monoclonal antibody.

与现有技术相比，本发明的有益效果如下：Compared with the prior art, the present invention has the following beneficial effects:

1)本发明的抗新型冠状病毒SARS-CoV-2的广谱抗体的制备流程简单快速，而且获得的广谱抗体为全人源单克隆抗体，无免疫原性。1) The preparation process of the broad-spectrum antibody against the new coronavirus SARS-CoV-2 of the present invention is simple and rapid, and the obtained broad-spectrum antibody is a fully human monoclonal antibody with no immunogenicity.

2)本发明的抗新型冠状病毒SARS-CoV-2的广谱抗体能够有效阻断新型冠状病毒RBD与宿主受体蛋白ACE2的结合，且对多种新型冠状病毒具有强效的结合能力。2) The broad-spectrum antibodies against the novel coronavirus SARS-CoV-2 of the present invention can effectively block the binding of the novel coronavirus RBD to the host receptor protein ACE2, and have a strong binding ability to a variety of novel coronaviruses.

3)本发明的抗新型冠状病毒SARS-CoV-2的广谱抗体对流行的新冠突变株(Alpha，Beta，Gamma，Delta以及Omicron)，特别是Omicron突变株，具有强效广谱的中和作用。3) The broad-spectrum antibodies against the novel coronavirus SARS-CoV-2 of the present invention have a potent and broad-spectrum neutralizing effect on the prevalent novel coronavirus mutant strains (Alpha, Beta, Gamma, Delta and Omicron), especially the Omicron mutant strain.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1是本发明中流式细胞术分选SARS-CoV-2S蛋白特异性记忆B细胞的流程图。FIG1 is a flow chart of the flow cytometry method for sorting SARS-CoV-2S protein-specific memory B cells in the present invention.

图2是本发明的抗体基因PCR产物凝胶电泳图。FIG. 2 is a gel electrophoresis diagram of the antibody gene PCR product of the present invention.

图3是本发明的抗新型冠状病毒SARS-CoV-2的广谱抗体的重链可变区的序列图。Figure 3 is a sequence diagram of the heavy chain variable region of the broad-spectrum antibody against the new coronavirus SARS-CoV-2 of the present invention.

图4是本发明的抗新型冠状病毒SARS-CoV-2的广谱抗体的轻链可变区的序列图。Figure 4 is a sequence diagram of the light chain variable region of the broad-spectrum antibody against the new coronavirus SARS-CoV-2 of the present invention.

图5是本发明的抗新型冠状病毒SARS-CoV-2的广谱抗体与新型冠状病毒株、突变株的结合情况图。Figure 5 is a diagram showing the binding of the broad-spectrum antibodies against the novel coronavirus SARS-CoV-2 of the present invention to the novel coronavirus strains and mutant strains.

图6是本发明的不同浓度抗新型冠状病毒SARS-CoV-2的广谱抗体与新型冠状病毒Omicron突变株的结合情况图。Figure 6 is a graph showing the binding of different concentrations of broad-spectrum antibodies against the novel coronavirus SARS-CoV-2 of the present invention to the novel coronavirus Omicron mutant strain.

图7是本发明的不同浓度抗新型冠状病毒SARS-CoV-2的中和抗体与新型冠状病毒Wuhan-hu-1、Alpha、Beta、Gamma、Delta、Omicron突变株的中和情况图。Figure 7 is a graph showing the neutralization of different concentrations of neutralizing antibodies against the novel coronavirus SARS-CoV-2 of the present invention against the novel coronavirus Wuhan-hu-1, Alpha, Beta, Gamma, Delta, and Omicron mutants.

具体实施方式Detailed ways

以下将结合实施例来详细说明本发明。需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。The present invention will be described in detail below in conjunction with the embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

实施例1Example 1

参见图1，获得新型冠状病毒SARS-CoV-2的广谱抗体(VSM12-37)的具体方法如下：Referring to Figure 1, the specific method for obtaining a broad-spectrum antibody (VSM12-37) against the novel coronavirus SARS-CoV-2 is as follows:

(1)流式单细胞分选SARS-CoV-2特异的Memory B细胞(1) Flow cytometry single-cell sorting of SARS-CoV-2-specific memory B cells

a.采集若干名新型冠状病毒SARS-CoV-2感染恢复患者外周血，分离得到PBMC，于液氮中冻存备用；a. Collect peripheral blood from several patients who have recovered from SARS-CoV-2 infection, separate PBMCs, and freeze them in liquid nitrogen for later use;

b.将收集的新型冠状病毒SARS-CoV-2感染恢复患者外周血PBMC从液氮复苏；b. Resuscitate the collected peripheral blood PBMCs from patients who have recovered from the novel coronavirus SARS-CoV-2 infection from liquid nitrogen;

c.加入完全培养基37℃静置过夜，其中，所述完全培养基为含10％胎牛血清(Gibco，货号：10270-106)的1640(Gibco，货号：C11875500CP)培养基；c. Add complete medium and incubate at 37°C overnight, wherein the complete medium is 1640 (Gibco, Catalog No.: C11875500CP) medium containing 10% fetal bovine serum (Gibco, Catalog No.: 10270-106);

d.随后进行Live/dead(ThermoFisher，货号：L34962)、CD3(BD Biosciences，货号：612752)、CD19(Biolegend，货号：302230)、IgD(Biolegend，货号：348240)、CD27(Biolegend，货号：356412)、IgG(BD Biosciences，货号：555787)以及SARS-CoV-2S探针的细胞染色，其中，所述探针为Alexa Fluor^TM488蛋白标记试剂盒(ThermoFisher，货号：A20181)标记的SARS-CoV-2S ECD蛋白(义翘神州，货号：40589-V08B1)；d. Then, cells were stained with Live/dead (ThermoFisher, Catalog No.: L34962), CD3 (BD Biosciences, Catalog No.: 612752), CD19 (Biolegend, Catalog No.: 302230), IgD (Biolegend, Catalog No.: 348240), CD27 (Biolegend, Catalog No.: 356412), IgG (BD Biosciences, Catalog No.: 555787), and SARS-CoV-2S probe, wherein the probe is SARS-CoV-2S ECD protein (Sino Biological, Catalog No.: 40589-V08B1) labeled with Alexa Fluor^™ 488 Protein Labeling Kit (ThermoFisher, Catalog No.: A20181);

e.流式分选Live/dead^-CD3^-CD19⁺IgD^-CD27⁺IgG⁺以及SARS-CoV-2S探针阳性的B细胞，获得SARS-CoV-2S蛋白特异的Memory B细胞。e. Flow cytometry sorting of Live/dead^- CD3^- CD19⁺ IgD^- CD27⁺ IgG⁺ and SARS-CoV-2S probe-positive B cells to obtain SARS-CoV-2S protein-specific Memory B cells.

