技术领域Technical Field
本发明涉及一种抗体广谱抗SARS-like冠状病毒和/或新冠突变株的抗体及其应用，属于生物医药领域。The present invention relates to an antibody with a broad spectrum of resistance to SARS-like coronavirus and/or novel coronavirus mutant strains and application thereof, belonging to the field of biomedicine.
背景技术Background technique
新冠病毒感染引发的肺炎(COVID-19)是一种名为严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的新型冠状病毒引起的呼吸道感染。SARS-CoV-2全球溯源研究结果表明，SARS-CoV-2起源于蝙蝠。事实上，在不到20年的时间里，动物冠状病毒对人类的蔓延已经导致了SARS、MERS和COVID-19。动物冠状病毒对人类的外溢是对人类健康和全球经济的重大威胁。动物冠状病毒在野生动物中无处不在，鉴于其在野生动物体内的大量储存、频繁重组和高基因组可塑性，未来可能会发生更多的动物冠状病传播给人类，特别是已经导致两次全球大流行的SARS-like冠状病毒。SARS-CoV-2和SARS-CoV都属于SARS-like冠状病毒，与蝙蝠、果子狸和穿山甲中发现的许多SARS-like冠状病毒具有共同起源，包括蝙蝠冠状病毒RaTG13、RsSHC014和WIV1，果子狸冠状病毒SZ3，以及穿山甲冠状病毒GD18和GX-P5L。这些动物冠状病毒与SARS-CoV或SARS-CoV-2非常相似，甚至能够使用相同的受体进入细胞并在原代人气道细胞中高效复制。此外，穿山甲冠状病毒GD18棘突蛋白中介导病毒进入细胞的受体结合基序(RBM)与SARS-CoV-2仅有一个氨基酸差异，这进一步突显了它们可能溢出到人类群体并引发新的流行病。COVID-19 is a respiratory infection caused by a new coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The results of global tracing studies of SARS-CoV-2 indicate that SARS-CoV-2 originated from bats. In fact, in less than 20 years, the spread of animal coronaviruses to humans has led to SARS, MERS, and COVID-19. The spillover of animal coronaviruses to humans is a major threat to human health and the global economy. Animal coronaviruses are ubiquitous in wild animals, and given their large storage, frequent recombination, and high genomic plasticity in wild animals, more animal coronaviruses may be transmitted to humans in the future, especially SARS-like coronaviruses that have caused two global pandemics. Both SARS-CoV-2 and SARS-CoV belong to SARS-like coronaviruses and share a common origin with many SARS-like coronaviruses found in bats, civets, and pangolins, including bat coronaviruses RaTG13, RsSHC014, and WIV1, civet coronavirus SZ3, and pangolin coronaviruses GD18 and GX-P5L. These animal coronaviruses are very similar to SARS-CoV or SARS-CoV-2, and are even able to use the same receptors to enter cells and replicate efficiently in primary human airway cells. In addition, the receptor binding motif (RBM) in the pangolin coronavirus GD18 spike protein that mediates viral entry into cells has only one amino acid difference from SARS-CoV-2, further highlighting their potential to spill over into the human population and cause new epidemics.
与SARS-like冠状病毒感染相关的死亡率和经济损失突出表明了制定广泛有效的应对措施的重要性，这可能是预防和缓解当前和未来人畜共患事件的关键。抗SARS-like冠状病毒的交叉中和抗体是一种有吸引力的对策，以预防或减轻当前和未来的溢出事件。事实上，从COVID19康复者和疫苗中分离出的几种抗SARS-CoV-2单克隆抗体具有广泛的抗SARS-like冠状病毒交叉中和活性。一般来说，广泛的交叉中和抗体靶向SARS-like冠状病毒保留的保守表位。因此，针对不同保守表位的多种交叉中和抗体的开发将为此类抗体用于预防和治疗提供更多选择。The mortality and economic losses associated with SARS-like coronavirus infections highlight the importance of developing broadly effective countermeasures, which may be key to preventing and mitigating current and future zoonotic events. Cross-neutralizing antibodies against SARS-like coronaviruses are an attractive countermeasure to prevent or mitigate current and future spillover events. In fact, several anti-SARS-CoV-2 monoclonal antibodies isolated from COVID19 survivors and vaccines have broad cross-neutralizing activity against SARS-like coronaviruses. In general, broadly cross-neutralizing antibodies target conserved epitopes retained by SARS-like coronaviruses. Therefore, the development of multiple cross-neutralizing antibodies targeting different conserved epitopes will provide more options for the use of such antibodies for prevention and treatment.
此外，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))。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单克隆抗体产生逃逸，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单克隆抗体产生了抵抗。因此，需要发掘能抵抗新冠突变株逃逸的强效广谱中和抗体以应对当前流行的Omicron突变株。In addition, 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 escaping host immunity and developing drug resistance (Duffy S, PLoS Biol, 2018 08; 16(8)). Currently, five new coronavirus mutants have been listed as mutants of concern (VOC) by the World Health Organization. In addition to the popular Omicron mutant, there are also Alpha, Beta, Gamma and Delta mutants. The biggest feature of the new coronavirus mutant is that it can escape vaccine immunity and monoclonal antibodies. At present, the new coronavirus Omicron mutant has developed resistance to multiple new coronavirus monoclonal antibodies that are on the market and in clinical trials. Therefore, it is necessary to discover potent and broad-spectrum neutralizing antibodies that can resist the escape of new coronavirus mutants to deal with the currently popular Omicron mutant.
