CN115304671B - 新冠病毒rbd特异性单克隆抗体及其应用 - Google Patents

新冠病毒rbd特异性单克隆抗体及其应用 Download PDF

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CN115304671B
CN115304671B CN202210272383.2A CN202210272383A CN115304671B CN 115304671 B CN115304671 B CN 115304671B CN 202210272383 A CN202210272383 A CN 202210272383A CN 115304671 B CN115304671 B CN 115304671B
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金艾顺
韩晓建
胡超
王应明
李婷婷
王建为
李胜龙
黄晶晶
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Chongqing Medical University
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Abstract

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

Description

新冠病毒RBD特异性单克隆抗体及其应用
技术领域
本发明属于单克隆抗体技术领域，尤其涉及新冠病毒RBD特异性单克隆抗体及其应用。
背景技术
抗体是由四条多肽链组成的免疫球蛋白分子，四条多肽链包括两条重链(H链)和两条轻链(L链)。H链由重链可变区(VH)和重链恒定区组成，重链恒定区由三个区CH1、CH2和CH3组成。L链由L链可变区(VL)和轻链恒定区组成，轻链恒定区由CL区组成。VH和VL可进一步分成称为互补决定区(CDRs)的高变区和称为构架区(FR)交替分布的保守区。
目前研究发现：新冠病毒(SARS-CoV-2)具有四种主要的结构蛋白，分别为刺突蛋白(S蛋白)、核衣壳蛋白(N蛋白)、膜蛋白(M蛋白)和包膜蛋白(E蛋白)，其中S蛋白有两个亚基：S1和S2，受体结合位点(RBD)位于S1亚基上，其主要功能是识别宿主细胞表面受体，介导与宿主细胞的融合。
目前针对新发病原体COVID-19尚无特效药物针对性治疗，疫苗研发尚需时日。近期治愈出院的患者血浆中含有高浓度特异性的抗原中和抗体，输入患者体内后，可以中和新冠病原体，介导有效的免疫反应，因此利用恢复期血浆有望为救治感染新冠病毒的患者提供有效的治疗手段，降低死亡率，保障患者生命安全。
申请公开号为CN111303280A的中国发明专利申请公开了一种高中和活性抗SARS-CoV-2全人源单克隆抗体，上述专利提供的是识别区域为S1非RBD区的全人源单克隆抗体，但是由于新冠病毒入侵宿主细胞是通过RBD与宿主细胞的ACE2结合，所以上述专利获得的全人源单克隆抗体对病毒的阻断效果有限，并且上述专利是通过标记浆细胞而获得抗体cDNA，但是与浆细胞相比，是记忆B细胞活化后迅速做出反应，所以记忆B细胞能够引发比初次反应更快，也更强烈的体液免疫反应，而浆细胞所引发的体液免疫反应有限。
发明内容
本发明的目的在于提供一种针对RBD，并且能够引发更强烈体液免疫反应的新冠病毒RBD特异性单克隆抗体及其应用。
为了达到上述目的，本发明提供了新冠病毒RBD特异性单克隆抗体，具体的，抗体的重链可变区氨基酸序列如SEQ ID NO:1所示；轻链可变区氨基酸序列如SEQ ID NO:2所示(单抗1-CQTS113)。重链可变区氨基酸序列还可如SEQ ID NO:3所示；轻链可变区氨基酸序列还可如SEQ ID NO:4所示(单抗2-CQTS114)。重链可变区氨基酸序列还可如SEQ ID NO:5所示；轻链可变区氨基酸序列还可如SEQ ID NO:6所示(单抗3-CQTS115)。重链可变区氨基酸序列还可如SEQ ID NO:7所示；轻链可变区氨基酸序列还可如SEQ ID NO:8所示(单抗4-CQTS116)。重链可变区氨基酸序列还可如SEQ ID NO:9所示；轻链可变区氨基酸序列还可如SEQ ID NO:10所示(单抗5-CQTS117)。重链可变区氨基酸序列还可如SEQ ID NO:11所示；轻链可变区氨基酸序列还可如SEQ ID NO:12所示(单抗6-CQTS118)。重链可变区氨基酸序列还可如SEQ ID NO:13所示；轻链可变区氨基酸序列还可如SEQ ID NO:14所示(单抗7-CQTS119)。重链可变区氨基酸序列还可如SEQ ID NO:15所示；轻链可变区氨基酸序列还可如SEQ ID NO:16所示(单抗8-CQTS120)。重链可变区氨基酸序列还可如SEQ ID NO:17所示；轻链可变区氨基酸序列还可如SEQ ID NO:18所示(单抗9-CQTS121)。重链可变区氨基酸序列还可如SEQ ID NO:19所示；轻链可变区氨基酸序列还可如SEQ ID NO:20所示(单抗10-CQTS122)。重链可变区氨基酸序列还可如SEQ ID NO:21所示；轻链可变区氨基酸序列还可如SEQ ID NO:22所示(单抗11-CQTS123)。重链可变区氨基酸序列还可如SEQ ID NO:23所示；轻链可变区氨基酸序列还可如SEQ ID NO:24所示(单抗12-CQTS124)。重链可变区氨基酸序列还可如SEQ ID NO:25所示；轻链可变区氨基酸序列还可如SEQ ID NO:26所示(单抗13-CQTS125)。重链可变区氨基酸序列还可如SEQ ID NO:27所示；轻链可变区氨基酸序列还可如SEQ ID NO:28所示(单抗14-CQTS126)。重链可变区氨基酸序列还可如SEQ ID NO:29所示；轻链可变区氨基酸序列还可如SEQ ID NO:30所示(单抗15-CQTS127)。重链可变区氨基酸序列还可如SEQ ID NO:31所示；轻链可变区氨基酸序列还可如SEQ ID NO:32所示(单抗16-CQTS128)。重链可变区氨基酸序列还可如SEQ ID NO:33所示；轻链可变区氨基酸序列还可如SEQ ID NO:34所示(单抗17-CQTS129)。重链可变区氨基酸序列还可如SEQ ID NO:35所示；轻链可变区氨基酸序列还可如SEQ ID NO:36所示(单抗18-CQTS130)。重链可变区氨基酸序列还可如SEQ ID NO:37所示；轻链可变区氨基酸序列还可如SEQ ID NO:38所示(单抗19-CQTS131)。重链可变区氨基酸序列还可如SEQ ID NO:39所示；轻链可变区氨基酸序列还可如SEQ ID NO:40所示(单抗20-CQTS132)。
本发明还提供了上述新冠病毒RBD特异性单克隆抗体，在制备检测或诊断SARS-CoV-2试剂或疫苗或药物中的应用，其中药物包括新冠病毒RBD特异性单克隆抗体和药学上可接受的赋形剂、稀释剂或载体；还提供了编码上述新冠病毒RBD特异性单克隆抗体的核酸分子；还提供了含有上述核酸分子的表达盒、重组载体、重组菌或转基因细胞系；还提供了上述表达盒、重组载体、重组菌或转基因细胞系在制备产品中的应用。
本发明还提供了产品，包括上述新冠病毒RBD特异性单克隆抗体；产品用途如下(b1)-(b4)中的任一种：(b1)结合新型冠状病毒SARS-CoV-2；(b2)检测结合新型冠状病毒SARS-CoV-2；(b3)结合新型冠状病毒SARS-CoV-2的S蛋白；(b4)检测新型冠状病毒SARS-CoV-2的S蛋白。
优选的，上述新冠病毒RBD特异性单克隆抗体均通过分选RBD特异性记忆B细胞，再通过RBD特异性记忆B细胞的mRNA获得抗体可变区cDNA。
本发明的原理和有益效果在于：
(1)本发明提供的单克隆抗体具有RBD特异性，与针对S1非RBD区的单可隆抗体相比，本发明提供的单克隆抗体与RBD结合，为抗体药物筛选，诊断、预防和治疗新冠肺炎提供了更加广泛的应用价值。
(2)本发明提供的单克隆抗体是通过分选RBD特异性记忆B细胞而得到，与通过分选浆细胞的现有技术相比，本发明制备的单克隆抗体能够引发更强烈的体液免疫反应。另外，本发明只针对RBD特异性记忆B细胞进行后续RT-PCR、巢式PCR和抗体功能分析，大大提高了单克隆抗体与RBD特异性结合能力。
附图说明
图1为采用流式细胞仪分析RBD特异性记忆B细胞的细胞分选图；
图2为采用流式细胞仪分析RBD特异性记忆B细胞的细胞分选图；
图3为RBD特异性检测结果图。
具体实施方式
下面将结合本发明实施例的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本实施例提供一种新冠病毒RBD特异性单克隆抗体，重链可变区氨基酸序列如SEQID NO:1所示；轻链可变区氨基酸序列如SEQ ID NO:2所示。
本实施例还提供了上述新冠病毒RBD特异性单克隆抗体，在制备检测或诊断SARS-CoV-2试剂或药物中的应用。
在实际生产时，可以采用本实施例得到RBD特异性单克隆抗体制备核酸分子，或者制备包含该核酸分子的表达盒、重组载体、重组菌或转基因细胞系，或者制备药物组合物，该药物组合物包括上述新冠病毒RBD特异性单克隆抗体和药学上可接受的赋形剂、稀释剂或载体。
在应用时，本实施例得到RBD特异性单克隆抗体制备的相关产品，可具有如下(b1)-(b4)中的任一种的用途：(b1)结合新型冠状病毒SARS-CoV-2；(b2)检测结合新型冠状病毒SARS-CoV-2；(b3)结合新型冠状病毒SARS-CoV-2的S蛋白；(b4)检测新型冠状病毒SARS-CoV-2的S蛋白。
实施例2-20
实施例2-20与实施例1的区别在于：RBD特异性单克隆抗体的可变区氨基酸序列不同，实施例2-20的可变区氨基酸序列如下表所示：
实验组 重链可变区氨基酸序列 轻链可变区氨基酸序列
实施例1-CQTS113 SEQ ID NO:1 SEQ ID NO:2