(2)扩增SARS-CoV-2特异的Memory B细胞Ig可变区序列(参照文献：Wardemann,H等，Methods Mol Biol,2019.1956:p.105-125.进行)(2) Amplification of SARS-CoV-2-specific memory B cell Ig variable region sequences (reference: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.)

a.将配好的细胞裂解液加入96孔PCR板内，体系如下；a. Add the prepared cell lysate into a 96-well PCR plate. The system is as follows;

b.将分选的SARS-CoV-2S蛋白特异Memory B细胞添加至加有上述细胞裂解液的96孔PCR板内；b. Add the sorted SARS-CoV-2S protein-specific Memory B cells to a 96-well PCR plate containing the above-mentioned cell lysate;

c.将96孔PCR板置于干冰上，速冻裂解细胞；c. Place the 96-well PCR plate on dry ice and quickly freeze the lysed cells;

d.68℃温育1分钟后，冰浴；d. Incubate at 68°C for 1 minute and place on ice;

e.配置cDNA合成体系，具体如下表所示：e. Configure the cDNA synthesis system as shown in the following table:

f.PCR扩增获得抗体可变区cDNA，其中，PCR反应条件为：42℃5min，25℃10min，50℃60min，94℃5min；f. PCR amplification to obtain antibody variable region cDNA, wherein the PCR reaction conditions are: 42°C for 5 min, 25°C for 10 min, 50°C for 60 min, and 94°C for 5 min;

g.Ig可变区第一轮扩增，反应体系如下：g. The first round of amplification of Ig variable region, the reaction system is as follows:

其中，上述5’First PCR primer mix和3’First PCR primer mix均参照文献：Wardemann,H等，Methods Mol Biol,2019.1956:p.105-125。Among them, the above-mentioned 5'First PCR primer mix and 3'First PCR primer mix are both referenced from the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.

h.PCR扩增获得第一轮PCR扩增产物，其中PCR反应条件为：94℃15min预变性；94℃30s，58℃30s(IgH和Igκ)或者60℃30s(Igλ)，72℃55s扩增50循环；72℃10min；h. PCR amplification to obtain the first round of PCR amplification products, wherein the PCR reaction conditions are: 94°C for 15 min pre-denaturation; 94°C for 30 s, 58°C for 30 s (IgH and Igκ) or 60°C for 30 s (Igλ), 72°C for 55 s amplification for 50 cycles; 72°C for 10 min;

i.Ig可变区第二轮扩增，反应体系如下：i. The second round of amplification of Ig variable region, the reaction system is as follows:

其中，上述5’Second PCR primer mix和3’Second PCR primer mix均参照文献：Wardemann,H等，Methods Mol Biol,2019.1956:p.105-125。Among them, the above-mentioned 5'Second PCR primer mix and 3'Second PCR primer mix are both referenced from the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.

j.PCR扩增获得第二轮PCR扩增产物，其中PCR反应条件为：94℃15min预变性；94℃30s，58℃30s(IgH和Igκ)或者60℃30s(Igλ)，72℃45s扩增50循环；72℃10min；j. PCR amplification to obtain the second round of PCR amplification products, wherein the PCR reaction conditions are: 94°C for 15 min pre-denaturation; 94°C for 30 s, 58°C for 30 s (IgH and Igκ) or 60°C for 30 s (Igλ), 72°C for 45 s amplification for 50 cycles; 72°C for 10 min;

k.通过琼脂糖凝胶DNA回收试剂盒(天根，货号：DP209-03)回收第二轮PCR扩增产物；k. Recover the second round of PCR amplification products using an agarose gel DNA recovery kit (Tian Gen, catalog number: DP209-03);

l.Ig可变区特异性扩增，反应体系如下：1. Ig variable region specific amplification, the reaction system is as follows:

其中，上述5’Specific PCR primer mix和3’Specific PCR primer mix均参照文献：Wardemann,H等，Methods Mol Biol,2019.1956:p.105-125。Among them, the above-mentioned 5'Specific PCR primer mix and 3'Specific PCR primer mix are both referenced from the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.

m.PCR反应条件：94℃15min预变性；94℃30s，58℃30s(IgH和Igκ)或者60℃30s(Igλ)，72℃45s扩增50循环；72℃10min；m. PCR reaction conditions: 94°C for 15 min pre-denaturation; 94°C for 30 s, 58°C for 30 s (IgH and Igκ) or 60°C for 30 s (Igλ), 72°C for 45 s for 50 cycles; 72°C for 10 min;

n.琼脂糖凝胶电泳检测扩增的特异性PCR产物，结果如图2所示；n. Detection of specific PCR products by agarose gel electrophoresis, the results are shown in Figure 2;

o.通过琼脂糖凝胶DNA回收试剂盒(天根，货号：DP209-03)回收特异性PCR产物。o. Recover specific PCR products using an agarose gel DNA recovery kit (Tian Gen, catalog number: DP209-03).

(3)构建表达质粒，体外转染、表达以及纯化抗体(3) Construction of expression plasmid, in vitro transfection, expression and purification of antibodies

3.1构建表达质粒3.1 Construction of expression plasmid

a.上述特异性PCR产物(IgH，Igκ和Igλ)各取30.4μL，分别加入3.4μL CutSmart缓冲液(NEB，货号：B7204S)混匀，获得相应的混合料；a. Take 30.4 μL of each of the above specific PCR products (IgH, Igκ and Igλ), add 3.4 μL of CutSmart buffer (NEB, catalog number: B7204S) and mix well to obtain the corresponding mixture;

b.配置酶切体系，具体如下：b. Configure the enzyme digestion system as follows:

其中，上述AgeI-HF、SalI-HF、BsiWI-HF以及XhoI为NEB限制性内切酶，货号分别为R3552L(AgeI-HF)、R3138L(SalI-HF)、R3553L(BsiWI-HF)和R0146L(XhoI)。Among them, the above-mentioned AgeI-HF, SalI-HF, BsiWI-HF and XhoI are NEB restriction endonucleases, and the product numbers are R3552L (AgeI-HF), R3138L (SalI-HF), R3553L (BsiWI-HF) and R0146L (XhoI), respectively.