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广谱抗SARS-like冠状病毒和/或新冠突变株的抗体及其应用。In view of the deficiencies in the prior art, the object of the present invention is to provide a broad-spectrum antibody against SARS-like coronaviruses and/or new coronavirus mutant strains 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:
一种广谱抗SARS-like冠状病毒和/或新冠突变株的抗体(VSM9-44)，所述抗体包括重链和轻链，所述抗体至少具有如下技术特征之一：An antibody (VSM9-44) with a broad spectrum of resistance to SARS-like coronavirus and/or novel coronavirus mutant strains, the antibody comprising a heavy chain and a light chain, and the 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：GFTFRSYA；(1) The heavy chain includes a heavy chain CDR1, whose amino acid sequence is: GFTFRSYA;
(2)所述重链包括重链CDR2，其氨基酸序列为：ISFDGNEK；(2) the heavy chain includes a heavy chain CDR2, the amino acid sequence of which is: ISFDGNEK;
(3)所述重链包括重链CDR3，其氨基酸序列为：AKDPVGSIVRATVVYSFYAMDV；(3) The heavy chain includes a heavy chain CDR3, whose amino acid sequence is: AKDPVGSIVRATVVYSFYAMDV;
(4)所述轻链包括轻链CDR1，其氨基酸序列为：SGSIASNY；(4) the light chain includes a light chain CDR1, whose amino acid sequence is: SGSIASNY;
(5)所述轻链包括轻链CDR2，其氨基酸序列为：EDN；(5) The light chain includes a light chain CDR2, whose amino acid sequence is: EDN;
(6)所述轻链包括轻链CDR3，其氨基酸序列为：QSSDSDTVV。(6) The light chain includes a light chain CDR3, whose amino acid sequence is: QSSDSDTVV.
进一步地，所述抗体至少具有如下技术特征之一：Furthermore, the 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：GFTFRSYA，重链CDR2的氨基酸序列为：ISFDGNEK，重链CDR3的氨基酸序列为：AKDPVGSIVRATVVYSFYAMDV；(a) the heavy chain comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is: GFTFRSYA, the amino acid sequence of heavy chain CDR2 is: ISFDGNEK, and the amino acid sequence of heavy chain CDR3 is: AKDPVGSIVRATVVYSFYAMDV;
(b)所述轻链包括轻链CDR1-3，轻链CDR1的氨基酸序列为：SGSIASNY，轻链CDR2的氨基酸序列为：EDN，轻链CDR3的氨基酸序列为：QSSDSDTVV。(b) The light chain includes light chain CDR1-3, the amino acid sequence of light chain CDR1 is: SGSIASNY, the amino acid sequence of light chain CDR2 is: EDN, and the amino acid sequence of light chain CDR3 is: QSSDSDTVV.
进一步地，所述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：GFTFRSYA；所述重链包括重链CDR2，其氨基酸序列为：ISFDGNEK；所述重链包括重链CDR3，其氨基酸序列为：AKDPVGSIVRATVVYSFYAMDV；所述轻链包括轻链CDR1，其氨基酸序列为：SGSIASNY；所述轻链包括轻链CDR2，其氨基酸序列为：EDN；所述轻链包括轻链CDR3，其氨基酸序列为：QSSDSDTVV。Furthermore, the antibody has the following technical characteristics: the heavy chain includes a heavy chain CDR1, whose amino acid sequence is: GFTFRSYA; the heavy chain includes a heavy chain CDR2, whose amino acid sequence is: ISFDGNEK; the heavy chain includes a heavy chain CDR3, whose amino acid sequence is: AKDPVGSIVRATVVYSFYAMDV; the light chain includes a light chain CDR1, whose amino acid sequence is: SGSIASNY; the light chain includes a light chain CDR2, whose amino acid sequence is: EDN; the light chain includes a light chain CDR3, whose amino acid sequence is: QSSDSDTVV.
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Furthermore, the heavy chain also includes heavy chain FR1-4, and the amino acid sequence of heavy chain FR1 is
QVQLVESGGGLVQPGRSLRLSCAAS，重链FR2的氨基酸序列为MHWVRQAPGKGLEWVAA，重链FR3的氨基酸序列为FFADSVKGRFSISRDNSKSTLFLQINRLRPEDTAVYYC，重链FR4的氨基酸序列为WGPGTTVTVSS。QVQLVESGGGLVQPGRSLRLSCAAS, the amino acid sequence of the heavy chain FR2 is MHWVRQAPGKGLEWVAA, the amino acid sequence of the heavy chain FR3 is FFADSVKGRFSISRDNSKSTLFLQINRLRPEDTAVYYC, and the amino acid sequence of the heavy chain FR4 is WGPGTTVTVSS.
