CN113943367B - 针对新型冠状病毒的单域抗体、试剂盒和药物 - Google Patents
针对新型冠状病毒的单域抗体、试剂盒和药物 Download PDFInfo
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Abstract
本发明公开了针对新型冠状病毒的单域抗体、试剂盒和药物，涉及抗体技术领域。本发明公开的单域抗体其具有互补决定区CDR1、CDR2和CDR3，该抗体能够结合新型冠状病毒的重组刺突S蛋白，针对新型冠状病毒具有较好的中和活性，可以用于检测新型冠状病毒以及用于预防或治疗由新型冠状病毒引起的相关疾病。
Description
技术领域
本发明涉及抗体技术领域，具体而言，涉及针对新型冠状病毒的单域抗体、试剂盒和药物。
背景技术
新型冠状病毒SARS-Cov-2能够引起严重的急性呼吸系统综合征。SARS-CoV-2与2002年出现的SARS-CoV均属于Sarbecovirus亚类(Betacoronavirus属，Coronaviridae家族)。这两种病毒都可以越过动物种属感染人类，并导致威胁生命的人类呼吸道疾病。目前，尚无批准可用于COVID-19治疗的靶向药物。研究证实，具有中和活性单克隆抗体靶向病毒表面蛋白是控制病毒传染病的有效治疗手段。冠状病毒中和抗体主要靶向病毒表面的三聚体刺突(S)糖蛋白，该糖蛋白的主要功能是介导病毒进入宿主细胞。新型冠状病毒SARS-Cov-2的S蛋白具有介导细胞附着的两个功能亚基(S1亚基，存在四个核心结构域S1A至S1D)以及病毒和细胞膜融合(S2亚基)。具有中和活性的单克隆抗体通常靶向S1中的受体相互作用位点，从而使病毒S蛋白与其细胞膜受体相互作用失效，阻止病毒在人体内的繁殖与复制。
但目前，缺乏针对新型冠状病毒的抗体。
鉴于此，特提出本发明。
发明内容
本发明的目的在于提供针对新型冠状病毒的单域抗体、试剂和试剂盒。本发明了一种新的单域抗体，其能够结合新型冠状病毒的重组刺突S蛋白，针对新型冠状病毒具有较好的中和活性，可以用于检测新型冠状病毒以及用于预防或治疗由新型冠状病毒引起的相关疾病。
本发明是这样实现的：
一方面，本发明提供一种针对新型冠状病毒的单域抗体，其具有互补决定区CDR1、CDR2和CDR3；
其中，CDR1的氨基酸序列如SEQ ID NO.1-13中任意一者所示；CDR2的氨基酸序列如SEQ ID NO.14-25中任意一者所示；CDR3的氨基酸序列如SEQ ID NO.26-34中任意一者所示。
具有上述的互补决定区的单域抗体能够结合新型冠状病毒的重组刺突S蛋白，本发明的提供的抗体针对新型冠状病毒具有较好的中和活性，可以用于检测新型冠状病毒以及用于预防或治疗由新型冠状病毒引起的相关疾病。
可选地，在本发明的一些实施方案中，所述单域抗体的各互补决定区的氨基酸如下表中(1)-(17)中的任意一项所示：
| CDR1 | CDR2 | CDR3 | |
| (1) | SEQ ID NO.1 | SEQ ID NO.14 | SEQ ID NO.26 |
| (2) | SEQ ID NO.2 | SEQ ID NO.15 | SEQ ID NO.26 |
| (3) | SEQ ID NO.3 | SEQ ID NO.16 | SEQ ID NO.26 |
| (4) | SEQ ID NO.4 | SEQ ID NO.17 | SEQ ID NO.26 |
| (5) | SEQ ID NO.5 | SEQ ID NO.18 | SEQ ID NO.26 |
| (6) | SEQ ID NO.4 | SEQ ID NO.19 | SEQ ID NO.27 |
| (7) | SEQ ID NO.6 | SEQ ID NO.19 | SEQ ID NO.26 |