实施例2-CQTS114 SEQ ID NO:3 SEQ ID NO:4
实施例3-CQTS115 SEQ ID NO:5 SEQ ID NO:6
实施例4-CQTS116 SEQ ID NO:7 SEQ ID NO:8
实施例5-CQTS117 SEQ ID NO:9 SEQ ID NO:10
实施例6-CQTS118 SEQ ID NO:11 SEQ ID NO:12
实施例7-CQTS119 SEQ ID NO:13 SEQ ID NO:14
实施例8-CQTS120 SEQ ID NO:15 SEQ ID NO:16
实施例9-CQTS121 SEQ ID NO:17 SEQ ID NO:18
实施例10-CQTS122 SEQ ID NO:19 SEQ ID NO:20
实施例11-CQTS123 SEQ ID NO:21 SEQ ID NO:22
实施例12-CQTS124 SEQ ID NO:23 SEQ ID NO:24
实施例13-CQTS125 SEQ ID NO:25 SEQ ID NO:26
实施例14-CQTS126 SEQ ID NO:27 SEQ ID NO:28
实施例15-CQTS127 SEQ ID NO:29 SEQ ID NO:30
实施例16-CQTS128 SEQ ID NO:31 SEQ ID NO:32
实施例17-CQTS129 SEQ ID NO:33 SEQ ID NO:34
实施例18-CQTS130 SEQ ID NO:35 SEQ ID NO:36
实施例19-CQTS131 SEQ ID NO:37 SEQ ID NO:38
实施例20-CQTS132 SEQ ID NO:39 SEQ ID NO:40
以上实施例1-20所提供的RBD特异性单克隆抗体均通过以下方法得到：首先从新冠肺炎康复患者的外周血中分选得到单个RBD特异性记忆B细胞，然后获得RBD特异性记忆B细胞的mRNA，再通过RT-PCR和巢式PCR构建抗体可变区基因表达盒，再将抗体可变区基因表达盒转导入293T细胞表达抗体并收集上清，用ELISA法检测上清的RBD特异性，筛选得到RBD特异性单克隆抗体。
具体包括以下步骤：
S1、采集若干名新冠肺炎康复患者外周血，分离得到PBMC，在-80℃的冰箱中冻存备用。
S2、首先采用去死细胞染料(Dead Dye)去除S1得到的PBMC的死细胞，然后采用CD19、mIg-G、mIg-D和S-RBD对PBMC中活的RBD特异性并且结合能力高的记忆B细胞染色标记，筛选出针对RBD特异性记忆B细胞；使用流式细胞分选仪将特异性记忆B细胞分选到96孔板上，每个孔内有一个特异性记忆B细胞，在-80℃的冰箱中冻存备用。
具体的，本实施例优选的Dead Dye染色时的浓度范围为1-2μg/mL，本实施例优选Dead Dye染色时的浓度为1.5μg/mL；CD19为Biolegend生产的B细胞标记物，染色时的浓度范围为1-2μg/mL，本实施例优选CD19染色时的浓度为1.5μg/mL。mIg-G为Biolegend生产的B细胞标表面受体，染色时的浓度范围为1-2μg/mL，本实施例优选mIg-G染色时的浓度为1.5μg/mL；mIg-D为Biolegend生产的B细胞表面受体，染色时的浓度范围为1-2μg/mL，本实施例优选mIg-D染色时的浓度为1.5μg/mL；S-RBD为sinobiological生产的新冠病毒是蛋白受体结构域，染色时的浓度范围为1-2μg/mL，本实施例优选S-RBD染色时的浓度为1.5μg/mL。
通过流式细胞仪分选RBD特异性记忆B细胞的，通过CD19、mIg-G、mIg-D和S-RBD对PBMC的细胞分选得到对S-RBD具有特异性记忆B细胞的细胞分选图如图1和图2所示，其中图2中的Batch ID 0428、0505、0522、0528是筛选批次。本实施例采用CD19、mIg-G、mIg-D和S-RBD筛选出针对RBD特异性记忆B细胞的原理在于：将PBMC用去死细胞染料(Dead Dye)、B细胞标记物CD19、记忆B细胞标记物mIg-G阳性和mIg-D阴性以及RBD特异性IgG表达的记忆B细胞进行染色，然后使用流式细胞分析仪将细胞群中CD19细胞群划分出来，再通过从CD19阳性细胞群中划分mIg-G+mIg-D-细胞群，再从mIg-G+mIg-D-细胞群划分RBD阳性的记忆B细胞，再通过流式细胞分选仪将RBD阳性的记忆B细胞进行分选。
S3、分选得到单个RBD特异性记忆B细胞的mRNA，采用RT-PCR扩增获得抗体可变区cDNA。具体的，使用RT-PCR扩增抗体可变区cDNA时，本实施例所设计的引物的引物前段设计有通用Leader(参见引物序列表一和引物序列表二)，有效提高了抗体基因的扩增率。
S4、采用巢式PCR扩增S1-S3得到的抗体可变区cDNA，构建抗体可变区基因表达盒。
S3和S4总共通过以下六部分进行：(1)提取RBD特异性记忆B细胞的mRNA；(2)单个细胞mRNA逆转录(RT)；(3)加G尾(TDT)；(4)第一轮PCR(1st PCR)；(5)第二轮PCR(2nd PCR)；(6)BCR-ORFPCR扩增构建基因表达盒；(7)CMV、WPRE-γ/κ/l片段扩增及CMV、BCR-Vγ/κ/l((6)的产物)、WPRE-γ/κ/l重叠PCR(Overlap PCR)预连接；(8)BCR-γORF、BCR-κORF、BCR-lPCR扩增。
各部分反应液配制及反应条件如下：
(1)采用DynabeadsTMmRNA DIRECTTMPurification Kit(ThermoFisherscientific)进行单细胞mRNA提取，具体包括以下步骤：
①离心：从-80℃冰箱中取出分选有单个RBD特异性记忆B细胞的96孔板后，600×g离心30s，使细胞离心于孔底部；
②清洗：将Dynabeads oligo(dT)25微球瓶取出后涡旋混匀，按照2μl/孔吸取足量微球，放置于磁铁块上，静置30s，弃上清，用500μl的Lysis Buffer重悬；
③配制：按照9μl/孔Lysis Buffer加入到50mL的离心管中，将上述500μl微球悬液加入其中，用枪吹匀；
④分装：用八连管分装微球，随后采用排枪将其按照9μl/孔加入到细胞板中；
⑤润洗：96孔板贴膜，随后润洗管壁四周，共2个循环；
⑥孵育：室温静置5min，使RBD特异性记忆B细胞的mRNA充分释放并结合到微球上，孵育结束后，600×g瞬时离心，使微球离心于孔底部。将96孔板放置于DynaMagTM-96sideMagnet磁板上，用排枪吸弃上清；
⑦Wash A清洗：按照8μl/孔加入Washing Buffer A，来回走板7-8次，使微球充分洗涤，弃上清；
⑧Wash B清洗：按照8μl/孔加入Washing Buffer B，来回走板7-8次，使微球充分洗涤，弃上清，随后按照10μl/孔加入预先配制的逆转录(RT)反应液。试剂配制及反应条件如下述(2)描述。
(2)逆转录(RT)(10μl体系)
所需配制的试剂如下表1所示：
试剂名称 体积
DEPC-H2O 4.5μl
5×primerscript Buffer 2.0μl
2.5mM dNTP 2.0μl
RNase Inhibitor 1μl
Sample beads
PrimerScriptⅡRTase 0.5μl
总体积 10μl
反应条件：42℃for 60min(每20min混合一次)；
反应结束后，600×g瞬时离心96孔板，然后将96孔板放置于DynaMagTM-96sideMagnet磁板上，用排枪吸弃上清，随后按照10μl/孔加入预先配制的TDT反应液，试剂配制及反应条件如下述(3)描述。
(3)加G尾(TDT)(10μl体系)
所需配制的试剂如下表2所示：
试剂名称 体积
H2O 6.4μl
5×TdT buffer 2.0μl
10mM dGTP 0.5μl
0.1％BSA 1.0μl
Sample beads
TdT 0.1μl
总体积 10μl
反应条件：37℃for 40min(每20min混合一次)。
反应结束，600×g瞬时离心96孔板，然后将其放置于DynaMagTM-96side Magnet磁板上，用排枪吸弃上清，随后按照10μl/孔加入预先配制的第一轮PCR(1st PCR)反应液，试剂配制及反应条件如下述(4)描述。
(4)1st PCR(10μl体系)(引物序列参见引物序列表)
所需配制的试剂如下表3所示：
基于PCR原理，1st PCR的实验反应条件为：①95℃预变性3min；②95℃变性15sec，60℃退火5sec，72℃延伸1min，30-35cycles，本实施例优选30cycles；③72℃循环外延伸5min，4℃保存。
(5)第二轮PCR(2nd PCR)(10μl体系)(引物序列参见引物序列表一和引物序列表二)
所需配制的试剂如下表4所示：
试剂名称 体积
H2O 1.5μl
2×GC Buffer 5μl
2.5mM dNTP 1μl
FP:MAC-AP3/AP3(10μM) 0.5μl
RP:Cg-nest/K20/CI-nest(10μM) 0.5μl
PrimesTAR 0.5μl
sample 1μl
总体积 10μl
基于PCR原理，2nd PCR的实验反应条件为：①95℃预变性3min；②95℃变性15sec，60℃退火5s，72℃延伸1min，30-35cycles，本实施例优选35cycles；72℃循环外延伸5min，4℃保存。
PCR结束后：每孔取4μl进行1.5％琼脂糖凝胶电泳。将Gamma链与Kappa链或Lamada链配对的细胞孔送测序。
(6)抗体表达盒(BCR-ORF)的扩增和构建
PCR扩增启动子区(CMV启动子)、WPRE-γ(抗体gamma链)和WPRE-κ(抗体kappa链)，PCR扩增体系如下表5所示：
PCR扩增条件为：①95℃预变性3min；②95℃变性15sec，56℃退火15sec，72℃延伸1min，30cycles；③72℃循环外延伸5min，12℃保存。