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Furthermore, the light chain also includes light chain FR1-4, and the amino acid sequence of light chain FR1 is
NFMLTQPHSVSESPEKTVIISCTRS，轻链FR2的氨基酸序列为VQWYQQRPGSSPTIIIQ，轻链FR3的氨基酸序列为QRPSGVPDRFSGSIDSSSNSASLTISRLKTEDEADYYC，轻链FR4的氨基酸序列为FGGGTKLTV。NFMLTQPHSVSESPEKTVIISCTRS, the amino acid sequence of the light chain FR2 is VQWYQQRPGSSPTIIIQ, the amino acid sequence of the light chain FR3 is QRPSGVPDRFSGSIDSSSNSASLTISRLKTEDEADYYC, and the amino acid sequence of the light chain FR4 is FGGGTKLTV.
进一步地，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：1所示；所述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 antibody is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 2.
进一步地，编码抗体的重链可变区的基因序列如SEQ ID NO：3所示；编码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 antibody is shown in SEQ ID NO: 3; the gene sequence encoding the light chain variable region of the antibody is shown in SEQ ID NO: 4.
编码如上所述的广谱抗SARS-like冠状病毒和/或新冠突变株的抗体的核酸分子。Nucleic acid molecules encoding broad-spectrum antibodies against SARS-like coronaviruses and/or new coronavirus mutants 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SARS-like冠状病毒和/或新冠突变株的抗体在制备用于诊断试剂或诊断试剂盒或药物中的应用。The use of the broad-spectrum antibodies against SARS-like coronavirus and/or new coronavirus mutant strains as described above in the preparation of diagnostic reagents or diagnostic kits or drugs.
进一步地，所述药物具有针对SARS-like冠状病毒的中和作用；优选地，所述SARS-like冠状病毒包括新型冠状病毒SARS-CoV-2病毒株、SARS-CoV病毒株、蝙蝠SARS-like冠状病毒RaTG13和WIV1、果子狸SARS-like冠状病毒SZ3和Civet007、穿山甲SARS-like冠状病毒GX-P5L和GD18、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 SARS-like coronavirus; preferably, the SARS-like coronavirus includes one or more of the new coronavirus SARS-CoV-2 strain, SARS-CoV strain, bat SARS-like coronavirus RaTG13 and WIV1, civet SARS-like coronavirus SZ3 and Civet007, pangolin SARS-like coronavirus GX-P5L and GD18, new coronavirus Alpha mutant strain, new coronavirus Beta mutant strain, new coronavirus Gamma mutant strain, new coronavirus Delta mutant strain, and new coronavirus Omicron mutant strain.
如上所述的核酸分子在制备广谱抗SARS-like冠状病毒和/或新冠突变株的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 SARS-like coronaviruses and/or new coronavirus mutant strains.
进一步地，所述药物具有针对SARS-like冠状病毒的中和作用；优选地，所述SARS-like冠状病毒包括新型冠状病毒SARS-CoV-2病毒株、SARS-CoV病毒株、蝙蝠SARS-like冠状病毒RaTG13和WIV1、果子狸SARS-like冠状病毒SZ3和Civet007、穿山甲SARS-like冠状病毒GX-P5L和GD18、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 SARS-like coronavirus; preferably, the SARS-like coronavirus includes one or more of the new coronavirus SARS-CoV-2 strain, SARS-CoV strain, bat SARS-like coronavirus RaTG13 and WIV1, civet SARS-like coronavirus SZ3 and Civet007, pangolin SARS-like coronavirus GX-P5L and GD18, new coronavirus Alpha mutant (B.1.1.7), new coronavirus Beta mutant (B.1.351), new coronavirus Gamma mutant (P1), new coronavirus Delta mutant (B.1.617.2), and new coronavirus Omicron mutant (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，本发明的广谱抗SARS-like冠状病毒和/或新冠突变株的抗体的制备方法包括如下步骤：Optionally, the method for preparing broad-spectrum antibodies against SARS-like coronavirus and/or novel coronavirus mutant strains 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MemoryB细胞；S1. SARS-CoV-2-specific MemoryB 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，获得广谱抗SARS-like冠状病毒和/或新冠突变株的抗体。S3. Construct expression plasmids, perform in vitro transfection, expression, and purification to obtain broad-spectrum antibodies against SARS-like coronaviruses and/or novel coronavirus mutant strains.
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谱抗SARS-like冠状病毒和/或新冠突变株的抗体(VSM9-44)。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 the PBMCs of recovered COVID-19 patients, and directly clones the antibody heavy chain and light chain variable region sequences, constructs an expression plasmid, and expresses and purifies it to obtain antibodies (VSM9-44) that are spectrally effective against SARS-like coronaviruses and/or COVID-19 mutant strains.
本发明的谱抗SARS-like冠状病毒和/或新冠突变株的抗体是特异性靶向RBD的抗体，能够特异性结合多种SARS-like冠状病毒以及新型冠状病毒突变株，具有强结合力，该特异性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 antibody against SARS-like coronavirus and/or novel coronavirus mutant strain of the present invention is an antibody that specifically targets RBD, can specifically bind to a variety of SARS-like coronaviruses and novel coronavirus mutant strains, and has strong binding force. This specific antibody can effectively block the binding of a variety of novel 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)本发明的广谱抗SARS-like冠状病毒和/或新冠突变株的抗体的制备流程简单快速，而且获得的抗体为全人源抗体，无免疫原性。1) The preparation process of the broad-spectrum antibodies against SARS-like coronavirus and/or new coronavirus mutant strains of the present invention is simple and rapid, and the obtained antibodies are fully human antibodies and have no immunogenicity.
