CN112409488B - 针对多种冠状病毒的单克隆抗体及应用 - Google Patents

针对多种冠状病毒的单克隆抗体及应用 Download PDF

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CN112409488B
CN112409488B CN202011143522.9A CN202011143522A CN112409488B CN 112409488 B CN112409488 B CN 112409488B CN 202011143522 A CN202011143522 A CN 202011143522A CN 112409488 B CN112409488 B CN 112409488B
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CN112409488A (zh
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王春河
陈玉宁
陈艺丽
陈淦均
王桂凤
王琪
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Dashi Pharmaceutical Guangdong Co ltd
Shanghai Institute of Materia Medica of CAS
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Dashi Pharmaceutical Guangdong Co ltd
Shanghai Institute of Materia Medica of CAS
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Abstract

本发明涉及针对多种冠状病毒的单克隆抗体及应用。所述单克隆抗体与氨基酸序列为SEQ ID NO:19的人ACE2在结合位点结合，所述结合位点选自Q18，E23，Q24，F28，K31，H34和Y83位中的任一个或两种以上的组合。所述单克隆抗体或其抗原结合片段可在临床上预防或治疗多种冠状病毒(包括但不限于SARS‑CoV‑2(D614和G614)、SARS‑CoV和HCoV‑NL63)相关疾病，亦可用于ACE2的检测或相关疾病分型。

Description

针对多种冠状病毒的单克隆抗体及应用
技术领域
本发明属于生物技术领域，具体涉及针对多种冠状病毒的单克隆抗体、其抗原结合片段及其应用。
背景技术
现有技术公开了严重急性呼吸系统综合征冠状病毒2(SARS-CoV-2)是一种引起世界范围内呼吸道流行病(COVID-19)的病毒，目前主要分为两种：野生型SARS-CoV-2，以及目前国外流行的感染力更强的变异株SARS-CoV-2-G614。近一年来，SARS-CoV-2已感染全球3850余万人，并导致109余万人死亡。目前本领域虽已研制出针对SARS-CoV-2的疫苗，但药效尚不可知，也无特效的治疗性药物。SARS-CoV-2、SARS-CoV和HCoV-NL63同属于冠状病毒。研究显示，这三种病毒都能通过各自的病毒刺突蛋白S1亚基与宿主受体人血管紧张素转化酶2(ACE2)结合，介导病毒的入侵，所以ACE2是该类病毒最主要的受体，而S1-ACE2结合被认为是有效的阻断靶标。抗体在抗感染治疗及紧急被动免疫预防方面扮演着重要的角色。因此，研制特异性SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63的抗体，用于紧急预防和治疗是十分必要和可行的。
发明内容
本发明的目的在于提供针对多种冠状病毒的单克隆抗体及应用。
一种疫苗、抗体或其抗原结合片段，其与氨基酸序列为SEQ ID NO:19的人ACE2在结合位点结合，所述结合位点选自SEQ ID NO:19中的Q18，E23，Q24，F28，K31，H34和Y83位中的任一个或两种以上的组合。
在具体实施方式中，所述结合位点为H34。
在具体实施方式中，所述抗体为单克隆抗体。
在具体实施方式中，所述单克隆抗体是人源化抗体或全人源抗体。
在具体实施方式中，所述单克隆抗体为IgG。
在具体实施方式中，所述抗原结合片段是scFv、Fv、Fab或F(ab)2。
在具体实施方式中，所述单克隆抗体包括重链可变区(VH)和轻链可变区(VL)，其中所述VH包括含序列SEQ ID NO:1的重链可变区CDR1，含序列SEQ ID NO:2的重链可变区CDR2，以及含序列SEQ ID NO:3的重链可变区CDR3，并且所述VL包括含序列SEQ ID NO:5的轻链可变区CDR1，含序列SEQ ID NO:6的轻链可变区CDR2，以及含序列SEQ ID NO:7的轻链可变区CDR3。
在具体实施方式中，所述重链可变区的氨基酸序列与所述VH具有至少90％的同一性，且所述轻链可变区的氨基酸序列与所述VL具有至少90％的同一性。
在具体实施方式中，所述重链可变区的氨基酸序列为SEQ ID NO:4，且所述轻链可变区的氨基酸序列为SEQ ID NO:8。
一种双特异性抗体，其含有前述单克隆抗体或其抗原结合片段。
一种前述单克隆抗体或其抗原结合片段，或前述双特异性抗体与一种效应分子的偶联物。
在具体实施方式中，所述效应分子是可检测标记。
在具体实施方式中，所述可检测标记是荧光标记、放射性标记、亲和素、生物素，或酶。
在具体实施方式中，所述效应分子是毒素或化疗剂。
在具体实施方式中，所述毒素是绿脓杆菌外毒素。
一种核酸分子，其编码前述单克隆抗体或抗原结合片段，前述双特异性抗体。
在具体实施方式中，所述核酸分子可操作地连接至启动子。
一种质粒，其含有前述核酸分子。
一种用于检测受试者的ACE2水平的试剂盒，其包括：前述单克隆抗体或其抗原结合片段、双特异性抗体或偶联物。