c.在步骤a中的相应混合料中分别加入对应的配置好的步骤b的混合料；c. Add the corresponding mixture of step b to the corresponding mixture in step a;

d.于37℃酶切2h；d. Enzyme digestion at 37°C for 2h;

e.使用琼脂糖凝胶DNA回收试剂盒(天根，货号：DP209-03)将酶切后的产物回收；e. Use agarose gel DNA recovery kit (Tian Gen, Cat. No.: DP209-03) to recover the digested product;

f.连接酶切后的特异性PCR产物与相应载体，连接体系如下：f. Connect the specific PCR product after enzyme digestion with the corresponding vector. The connection system is as follows:

其中，上述IgH载体为AbVec2.0-IGHG1(AddGene，货号：80795)，Igκ载体为AbVec1.1-IGKC(AddGene，货号：80796)，Igλ载体为AbVec1.1-IGLC2-XhoI(AddGene，货号：99575))。Among them, the above-mentioned IgH vector is AbVec2.0-IGHG1 (AddGene, catalog number: 80795), the Igκ vector is AbVec1.1-IGKC (AddGene, catalog number: 80796), and the Igλ vector is AbVec1.1-IGLC2-XhoI (AddGene, catalog number: 99575).

g.于16℃连接过夜；g. Connect at 16°C overnight;

h.将上述连接过夜的连接产物加到含有100μL DH5α感受态细胞(天根，货号：CB101-02)的离心管中，置于冰上冰浴30min；h. Add the overnight ligation product to a centrifuge tube containing 100 μL DH5α competent cells (Tian Gen, Catalog No.: CB101-02) and place on ice for 30 minutes;

i.将步骤h中的连接产物与感受态细胞的混合物置于42℃水浴锅中，热击90s；i. Place the mixture of the ligation product and competent cells in step h in a 42°C water bath and heat shock for 90 seconds;

j.取出置于冰上3-5min后加入900μL TB培养基(ThermoFisher，货号：22711022)；j. Take out and place on ice for 3-5 minutes, then add 900 μL TB medium (ThermoFisher, catalog number: 22711022);

k.震荡培养45-60min后，6000rpm离心1min；k. After shaking culture for 45-60 minutes, centrifuge at 6000 rpm for 1 minute;

l.吸去800μL上清，并使用剩余的液体将细菌(源自前述感受态细胞)吹打混匀；1. Aspirate 800 μL of supernatant and use the remaining liquid to mix the bacteria (derived from the competent cells described above);

m.将步骤l中的细菌混悬液均匀的涂抹在加有氨苄的琼脂板上；m. The bacterial suspension in step l is evenly applied on an agar plate with ampicillin;

n.将琼脂板倒置于37℃细菌孵育箱中培养16h；n. Place the agar plate upside down in a 37°C bacterial incubator and culture for 16 hours;

o.挑取琼脂板上单个饱满的菌落与第二轮PCR产物一同送去公司测序；o. Pick a single full colony on the agar plate and send it to the company for sequencing together with the second round of PCR products;

p.选取与第二轮PCR产物序列100％匹配的菌落，使用无内毒素质粒小提中量试剂盒(天根，货号：DP118-02)提取质粒，分别获得重链质粒与轻链质粒。p. Select the colonies that match the second round PCR product sequence 100%, use the endotoxin-free plasmid mini-extraction kit (Tian Gen, catalog number: DP118-02) to extract the plasmids, and obtain the heavy chain plasmid and the light chain plasmid respectively.

3.2体外转染、表达3.2 In vitro transfection and expression

a.转染前一天使用FreeStyle^TM293表达培养基(Gibco，货号：12338018)将293F细胞密度调整为1×10⁶个/mL；a. One day before transfection, the density of 293F cells was adjusted to 1×10⁶ cells/mL using FreeStyle^™ 293 Expression Medium (Gibco, Cat. No.: 12338018);

b.转染当天对细胞计数，并使用新鲜的FreeStyle^TM293表达培养基(Gibco，货号：12338018)将293F细胞密度调整为2×10⁶个/mL；b. Count the cells on the day of transfection and adjust the 293F cell density to 2×10⁶ cells/mL using fresh FreeStyle^™ 293 Expression Medium (Gibco, Catalog No.: 12338018);

c.配制PEI混合液：将Polyethylenimine(PEI)(Polysciences，货号：23996-2)加入到OptiPRO^TMSMF培养基(ThermoFisher，货号：12309019)中，使转染时浓度为4μg/mL；c. Prepare PEI mixed solution: Add Polyethylenimine (PEI) (Polysciences, Catalog No.: 23996-2) to OptiPRO^TM SMF medium (ThermoFisher, Catalog No.: 12309019) to a concentration of 4 μg/mL during transfection;

d.配制质粒混合液：将提取好的配对重链质粒与轻链质粒按1：2的质量比加入到OptiPRO^TMSFM培养基(ThermoFisher，货号：12309019)中混匀，使转染时总质粒浓度为1μg/mL；d. Prepare plasmid mixture: add the extracted paired heavy chain plasmid and light chain plasmid into OptiPRO^TM SFM medium (ThermoFisher, catalog number: 12309019) at a mass ratio of 1:2 and mix well to make the total plasmid concentration at the time of transfection to be 1 μg/mL;

e.最后将PEI混合液加到质粒混合液中轻柔混匀，配成转染混合体系，室温静置20min；e. Finally, add the PEI mixture to the plasmid mixture and mix gently to form a transfection mixture system, and let it stand at room temperature for 20 minutes;

f.将步骤e中的转染混合体系加入到步骤b中的293F细胞中，轻柔摇晃混匀；f. Add the transfection mixture in step e to the 293F cells in step b and shake gently to mix;

g.将步骤f的293F细胞置于含8％CO₂的37℃悬浮培养箱中，于125rpm条件下悬浮培养7天。g. The 293F cells from step f were placed in a 37°C suspension incubator containing 8% CO₂ and cultured at 125 rpm for 7 days.