| (8) | SEQ ID NO.7 | SEQ ID NO.20 | SEQ ID NO.28 |
| (9) | SEQ ID NO.8 | SEQ ID NO.21 | SEQ ID NO.29 |
| (10) | SEQ ID NO.9 | SEQ ID NO.22 | SEQ ID NO.30 |
| (11) | SEQ ID NO.10 | SEQ ID NO.23 | SEQ ID NO.26 |
| (12) | SEQ ID NO.4 | SEQ ID NO.19 | SEQ ID NO.31 |
| (13) | SEQ ID NO.8 | SEQ ID NO.19 | SEQ ID NO.31 |
| (14) | SEQ ID NO.11 | SEQ ID NO.24 | SEQ ID NO.32 |
| (15) | SEQ ID NO.12 | SEQ ID NO.24 | SEQ ID NO.33 |
| (16) | SEQ ID NO.12 | SEQ ID NO.24 | SEQ ID NO.32 |
| (17) | SEQ ID NO.13 | SEQ ID NO.25 | SEQ ID NO.34 |
。
可选地，在本发明的一些实施方案中，所述单域抗体的氨基酸序列如SEQ IDNO.35-52中任意一者所示。
SEQ ID NO.35-52所示的单域抗体能够结合新型冠状病毒的重组刺突S蛋白，针对新型冠状病毒具有较好的中和活性，可以用于检测新型冠状病毒以及用于预防或治疗由新型冠状病毒引起的相关疾病。
另一方面，本发明提供一种针对新型冠状病毒的传统抗体或其功能性片段，所述传统抗体或其功能性片段的重链可变区由如上任一项所述的单域抗体组成。
传统抗体由两条相同的重链和两条相同的轻链组成，呈“Y”字型结构，其轻链具有轻链可变区(VL)和轻链恒定区(CL)；其重链具有重链可变区(VH)和重链恒定区(CH)。本领域技术人员在本发明公开的单域抗体的CDR序列或全长序列的基础上，容易想到将该单域抗体的CDR序列组装到传统抗体的重链可变区尤其是重链上的互补决定区中，以使改造后的传统抗体也能够结合新型冠状病毒的S蛋白；基于此，使用本发明上述的单域抗体的CDR区或全长序列改造所得到的传统抗体或该传统抗体的功能性片段也是属于本发明的保护范围。
其中，功能性片段包括但不限于选自传统抗体的Fab、Fab’、(Fab’)2、Fv、scFv或sdFv等具有与S蛋白结合特异性的结构。
另一方面，本发明提供一种融合蛋白，其包括如上任一项所述的单域抗体或如上所述的传统抗体或其功能性片段。
本发明提供上述单域抗体或传统抗体或其功能性片段可以与其他的功能性蛋白、多肽、或酶等融合以形成融合蛋白。该功能性蛋白、多肽、或酶可以根据使用的目的或需要合理选择。
例如，可以融合荧光蛋白(例如绿色荧光蛋白EGFG、AcGFP、TurboGFP、Emerald、Azani Green和ZsGreen等，例如蓝色荧光蛋白EBFP、Sapphire和T-Sapphire等，再如青色荧光蛋白ECFP、mCFP、Cerulean、CyPet、AmCyan1、Midori-Ishi Cyan和mTFP1(Teal)等，又如橙色和红色荧光蛋白Kusabira Orange、mOrange、dTomato、dTomato-Tandem、DsRed、DsRed2、DsRed-Express(T1)、DSRed-Monomer、mTangerine、mStrawberry、AsRed2、mRFP1、Jred、mCherry、HcRed1、mRaspberry、HcRed-Tandem、mPlum和AQ143等)便于检测等。例如，还可以融合Fc段便于纯化；例如，融合催化底物显色的酶(碱性磷酸酶)便于观察等。无论以何种功能性蛋白、多肽、或酶与本发明的单域抗体或传统抗体或其功能性片段融合，所得到融合蛋白均属于本发明的保护范围。