(7)CMV、WPRE-γ/κ/l片段扩增及CMV、BCR-Vγ/κ/l、WPRE-γ/κ/l重叠PCR(Overlap PCR)预连接
实验体系如下表6所示：
PCR扩增条件为：95℃预变性3min；95℃变性15sec，50℃退火15sec，72℃延伸1.5min，10cycles；72℃循环外延伸5min，12℃保存。
(8)BCR-γORF、BCR-κORF、BCR-l PCR扩增
实验体系如下表7所示：
PCR扩增程序：95℃预变性3min；95℃变性15sec，58℃退火15sec，72℃延伸1.5min，30cycles；72℃循环外延伸5min，12℃保存。
BCR-γORF和BCR-κ/ORF乙醇沉淀：BCR-γORF和BCR-κORF的PCR产物各取30μl置于8连管中，再加入120μl无水乙醇，6μl醋酸钠溶液，充分混匀，-80℃静置30min；10000rpm，离心20min，弃上清，依次用200μl的70％乙醇和无水乙醇各漂洗一次，于56℃乙醇充分挥发，加入40μl无菌水，振荡，使沉淀充分溶解，检测抗体可变区基因的浓度。
S3和S4所用到的Leader引物参见如下的引物序列表一：
S3和S4所用到的所用到J-region引物参见如下的引物序列表二：
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S5、将S4得到的抗体可变区基因表达盒转导入293T细胞48小时内表达抗体并收集上清，用ELISA法检测上清的RBD特异性，筛选RBD特异性全人源单克隆抗体。
(A)使用PBS稀释抗原(终浓度2μg/mL)，10μl/孔，包被384孔ELISA板4℃过夜或37℃包被2h(本实施例优选4℃过夜)。NOTE：加完后瞬时离心保证液体在底部。
实验体系如下表8所示：
试剂名称 货号 原浓度 终浓度 稀释比
SARS-COV-2RBD Cat:40592-V08H 200μg/mL 2μg/mL 1:100
Goat pab to Hu IgG－ALP Cat:ab97221 1mg/mL 2μg/mL 1:500
(B)配制PBST(0.05％ Tween 20，Cat#TB220)：1L的PBS加入0.5mL的Tween 20；
PBST机洗板子(Thermoscientific wellwash versa)或者手洗(机洗完的板子依然要手动拍板/使用微孔板离心机(MPC-P25)离心1min，使板子看不见有水和气泡)。
封闭：80μl的5％BSA(BioFroxx，Cat.NO:4240GR100)(PBST配制)加入上述洗好的板子里，放置于37℃的孵育箱孵育1h。PBST机洗板子或者手洗。
(C)加样及标准品。其中，标准品：10μl/well原浓度1μg/mL，梯度稀释为250ng/mL、125ng/mL、62.5ng/mL、31.25ng/mL、15.63ng/mL、7.81ng/mL、3.9ng/mL和1.95ng/mL。(封闭液稀释)；样品：转染抗体基因的细胞上清液。阴性对照/空白孔：封闭液10μl/well。
在37℃孵育30min。PBST机洗板子或者手洗。
(D)加二抗，加入的浓度为10μl/well，然后在37℃下孵育30min。
实验体系如下表9所示：
二抗名称 货号 原浓度 终浓度 稀释比
goat-anti-human IgG-ALP A18808 1.5mg/ml 0.3μg/ml 1:5000
Goat pab to Hu IgG-ALP Ab98532 0.5mg/ml 0.25μg/ml 1:2000
PBST机洗板子或者手洗。10μl/well的PNPP(对硝基苯磷酸二钠)，使用(Thermoscientific Muttiskan GO)检测5min、10min、15min、20min、25min、30min、35min、40min、45min、50min、55min和60min的OD(450mm)值。50mg的PNPP粉末(Thermo，Prod#34045)+40mL的ddH2O+10mL的Diethanol aminesubstrate Buffer(5X)，PNPP避光4℃储存。
实验结果如图3所示，OD值大于0.1为阳性。
以上所述仅为本发明的优选实施例，对本发明而言仅是说明性的，而非限制性的；本领域普通技术人员理解，在本发明权利要求所限定的精神和范围内可对其进行许多改变，修改，甚至等效变更，但都将落入本发明的保护范围内。
<110>重庆医科大学，冯玉林
<120>新冠病毒RBD特异性单克隆抗体及其应用
<160>40
<210>1
<211>123
<212>PRT
<213>人工序列（Artificial Sequence）
<400>1
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly
1 5 10 15
Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser
20 25 30
Thr Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Val Trp Val Ala Phe Ile Ser Tyr Asp Glu Ser Asn Lys Tyr Tyr
50 55 60
Thr Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser
65 70 75
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
80 85
Asp Thr Ala Val Tyr Tyr Cys Ala Gly Gly Gly Val Leu Val Thr
90 95 100
Ser Asp Pro Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
105 110 115
Thr Val Ser Ser
120
<210>2
<211>107
<212>PRT
<213>人工序列（Artificial Sequence）
<400>2
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser
20 25 30
Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Phe Thr Ile
65 70 75
Ser Ser Leu Gln Pro Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Gln
80 85 90
Ser Asp Asn Val Pro Val Thr Phe Gly Pro Gly Thr Lys Val Asp
95 100 105
Ile Lys
<210>3
<211>122
<212>PRT
<213>人工序列（Artificial Sequence）
<400>3
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser
20 25 30
Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Gly Arg Ile Lys Phe Thr Thr Asp Gly Gly Thr Ser
50 55 60
Asp Tyr Ala Ala Pro Val Arg Gly Arg Phe Thr Ile Ser Arg Asp
65 70 75
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
80 85
Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ser Thr Thr Asn Asp Tyr
90 95 100
Gly Asp Tyr Ser Ala Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr
105 110 115
Val Ser Ser
120
<210>4
<211>107
<212>PRT
<213>人工序列（Artificial Sequence）
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
20 25 30
Arg Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
80 85 90
Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
95 100 105
Ile Lys
<210>5
<211>122
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<213>人工序列（Artificial Sequence）
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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr
20 25 30
Arg Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu
35 40 45
Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr
50 55 60
Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser
65 70 75
Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
80 85 90