2)本发明的广谱抗SARS-like冠状病毒和/或新冠突变株的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SARS-like冠状病毒具有强效的结合能力。2) The broad-spectrum antibodies against SARS-like coronaviruses and/or new coronavirus mutants of the present invention can effectively block the binding of the new coronavirus RBD to the host receptor protein ACE2, and have a strong binding ability to a variety of SARS-like coronaviruses.
3)本发明的广谱抗SARS-like冠状病毒和/或新冠突变株的抗体对多种SARS-like冠状病毒以及流行的新冠突变株(Alpha，Beta，Gamma，Delta以及Omicron)，具有强效广谱的中和作用。3) The broad-spectrum anti-SARS-like coronavirus and/or novel coronavirus mutant antibodies of the present invention have a strong and broad-spectrum neutralizing effect on a variety of SARS-like coronaviruses and popular novel coronavirus mutants (Alpha, Beta, Gamma, Delta and Omicron).
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-2 S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-2 S protein-specific memory B cells in the present invention.
图2是本发明的广谱抗SARS-like冠状病毒和/或新冠突变株的抗体的重链可变区的序列图。Figure 2 is a sequence diagram of the heavy chain variable region of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant antibody of the present invention.
图3是本发明的广谱抗SARS-like冠状病毒和/或新冠突变株的抗体的轻链可变区的序列图。Figure 3 is a sequence diagram of the light chain variable region of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant antibody of the present invention.
图4是本发明的广谱抗SARS-like冠状病毒和/或新冠突变株的抗体与SARS-CoV-2S蛋白不同亚基的结合情况图。Figure 4 is a diagram showing the binding of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant antibodies of the present invention to different subunits of the SARS-CoV-2S protein.
图5是本发明的广谱抗SARS-like冠状病毒和/或新冠突变株的抗体与SARS-like冠状病毒的结合情况图，其中横坐标为log转换后的抗体稀释浓度，纵坐标为450nm波长处吸光度。Figure 5 is a diagram of the binding of the broad-spectrum anti-SARS-like coronavirus and/or new coronavirus mutant strain antibody of the present invention to the SARS-like coronavirus, wherein the abscissa is the antibody dilution concentration after log conversion, and the ordinate is the absorbance at a wavelength of 450nm.
图6是本发明的不同浓度广谱抗SARS-like冠状病毒和/或新冠突变株的抗体对SARS-like冠状病毒的中和情况图，其中横坐标为抗体浓度，纵坐标为抗体抑制率。Figure 6 is a graph showing the neutralization of SARS-like coronavirus by antibodies with different concentrations of broad-spectrum anti-SARS-like coronaviruses and/or new coronavirus mutant strains of the present invention, wherein the horizontal axis is the antibody concentration and the vertical axis is the antibody inhibition rate.
图7是本发明的广谱抗SARS-like冠状病毒和/或新冠突变株的抗体对新型冠状病毒原始株、Alpha、Beta、Gamma、Delta、Omicron突变株的中和情况图，其中横坐标为抗体浓度，纵坐标为抗体抑制率。Figure 7 is a graph showing the neutralization of the novel coronavirus original strain, Alpha, Beta, Gamma, Delta, and Omicron mutant strains by the broad-spectrum anti-SARS-like coronavirus and/or novel coronavirus mutant antibodies of the present invention, wherein the horizontal axis represents the antibody concentration and the vertical axis represents the antibody inhibition rate.
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，获得广谱抗SARS-like冠状病毒和/或新冠突变株的抗体(VSM9-44)的具体方法如下：Referring to Figure 1, the specific method for obtaining broad-spectrum antibodies (VSM9-44) against SARS-like coronavirus and/or novel coronavirus mutant strains 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-2 S探针的细胞染色，所述探针为Alexa FluorTM 488蛋白标记试剂盒(ThermoFisher，货号：A20181)标记的SARS-CoV-2 S 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-2 S probe, which was SARS-CoV-2 S 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-2 S探针阳性的B细胞，获得SARS-CoV-2 S蛋白特异的Memory B细胞。e. Flow cytometry sorting of Live/dead- CD3- CD19+ IgD- CD27+ IgG+ and SARS-CoV-2 S probe-positive B cells to obtain SARS-CoV-2 S 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-2 S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-2 S protein-specific Memory B cells to the 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℃5 min，25℃10 min，50℃60min，94℃5 min；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:
上述5’First PCR primer mix和3’First PCR primer mix参照文献：Wardemann,H等，Methods MolBiol,2019.1956:p.105-125。The above 5’First PCR primer mix and 3’First PCR primer mix refer to the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.
第一轮PCR扩增获得第一轮PCR扩增产物，其中PCR反应条件为：94℃15 min预变性；94℃30 s，58℃30 s(IgH和Igκ)或者60℃30 s(Igλ)，72℃55 s扩增50循环；72℃10min；The first round of PCR amplification obtained the first round of PCR amplification products, wherein the PCR reaction conditions were: 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 for 50 cycles; 72°C for 10 min;
h.Ig可变区第二轮扩增，反应体系如下：h. 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.