另一方面，本发明提供一种缀合物，其包括如上任一项所述的单域抗体、或如上所述的传统抗体或其功能性片段。
本发明提供的单域抗体、或传统抗体或其功能性片段可以与多种功能物质缀合，形成抗体缀合物，例如可以与对新型冠状病毒具有抑制活性的药物缀合以增强抗病毒作用。本领域技术人员可以根据需要，选择合适的物质(例如稳定抗体结构的物质，延长半衰期的物质等)与本发明的单域抗体、或传统抗体或其功能性片段缀合形成缀合物，任何缀合的物质都属于本发明的保护范围。
另一方面，本发明提供一种检测新型冠状病毒的试剂或试剂盒，其包括如上任一项所述的单域抗体、或如上所述的传统抗体或其功能性片段、或如上所述的融合蛋白、或如上所述的缀合物。
另一方面，本发明提供一种预防或治疗由新型冠状病毒引起的疾病的药物，其包括如上任一项所述的单域抗体、或如上所述的传统抗体或其功能性片段、或如上所述的融合蛋白、或如上所述的缀合物。
另一方面，本发明提供分离的核酸分子，其编码如上任一项所述的单域抗体、或如上所述的传统抗体或其功能性片段。
另一方面，本发明提供一种含有如上所述的核酸分子的载体或重组细胞。
另一方面，本发明提供一种制备如上任一项所述的单域抗体、或如上所述的传统抗体或其功能性片段的方法，其包括：培养如上所述的重组细胞，从培养产物中分离纯化得到所述单域抗体或传统抗体或其功能性片段。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明实施例中的各单域抗体与刺突S蛋白的ELISA结合验证结果。
图2为本发明实施例中表1中的单域抗体(1)-(6)与膜表达刺突S蛋白的FACS结合验证结果。
图3为本发明实施例中表1中的单域抗体(7)-(12)与膜表达刺突S蛋白的FACS结合验证结果。
图4为本发明实施例中表1中的单域抗体(13)-(18)与膜表达刺突S蛋白的FACS结合验证结果。
图5为本发明实施例中的各单域抗体对假病毒的中和活性检测结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
单域抗体的筛选
1重组刺突S蛋白制备
根据NCBI及文献报道的新冠病毒SARS-Cov-2的重组刺突S蛋白的编码区序列信息(GenBank:MN908947)，选择其胞外结构域的1AA-1208AA,在其C端添加human IgG1FC(CH2-CH3)标签，根据CHO细胞的密码子偏好性进行密码子优化，并进行全基因合成，将合成后的基因亚克隆至真核表达载体pcDNA3.4中，经Sanger测序验证插入的目的S蛋白核酸序列后，使用Qiagen质粒大抽试剂盒，提取重组质粒，采用PEI瞬时转染至CHO-S无血清悬浮培养细胞中，连续培养5天后，12000g、4℃离心15分钟，去除细胞沉淀，收集上清，使用Protein A柱亲和纯化重组S蛋白，并进行SDS-PAGE凝胶电泳，鉴定目标蛋白的纯度后，用于后续的羊驼免疫。
2重组HEK293T刺突S蛋白过表达细胞株构建
根据NCBI及文献报道的新冠病毒SARS-Cov-2的重组刺突S蛋白的编码区全长序列(GenBank:MN908947)，根据HEK293细胞密码子偏好性进行密码子优化后，合成S蛋白全长核酸序列，并亚克隆至慢病毒表达载体Lenti-CMV-Puro中，经Sanger测序验证无误后，使用Qiagen质粒大抽试剂盒进行质粒抽提，并包装慢病毒，感染HEK293T细胞后，加入嘌呤霉素筛选7天，获得稳定过表达刺突S蛋白的重组HEK293T细胞，用于后续的S蛋白单域抗体筛选。
3羊驼免疫