Thr Ala Met Tyr Tyr Cys Ala Arg Ile Glu Tyr Tyr Asn Asp Ser
95 100 105
Ser Gly Tyr Tyr Gln Phe Trp Gly Gln Gly Thr Leu Val Thr Val
110 115 120
Ser Ser
<210>6
<211>107
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Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro
1 5 10 15
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
20 25 30
Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
80 85 90
Gln Tyr Gly Ser Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu
95 100 105
Ile Lys
<210>7
<211>126
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<213>人工序列（Artificial Sequence）
<400>7
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
Ser Tyr Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu
35 40 45
Glu Trp Met Gly Trp Met Asn Pro Lys Ser Val Tyr Thr Gly Tyr
50 55 60
Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asn Thr
65 70
Ser Ile Arg Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
75 80 85
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Leu Trp Phe Gly
90 95 100
Asp Leu Thr Arg Thr Lys Tyr Asn Trp Phe Asp Pro Trp Gly
105 110 115
Gln Gly Thr Leu Val Thr Val Ser Ser
120 125
<210>8
<211>107
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<400>8
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser
20 25 30
Ser Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Asn Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
80 85 90
Phe Asn Asn Phe Leu Leu Thr Phe Gly Gly Gly Thr Lys Val
95 100
Glu Ile Lys
105
<210>9
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<400>9
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Thr Phe Ser
20 25 30
Asn Tyr Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ser Ala Ile Ser Gly Ser Ala Gly Ser Ile Tyr Tyr
50 55 60
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
65 70 75
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp
80 85
Asp Thr Ala Val Tyr Phe Cys Ala Lys Gly Gln Arg Gly Ser Pro
90 95 100
Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
105 110 115
Ser
120
<210>10
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<400>10
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
20 25 30
Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Tyr Asp Ser Ser Ser Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
80 85 90
Tyr Asn Ser Tyr Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
95 100 105
Glu Ile Lys
<210>11
<211>117
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Glu Val Leu Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Val Ser
20 25 30
Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala
50 55 60
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
65 70 75
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
80 85
Thr Ala Val Tyr Tyr Cys Ala Arg Asp Leu Val Thr Trp Gly Leu
90 95 100
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
105 110 115
<210>12
<211>107
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<400>12
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser
20 25 30
Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu
80 85 90
Leu Asn Thr Asp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu
95 100 105
Ile Lys
<210>13
<211>116
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<400>13
Gln Val Gln Ile Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
Ser Tyr Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu
35 40 45
Glu Trp Met Gly Trp Ile Asn Thr Gly Asn Gly Asn Thr Lys Tyr
50 55 60
Ser Gln Asn Phe Gln Asp Arg Val Thr Ile Thr Arg Asp Thr Ser
65 70 75
Ala Thr Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Pro Glu Asp
80 85 90
Thr Ala Val Tyr Tyr Cys Ala Arg Ala Gly Trp Glu Leu Asn Tyr
95 100 105
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
110 115
<210>14
<211>112
<212>PRT
<213>人工序列（Artificial Sequence）
<400>14
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
1 5 10 15
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu
20 25 30
Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
35 40 45
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
50 55 60
Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
65 70 75
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
80 85 90
Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Phe Trp Ala Phe Gly Gln
95 100 105
Gly Thr Lys Val Glu Ile Lys
110
<210>15