3.3广谱抗体的纯化3.3 Purification of broad-spectrum antibodies

a.将所述转染后悬浮培养液于4000rpm离心15min，收集表达上清；a. The transfected suspension culture was centrifuged at 4000 rpm for 15 min to collect the expression supernatant;

b.上清使用0.22μm滤器(JET，货号：FPE204030)过滤；b. The supernatant was filtered using a 0.22 μm filter (JET, catalog number: FPE204030);

c.打开蛋白纯化仪，使用PBS将Protein A柱子平衡，流速3mL/min；c. Open Protein purification instrument, balance the Protein A column with PBS, flow rate 3mL/min;

d.随后将过滤后的表达上清加载Protein A柱，流速3mL/min；d. The filtered expression supernatant was then loaded onto the Protein A column at a flow rate of 3 mL/min;

e.使用PBS洗去Protein A柱上非特异性结合蛋白，流速3mL/min；e. Use PBS to wash away non-specific binding proteins on the Protein A column at a flow rate of 3 mL/min;

f.使用pH＝3.0的甘氨酸缓冲液洗脱Protein A柱上结合的抗体，流速1mL/min；f. Use glycine buffer at pH 3.0 to elute the antibody bound to the Protein A column at a flow rate of 1 mL/min;

g.收集洗脱液，获得纯化后的抗体。g. Collect the eluate to obtain the purified antibody.

(4)ELISA筛选与新型冠状病毒SARS-CoV-2 Wuhan-Hu-1 S蛋白结合的抗体(4) ELISA screening of antibodies binding to the novel coronavirus SARS-CoV-2 Wuhan-Hu-1 S protein

a.将SARS-CoV-2 Wuhan-Hu-1 S蛋白按2μg/mL(100μL/孔)的浓度包被酶标板；a. Coat the ELISA plate with SARS-CoV-2 Wuhan-Hu-1 S protein at a concentration of 2 μg/mL (100 μL/well);

b.4℃孵育过夜之后使用1×PBST洗去未结合蛋白；b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

c.使用含2％FBS(Gibco，货号：10270-106)和2％BSA(Sigma，货号：V900933)的封闭液常温封闭2h；c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

d.使用1×PBST洗去封闭液后，将上述纯化后的抗体按1μg/mL的浓度加至酶标板中，37℃孵育1h；d. After washing away the blocking solution with 1×PBST, add the purified antibody to the ELISA plate at a concentration of 1 μg/mL and incubate at 37°C for 1 hour;

e.使用1×PBST洗去未结合的抗体后，加入RHP标记的抗人IgG抗体(Jacksonimmunoresearc，货号：109-035-003)，37℃孵育1h；e. After washing away the unbound antibody with 1×PBST, add RHP-labeled anti-human IgG antibody (Jacksonimmunoresearch, catalog number: 109-035-003) and incubate at 37°C for 1 h;

f.使用1×PBST洗去未结合的抗人IgG抗体后，加入100μL TMB显色液(ThermoFisher，货号：002023)常温孵育5min；f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

g.最后加入50μL 1M的硫酸终止反应；g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

h.使用Varioskan Flash全波长扫描式多功能读数仪(ThermoFisherScientific)测量OD值，并通过OD值来反应抗体的结合强度。h. The OD value was measured using a Varioskan Flash full wavelength scanning multifunctional reader (Thermo Fisher Scientific), and the OD value was used to reflect the binding strength of the antibody.

根据抗体与SARS-CoV-2S蛋白的结合强度选出抗新型冠状病毒SARS-CoV-2的广谱抗体(VSM12-37)。A broad-spectrum antibody (VSM12-37) against the new coronavirus SARS-CoV-2 was selected based on the binding strength of the antibody to the SARS-CoV-2S protein.

经测序鉴定，该抗新型冠状病毒SARS-CoV-2的广谱抗体的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：2所示。编码广谱抗体的重链可变区的基因序列如SEQ ID NO：3所示；编码广谱抗体的轻链可变区的基因序列如SEQID NO：4所示。广谱抗体的重链全长的氨基酸序列如SEQ ID NO：5所示；广谱抗体的轻链全长的氨基酸序列如SEQ ID NO：6所示。After sequencing and identification, the amino acid sequence of the heavy chain variable region of the broad-spectrum antibody against the new coronavirus SARS-CoV-2 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2. The gene sequence encoding the heavy chain variable region of the broad-spectrum antibody is shown in SEQ ID NO: 3; the gene sequence encoding the light chain variable region of the broad-spectrum antibody is shown in SEQ ID NO: 4. The amino acid sequence of the full-length heavy chain of the broad-spectrum antibody is shown in SEQ ID NO: 5; the amino acid sequence of the full-length light chain of the broad-spectrum antibody is shown in SEQ ID NO: 6.

SEQ ID NO：1：SEQ ID NO: 1:

EVQLVESGGGLVQPGGSLRLSCAVSGFTVSSNYMSWVRQAPGKGLEWVSLIYSGGSTYYADSVKGRFTVSRDRSKNTLYLQMNSLRAEDTALYYCARDNPRVGSNYWGQGTLVTVSS。EVQLVESGGGLVQPGGSLRLSCAVSGFTVSSNYMSWVRQAPGKGLEWVSLIYSGGSTYYADSVKGRFTVSRDRSKNTLYLQMNSLRAEDTALYYCARDNPRVGSNYWGQGTLVTVSS.

SEQ ID NO：2：SEQ ID NO: 2:

DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAPKLLIYDASNLEAGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQCDNVPLTFGGGTKVEIK。DIQMTQSPSSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAPKLLIYDASNLEAGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQCDNVPLTFGGGTKVEIK.

SEQ ID NO：3：SEQ ID NO: 3:

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGATTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACTTATTTATAGCGGTGGCAGCACATACTACGCAGACTCCGTGAAGGGCAGATTCACCGTCTCCAGAGACAGGTCCAAGAACACGCTGTATCTTCAGATGAACAGCCTGAGAGCCGAGGACACGGCTCTCTATTACTGTGCGAGAGATAATCCCCGGGTGGGTTCAAACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA。GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGATTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACTTATTTATAGCGGTGGCAGCACATACTACGCAGACTCCGTGAAGGGCAGATTCACCGTCTCCAGAGACAGGTCCAAGAACACGCTGTATCTTCAGATGAACAGCCTGA GAGCCGAGGACACGGCTCCTATTACTGTGCGAGAGATAATCCCCGGGTGGGTTCAAACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA.

SEQ ID NO：4：SEQ ID NO: 4:

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAACAATTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAGCAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTGTGATAATGTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA。GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAACAATTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAGCAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACAT ATTACTGTCAACAGTGTGATAATGTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA.

SEQ ID NO：5：SEQ ID NO: 5:

MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAVSGFTVSSNYMSWVRQAPGKGLEWVSLIYSGGSTYYADSVKGRFTVSRDRSKNTLYLQMNSLRAEDTALYYCARDNPRVGSNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAVSGFTVSSNYMSWVRQAPGKGLEWVSLIYSGGSTYYADSVKGRFTVSRDRSKNTLYLQMNSLRAEDTALYYCARDNPRVGSNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

SEQ ID NO：6：SEQ ID NO: 6:

MGWSCIILFLVATAVHSDIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAPKLLIYDASNLEAGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQCDNVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。MGWSCIILFLVATAVHSDIQMTQSPSSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAPKLLIYDASNLEAGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQCDNVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC.