选一只3岁龄的羊驼，将上述制备的重组S蛋白与佐剂混匀后，进行皮下免疫，共免疫4次，每次免疫剂量为500ug重组S蛋白，免疫时间间隔为14天；第三次免疫结束后，通过颈静脉采集5mL外周血，分离血清后，采用ELISA检测免疫效价。
4单域抗体噬菌体展示库构建
(1)通过颈静脉采集羊驼外周血，取出淋巴细胞分离液，向一新的50mL离心管中加入15mL淋巴细胞分离液，然后使用移液器小心将血样沿管壁缓慢添加至淋巴细胞分离试剂的上层，800g、20℃，离心20分钟，用移液器将处于中间的白色膜层吸至一个新的无菌50mL离心管中，加入PBS至50mL，旋紧离心管盖，上下颠倒混匀，800g，离心10分钟，离心结束后，弃去上清，用RNAiso plus吹打裂解细胞。加入200μL氯仿/1mL RNAiso plus裂解液，盖紧离心管盖，混合至溶液乳化呈乳白色，12,000g、4℃离心15分钟。从离心机中小心取出离心管，小心吸取上清液转移至另一新的离心管中；向上清中加入500μL异丙醇/管，上下颠倒离心管充分混匀后，12,000g、4℃离心10分钟；小心弃去上清,加入1mL/管75％乙醇，轻轻上下颠倒洗涤离心管管壁，然后将样品置于离心机中，7,500g、4℃离心5分钟，小心弃去上清。
(2)cDNA制备
1)预热PCR仪，设置为65℃保温，体系100μL。
2)在200μL Microtube中配制下列反应混合液MIX1
| 试剂 | 用量 | 加样顺序 |
| Oligo dT Primer(50μM) | 8μL | 4 |
| dNTP Mixture(10mM each) | 8μL | 3 |
| 总RNA样品 | 20μg | 2 |
| RNase-Free水 | Up to 80μL | 1 |
3)65℃保温5min后，冰上迅速冷却。
4)在上述Microtube管中配制下列反转录反应液MIX2
5)用移液器上下吹打混匀后，分装成80μL/管，置于PCR仪中运行下列程序：
6)42℃反转录1小时，70℃热失活15分钟，4℃保温。
7)最后将cDNA样品置于冰上或-20℃长期保存。进行下游PCR时，每25μL PCR反应体系，使用1μL cDNA样品作为模板。
(3)VHH单域抗体PCR克隆
1)配置第一轮PCR反应体系
| 成分 | 用量 |
| 上游引物(5μM) | 16 |
| 下游引物(10μM) | 8 |
| NuHi Power mix(2×) | 200 |
| cDNA模板 | 16 |
| 无菌水 | 160 |
将配置好的PCR混合物置于PCR仪上运行以下程序：
2)PCR产物的琼脂糖电泳
取PCR产物，加入1/10体积10×loading buffer，使用1％的琼脂糖进行电泳分析，从胶上切取700bp目的条带，用Qiagen胶回收试剂盒回收PCR产物，并用NanoDrop测定浓度，至少需要2-4μg PCR产物。
3)配置二轮PCR反应体系
| 成分 | 用量 |
| 2nd F引物mix(4.5μM each) | 28 |
| 2nd R引物mix(5μM each) | 28 |
| NuHi Power mix(2×) | 350 |
| 一轮PCR回收产物 | 2800ng |
| 无菌水 | 补至700μL |
将配置好的PCR混合物分装成50μL/管，置于PCR仪上运行以下程序：
4)二轮PCR产物的琼脂糖电泳分析
取PCR产物，加入1/10体积10×loading buffer，使用1％的琼脂糖进行电泳分析，从胶上切下350bp目的条带，用Qiagen胶回收试剂盒纯化PCR产物，并用NanoDrop测定浓度。
(4)酶切VHH产物及与pCANTab5f载体的连接
1)SfiI酶切pCANTab5f载体
pCANTab5f载体的酶切体系如下：