<211>119
<212>PRT
<213>人工序列（Artificial Sequence）
<400>15
Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr
1 5 10 15
Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser
20 25 30
Thr Ser Gly Met Cys Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
35 40 45
Ala Leu Glu Trp Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Tyr
50 55 60
Tyr Ser Thr Ser Leu Glu Thr Arg Leu Thr Ile Ser Lys Asp Thr
65 70 75
Ser Lys Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val
80 85 90
Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Ile Thr Pro His Leu Val
95 100 105
Tyr Asp Tyr Trp Ser Gln Gly Thr Leu Val Thr Val Ser Ser
110 115
<210>16
<211>107
<212>PRT
<213>人工序列（Artificial Sequence）
<400>16
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
20 25 30
Arg Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Ser Leu Ile Tyr Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
80 85 90
Ser Tyr Ser Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu
95 100 105
Ile Lys
<210>17
<211>118
<212>PRT
<213>人工序列（Artificial Sequence）
<400>17
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser
20 25 30
Thr Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu
35 40 45
Glu Trp Met Gly Ile Ile Tyr Pro Tyr Asp Ser Asp Thr Arg Tyr
50 55 60
Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser
65 70 75
Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
80 85 90
Thr Ala Ile Tyr Tyr Cys Ala Arg Gln Glu Ser Gly Trp Ser Phe
95 100 105
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
110 115
<210>18
<211>108
<212>PRT
<213>人工序列（Artificial Sequence）
<400>18
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly
1 5 10 15
Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr
20 25 30
Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu
35 40 45
Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg
50 55 60
Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr
65 70 75
Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Ser Cys Asn Ser
80 85
Arg Asp Ser Ser Gly Asn His Trp Val Phe Gly Gly Gly Thr Lys
90 95 100
Leu Thr Val Leu
105
<210>19
<211>122
<212>PRT
<213>人工序列（Artificial Sequence）
<400>19
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser
1 5 10 15
Glu Thr Leu Ser Leu Thr Cys Ala Val His Gly Gly Ser Phe Ser
20 25 30
Gly Tyr Leu Trp Thr Trp Ile Arg Leu Pro Pro Gly Lys Gly Leu
35 40 45
Glu Trp Leu Gly Glu Ile Asn Gln Ser Gly Asn Thr Asn Tyr Asn
50 55 60
Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys
65 70 75
Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
80 85 90
Ala Val Tyr Tyr Cys Ala Arg Asp Asp Ser Ser Ser Ser Gly Val
95 100 105
Gly Thr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
110 115 120
Ser Ser
<210>20
<211>110
<212>PRT
<213>人工序列（Artificial Sequence）
<400>20
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly
1 5 10 15
Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
20 25 30
Ile Asn Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
65 70 75
Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
80 85 90
Val Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr
95 100 105
Lys Leu Thr Val Leu
110
<210>21
<211>123
<212>PRT
<213>人工序列（Artificial Sequence）
<400>21
Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr
1 5 10 15
Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser
20 25 30
Thr Ser Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys
35 40 45
Ala Leu Glu Trp Leu Ala Val Ile Tyr Trp Asp Asp Asp Lys Arg
50 55 60
Tyr Ser Pro Ser Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr
65 70 75
Ser Lys Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val
80 85 90
Asp Thr Ala Thr Tyr Tyr Cys Ala His Thr Thr Trp Tyr Tyr Tyr
95 100 105
Gly Ser Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr
110 115 120
Val Ser Ser
<210>22
<211>110
<212>PRT
<213>人工序列（Artificial Sequence）
<400>22
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly
1 5 10 15
Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
20 25 30
Ser Asn Asn Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
35 40 45
Lys Val Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
65 70 75
Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
80 85 90
Ala Trp Asp Asp Ser Leu Asn Gly His Val Phe Gly Gly Gly
95 100
Thr Lys Leu Thr Val Leu
105 110
<210>23