第二轮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；The second round of PCR amplification obtained the second round of PCR amplification products, wherein the PCR reaction conditions were: 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;
i.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；i. Recover the second round of PCR amplification products using an agarose gel DNA recovery kit (Tian Gen, Cat. No.: DP209-03);
j.Ig可变区特异性扩增，获得特异性PCR产物，反应体系如下：j. Specific amplification of Ig variable region to obtain specific PCR products, the reaction system is as follows:
其中，上述5’Specific PCR primer mix和3’Specific PCR primer mix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 refer to the literature: Wardemann, H et al., Methods Mol Biol, 2019.1956: p.105-125.
k.特异性扩增的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；k. PCR reaction conditions for specific amplification: pre-denaturation at 94°C for 15 min; 50 cycles of amplification at 94°C for 30 s, 58°C for 30 s (IgH and Igκ) or 60°C for 30 s (Igλ), and 72°C for 45 s; 72°C for 10 min;
l.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；1. The specific PCR product amplified was detected by agarose gel electrophoresis, and the result is shown in FIG2 ;
m.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。m. 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中的各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 configured mixture of step b to each 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 overnight at 16°C;
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；l. 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 100% of the sequence of the second-round PCR product, and use the endotoxin-free plasmid mini-extraction kit (Tian Gen, catalog number: DP118-02) to extract the plasmids to 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FreeStyleTM 293表达培养基(Gibco，货号：12338018)将293 F细胞密度调整为1×106个/mL；a. One day before transfection, the density of 293 F 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FreeStyleTM 293表达培养基(Gibco，货号：12338018)将293 F细胞密度调整为2×106个/mL；b. Count the cells on the day of transfection and adjust the 293 F cell density to 2×106 cells/mL using fresh FreeStyle™ 293 Expression Medium (Gibco, Cat. No.: 12338018);
c.配制PEI混合液：将Polyethylenimine(PEI)(Polysciences，货号：23996-2)加入到OptiPROTM SMF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 OptiPROTM 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OptiPROTM SFM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 OptiPROTM 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293 F细胞中，轻柔摇晃混匀；f. Add the transfection mixture in step e to the 293 F cells in step b and shake gently to mix;
g.将步骤f的293 F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. Place the 293 F cells from step f in a 37°C suspension incubator containing 8% CO2 and culture at 125 rpm for 7 days.
3.3抗体的纯化3.3 Antibody Purification
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 medium 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. Elute the antibody bound to the Protein A column using glycine buffer at pH 3.0 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原始株S蛋白结合的抗体(4) ELISA screening of antibodies that bind to the S protein of the original strain of the new coronavirus SARS-CoV-2
a.将SARS-CoV-2 Wuhan-Hu-1 S蛋白(Sino Biological，货号：40589-V08B1)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 (Sino Biological, Cat. No. 40589-V08B1) 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 antibodies 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-2 S蛋白的结合强度选出广谱抗SARS-like冠状病毒和/或新冠突变株的抗体(VSM9-44)。Antibodies (VSM9-44) with a broad spectrum of activity against SARS-like coronaviruses and/or new coronavirus mutants were selected based on the binding strength of the antibodies to the SARS-CoV-2 S protein.
经测序鉴定，该广谱抗SARS-like冠状病毒和/或新冠突变株的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。编码抗体的重链可变区的基因序列如SEQ ID NO：3所示；编码抗体的轻链可变区的基因序列如SEQID NO：4所示。抗体的重链全长的氨基酸序列如SEQ ID NO：5所示；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 anti-SARS-like coronavirus and/or new coronavirus mutant strain antibody 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 antibody is shown in SEQ ID NO: 3; the gene sequence encoding the light chain variable region of the antibody is shown in SEQ ID NO: 4. The amino acid sequence of the full-length heavy chain of the antibody is shown in SEQ ID NO: 5; the amino acid sequence of the full-length light chain of the antibody is shown in SEQ ID NO: 6.
SEQ ID NO：1：SEQ ID NO: 1:
QVQLVESGGGLVQPGRSLRLSCAASGFTFRSYAMHWVRQAPGKGLEWVAAISFDGNEKFFADSVKGRFSISRDNSKSTLFLQINRLRPEDTAVYYCAKDPVGSIVRATVVYSFYAMDVWGPGTTVTVSS。QVQLVESGGGLVQPGRSLRLSCAASGFTFRSYAMHWVRQAPGKGLEWVAAISFDGNEKFFADSVKGRFSISRDNSKSTLFLQINRLRPEDTAVYYCAKDPVGSIVRATVVYSFYAMDVWGPGTTVTVSS.
SEQ ID NO：2：SEQ ID NO: 2:
NFMLTQPHSVSESPEKTVIISCTRSSGSIASNYVQWYQQRPGSSPTIIIQEDNQRPSGVPDRFSGSIDSSSNSASLTISRLKTEDEADYYCQSSDSDTVVFGGGTKLTV。NFMLTQPHSVSESPEKTVIISCTRSSGSIASNYVQWYQQRPGSSPTIIIQEDNQRPSGVPDRFSGSIDSSSNSASLTISRLKTEDEADYYCQSSDSDTVVFGGGTKLTV.