将上述酶切体系置于PCR仪上50℃酶切过夜；
将酶切产物加入1/10体积的10×loading buffer，用1％的琼脂糖凝胶分离载体片段，从胶上切下目的载体片段，并用Qiagen胶回收试剂盒进行回收。
2)SfiI酶切单域抗体VHH PCR的产物。
酶切体系如下：
将上述酶切体系置于PCR仪上50℃酶切过夜；
使用TakaRa的DNA片段回收试剂盒纯化PCR酶切产物，具体参照供应商说明书。
3)载体和片段的连接
将酶切好的载体和片段按照1:3的摩尔比进行混合，在T4DNA ligase的作用下进行连接反应。
连接体系如下：
| 成分 | 用量 |
| pCANTab5f(SfiI-digested) | 2μg |
| 第二轮PCR产物(SfiI-digested) | 235ng |
| 10×T4buffer | 20μL |
| T4ligase | 10μL |
| 无菌水 | 补足至200μL |
将上述连接产物置于PCR仪上16℃连接过夜后，将连接产物合并为一管，每管200μL，加入22μL的3M醋酸钠，2μg/μL糖原(Glycogen)，充分混匀后，再加入888μL的无水乙醇，充分混匀后，-80℃放置2小时或过夜。将沉淀的连接产物置于离心机中，12,000g、4℃离心15分钟，去掉上清。在沉淀管中加入1mL 70％的乙醇后，轻轻上下颠倒数次。将产物再次置于离心机中12,000g离心10分钟，小心去掉上清；加入15μL无菌水溶解DNA沉淀。
(5)电转化获得大肠杆菌库
1)将电转杯、连接产物置于冰上10分钟；同时在冰上解冻电转感受态300μL。
2)取300μL电转感受态加入到15μL连接产物中，快速吹打3次，混匀感受态和连接产物，并转移到电击杯中，置于冰上1分钟。
3)将电击杯置于BioRad电转仪上，用电转程序2500V，200Ω，25μF，5ms；进行电击。
4)电击结束后，立刻用2mL预热至37℃的SOC培养基冲洗电转杯，并将菌液转移至一个无菌离心管中，37℃恢复培养1小时。
5)向菌液中加入终浓度为100μg/mL的氨苄青霉素，37℃选择培养4h，将选择培养后的电转产物离心后浓缩，加入等体积的50％甘油，均匀混合后保存在-80℃。
(6)噬菌体展示库的复苏和拯救
1)将选择培养后的转化产物50mL(总体积为100mL)，置于恒温摇床中，37℃，225rpm培养，至OD600为0.5，投入辅助噬菌体M13KO7，轻轻摇晃混匀噬菌体和菌液混合物，置于37℃静置侵染30min；
2)将浸染结束的混合物置于恒温摇床中，37℃，225rpm培养1h；
3)将菌液转移至无菌离心瓶中，8000rpm离心10min，弃去上清，菌体用200mL 2YT-AK培养基重悬，重置于摇床中，25℃，225rpm培养过夜；
4)将扩增过夜的噬菌体库菌液转移至200mL离心瓶中，10000rpm，4℃，离心10min；
5)将上清按10mL PEG-NaCl/40mL，混匀后放置冰上，静置1h；
6)将冰上沉淀的噬菌体上清置于离心机中，3900rpm，4℃，离心30min；弃去上清，每个50ml离心管的沉淀(噬菌体)用1ml无菌PBS重悬；
7)将重悬的噬菌体转移至1.5mLEP管中，置于离心机中，12000g，4℃，离心5min；
8)将1mL上清转移至新的1.5ml EP管，每管加入250μl的PEG/NaCl，混匀后4℃静置10min；
9)将沉淀后的噬菌体置于离心机中，12000g，4℃，离心10min，弃上清；
10)加入1ml PBS重悬噬菌体沉淀；12000g，4℃，离心5min，将上清转移至新的1.5ml EP管。
(7)噬菌体展示库的淘选
1)用CBS稀释目标抗原S蛋白至50μg/ml，150μl/well，共包被3well，同时用CBS包被对照蛋白共6孔，用1％PBSA包被1个1.5mlEP管，置于4℃过夜；