一种药用组合物，其含有有效预防剂量的前述单克隆抗体或抗原结合片段，前述双特异性抗体，前述偶联物，前述核酸，和任选地，药学上可接受载体。
前述单克隆抗体或其抗原结合片段，双特异性抗体，或偶联物，或前述药用组合物在制备用于预防或治疗或诊断与ACE2相关疾病的药物中的用途。
在具体实施方式中，所述疾病选自冠状病毒感染或高血压。
在具体实施方式中，所述冠状病毒包括但不限于SARS-CoV-2(D614和G614)、SARS-CoV或HCoV-NL63。
有益效果
本发明创造性地提出了ACE2的结合位点Q18，E23，Q24，F28，K31，H34和Y83位在制备ACE2相关的诊断、预防或治疗制剂中的应用。
进一步地，本发明的特异性抗体3E8显示出对于SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63的中和能力，同时其对于ACE2的催化活性没有影响，因此，其可在临床上预防或治疗与上述病毒相关的疾病，亦可用于ACE2的检测或相关疾病的分型。
附图说明
图1：利用ELISA(A)、生物膜干涉技术(BLI)(B)和流式细胞术(FACS)(C)法检测本发明的特异性抗体3E8与ACE2的结合能力。
图2：利用ELISA法检测本发明的特异性抗体3E8对SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1的中和能力(A：SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1与ACE2结合，B：单克隆抗体3E8对SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1破坏前述结合)。
图3：利用ELISA法检测本发明的特异性抗体3E8对SARS-CoV-2(D614(B)和G614(A))、SARS-CoV(C)和HCoV-NL63(D)假病毒的中和能力。
图4：qRT-PCR法检测本发明的特异性抗体3E8在体外(A)和体内(B)对活病毒SARS-CoV-2(D614)的中和能力。
图5：本发明的特异性抗体3E8在分子水平(A)和细胞水平(B)对ACE2的催化活性的影响。
图6：示意性示出特异性抗体3E8竞争性结合ACE2的位点。
具体实施方式
以下通过具体实施例详细描述本发明，然而下述实施例并非用于限制本发明的范围。
本发明实例中未明确具体条件的实验方法，均为常规方法或按厂商推荐条件。未注明具体来源的试剂，为市场中的常规试剂。
实施例1 ACE2蛋白的产生
将编码全长人ACE2的质粒中编码胞外hACE2(19-740位残基)的DNA片段亚克隆到哺乳动物表达载体pTT5(his-tag)和pINFUSE-hIgG1-Fc1(Fc-tag)，将纯化的质粒转染细胞进行表达。当抗体富集到一定程度后，通过protein A色谱法纯化。
实施例2免疫小鼠获得杂交瘤细胞
免疫小鼠选用雌性balb/c小鼠，将免疫所需抗原及佐剂制备匀浆，采用脚趾注射和皮下多点注射法。初次免疫后，再多次加强免疫。初次免疫和加强免疫用ACE2-Fc+CpGODN+弗氏不完全佐剂。免疫完成后，提取小鼠B细胞，将其与骨髓瘤细胞SP2/0融合，制备杂交瘤细胞。
实施例3抗体分子的人源化
抗单克隆抗体，其可变区来自于杂交瘤筛选获得，恒定区来自于人源抗体IgG。嵌合抗体需要进行人源化，通过互补决定区(complementarity determining region，CDR)移植、特异性决定区(specificity-determining region，SDR)移植、超级人源化(superhumanization)或框架区轮换(framework shuffling)、导向性选择三种策略进行，以有效降低抗体在临床使用中的免疫源性，并对抗体分子的CDR区进行优化，提高抗体的亲和力。
实验实施例1单克隆抗体3E8与靶点ACE2的结合。
根据测试结果选择抗体3E8克隆，具体为：将用于表达3E8的重链和轻链的质粒转入HEK293F细胞。5天后，用protein A色谱法纯化，用分子排阻色谱分析蛋白纯度。
抗体3E8的重链和轻链可变区序列分别为：SEQ ID NO:4和SEQ ID NO:8，重链和轻链恒定区序列分别为：SEQ ID NO:9和SEQ ID NO:10，重链和轻链恒定区的核苷酸序列分别为：SEQ ID NO:11和SEQ ID NO:12，重链和轻链全长序列分别为：SEQ ID NO:13和SEQ IDNO:14，重链和轻链可变区的核苷酸序列(原始)分别为：SEQ ID NO:15和SEQ ID NO:16，重链和轻链恒定区的核苷酸序列(优化)分别为：SEQ ID NO:17和SEQ ID NO:18，分别用ELISA法、FACS技术和生物膜干涉技术(BLI)检测单克隆抗体3E8与ACE2的结合能力。
ELISA法检测单克隆抗体3E8与细胞表面ACE2的结合能力：将96孔板包被纯化的重组hACE2-his蛋白，4℃过夜。在室温下用1％casein蛋白封闭1小时后，将板用含0.05％Tween-20的磷酸盐缓冲液(PBST)洗涤，并加入梯度稀释的3E8孵育一小时。PBST洗涤后，加入HRP标记的山羊抗人IgG，孵育1小时。PBST洗涤，加入TMB底物，并用2M的H2SO4终止，再用SpectraMax M5e酶标仪检测OD450。为了测量S1蛋白与hACE2的结合能力，将2μg/ml的各种S1蛋白包被到平板中，再加入梯度稀释的ACE2-Fc，利用辣根过氧化物酶(HRP)标记的山羊抗人IgG检测结合的ACE2-Fc。
FACS法检测单克隆抗体3E8与细胞表面ACE2的结合能力：取5×105个稳定转染ACE2的HEK293F细胞或内源表达ACE2的Vero E6细胞于96孔板中，PBS清洗，将梯度稀释的3E8抗体加入到板中，加入PE标记的山羊抗人IgG，4℃孵育40分钟，PBS洗，流式细胞仪上机检测。