SEQ ID NO：3：SEQ ID NO: 3:
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGGAGTTATGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTATCTCTTTTGATGGAAATGAGAAGTTCTTTGCAGACTCCGTGAAGGGCCGATTCAGCATCTCCAGAGACAATTCCAAGAGCACATTGTTTCTGCAAATAAACCGCCTGAGACCTGAGGACACGGCTGTTTATTACTGTGCGAAAGACCCCGTTGGGAGTATAGTTCGGGCAACTGTTGTTTACTCCTTCTACGCTATGGACGTCTGGGGCCCAGGGACCACGGTCACCGTCTCCTCAGC。CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGGAGTTATGCCATGCACTGGGTCCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTATCTCTTTTGATGGAAATGAGAAGTTCTTTGCAGACTCCGTGAAGGGCCGATTCAGCATCTCCAGAGACAATTCCAAGAGCACATTGTTTCTGCAAATAAACCGC CTGAGACCTGAGGACACGGCTGTTTATTACTGTGCGAAAGACCCCGTTGGGAGTATAGTTCGGGCAACTGTTGTTTACTCCTTCTACGCTATGGACGTCTGGGGCCCAGGGACCACGGTCACCGTCTCCTCAGC.
SEQ ID NO：4：SEQ ID NO: 4:
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGAGAAGACGGTAATCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCATTATAATCCAAGAGGATAATCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTAGACTGAAGACTGAGGACGAGGCTGACTACTATTGTCAGTCTTCTGATAGCGACACTGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA。AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGAGAAGACGGTAATCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCATTATAATCCAAGAGGATAATCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGTCCCTCCAACTCTGCCTCCCTCACCATCTCTAGACTGAAGACTGAGGACGAGGCTG ACTACTATTGTCAGTCTTCTGATAGCGACACTGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA.
SEQ ID NO：5：SEQ ID NO: 5:
MGWSCIILFLVATATGVHSQVQLVESGGGLVQPGRSLRLSCAASGFTFRSYAMHWVRQAPGKGLEWVAAISFDGNEKFFADSVKGRFSISRDNSKSTLFLQINRLRPEDTAVYYCAKDPVGSIVRATVVYSFYAMDVWGPGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。MGWSCIILFLVATATGVHSQVQLVESGGGLVQPGRSLRLSCAASGFTFRSYAMHWVRQAPGKGLEWVAAISFDGNEKFFADSVKGRFSISRDNSKSTLFLQINRLRPEDTAVYYCAKDPVGSIVRATVVYSFYAMDVWGPGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
SEQ ID NO：6：SEQ ID NO: 6:
MGWSCIILFLVATATGSWANFMLTQPHSVSESPEKTVIISCTRSSGSIASNYVQWYQQRPGSSPTIIIQEDNQRPSGVPDRFSGSIDSSSNSASLTISRLKTEDEADYYCQSSDSDTVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS。MGWSCIILFLVATATGSWANFMLTQPHSVSESPEKTVIISCTRSSGSIASNYVQWYQQRPGSSPTIIIQEDNQRPSGVPDRFSGSIDSSSNSASLTISRLKTEDEADYYCQSSDSDTVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS.
实施例2：结合实验Example 2: Binding Experiment
(1)ELISA确定广谱抗SARS-like冠状病毒和/或新冠突变株的抗体与SARS-CoV-2S蛋白结合的靶点(1) ELISA to determine the targets of broad-spectrum antibodies against SARS-like coronaviruses and/or new coronavirus mutants and SARS-CoV-2 S protein binding
a.将SARS-CoV-2 S1蛋白(Sino Biological，货号：40591-V08H)或SARS-CoV-2 S2蛋白(Sino Biological，货号：40590-V08B)按2μg/mL(100μL/孔)的浓度包被酶标板；a. Coat the ELISA plate with SARS-CoV-2 S1 protein (Sino Biological, Catalog No.: 40591-V08H) or SARS-CoV-2 S2 protein (Sino Biological, Catalog No.: 40590-V08B) 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以及对照抗体按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 above purified antibodies and control antibodies 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 antibodies 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.
结果如图4所示，VSM9-44对SARS-CoV-2 S1蛋白展现出强效的结合能力，而对SARS-CoV-2 S2没有结合，表明该抗体是靶向S1的抗体。The results are shown in Figure 4. VSM9-44 exhibited strong binding ability to SARS-CoV-2 S1 protein, but had no binding to SARS-CoV-2 S2, indicating that the antibody is an antibody targeting S1.