2)去掉包被孔内的蛋白，用3％MPBS室温封闭1h，每孔加200μL的MPBS；
3)将噬菌体库150μL，用300μl 1％PBSA稀释，将封闭的三个对照蛋白孔中的MPBS吸去，每孔投入150μL的稀释好的噬菌体，室温孵育0.5h；
4)将对照蛋白孔中的噬菌体转至包被S蛋白的孔中，室温孵育1h；
5)将包被S蛋白孔中的噬菌体吸去，用0.05％PBST洗涤10次，每次2-3min，再用PBS洗涤5次，每次2-3min，同时用PBS洗涤事先封闭的1.5ml EP管；
6)配置1×TEA，每孔200μL，用1xTEA洗脱5min，收集洗脱下来的产物至事先封闭过的EP管中，再加入300μL pH7.6、1M Tris-HCl中和；
7)取10μl上述洗脱的噬菌体产物加入至90μl 2YT培养基，记做100，依次往后10倍稀释至10-2，取10-1、10-2两个梯度各20μL稀释好的样品投入200μL事先准备好的OD600为0.5的ER2738，混匀后置于37℃水浴中，静置侵染10min，每108μl涂1块LB-AMP平板，37℃倒置培养过夜，第二天计算克隆数，以确定获取到的淘选后噬菌体滴度。
(8)淘选噬菌体的扩增
1)向事先准备的20mL OD600＝0.5的ER2738菌中，投入一半体积的噬菌体淘选产物，混匀后37℃水浴30min；
2)将20mL的2YT培养基加入20mL混有淘选后噬菌体的菌液中，37℃，225rpm培养30min；
3)当菌液的OD600约为0.5时，投入M13KO7，摇匀后37℃静置30min，然后再按1：1000比例加入AMP，37℃，225rpm培养45min；
4)将菌液3900rpm，离心20min，弃去上清，用40ml 2YT-AK培养基重悬，30℃，210rpm过夜培养后，进行噬菌体沉淀。
重复噬菌体库淘选步骤4次，富集最终需要的单克隆噬菌体。
(9)单克隆的噬菌体ELISA
1).分装2YT-A培养基至96孔深孔板，每孔200μL，用灭菌牙签挑取噬菌体淘选平板上的单克隆，37℃，225rpm培养过夜；
2).同时用pH9.6的CBS包被抗原至ELISA板，抗原包被浓度为1μg/ml，100μL/well，4℃，包被过夜；
3).第二天，取一块新的96深孔板，分装2YT-A培养基，每孔150μL，用排枪依次吸取过夜培养的菌液20μl至新分装的96孔板中，37℃，225rpm培养至OD600约为0.5(约需要1.5h)；
4).在OD600为0.5的菌液中，加入M13KO7，混匀后放于37℃，静置15min；
5).将侵染结束的菌液置于摇床上，37℃，225rpm培养45min；
6).将菌液置于离心机中，3900rpm离心10min,弃去上清，用2YT-AK培养基重悬，每孔500μl，重置于摇床中，30℃，220rpm过夜培养；
7).同时ELISA板甩掉抗原，用PBST洗液洗三遍后，用3％MPBS封闭250μL/well，4℃过夜；并额外封闭一块空白板，作为Negative control；
8).第三天，将96孔深孔板置于离心机中，3900rpm离心10min；上清中含有展示的噬菌体抗体颗粒，待用；
9).将ELISA板中的封闭缓冲液弃去，用200μL 0.05％PBST洗涤4次；在每孔先加入50μL的0.1％的PBST，再一一对应加入50μL离心后的噬菌体上清，置4℃孵育1h；
10).弃去上清，并用0.05％PBST洗涤5次；
11).用0.05％PBST 1：5000稀释Anti M13-HRP抗体，每孔100μL，4℃孵育45min后；
12).弃去上清，0.05％PBST洗5次，加入100μL TMB常温显色10min，50μL 0.2M盐酸终止显色反应，读取OD450的吸光值；