<211>117
<212>PRT
<213>人工序列（Artificial Sequence）
<400>23
Gln Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly
1 5 10 15
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Thr Tyr Thr Phe Thr
20 25 30
Asn Phe Gly Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
35 40 45
Glu Trp Met Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr
50 55 60
Ala Gln Lys Leu Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser
65 70 75
Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp
80 85
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val Gln Glu Phe Trp Leu
90 95 100
Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
105 110 115
<210>24
<211>108
<212>PRT
<213>人工序列（Artificial Sequence）
<400>24
Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly
1 5 10 15
Lys Thr Ala Ser Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys
20 25 30
Ser Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu
35 40 45
Val Ile Tyr Tyr Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu Arg
50 55 60
Phe Ser Gly Ser Asn Ser Val Asn Thr Ala Thr Leu Thr Ile Ser
65 70 75
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp
80 85 90
Asp Ser Ser Ser Asp His Pro Leu Phe Gly Gly Gly Thr Lys Leu
95 100 105
Thr Val Leu
<210>25
<211>126
<212>PRT
<213>人工序列（Artificial Sequence）
<400>25
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
Ser Tyr Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu
35 40 45
Glu Trp Met Gly Trp Met Asn Pro Lys Ser Val Tyr Thr Gly Tyr
50 55 60
Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asn Thr
65 70
Ser Ile Arg Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
75 80 85
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Leu Trp Phe Gly
90 95 100
Asp Leu Thr Arg Thr Lys Tyr Asn Trp Phe Asp Pro Trp Gly
105 110 115
Gln Gly Thr Leu Val Thr Val Ser Ser
120 125
<210>26
<211>110
<212>PRT
<213>人工序列（Artificial Sequence）
<400>26
Arg Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly
1 5 10 15
Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
20 25 30
Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Asn Asn Asn Gln Arg Pro Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
65 70 75
Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Ser Cys Ala
80 85 90
Ala Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly
95 100
Thr Lys Leu Thr Val Leu
105 110
<210>27
<211>123
<212>PRT
<213>人工序列（Artificial Sequence）
<400>27
Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly
1 5 10 15
Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser
20 25 30
Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln His Leu
35 40 45
Glu Trp Ile Gly Trp Ile Val Val Gly Ser Gly Asn Thr Asn Tyr
50 55 60
Ala Gln Lys Phe Gln Glu Arg Val Thr Leu Thr Arg Asp Met
65 70
Ser Thr Arg Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
75 80 85
Asp Thr Ala Val Tyr Tyr Cys Ala Ala Pro Asn Cys Asn Ser Thr
90 95 100
Thr Cys His Asp Gly Phe Asp Ile Trp Gly Gln Gly Thr Val Val
105 110 115
Thr Val Ser Ser
120
<210>28
<211>108
<212>PRT
<213>人工序列（Artificial Sequence）
<400>28
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro
1 5 10 15
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg
20 25 30
Ser Ser Tyr Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75
Ile Ser Arg Leu Glu Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln
80 85 90
Gln Tyr Asp Asn Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
95 100 105
Glu Ile Lys
<210>29
<211>122
<212>PRT
<213>人工序列（Artificial Sequence）
<400>29
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30
Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ser Thr Ile Ser Ala Ser Gly Asp Ser Thr Tyr Tyr
50 55 60
Ala Asp Phe Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
65 70 75
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
80 85
Asp Thr Ala Ile Tyr Tyr Cys Ala Lys Glu Pro Ser Phe Gly Leu
90 95 100
Trp Phe Gly Glu Leu Ser Gly Trp Gly Gln Gly Thr Leu Val Thr
105 110 115
Val Ser Ser
120
<210>30
<211>109
<212>PRT
<213>人工序列（Artificial Sequence）
<400>30
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro
1 5 10 15
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser
20 25 30
Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
80 85 90
Gln Tyr Gly Ser Ser Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys
95 100 105
Leu Glu Ile Lys
<210>31
<211>129
<212>PRT
<213>人工序列（Artificial Sequence）
<400>31
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
1 5 10 15
Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ile
20 25 30
Thr Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
35 40 45
Glu Trp Ile Gly Tyr Ile Asp Tyr Ser Gly Ser Thr Asn Tyr Asn
50 55 60
Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys
65 70 75
Asn Gln Phe Ser Leu Glu Leu Ser Ser Val Thr Ala Ala Asp Thr
80 85 90
Ala Val Tyr Tyr Cys Ala Arg Thr Leu Gly Ala Tyr Tyr Asp Ile
95 100 105
Leu Thr Gly Phe Arg Thr Pro Gly Gly Trp Phe Ala Pro Trp Gly
110 115 120
Gln Gly Thr Leu Val Thr Val Ser Ser
125
<210>32
<211>108
<212>PRT
<213>人工序列（Artificial Sequence）
<400>32
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro
1 5 10 15
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Gln Ser Val Ser
20 25 30
Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Val Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
80 85 90
Gln Tyr Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
95 100 105
Glu Ile Lys
<210>33
<211>130
<212>PRT
<213>人工序列（Artificial Sequence）
<400>33
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30
Asp Tyr Tyr Met Ile Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ala Thr Ile Tyr Tyr
50 55 60
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
65 70 75
Lys Asn Ser Leu Tyr Leu Gln Met Thr Ser Leu Arg Ala Glu
80 85
Asp Thr Ala Val Tyr Tyr Cys Ala Ser Pro Leu Leu Ser His Asn
90 95 100
Tyr Gly Ser Gly Ser Tyr Tyr Asn Val Tyr Trp Tyr Phe Glu Leu
105 110 115
Trp Gly Arg Gly Thr Leu Val Ser Val Ser Ser
120 125 130
<210>34
<211>103
<212>PRT
<213>人工序列（Artificial Sequence）
<400>34
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
1 5 10 15
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
20 25 30
Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
35 40 45
Leu Leu Ile Phe Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
80 85 90
Leu Gly Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
95 100
<210>35
<211>123
<212>PRT
<213>人工序列（Artificial Sequence）
<400>35
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Phe Leu Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30
Ser Asp Asp Met His Trp Val Arg Gln Val Thr Gly Lys Gly Leu
35 40 45
Glu Trp Val Ser Ala Ile Gly Thr Ser Gly Asp Thr Tyr Tyr Ser
50 55 60
Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys
65 70 75
Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Val Gly Asp
80 85
Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser Leu Arg Gly Gly Ile
90 95 100
Leu Ser Gly Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
105 110 115
Thr Val Ser Ser
120
<210>36
<211>108
<212>PRT
<213>人工序列（Artificial Sequence）
<400>36
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Thr Val Thr Ile Thr Cys Arg Ala Ser Gln Ile Ile Asn
20 25 30
Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
80 85 90
Ser Tyr Ser Ser Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
95 100 105
Glu Ile Lys
<210>37
<211>117
<212>PRT
<213>人工序列（Artificial Sequence）
<400>37
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Ile Val Ser
20 25 30
Arg Asn Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
35 40
Leu Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr
45 50 55
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
60 65 70
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
75 80 85
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Leu Glu Asn Gly
90 95 100
Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Phe Ser
105 110 115
<210>38
<211>106
<212>PRT
<213>人工序列（Artificial Sequence）
<400>38
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser
20 25 30
Lys Trp Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Ile Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
80 85 90
Thr Asn Ser Phe Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
95 100 105
Lys
<210>39
<211>119
<212>PRT
<213>人工序列（Artificial Sequence）
<400>39
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Asp Ser Phe Pro
20 25 30
Ser Tyr Trp Ile Glu Trp Val Arg Gln Met Pro Gly Lys Gly Leu
35 40 45
Glu Trp Met Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr
50 55 60
Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser
65 70 75