(2)SARS-like S1蛋白的表达和纯化(2) Expression and purification of SARS-like S1 protein
a.转染前一天使用FreeStyleTM 293表达培养基(Gibco，货号：12338018)将293 F细胞密度调整为1×106个/mL；a. One day before transfection, the density of 293 F 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FreeStyleTM 293表达培养基(Gibco，货号：12338018)将293 F细胞密度调整为2×106个/mL；b. Count the cells on the day of transfection and adjust the 293 F cell density to 2×106 cells/mL using fresh FreeStyle™ 293 Expression Medium (Gibco, Cat. No.: 12338018);
c.编码SARS-like冠状病毒RaTG13、WIV1、GX-P5L、GD18、SZ3、Civet007 S1蛋白的基因序列，由南京金斯瑞公司合成并插入到带有Strep-Tactin标签(SA-WSHPQFEK-(GGGS)2-GGSAWSHPQFEK)的pcDNA3.1载体中。其中，上述SARS-like冠状病毒的基因编号为：RaTG13(Genebank:MN996532.2)、WIV1(Gendbank:KC881007.1)、GX-P5L(Genebrank:MT040335.1)、GD18(Genebbank:MT799524.1)、SZ3(Geneban k:AY304486.1)和Civet007(Genebang:AY572034.1)。c. The gene sequences encoding the SARS-like coronavirus RaTG13, WIV1, GX-P5L, GD18, SZ3, and Civet007 S1 proteins were synthesized by Nanjing GenScript and inserted into the pcDNA3.1 vector with a Strep-Tactin tag (SA-WSHPQFEK-(GGGS)2-GGSAWSHPQFEK). The gene numbers of the above SARS-like coronaviruses are: RaTG13 (Genebank: MN996532.2), WIV1 (Gendbank: KC881007.1), GX-P5L (Genebrank: MT040335.1), GD18 (Genebbank: MT799524.1), SZ3 (Geneban k: AY304486.1), and Civet007 (Genebang: AY572034.1).
d.配制转染混合液：将步骤c合成的质粒加入到Opti-MEMTM(ThermoFisher，货号：31985062)培养基中混匀，使转染质粒浓度为1μg/mL，再加入Polyethylenimine(PEI)(Polysciences，货号：23996-2)混匀，使PEI转染时浓度为4μg/mL；d. Prepare transfection mixture: add the plasmid synthesized in step c to Opti-MEMTM (ThermoFisher, Catalog No.: 31985062) medium and mix well to make the transfection plasmid concentration 1 μg/mL, then add Polyethylenimine (PEI) (Polysciences, Catalog No.: 23996-2) and mix well to make the PEI transfection concentration 4 μg/mL;
e.将步骤d中的转染混合体系加入到步骤a中的293 F细胞中，轻柔摇晃混匀；e. Add the transfection mixture in step d to the 293 F cells in step a and shake gently to mix;
f.将步骤e的293 F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；f. The 293 F cells from step e were placed in a 37°C suspension incubator containing 8% CO2 and cultured at 125 rpm for 7 days;
g.将所述转染后悬浮培养液于4000rpm离心15min收集表达上清；g. The transfected suspension culture was centrifuged at 4000 rpm for 15 min to collect the expression supernatant;
h.上清使用0.22μm滤器(JET，货号：FPE204030)过滤；h. The supernatant was filtered using a 0.22 μm filter (JET, catalog number: FPE204030);
i.打开蛋白纯化仪，使用Buffer W(IBA Lifesciences，货号：2-1003-100)将Strep-high capacity cartridge(IBA Lifesciences，货号：2-1237-001)柱子平衡，流速1mL/min；i. Open Protein purification instrument, using Buffer W (IBA Lifesciences, catalog number: 2-1003-100) to separate Strep- The column was balanced with a high capacity cartridge (IBA Lifesciences, catalog number: 2-1237-001) at a flow rate of 1 mL/min.
j.随后将过滤后的表达上清加载Strep-high capacitycartridge柱，流速1mL/min；j. Then the filtered expression supernatant was loaded with Strep- High capacity cartridge column, flow rate 1mL/min;
k.使用Buffer W洗去柱上非特异性结合蛋白，流速1mL/min；k. Use Buffer W to wash away non-specific binding proteins on the column at a flow rate of 1 mL/min;
l.使用Buffer BXT(IBA Lifesciences，货号：2-1042-025)洗脱柱上结合的蛋白，流速1mL/min；l. Use Buffer BXT (IBA Lifesciences, Catalog No.: 2-1042-025) to elute the protein bound to the column at a flow rate of 1 mL/min;
m.收集洗脱液，获得纯化后的SARS-like S1蛋白。m. Collect the eluate to obtain the purified SARS-like S1 protein.
(3)ELISA检测构建抗体与新型冠状病毒SARS-CoV-2和SARS-like S1蛋白的半数效应浓度(EC50)(3) ELISA test of the half effective concentration (EC50 ) of the constructed antibody against the novel coronavirus SARS-CoV-2 and SARS-like S1 protein
a.将上述纯化的SARS-like S1蛋白或SARS-CoV-2 S1蛋白(Sino Biological，货号：40591-V08H)或SARS-CoV S1蛋白(Sino Biological，货号：40150-V08B1)蛋白按2μg/mL(100μL/孔)的浓度包被酶标板；a. Coat the ELISA plate with the above-mentioned purified SARS-like S1 protein or SARS-CoV-2 S1 protein (Sino Biological, Catalog No.: 40591-V08H) or SARS-CoV S1 protein (Sino Biological, Catalog No.: 40150-V08B1) 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，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(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5所示，VSM9-44抗体对新型冠状病毒SARS-CoV-2和SARS-CoV以及动物来源的SARS-like冠状病毒S1都展现出强效的结合能力，EC50在16.12-22.80ng/mL之间。The results are shown in Figure 5. The VSM9-44 antibody exhibited strong binding ability to the new coronavirus SARS-CoV-2 and SARS-CoV as well as the animal-derived SARS-like coronavirus S1, with an EC50 between 16.12-22.80 ng/mL.