实施例2：结合实验Example 2: Binding Experiment

(1)ELISA检测构建抗体与新型冠状病毒突变株S蛋白的结合能力(1) ELISA test of the binding ability of the constructed antibody to the S protein of the mutated strain of the new coronavirus

a.将SARS-CoV-2 Wuhan-Hu-1(Sino Biological，货号：40589-V08B1)或Alpha突变株(Sino Biological，货号：40589-V08B1)或Beta突变株(Sino Biological，货号：40589-V08B1)或Gamma突变株(Sino Biological，货号：40589-V08B1)或Delta突变株(SinoBiological，货号：40589-V08B1)或者Omicron BA.1突变株(Sino Biological，货号：40589-V08B1)或者Omicron BA.2突变株(Sino Biological，货号：40589-V08B1)S蛋白按2μg/mL(100μL/孔)的浓度包被酶标板；a. Coat the SARS-CoV-2 Wuhan-Hu-1 (Sino Biological, Catalog No.: 40589-V08B1) or Alpha mutant (Sino Biological, Catalog No.: 40589-V08B1) or Beta mutant (Sino Biological, Catalog No.: 40589-V08B1) or Gamma mutant (Sino Biological, Catalog No.: 40589-V08B1) or Delta mutant (Sino Biological, Catalog No.: 40589-V08B1) or Omicron BA.1 mutant (Sino Biological, Catalog No.: 40589-V08B1) or Omicron BA.2 mutant (Sino Biological, Catalog No.: 40589-V08B1) S protein on the ELISA plate at a concentration of 2 μg/mL (100 μL/well);

b.4℃孵育过夜之后使用1×PBST洗去未结合蛋白；b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

c.使用含2％FBS(Gibco，货号：10270-106)和2％BSA(Sigma，货号：V900933)的封闭液常温封闭2h；c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

d.使用1×PBST洗去封闭液后，加入1μg/mL实施例1所得的纯化后的抗体，37℃孵育1h；d. After washing away the blocking solution with 1×PBST, 1 μg/mL of the purified antibody obtained in Example 1 was added and incubated at 37°C for 1 h;

e.使用1×PBST洗去未结合的抗体，随后加入RHP标记的抗人IgG抗体(JacksonImmunoResearch，货号：109-035-003)，37℃孵育1h；e. Unbound antibodies were washed away with 1× PBST, and then RHP-labeled anti-human IgG antibody (Jackson ImmunoResearch, Catalog No.: 109-035-003) was added and incubated at 37°C for 1 h;

f.使用1×PBST洗去未结合的抗人IgG抗体后，加入100μL TMB显色液(ThermoFisher，货号：002023)常温孵育5min；f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

g.最后加入50μL 1M的硫酸终止反应；g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

h.使用Varioskan Flash全波长扫描式多功能读数仪(ThermoFisherScientific)测量OD值。h. OD values were measured using a Varioskan Flash full wavelength scanning multifunctional reader (Thermo Fisher Scientific).

结果如图5所示，VSM12-37对所有新型冠状病毒VOC突变株都展现出强效的结合能力。The results are shown in Figure 5. VSM12-37 exhibits strong binding ability to all novel coronavirus VOC mutant strains.

(2)ELISA检测构建抗体与新型冠状病毒SARS-CoV-2 Wuhan-Hu-1和Omicron突变株S蛋白的半数效应浓度(EC₅₀)(2) ELISA test of the half effective concentration (EC₅₀ ) of the constructed antibody against the S protein of the new coronavirus SARS-CoV-2 Wuhan-Hu-1 and Omicron mutant strains

a.将SARS-CoV-2 Wuhan-Hu-1(Sino Biological，货号：40589-V08B1)或OmicronBA.1(Sino Biological，货号：40589-V08B33)或Omicron BA.2(Sino Biological，货号：40589-V08H28)突变株S蛋白按2μg/mL(100μL/孔)的浓度包被酶标板；a. Coat the ELISA plate with SARS-CoV-2 Wuhan-Hu-1 (Sino Biological, Catalog No.: 40589-V08B1) or Omicron BA.1 (Sino Biological, Catalog No.: 40589-V08B33) or Omicron BA.2 (Sino Biological, Catalog No.: 40589-V08H28) mutant strain S protein at a concentration of 2 μg/mL (100 μL/well);

b.4℃孵育过夜之后使用1×PBST洗去未结合蛋白；b. After incubation at 4°C overnight, unbound proteins were washed away with 1× PBST;

c.使用含2％FBS(Gibco，货号：10270-106)和2％BSA(Sigma，货号：V900933)的封闭液常温封闭2h；c. Use blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933) to block for 2 hours at room temperature;

d.使用1×PBST洗去封闭液后，将实施例1中纯化的抗体稀释(使用含2％FBS(Gibco，货号：10270-106)和2％BSA(Sigma，货号：V900933)的封闭液进行稀释)，获得浓度分别为10、3.33、1.11、0.37、0.123、0.041、0.0137、0.0046、0.0015、0.0005、0.0017以及0μg/mL的抗体溶液，将抗体溶液分别加入到酶标板中，37℃孵育1h；d. After washing away the blocking solution with 1×PBST, the purified antibody in Example 1 was diluted (using a blocking solution containing 2% FBS (Gibco, Catalog No.: 10270-106) and 2% BSA (Sigma, Catalog No.: V900933)) to obtain antibody solutions with concentrations of 10, 3.33, 1.11, 0.37, 0.123, 0.041, 0.0137, 0.0046, 0.0015, 0.0005, 0.0017 and 0 μg/mL, respectively. The antibody solutions were added to the ELISA plate and incubated at 37°C for 1 h.

e.使用1×PBST洗去未结合的抗体后，随后加入RHP标记的抗人IgG抗体(JacksonImmunoResearch，货号：109-035-003)，37℃孵育1h；e. After washing away the unbound antibody with 1×PBST, RHP-labeled anti-human IgG antibody (Jackson ImmunoResearch, Catalog No.: 109-035-003) was added and incubated at 37°C for 1 h;

f.使用1×PBST洗去未结合的抗人IgG抗体后，加入100μL TMB显色液(ThermoFisher，货号：002023)常温孵育5min；f. After washing away the unbound anti-human IgG antibody with 1×PBST, add 100 μL TMB colorimetric solution (ThermoFisher, catalog number: 002023) and incubate at room temperature for 5 min;

g.最后加入50μL 1M的硫酸终止反应；g. Finally, add 50 μL 1M sulfuric acid to terminate the reaction;

h.使用Varioskan Flash全波长扫描式多功能读数仪(ThermoFisher)测量OD值；h. OD values were measured using Varioskan Flash full wavelength scanning multifunctional reader (ThermoFisher);

i.数据经过Prism 8.0软件(GraphPad)计算，获得抗体对新型冠状病毒SARS-CoV-2 Wuhan-Hu-1和Omicron突变株的半数效浓度(EC₅₀)。i. The data were calculated using Prism 8.0 software (GraphPad) to obtain the half effective concentration (EC₅₀ ) of the antibody against the novel coronavirus SARS-CoV-2 Wuhan-Hu-1 and Omicron mutants.