生物膜层光学干涉(BLI)技术：使用Octet Red96通过BLI技术测量结合能力。针对3E8，利用Ni-NTA生物传感器捕获15μg/ml ACE2-his蛋白，并与不同梯度的3E8进行结合。针对与ACE2结合的各种S1蛋白，将15μg/ml的S1-his蛋白固定在Ni-NTA生物传感器上，并与不同梯度的ACE2-Fc进行结合。用含0.05％Tween 20的PBS持续60s进行平衡。结合和解离时间均为400s。根据所有结合曲线的整体拟合度(1：1)，计算结合亲和力的平均Kon，Koff和表观KD值。
实验结果如图1所示，从图1可以看出，在分子和细胞水平上，通过不同方法都证明了3E8能特异性地结合ACE2，且结合能力较强。
实验实施例2单克隆抗体3E8对SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1的中和能力。
具体为：将2μg/ml的各种S1蛋白在4℃下包被过夜。将梯度稀释的3E8或同型对照与5μg/ml ACE2-Fc在室温下预孵育30分钟，再将混合物加入包被的板中孵育1小时。利用HRP标记的山羊抗人IgG检测结合的ACE2-Fc。
实验结果如图2所示，从图2可以看出，SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1都能和ACE2结合(图2A和下表1)，而这种结合能够被3E8竞争破坏，即单克隆抗体3E8对SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1具有很好的中和能力。
表1
Figure GDA0003024174810000071
实验实施例3单克隆抗体3E8对SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63假病毒的中和能力。
构建假病毒SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63：将两个质粒共转HEK293T细胞：其中一个质粒表达Env缺陷型HIV-1(pNL4-3.luc.RE)(带有荧光素酶报告基因)，另一个质粒表达SARS-CoV-2(D614和G614)、SARS-CoV或HCoV-NL63的全长S蛋白。转染48小时后收集含有病毒颗粒的上清液，0.45μm过滤膜过滤。
在96孔板中，每孔接种1.2×104个HEK293F/hACE2/EGFP细胞。将细胞与50μl梯度稀释的抗体或ACE2-Fc、B38(作为阳性对照)、Isotype(同型对照)在37℃温育1小时，加入各种相同体积的假病毒。100μl含10％FBS的DMEM作为阴性对照，50μl假病毒和同体积的DMEM设为阳性对照。24小时后，更换新鲜培养基并再温育48小时。利用萤火虫荧光素酶报告基因检测试剂盒测量相对荧光单位(RLU)。中和活性(％)[1-(RFU样品–RFU阴性对照)/(RFU阳性对照–RFU阴性对照)]×100％。通过GraphPad Prism 8中的非线性回归计算IC50值。
实验结果如图3和表2所示，从图3可以看出，单克隆抗体3E8能够浓度依赖性地竞争SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63假病毒与ACE2的结合，即3E8对各种假病毒具有很好的中和能力。
表2
Figure GDA0003024174810000081
实验实施例4特异性抗体3E8在体内外对活病毒SARS-CoV-2(D614)的中和能力。
细胞水平：在24孔板中，每孔接种1×105个Vero E6细胞，24小时后，将梯度稀释的抗体和SARS-CoV-2(D614)加入板中。24小时后，收集病毒上清，提取RNA，用qRT-PCR法检测病毒RNA水平，计算各抗体对病毒的抑制率。
动物水平：6-8周龄的Balb/c雌性小鼠(n＝5)用avertin(250mg/ml)麻醉后，首先以106FFU VRP-hACE2感染每只小鼠。12小时后，腹腔注射抗体(3E8/B38/Isotype)，再12小时后将105PFU SARS-CoV-2通过滴鼻的方式感染小鼠。感染3天后，取每只小鼠的肺组织，检测各组SARS-CoV-2的RNA水平。引物序列为：RBD-qF1：5’-caatggtttaacaggcacagg-3’(SEQID NO:20),RBD-qR1：5’-ctcaagtgtctgtggatcacg-3’(SEQ ID NO:21)，Probe：acagcatcagtagtgtcagcaatgtctc(SEQ ID NO:22)。
实验结果如图4，表3和表4所示，特异性抗体3E8能够在细胞和动物水平显著降低活SARS-CoV-2-D614的RNA水平，并优于阳性对照B38。
表3细胞水平的抗新冠病毒活性
Figure GDA0003024174810000091
表4动物水平的抗新冠病毒活性
Figure GDA0003024174810000092
实验实施例5特异性抗体3E8对于ACE2的催化活性的影响。
分子水平ACE2酶活性检测：将稀释的抗体(AF933/3E8/isotype)与2μg/ml ACE2-his在振荡器上室温孵育1小时后，用活性缓冲液稀释5倍，再加到含有50μl 200mM底物的平板中。37℃孵育20分钟，读取325nm/393nm数值。
细胞水平ACE2酶活性检测：将Vero E6细胞以每孔1×105个细胞铺在96孔板中，37℃过夜。弃上清，用PBS洗细胞，再加入浓度为20μg/ml的AF933/3E8/isotype，每孔100μl，37℃孵育1小时；弃去板中液体，加50μl活性缓冲液和50μl 200mM底物，于37℃孵育20分钟，读取325nm/393nm数值。
稀释缓冲液和活性缓冲液的配制如下：稀释缓冲液：100mM甘氨酸，50μMZnCl2、150mM NaCl，1％BSA；活性缓冲液：75mM Tris，10μM ZnCl2、150mM NaCl，0.01％TritonX-100，pH7.2。