Ile Ser Thr Ala Tyr Leu Gln Leu Ser Ser Leu Lys Ala Ser Asp
80 85 90
Thr Ala Met Tyr Phe Cys Ala Arg His Pro Val Leu Arg Gly
95 100
Asn Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Ile Val Ser Ser
105 110 115
<210>40
<211>111
<212>PRT
<213>人工序列（Artificial Sequence）
<400>40
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly
1 5 10 15
Gln Arg Val Thr Ile Ser Cys Thr Ala Ser Ser Ser Asn Ile Gly
20 25 30
Ala Asp Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Val
35 40 45
Pro Lys Leu Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
65 70 75
Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
80 85 90
Gln Ser Tyr Asp Ser Ser Leu Arg Gly Ser Val Phe Gly Gly Gly
95 100 105
Thr Lys Leu Thr Val Leu
110

Claims (8)

1.新冠病毒RBD特异性单克隆抗体，其特征在于，重链可变区氨基酸序列如SEQ ID NO:7所示；轻链可变区氨基酸序列如SEQ ID NO:8所示。
2.新冠病毒RBD特异性单克隆抗体，其特征在于，重链可变区氨基酸序列如SEQ ID NO:19所示；轻链可变区氨基酸序列如SEQ ID NO:20所示。
3.新冠病毒RBD特异性单克隆抗体，其特征在于，重链可变区氨基酸序列如SEQ ID NO:31所示；轻链可变区氨基酸序列如SEQ ID NO:32所示。
4.根据权利要求1-3中任一权利要求所述的新冠病毒RBD特异性单克隆抗体在制备检测或诊断SARS-CoV-2试剂或药物中的应用，其中药物包括权利要求1-3任一权利要求所述的新冠病毒RBD特异性单克隆抗体和药学上可接受的赋形剂、稀释剂或载体。
5.编码权利要求1-3中任一权利要求所述的新冠病毒RBD特异性单克隆抗体的核酸分子。
6.含有权利要求5所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
7.权利要求6所述的表达盒、重组载体、重组菌或转基因细胞系在制备产品中的应用，其特征在于，产品用途如下（b1）-（b2）中的任一种：（b1）结合新型冠状病毒SARS-CoV-2；（b2）检测新型冠状病毒SARS-CoV-2。
8.权利要求6所述的表达盒、重组载体、重组菌或转基因细胞系在制备产品中的应用，其特征在于，产品用途如下（b1）-（b2）中的任一种：（b1）结合新型冠状病毒SARS-CoV-2的S蛋白；（b2）检测新型冠状病毒SARS-CoV-2的S蛋白。
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Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10787501B1 (en) 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
WO2022053054A1 (en) * 2020-09-14 2022-03-17 Vazyme Biotech Co., Ltd. Neutralizing antibodies against sars-cov-2
CN114685652B (zh) * 2020-12-31 2023-11-03 中国科学院分子细胞科学卓越创新中心 针对SARS-CoV-2和SARS-CoV的全人广谱交叉中和抗体及其应用
CN114716541B (zh) * 2021-01-05 2023-07-21 中国科学院分子细胞科学卓越创新中心 抗冠状病毒的全人广谱中和抗体76e1及其应用
CN115427441B (zh) * 2021-01-27 2023-09-05 保诺生物科技(江苏)有限公司 针对sars-cov-2的抗体
CN114805556A (zh) * 2021-01-29 2022-07-29 中国科学院上海巴斯德研究所 一种针对SARS-CoV-2病毒的中和性抗体
CN115109150A (zh) * 2021-01-31 2022-09-27 中南大学湘雅医院 新型冠状病毒单克隆抗体xy10及其应用
WO2022179561A1 (en) * 2021-02-24 2022-09-01 The University Of Hong Kong Neutralizing antibodies against covid-19 and methods of use thereof
CN113651884B (zh) * 2021-05-25 2022-08-26 中国科学院微生物研究所 人源化抗SARS-CoV-2单克隆抗体及其应用
TW202337497A (zh) 2022-02-18 2023-10-01 中國大陸商重慶明道浩悅生物科技有限公司 鼻內調配物及抗sars-cov-2棘蛋白抗體

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB202003632D0 (en) * 2020-03-12 2020-04-29 Harbour Antibodies Bv SARS-Cov-2 (SARS2, COVID-19) antibodies
CN111537743A (zh) * 2020-04-03 2020-08-14 广州医科大学附属第一医院（广州呼吸中心） SARS-CoV-2新冠病毒抗体检测试剂盒

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2997444A1 (en) * 2015-09-29 2017-04-06 Amgen Inc. Asgr inhibitors for reducing cholesterol levels
CN111153991A (zh) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 一种人SARS-CoV-2单克隆抗体及其制备方法和应用
CN111303280B (zh) * 2020-03-22 2022-01-07 中国人民解放军军事科学院军事医学研究院 高中和活性抗SARS-CoV-2全人源单克隆抗体及应用
CN111474345A (zh) * 2020-03-25 2020-07-31 北京博奥森生物技术有限公司 一种SARS-CoV-2抗体检测方法
RU2723008C9 (ru) * 2020-05-19 2021-02-09 федеральное государственное бюджетное учреждение «Национальный исследовательский центр эпидемиологии и микробиологии имени почетного академика Н.Ф. Гамалеи» Министерства здравоохранения Российской Федерации Способ получения штамма клеток яичника китайского хомячка, продуцента рекомбинантного белка RBD вируса SARS-CoV-2, штамм клеток яичника китайского хомячка, продуцент рекомбинантного белка RBD вируса SARS-CoV-2, способ получения рекомбинантного белка RBD вируса SARS-CoV-2, тест-система для иммуноферментного анализа сыворотки или плазмы крови человека и ее применение

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB202003632D0 (en) * 2020-03-12 2020-04-29 Harbour Antibodies Bv SARS-Cov-2 (SARS2, COVID-19) antibodies
CN111537743A (zh) * 2020-04-03 2020-08-14 广州医科大学附属第一医院（广州呼吸中心） SARS-CoV-2新冠病毒抗体检测试剂盒

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2 receptor;Xiangyu Chen等;《Cell Mol Immunol》;第17卷(第6期);第649页左栏 *
immunoglobulin heavy chain VHDJ region, partial [Homo sapiens];GenBank;《GenBank》;GenBank: BAC02199.1 *
新型冠状病毒高效价中和血清的快速制备及应用;徐康维等;《中国生物制品学杂志》;第33卷(第8期);第918-920页 *

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