结果如图6所示，VSM12-37抗体对新型冠状病毒SARS-CoV-2 Wuhan-Hu-1和Omicron BA.2展现出强效的结合能力，EC₅₀达到纳克级别，强于许多目前上市和正在进行临床实验的新冠抗体。而对SARS-CoV-2 Omicron BA.1突变株则展现出相对较弱的结合能力，EC₅₀达到5μg/mL以上。The results are shown in Figure 6. The VSM12-37 antibody showed strong binding ability to the new coronavirus SARS-CoV-2 Wuhan-Hu-1 and Omicron BA.2, with an EC₅₀ of nanograms, which is stronger than many new coronavirus antibodies currently on the market and in clinical trials. However, it showed relatively weak binding ability to the SARS-CoV-2 Omicron BA.1 mutant strain, with an EC₅₀ of more than 5μg/mL.

实施例3：病毒中和实验Example 3: Virus Neutralization Experiment

(1)假病毒包被：(1) Pseudovirus coating:

a.感染前一天以5×10⁶个/孔密度将293T细胞接种到10cm的细胞培养皿中；a. One day before infection, 293T cells were seeded into a 10 cm cell culture dish at a density of 5 × 10⁶ cells/well;

b.编码SARS-CoV-2 Wuhan-Hu-1或Alpha突变株或Beta突变株或Gamma突变株或Delta突变株或者Omicron突变株S蛋白的基因序列，由南京金斯瑞公司合成并插入到pcDNA3.1载体中。其中，上述突变株的基因编号为：SARS-CoV-2 Wuhan-Hu-1(Genebank：YP_009724390.1)、SARS-CoV-2Alpha(Genebank：OV054768.1)、SARS-CoV-2Beta(Genebank：MZ433432.1)、SARS-CoV-2Gamm(Genebank：MZ427312.1)、SARS-CoV-2Delta(Genebank：OK091006.1)、SARS-CoV-2 Omicron BA.1(GISAID ID：EPI_ISL_6590782.2)和SARS-CoV-2Omicron BA.2(Genebank：UKS51680.1)、SARS-CoV-2 Omicron BA.3spike(GISAID：EPI_ISL_7605589)和SARS-CoV-2 Omicron BA.4/5spike(GISAID:EPI_ISL_12268493.2)；b. The gene sequence encoding the S protein of SARS-CoV-2 Wuhan-Hu-1 or Alpha mutant strain or Beta mutant strain or Gamma mutant strain or Delta mutant strain or Omicron mutant strain was synthesized by Nanjing GenScript and inserted into the pcDNA3.1 vector. Among them, the gene numbers of the above mutants are: SARS-CoV-2 Wuhan-Hu-1 (Genebank: YP_009724390.1), SARS-CoV-2Alpha (Genebank: OV054768.1), SARS-CoV-2Beta (Genebank: MZ433432.1), SARS-CoV-2Gamm (Genebank: MZ427312.1), SARS-CoV-2Delta (Genebank: OK091006.1), SARS-CoV-2 Omicron BA.1 (GISAID ID: EPI_ISL_6590782.2) and SARS-CoV-2Omicron BA.2 (Genebank: UKS51680.1), SARS-CoV-2 Omicron BA.3spike (GISAID: EPI_ISL_7605589) and SARS-CoV-2 Omicron BA.4/5spike(GISAID:EPI_ISL_12268493.2);

c.配制质粒混合液：将步骤b合成的质粒与质粒PNL4-3(Genebank：AF324493.2)按质量比为SARS-CoV-2S：PNL4-3＝1：3加入到Opti-MEM^TM(ThermoFisher，货号：31985062)培养基中混匀，使总质粒浓度浓度为1μg/mL；c. Prepare a plasmid mixture: add the plasmid synthesized in step b and plasmid PNL4-3 (Genebank: AF324493.2) at a mass ratio of SARS-CoV-2S:PNL4-3=1:3 to Opti-MEM^TM (ThermoFisher, catalog number: 31985062) medium and mix well to make the total plasmid concentration 1 μg/mL;

d.配制PEI混合液：再将Polyethylenimine(PEI)(Polysciences，货号：23996-2)加入到Opti-MEM^TM培养基(ThermoFisher，货号：31985062)中，使转染时浓度为4μg/mL；d. Prepare PEI mixed solution: Add Polyethylenimine (PEI) (Polysciences, Catalog No.: 23996-2) to Opti-MEM^TM medium (ThermoFisher, Catalog No.: 31985062) to a concentration of 4 μg/mL during transfection;

e.最后将PEI混合液加到质粒混合液中轻柔混匀，配成转染混合体系，室温静置20min；e. Finally, add the PEI mixture to the plasmid mixture and mix gently to form a transfection mixture system, and let it stand at room temperature for 20 minutes;

f.弃去步骤a中的293T细胞培养基，将步骤d中的转染混合体系加入到293T细胞中；f. Discard the 293T cell culture medium in step a, and add the transfection mixture in step d to the 293T cells;

g.将步骤f的293T细胞置于含5％CO2的37℃细胞培养箱中培养6h；g. The 293T cells from step f were placed in a 37°C cell culture incubator containing 5% CO2 for 6 h;

h.将转染混合体系吸出，置换为新鲜配置的含有10％FBS(Gibco，货号：10270-106)的DMEM培养基(Gibco，货号：11995065)；h. Aspirate the transfection mixture and replace with freshly prepared DMEM medium (Gibco, catalog number: 11995065) containing 10% FBS (Gibco, catalog number: 10270-106);

i.置于含5％CO₂的37℃细胞培养箱中培养72h后，12000rpm离心15min收集上清，所述上清即为包装好的假病毒。i. After culturing for 72 hours in a 37°C cell culture incubator containing 5% CO₂ , the supernatant was collected by centrifugation at 12000 rpm for 15 minutes. The supernatant was the packaged pseudovirus.