实验结果如图5所示，从图5可以看出，3E8在高浓度下(分子水平100μg/ml；细胞水平20μg/ml)，均未对ACE2的酶催化活性产生影响，安全性很好。其中AF933作为阳性对照，能够显著抑制ACE2的酶活。
实验实施例6特异性抗体3E8结合ACE2的结构基础。
利用冷冻电镜法对3E8与ACE2的结合位点进行分析和数据处理，最终发现3E8的CDR2与ACE2的H34、K31、E23和Q18作用，3E8的CDR3与ACE2的Q24、Y83和F28作用，从而阻断了SARS-CoV-2、SARS-CoV-2和HCoV-NL63与ACE2的结合(参见图6)，其中ACE2上H34位点起着关键作用，能够同时抑制多种冠状病毒与ACE2的结合。
人ACE2氨基酸序列为SEQ ID NO:19。
序列表
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<120> 针对多种冠状病毒的单克隆抗体及应用
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<212> PRT
<213> 人工序列
<220>
<223> 轻链
<400> 14
Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly
1 5 10 15
Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Arg Asp Tyr
20 25 30
Leu Tyr Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro
65 70 75 80
Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200
<210> 15
<211> 363
<212> DNA
<213> 人工序列
<220>
<223> 重链可变区核苷酸序列 (原始)
<400> 15
gaggtgcagc tggaggagtc tggggctgag ctggtgaggc ctggggcctc agtgaagatt 60
tcctgcaagg cttttggcta caccttcaca aaccatcata taaactggat gaaacagagg 120
cctggacagg gcctggactg gattggatat gttaatcctt ataatgatta tactaagtac 180
agccagaact tcaagggcaa ggccacattg tctgtagaca gatcctccag cacagcctat 240
atggagctta gcagcctgac atctgaggac tctgcagtct attactgtgc aagatggagg 300
gattacgaca gggactggta cttcgatgtc tggggcgcag ggaccacggt catcgtctcc 360
tca 363
<210> 16
<211> 324
<212> DNA
<213> 人工序列
<220>
<223> 轻链可变区核苷酸序列 (原始)
<400> 16
gacattgtga tgacccagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60
ctttcctgca gggccagcca gagtattagg gactatttat attggtatca acaaaaatca 120
catgagtctc caaggcttct catcaaatat gcctcccaat ccatctctgg gatcccctcc 180
aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct 240
gaagatgttg gagtgtatta ctgtcaaaat ggtcacagct ttccgtacac gttcggaggg 300
gggaccaagc tggaaataaa acgg 324
<210> 17
<211> 360
<212> DNA
<213> 人工序列
<220>
<223> 重链可变区核苷酸序列 (优化)
<400> 17
gaggtgcagc tggaagagag cggcgccgag ctggtgcggc ctggagccag cgttaagatc 60
agctgtaaag cctttggata tacattcacc aatcaccaca tcaactggat gaagcaaaga 120
cctggccagg gcctggattg gatcggctac gtgaacccct acaacgacta caccaagtac 180
agccagaact tcaagggcaa ggctacactg agcgtcgaca gaagcagctc tacagcctac 240
atggaactgt cttctctgac cagcgaggac agcgccgtgt actactgcgc cagatggcgg 300
gattacgaca gagattggta cttcgacgtg tggggcgctg gcaccaccgt gatcgtgtcc 360
<210> 18
<211> 324
<212> DNA
<213> 人工序列
<220>
<223> 轻链可变区核苷酸序列 (优化)
<400> 18
gatatcgtga tgacacagag ccctgctaca ctgtccgtca cccctggaga tagagtgtcc 60
ctgtcttgta gagccagcca gagcatccgg gactacctgt actggtatca gcagaaaagc 120
cacgagagcc ccagactgct gatcaagtac gcctctcaat ctatcagcgg catccccagc 180
agattcagcg gctccggctc tggcagcgac ttcaccctga gcattaacag cgtggaacct 240
