CN114920832A - 新冠病毒rbd特异性单克隆抗体和应用 - Google Patents
新冠病毒rbd特异性单克隆抗体和应用 Download PDFInfo
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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
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特异性单克隆抗体和应用。
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特异性单克隆抗体和应用。
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，抗体的重链氨基酸序列如SEQ ID NO:21所示；轻链氨基酸序列可如SEQ ID NO:22所示(单抗11-CQTS011)。重链氨基酸序列还可如SEQ ID NO:23所示；轻链氨基酸序列还可如SEQ ID NO:24所示(单抗12-CQTS012)。重链氨基酸序列还可如SEQ ID NO:25所示；轻链氨基酸序列还可如SEQ ID NO:26所示(单抗13-CQTS013)。重链氨基酸序列还可如SEQ ID NO:27所示；轻链氨基酸序列还可如SEQ ID NO:28所示(单抗14-CQTS014)。重链氨基酸序列还可如SEQ IDNO:29所示；轻链氨基酸序列还可如SEQ ID NO:30所示(单抗15-CQTS015)。重链氨基酸序列还可如SEQ ID NO:31所示；轻链氨基酸序列还可如SEQ ID NO:32所示(单抗16-CQTS016)。重链氨基酸序列还可如SEQ ID NO:33所示；轻链氨基酸序列还可如SEQ ID NO:34所示(单抗17-CQTS017)。重链氨基酸序列还可如SEQ ID NO:35所示；轻链氨基酸序列还可如SEQ IDNO:36所示(单抗18-CQTS018)。重链氨基酸序列还可如SEQ ID NO:37所示；轻链氨基酸序列还可如SEQ ID NO:38所示(单抗19-CQTS019)。重链氨基酸序列还可如SEQ ID NO:39所示；轻链氨基酸序列还可如SEQ ID NO:40所示(单抗20-CQTS020)。
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，其中药物包括权利要求1-10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；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记忆B细胞的细胞分选图；
图2为采用流式细胞仪分析RBD特异性记忆B细胞的细胞分选图；
图3为单细胞抗体基因PCR产物凝胶电泳图；
图4为PCR扩增包含CMV启动子、WPRE-γ或WPRE-κ元件抗体基因表达盒后的琼脂糖凝胶电泳图；
图5为RBD特异性的实验结果图。
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，重链氨基酸序列如SEQ IDNO:21所示；轻链氨基酸序列如SEQ ID NO:22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2-10
实施例2-10与实施例1的区别在于：RBD特异性单克隆抗体的氨基酸序列不同，实施例2-10的氨基酸序列如下表所示：
以上实施例1-10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，实验结果如图3所示。
S4、采用巢式PCR扩增S1-S3得到的抗体可变区cDNA，构建抗体可变区基因表达盒。
S3和S4总共通过以下六部分进行：(1)提取RBD特异性记忆B细胞的mRNA；(2)单个细胞mRNA逆转录(RT)；(3)加G尾(TDT)；(4)第一轮PCR(1st PCR)；(5)第二轮PCR(2nd PCR)；(6)BCR-ORF PCR扩增构建基因表达盒；(7)CMV、WPRE-γ/κ/l片段扩增及CMV、BCR-Vγ/κ/l((6)的产物)、WPRE-γ/κ/l重叠PCR(Overlap PCR)预连接；(8)BCR-γORF、BCR-κORF、BCR-lPCR扩增。
各部分反应液配制及反应条件如下：
(1)采用DynabeadsTM mRNA DIRECTTM Purification 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。
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 |
| H<sub>2</sub>O | 6.4μl |
| 5×TdTbuffer | 2.0μl |
| 10mMdGTP | 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(2ndPCR)(10μl体系)(引物序列参见引物序列表一和引物序列表二)：所需配制的试剂如下表4所示：
| 试剂名称 | 体积 |
| H<sub>2</sub>O | 1.5μl |
| 2×GCBuffer | 5μl |
| 2.5mMdNTP | 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，2ndPCR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。
扩增后，采用琼脂糖凝胶电泳，凝胶成像分析得到的抗体可变区基因大小是否正确，实验结果如图4所示，Marker在中间位置，条带在5000bp处。
BCR-γORF和BCR-κ/ORF乙醇沉淀：BCR-γORF和BCR-κORF的PCR产物各取30μl置于8连管中，再加入120μl无水乙醇，6μl醋酸钠溶液，充分混匀，-80℃静置30min；10000rpm，离心20min，弃上清，依次用200μl的70％乙醇和无水乙醇各漂洗一次，于56℃乙醇充分挥发，加入40μl无菌水，振荡，使沉淀充分溶解，检测抗体可变区基因的浓度。
S3和S4所用到的Leader引物参见如下的引物序列表一：
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：
| primerID | sequence |
| IGHJ_01 | GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCAGGGTGCCCTGGCCCCAGT |
| IGHJ_02 | GATGGGCCCTTGGTGGAGGGTGAGGAGACAGTGACCAGGGTGCCACGGCCCCAGA |
| IGHJ_03 | GATGGGCCCTTGGTGGAGGGTGAAGAGACGGTGACCATTGTCCCTTGGCCCCAGA |
| IGHJ_04 | GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCGTGGTCCCTTGCCCCCAGA |
| IGKJ_01 | GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC |
| IGKJ_02 | GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC |
| IGKJ_03 | GATGGTGCAGCCACAGTTCGTTTGATATCCACTTTGGTCCCAGGGCCGAAAGTGA |
| IGKJ_04 | GATGGTGCAGCCACAGTTCGTTTGATCTCCACCTTGGTCCCTCCGCCGAAAGTGA |
| IGKJ_05 | GATGGTGCAGCCACAGTTCGTTTAATCTCCAGTCGTGTCCCTTGGCCGAAGGTGA |
| IGLJ_01 | GGGGCAGCCTTGGGCTGACCTAGGACGGTGACCTTGGTCCCAGTTCCGAAGACAT |
| IGLJ_02 | GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTTGGTCCCTCCGCCGAATACCA |
| IGLJ_03 | GGGGCAGCCTTGGGCTGACCTAAAATGATCAGCTGGGTTCCTCCACCAAATACAA |
| IGLJ_04 | GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCGGTCCCCTCACCAAACACCC |
| IGLJ_05 | GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCCGTCCCCTCACCAAACACCC |
| IGLJ_06 | GGGGCAGCCTTGGGCTGACCGAGGACGGTCACCTTGGTGCCACTGCCGAACACAT |
| IGLJ_07 | GGGGCAGCCTTGGGCTGACCGAGGACGGTCAGCTGGGTGCCTCCTCCGAACACAG |
| IGLJ_08 | GGGGCAGCCTTGGGCTGACCGAGGGCGGTCAGCTGGGTGCCTCCTCCGAACACAG |
S5、将S4得到的抗体可变区基因表达盒转导入293T细胞48小时内表达抗体并收集上清，用ELISA法检测上清的RBD特异性，筛选RBD特异性全人源单克隆抗体。
(A)使用PBS稀释抗原(终浓度2μg/mL)，10μl/孔，包被384孔ELISA板4℃过夜或37℃包被2h(本实施例优选4℃过夜)。NOTE：加完后瞬时离心保证液体在底部。
实验体系如下表8所示：
| 试剂名称 | 货号 | 原浓度 | 终浓度 | 稀释比 |
| SARS-COV-2RBD | Cat:40592-V08H | 200μg/mL | 2μg/mL | 1:100 |
| GoatpabtoHuIgG－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-humanIgG-ALP | A18808 | 1.5mg/ml | 0.3μg/ml | 1:5000 |
| GoatpabtoHuIgG-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5所示，图5中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。
序列表
<110> 重庆医科大学,冯玉林
<120> 新冠病毒RBD特异性单克隆抗体和应用
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Ser Tyr Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Thr Thr His Tyr Asp Pro Ser
50 55 60
Leu Lys Ser Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asp Gln Phe
65 70 75 80
Ser Leu Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Thr Leu Glu Trp Leu Arg Ala Pro Phe Asp Asp Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 28
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Asp Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser Asp
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val
35 40 45
Ile Tyr Glu Asp Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asp Ser Ala Ser Leu Thr Ile Ser Gly
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser
85 90 95
Ser Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 29
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45
Trp Leu Ala Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Ser Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asp Gln Val
65 70 75 80
Val Leu Thr Met Thr Thr Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala His Arg Pro Leu Phe Ser Gly Pro Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 30
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Ser Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser Asp
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Leu
35 40 45
Ile Tyr Glu Asp Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asp Ser Ala Ser Leu Thr Ile Ser Gly
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser
85 90 95
Ser Thr Ile Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 31
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ser Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Pro Ile Leu Gly Val Ala Asp Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Thr Thr Ile Thr Glu Tyr Phe Gln Gln Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 32
<211> 111
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Asp Ser Asp Ile Gly Ala Gly
20 25 30
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asp Ser Asp Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95
Leu Thr Gly Ser Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu
100 105 110
<210> 33
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Arg Ser Phe Ser Gly Asp
20 25 30
Ser Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asp His Ser Gly Ser Thr Asp Tyr Asp Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asp Gln Phe Ser Leu
65 70 75 80
Lys Leu Asp Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Trp Thr Val Pro Pro Leu Trp Val Leu Asp Trp Phe Asp Pro
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 34
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asp Gly Tyr Asp Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Val Ile Tyr Leu Gly Ser Asp Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 35
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45
Trp Leu Ala Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Ser Pro Ser