(2)假病毒中和实验检测抗体对新型冠状病毒SARS-CoV-2 Wuhan-Hu-1、Alpha、Beta、Gamma、Delta、Omicron BA.1、Omicron BA.2、Omicron BA.3、Omicron BA.4/5的半数抑制浓度(IC₅₀)(2) Pseudovirus neutralization assay to detect the half inhibitory concentration (IC 50 ) of antibodies against the novel coronavirus SARS-CoV-2 Wuhan-Hu-1, Alpha, Beta, Gamma, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.3, and Omicron BA.4/₅

a.感染前一天以0.8×10⁵个/mL密度将293T/hACE2细胞按每孔100μL接种到96孔板(所用培养基为：含10％FBS的DMEM)中；a. One day before infection, 293T/hACE2 cells were seeded into 96-well plates at a density of 0.8×10⁵ cells/mL at 100 μL per well (the culture medium used was DMEM containing 10% FBS);

b.感染当天，将实施例1所得的纯化的抗体分别与上述包装好的假病毒混合，稀释，获得多组混合液；b. On the day of infection, the purified antibodies obtained in Example 1 were mixed with the packaged pseudoviruses described above, and diluted to obtain multiple mixed solutions;

其中，各组混合液包括抗体浓度分别为100、33.3、11.1、3.7、1.23、0.41、0.137、0.046、0.015、0.005、0.017以及0μg/mL等不同浓度的混合液；用含10％FBS(Gibco，货号：10270-106)的DMEM培养基(Gibco，货号：11995065)进行稀释；Each group of mixed solutions includes mixed solutions with antibody concentrations of 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.137, 0.046, 0.015, 0.005, 0.017 and 0 μg/mL; diluted with DMEM medium (Gibco, catalog number: 11995065) containing 10% FBS (Gibco, catalog number: 10270-106);

c.将步骤b获得的抗体与假病毒的混合液置于37℃孵育1h；c. Incubate the mixture of the antibody and pseudovirus obtained in step b at 37°C for 1 h;

d.弃去步骤a中96孔板中的培养基，加入步骤c中的抗体与病毒的混合液，800g离心30min；d. Discard the culture medium in the 96-well plate in step a, add the antibody and virus mixture in step c, and centrifuge at 800g for 30 minutes;

e.置于37℃细胞培养箱孵育6-8h后，弃去抗体与病毒的混合液，加入新鲜配置的含10％FBS(Gibco，货号：10270-106)的DMEM培养基(Gibco，货号：11995065)；e. After incubation at 37°C in a cell culture incubator for 6-8 hours, the antibody and virus mixture was discarded and freshly prepared DMEM medium (Gibco, catalog number: 11995065) containing 10% FBS (Gibco, catalog number: 10270-106) was added;

f.细胞继续培养48h后，于96孔培养板中每孔加入50μL的细胞裂解液(Promega，货号：E153A)，37℃裂解2min；f. After the cells were cultured for 48 h, 50 μL of cell lysis buffer (Promega, catalog number: E153A) was added to each well of the 96-well culture plate and lysed at 37°C for 2 min;

g.随后将96孔培养板置于-40℃冷冻30min；g. The 96-well culture plate was then placed in a -40°C freezer for 30 min;

h.冷冻后，将96孔培养板取出置于37℃裂解3min，再2000rpm离心1min，获得细胞裂解液；h. After freezing, the 96-well culture plate was taken out and placed at 37°C for 3 min for lysis, and then centrifuged at 2000 rpm for 1 min to obtain cell lysate;

i.吸40μL上述细胞裂解液加入96孔黑色平地板中；i. Pipette 40 μL of the above cell lysate and add it into the 96-well black flat floor;

j.再加入50μL荧光素酶检测试剂(Promega，货号：E1501)，通过Varioskan Flash全波长扫描式多功能读数仪(ThermoFisher)测量OD值；j. Add 50 μL of luciferase detection reagent (Promega, catalog number: E1501) and measure the OD value using a Varioskan Flash full wavelength scanning multifunctional reader (ThermoFisher);

k.计算中和抑制率：抑制率＝[1－(加有抗体与病毒混合物孔OD值－空白孔OD值)/(未加抗体，只加病毒孔OD值－空白孔OD值]×100％。k. Calculate the neutralization inhibition rate: inhibition rate = [1 - (OD value of the well with the mixture of antibody and virus - OD value of the blank well) / (OD value of the well with only virus but no antibody added - OD value of the blank well] × 100%.

l.根据中和抑制率的结果，利用Prism 8.0软件(GraphPad)计算抗体的IC₅₀。1. Based on the results of neutralization inhibition rate, IC₅₀ of the antibody was calculated using Prism 8.0 software (GraphPad).

结果如图7所示，VSM12-37对新型冠状病毒SARS-CoV-2 Wuhan-Hu-1以及Alpha、Beta、Gamma、Delta、Omicron BA.1、Omicron BA.2和Omicron BA.4/5突变株假病毒展现出强效的中和能力，IC₅₀达到纳克级别。此外VSM7-80还对新型冠状病毒SARS-CoV-2 OmicronBA.3突变株假病毒也具有很强中和能力。相比而言，许多上市以及正进行临床实验的新型冠状病毒中和抗体对多种新型冠状病毒SARS-CoV-2VOC突变株出现了逃逸(PMID:35594867,34237773,35790190,35921836,35772405,35714668)，而VSM12-37对多种新型冠状病毒SARS-CoV-2的突变株都具有很好的中和活性。As shown in Figure 7, VSM12-37 showed strong neutralizing ability against the novel coronavirus SARS-CoV-2 Wuhan-Hu-1 and Alpha, Beta, Gamma, Delta, Omicron BA.1, Omicron BA.2 and Omicron BA.4/5 mutant pseudoviruses, with IC₅₀ reaching the nanogram level. In addition, VSM7-80 also has a strong neutralizing ability against the novel coronavirus SARS-CoV-2 OmicronBA.3 mutant pseudovirus. In comparison, many new coronavirus neutralizing antibodies that are on the market and undergoing clinical trials have escaped from a variety of new coronavirus SARS-CoV-2 VOC mutants (PMID: 35594867, 34237773, 35790190, 35921836, 35772405, 35714668), while VSM12-37 has good neutralizing activity against a variety of new coronavirus SARS-CoV-2 mutants.