实施例3：病毒中和实验Example 3: Virus Neutralization Experiment
(1)假病毒包被(1) Pseudovirus Encapsulation
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或SARS-CoV或动物来源SARS-like病毒RaTG13或WIV1或GX-P5L或GD18或SZ3或Civet007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SARS-like冠状病毒以及突变株的基因编号为：SARS-CoV-2(Genebank：YP_009724390.1)、SARS-CoV(GenBank：Y463060.1)、RaTG13(Genebank:MN996532.2)、WIV1(Gendbank:KC881007.1)、GX-P5L(Genebrank:MT040335.1)、GD18(Genebbank:MT799524.1)、SZ3(Geneban k:AY304486.1)、Civet007(Genebang:AY572034.1)、SARS-CoV-2 Alpha(Genebank：OV054768.1)、SARS-CoV-2 Beta(Genebank：MZ433432.1)、SARS-CoV-2Gamm(Genebank：MZ427312.1)、SARS-CoV-2 Delta(Genebank：OK091006.1)、SARS-CoV-2Omicron BA.1(GISAID ID：EPI_ISL_6590782.2)和SARS-CoV-2 Omicron BA.2(Genebank：UKS51680.1)、SARS-CoV-2 Omicron BA.3spike(GISAID：EPI_ISL_7605589)和SARS-CoV-2Omicron BA.4/5spike(GISAID:EPI_ISL_12268493.2)；b. The gene sequence encoding the S protein of SARS-CoV-2 or SARS-CoV or animal-derived SARS-like virus RaTG13 or WIV1 or GX-P5L or GD18 or SZ3 or Civet007 or Alpha mutant or Beta mutant or Gamma mutant or Delta mutant or Omicron mutant was synthesized by Nanjing GenScript and inserted into the pcDNA3.1 vector. Among them, the gene numbers of the above-mentioned SARS-like coronaviruses and mutants are: SARS-CoV-2 (Genebank: YP_009724390.1), SARS-CoV (GenBank: Y463060.1), RaTG13 (Genebank: MN996532.2), WIV1 (Gendbank: KC881007.1), GX-P5L (Genebrank: MT040335.1), GD18 (Genebbank: MT799524.1), SZ3 (Geneban k: AY304486.1), Civet007 (Genebang: AY572034.1), SARS-CoV-2 Alpha (Genebank: OV054768.1), SARS-CoV-2 Beta (Genebank: MZ433432.1), SARS-CoV-2 Gamm (Genebank: MZ427312.1), SARS-CoV-2 Delta (Genebank: OK091006.1), SARS-CoV-2 Omicron BA.1 (GISAID ID: EPI_ISL_6590782.2) and SARS-CoV-2 Omicron BA.2 (Genebank: UKS51680.1), SARS-CoV-2 Omicron BA.3spike (GISAID: EPI_ISL_7605589) and SARS-CoV-2Omicron 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1：3的质量比加入到Opti-MEMTM(ThermoFisher，货号：31985062)培养基中混匀，使总质粒浓度为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 1:3 to Opti-MEMTM (ThermoFisher, catalog number: 31985062) culture 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-MEMTM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-MEMTM 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、SARS-CoV、动物SARS-like冠状病毒RaTG13、WIV1、GX-P5L、GD18、SZ3、Civet007以及新冠病毒Alpha、Beta、Gamma、Delta、Omicron BA.1、Omicron BA.2、Omicron BA.3、Omicron BA.4/5突变株的半数抑制浓度(IC50)(2) Pseudovirus neutralization experiments were performed to detect the half inhibitory concentration (IC 50 ) of antibodies against the novel coronavirus SARS-CoV-2, SARS-CoV, animal SARS-like coronaviruses RaTG13, WIV1, GX-P5L, GD18, SZ3, Civet007, and novel coronavirus Alpha, Beta, Gamma, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.3, and Omicron BA.4/5 mutants.
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
为：含10％FBS的DMEM)中；For: 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 medium in the 96-well plate in step a, add the antibody and virus mixture in step c, and centrifuge at 800g for 30min;
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6所示，VSM9-44对新型冠状病毒SARS-CoV-2和SARS-CoV以及动物来源的SARS-like冠状病毒都展现出强效的中和能力，其中对GX-P5L、GD18、SZ3、Civet007、RaTG13以及WIV1的IC50介于10.06-79.08ng/mL之间。而且如图7所示，VSM9-44对新冠病毒Alpha、Beta、Gamma以及Delta突变株也具有强效的中和能力，其中对Gamma突变株IC50为11.47ng/mL。而对最近流行的Omicron突变株BA.1、BA.2、BA.3以及BA.4/5在高浓度下也具有中和能力。As shown in Figure 6, VSM9-44 showed strong neutralizing ability against the new coronavirus SARS-CoV-2 and SARS-CoV, as well as SARS-like coronaviruses from animals, with IC50 values for GX-P5L, GD18, SZ3, Civet007, RaTG13, and WIV1 ranging from 10.06 to 79.08 ng/mL. And as shown in Figure 7, VSM9-44 also had strong neutralizing ability against the Alpha, Beta, Gamma, and Delta mutants of the new coronavirus, with an IC50 of 11.47 ng/mL for the Gamma mutant. It also had neutralizing ability against the recently popular Omicron mutants BA.1, BA.2, BA.3, and BA.4/5 at high concentrations.
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.
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