50 55 60
Leu Lys Thr Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asp Gln Val
65 70 75 80
Val Leu Thr Leu Thr Asp Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala His Arg Pro Ala Gly Phe Trp Ser Ala His Phe Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<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 Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Ala
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<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 Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Ile Ile Val Ser Ser Asp
20 25 30
Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Leu Leu Tyr Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asp Thr Leu Tyr Leu
65 70 75 80
Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Leu Gly Pro Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ser Ser
115
<210> 38
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asp Ser Tyr Pro Gln
85 90 95
Ala Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 39
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Glu Val Gln Leu Val Gly Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asp
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Leu Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asp Thr Leu Phe Leu
65 70 75 80
Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Gly Arg Ala Val Ala Gly Thr Asp Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 40
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asp Asp Tyr
20 25 30
Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asp Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Val Asp Ile Ala Thr Tyr Tyr Cys His Gln Tyr Asp Asp Leu Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
Claims (10)
1.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:21所示；轻链氨基酸序列还可如SEQ ID NO:22所示。
2.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:23所示；轻链氨基酸序列还可如SEQ ID NO:24所示。
3.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:25所示；轻链氨基酸序列还可如SEQ ID NO:26所示。
4.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:27所示；轻链氨基酸序列还可如SEQ ID NO:28所示。
5.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:29所示；轻链氨基酸序列还可如SEQ ID NO:30所示。
6.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:31所示；轻链氨基酸序列还可如SEQ ID NO:32所示。
7.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:33所示；轻链氨基酸序列还可如SEQ ID NO:34所示。
8.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:35所示；轻链氨基酸序列还可如SEQ ID NO:36所示。
9.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:37所示；轻链氨基酸序列还可如SEQ ID NO:38所示。
10.根据权利要求1所述的新冠病毒RBD特异性单克隆抗体，其特征在于，重链氨基酸序列还可如SEQ ID NO:39所示；轻链氨基酸序列还可如SEQ ID NO:40所示。
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| CN114920832B (zh) * | 2020-08-19 | 2023-10-13 | 重庆医科大学 | 新冠病毒rbd特异性单克隆抗体和应用 |
| WO2022102744A1 (ja) * | 2020-11-13 | 2022-05-19 | 株式会社ハカレル | ＳＡＲＳ－ＣｏＶ－２のスパイクタンパク質に対する抗体 |
| WO2022112392A1 (en) * | 2020-11-26 | 2022-06-02 | Memo Therapeutics Ag | Anti-sars-cov-2 antibody molecules |
| WO2022179561A1 (en) * | 2021-02-24 | 2022-09-01 | The University Of Hong Kong | Neutralizing antibodies against covid-19 and methods of use thereof |
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| CN113388030B (zh) * | 2021-08-17 | 2021-11-23 | 上海浙江大学高等研究院 | 单克隆抗体32c7及其制备方法和用途 |
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| WO2023155902A1 (en) | 2022-02-18 | 2023-08-24 | Chongqing Mingdao Haoyue Biotechnology Co., Ltd. | Intranasal formulations and anti-sars-cov-2-spike protein antibodies |
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| CN117736313A (zh) | 2024-03-22 |
| CN111909263A (zh) | 2020-11-10 |
| CN115925896A (zh) | 2023-04-07 |
| CN118085068A (zh) | 2024-05-28 |
| CN117003862A (zh) | 2023-11-07 |
| WO2022037616A1 (zh) | 2022-02-24 |
| CN116813760A (zh) | 2023-09-29 |
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