上述实施例阐明的内容应当理解为这些实施例仅用于更清楚地说明本发明，而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落入本申请所附权利要求所限定的范围。The contents explained in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

Claims (10)

Translated fromChinese

1.一种抗新型冠状病毒SARS-CoV-2的广谱抗体，所述广谱抗体包括重链和轻链，其特征在于，所述广谱抗体至少具有如下技术特征之一：1. A broad-spectrum antibody against the novel coronavirus SARS-CoV-2, the broad-spectrum antibody comprising a heavy chain and a light chain, characterized in that the broad-spectrum antibody has at least one of the following technical features:(1)所述重链包括重链CDR1，其氨基酸序列为：GFTVSSNY；(1) The heavy chain includes a heavy chain CDR1, whose amino acid sequence is: GFTVSSNY;(2)所述重链包括重链CDR2，其氨基酸序列为：IYSGGST；(2) the heavy chain includes a heavy chain CDR2, the amino acid sequence of which is: IYSGGST;(3)所述重链包括重链CDR3，其氨基酸序列为：ARDNPRVGSNY；(3) the heavy chain includes a heavy chain CDR3, the amino acid sequence of which is: ARDNPRVGSNY;(4)所述轻链包括轻链CDR1，其氨基酸序列为：QDINNY；(4) the light chain includes a light chain CDR1, whose amino acid sequence is: QDINNY;(5)所述轻链包括轻链CDR2，其氨基酸序列为：DAS；(5) The light chain includes a light chain CDR2, whose amino acid sequence is: DAS;(6)所述轻链包括轻链CDR3，其氨基酸序列为：QQCDNVPLT。(6) The light chain includes a light chain CDR3, whose amino acid sequence is: QQCDNVPLT.2.根据权利要求1所述的广谱抗体，其特征在于，所述广谱抗体至少具有如下技术特征之一：2. The broad-spectrum antibody according to claim 1, characterized in that the broad-spectrum antibody has at least one of the following technical features:(a)所述重链包括重链CDR1-3，重链CDR1的氨基酸序列为：GFTVSSNY，重链CDR2的氨基酸序列为：IYSGGST，重链CDR3的氨基酸序列为：ARDNPRVGSNY；(a) the heavy chain includes heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is: GFTVSSNY, the amino acid sequence of heavy chain CDR2 is: IYSGGST, and the amino acid sequence of heavy chain CDR3 is: ARDNPRVGSNY;(b)所述轻链包括轻链CDR1-3，轻链CDR1的氨基酸序列为：QDINNY，轻链CDR2的氨基酸序列为：DAS，轻链CDR3的氨基酸序列为：QQCDNVPLT。(b) The light chain includes light chain CDR1-3, the amino acid sequence of light chain CDR1 is: QDINNY, the amino acid sequence of light chain CDR2 is: DAS, and the amino acid sequence of light chain CDR3 is: QQCDNVPLT.3.根据权利要求1所述的广谱抗体，其特征在于，所述广谱抗体的重链可变区的氨基酸序列如SEQ ID NO：1所示；所述广谱抗体的轻链可变区的氨基酸序列如SEQ ID NO：2所示。3. The broad-spectrum antibody according to claim 1, characterized in that the amino acid sequence of the heavy chain variable region of the broad-spectrum antibody is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the broad-spectrum antibody is shown in SEQ ID NO: 2.4.编码如权利要求1-3任一项所述的广谱抗体的核酸分子。4. A nucleic acid molecule encoding the broad-spectrum antibody according to any one of claims 1 to 3.5.根据权利要求4所述的核酸分子，其特征在于，所述核酸分子包括核苷酸序列SEQ IDNO：3和/或核苷酸序列SEQ ID NO：4。5 . The nucleic acid molecule according to claim 4 , characterized in that the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 3 and/or the nucleotide sequence SEQ ID NO: 4.6.如权利要求1-3任一项所述的广谱抗体在制备用于诊断试剂或诊断试剂盒或药物中的应用。6. Use of the broad-spectrum antibody according to any one of claims 1 to 3 in the preparation of a diagnostic reagent, a diagnostic kit or a drug.7.根据权利要求6所述的应用，其特征在于，所述药物具有针对新型冠状病毒SARS-CoV-2的中和性抗病毒作用；优选地，所述新型冠状病毒SARS-CoV-2包括新型冠状病毒SARS-CoV-2 Wuhan-Hu-1病毒株、新型冠状病毒Alpha突变株、新型冠状病毒Beta突变株、新型冠状病毒Gamma突变株、新型冠状病毒Delta突变株、新型冠状病毒Omicron突变株中的一种或几种。7. The use according to claim 6, characterized in that the drug has a neutralizing antiviral effect against the new coronavirus SARS-CoV-2; preferably, the new coronavirus SARS-CoV-2 includes one or more of the new coronavirus SARS-CoV-2 Wuhan-Hu-1 virus strain, the new coronavirus Alpha mutant strain, the new coronavirus Beta mutant strain, the new coronavirus Gamma mutant strain, the new coronavirus Delta mutant strain, and the new coronavirus Omicron mutant strain.8.如权利要求4或5所述的核酸分子在制备抗新型冠状病毒SARS-CoV-2的广谱抗体或药物中的应用。8. Use of the nucleic acid molecule as described in claim 4 or 5 in the preparation of broad-spectrum antibodies or drugs against the new coronavirus SARS-CoV-2.9.根据权利要求8所述的应用，其特征在于，所述药物具有针对新型冠状病毒SARS-CoV-2的中和作用；优选地，所述新型冠状病毒SARS-CoV-2包括新型冠状病毒SARS-CoV-2Wuhan-Hu-1病毒株、新型冠状病毒Alpha突变株、新型冠状病毒Beta突变株、新型冠状病毒Gamma突变株、新型冠状病毒Delta突变株、新型冠状病毒Omicron突变株中的一种或几种。9. The use according to claim 8, characterized in that the drug has a neutralizing effect on the new coronavirus SARS-CoV-2; preferably, the new coronavirus SARS-CoV-2 includes one or more of the new coronavirus SARS-CoV-2 Wuhan-Hu-1 virus strain, the new coronavirus Alpha mutant strain, the new coronavirus Beta mutant strain, the new coronavirus Gamma mutant strain, the new coronavirus Delta mutant strain, and the new coronavirus Omicron mutant strain.10.含有如权利要求4或5所述的核酸分子的表达盒、重组载体、重组菌或转基因细胞系。10. An expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule according to claim 4 or 5.