gaggacgtgg gcgtgtacta ctgccagaac ggccacagct ttccatacac cttcggcgga 300
ggcaccaagc tggaaatcaa gcgg 324
<210> 19
<211> 805
<212> PRT
<213> 人工序列
<220>
<223> ACE2
<400> 19
Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe
20 25 30
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
35 40 45
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
50 55 60
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
65 70 75 80
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
85 90 95
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
100 105 110
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
115 120 125
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
130 135 140
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
145 150 155 160
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
165 170 175
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
180 185 190
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
195 200 205
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
210 215 220
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
225 230 235 240
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
260 265 270
Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
275 280 285
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
290 295 300
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
305 310 315 320
Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro
325 330 335
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
340 345 350
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
355 360 365
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
370 375 380
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
385 390 395 400
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
405 410 415
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
420 425 430
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
450 455 460
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
465 470 475 480
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
485 490 495
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
500 505 510
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
515 520 525
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
530 535 540
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
545 550 555 560
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
565 570 575
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
580 585 590
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
595 600 605
Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
610 615 620
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
625 630 635 640
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
645 650 655
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
660 665 670
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
675 680 685
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
690 695 700
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
705 710 715 720
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
725 730 735
Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val
740 745 750
Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg
755 760 765
Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile
770 775 780
Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp
785 790 795 800
Val Gln Thr Ser Phe
805
<210> 20
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> RBD-qF1
<400> 20
caatggttta acaggcacag g 21
<210> 21
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> RBD-qR1
<400> 21
ctcaagtgtc tgtggatcac g 21
<210> 22
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 探针
<400> 22
acagcatcag tagtgtcagc aatgtctc 28

Claims (17)

1.一种抗体或其抗原结合片段，其与氨基酸序列为SEQ ID NO:19的人ACE2在结合位点结合，所述结合位点选自SEQ ID NO:19中的Q18，E23，Q24，F28，K31，H34和Y83位中的任一个或两种以上的组合，
其中，所述抗体包括重链可变区（VH）和轻链可变区（VL），其中所述VH包括序列SEQ IDNO: 1的重链可变区CDR1，序列SEQ ID NO: 2的重链可变区CDR2，以及序列SEQ ID NO: 3的重链可变区CDR3，并且所述VL包括序列SEQ ID NO: 5的轻链可变区CDR1，序列SEQ ID NO:6的轻链可变区CDR2，以及序列SEQ ID NO: 7的轻链可变区CDR3。
2. 根据权利要求1所述的抗体或其抗原结合片段，其中，所述重链可变区的氨基酸序列为SEQ ID NO:4，且所述轻链可变区的氨基酸序列为SEQ ID NO:8。
3.根据权利要求1或2所述的抗体或其抗原结合片段，其中，所述结合位点为H34。
4.根据权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体为单克隆抗体。
5.根据权利要求4所述的抗体或其抗原结合片段，其中，所述单克隆抗体是人源化抗体或全人源抗体。
6.根据权利要求4所述的抗体或其抗原结合片段，其中，所述单克隆抗体为IgG。
7.根据权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗原结合片段是scFv、Fv、Fab或F(ab)2。
8.一种双特异性抗体，其含有如权利要求1至7任一项所述的抗体或其抗原结合片段。
9.一种如权利要求1至7任一项所述的抗体或其抗原结合片段，或如权利要求8所述的双特异性抗体与一种效应分子的偶联物。
10.根据权利要求9所述的偶联物，其中，所述效应分子是可检测标记。
11.根据权利要求10所述的偶联物，其中，所述可检测标记是荧光标记、放射性标记、亲和素、生物素，或酶。
12.根据权利要求9所述的偶联物，其中，所述效应分子是毒素或化疗剂。
13.根据权利要求12所述的偶联物，其中，所述毒素是绿脓杆菌外毒素。
14.一种核酸分子，其编码如权利要求1至7任一项所述的抗体或抗原结合片段，或如权利要求8所述的双特异性抗体。
15.一种用于检测受试者的ACE2水平的试剂盒，其包括：如权利要求1至7任一项所述的抗体或其抗原结合片段、如权利要求8所述的双特异性抗体，或如权利要求9-13任一项所述的偶联物。
16.一种药用组合物，其含有有效预防剂量的如权利要求1至7任一项所述的抗体或抗原结合片段，如权利要求8所述的双特异性抗体，如权利要求9-13任一项所述的偶联物或如权利要求14所述的核酸分子，和任选地，药学上可接受载体。
17.如权利要求1至7任一项所述的抗体或其抗原结合片段，如权利要求8所述的双特异性抗体，如权利要求9-13任一项所述的偶联物，或如权利要求16所述的药用组合物在制备用于预防或治疗或诊断与ACE2相关疾病的药物中的用途，其中所述疾病为冠状病毒感染，并且所述冠状病毒选自SARS-CoV-2、SARS-CoV-2-G614、SARS-CoV和HCoV-NL63。
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