计算样品孔OD值/阴性对照孔OD的数值，选取S/N比大值的克隆，进行Sanger测序，获取能够与目标抗原结合的抗体序列，共18个单域抗体，各单域抗体的全长氨基酸分别如SEQ ID NO.35所示，各单域抗体的CDR区序列如下表1所示：
表1
各抗体的氨基酸序列如下(下划线为CDR区)：
1>iCA20-2-17-1-H1:
MAAVQLVDSGGGLVQPGGSLTLSCSASGFFFNGYNMGWYRQAPGKQRELVATISEAGTTGYADSVKGRFTISRDNVKNTVDLHMNSLKPEDTAVYYCKRELGPFSSWGQGTQVTVSS(SEQ ID NO.35)；
2>iCA20-2-17-1-A5:
MAAVQLVDSGGGLVQPGGSLRLSCAASGFAFNIYNMGWYRQAPGKQRELVATIAADGSTGYADSVKGRFTISRDNVKNKVDLQMNSLKPEDTAVYYCKRELGPFSSWGQGTQVTVSS(SEQ ID NO.36)；
3>iCA20-2-17-1-D3:
QAQLVESGGGLVQPGGSLRLSCAASGFNFNMYAMGWYRQAPGKERELVATVAAGGSTGYADSVKGRFTISRDNVKMKVDLQMNSLKLEDTAVYYCKRELGPFSSWGQGTQVTVSS(SEQ ID NO.37)；
4>iCA20-2-17-4-1:
MAAVQLVDSGGGMVQPGGSLRLSCAASGFNFNIYAMGWYRQAPGKQRELVATIAAGGNTGYADSVKGRFTISRDNVKNKVNLQMNNLKPEDTAVYYCKRELGPFSSWGQGTQVTVSS(SEQ ID NO.38)；
5>iCA20-2-17-4-2:
QVQLVESGGGLVQPGGSLRLSCAASGFIFKMYAMGWYRQAPGLQRESVASIAQDGSASYADSVKGRFTISRDNVKNTVDLQMNSLKPEDTAHYYCKRELGPFSSWGQGTQVTVSS(SEQ ID NO.39)；
6>iCA20-2-17-4-11:
AVQLVESGGGLVQPGGSLRLSCAASGFNFNIYAMGWYRQAPGKQRELLATIAAGGSTNYADSVKGRFTISRDNIKNKVDLQMNSLKPEDTAVYYCKRELGSFSSWGQGTQVTVSS(SEQ ID NO.40)；
7>iCA20-2-17-4-14:
MAQVKLEESGGGLVQPGGSLRVSCAASGFNFNIYNMGWYRQAPGKQRELVATIAAGGSTGYADPVKGRFAISRDNVKNTVDLQMNSLKPEDTAVYYCKRELGPFSSWGQGTQVTVSS(SEQ ID NO.41)；
8>iCA20-2-17-4-32:
QVQLVESGGGLVQPGGSLRLSCAASGFVFNMYAMGWYRQAPGKQRELVATIAKDGSTGYAISVKGRFTISRDNVKNTVDLQMNSLKPEDTAVYYCKRELGPYGSWGQGTQVTVSS(SEQ ID NO.42)；
9>iCA20-2-17-4-39:
AVQLVESGGGLVQPGGSLRLSCAASGFNFNFYAMGWYRQAPGKQRELVATIAADGSAAYADSVKGRFTISRHNEKNTVDLQMNSLKPEDTGVYYCKRELGPFGSWGQGTQVTVSS(SEQ ID NO.43)；
10>iCA20-2-17-4-43:
AVQLVDSGGGLVQPGGSLRLSCAASGFGFNMYAMGWYRQAPGKQRELVATIARDGSTGYAISVKGRFTISRDNVKNTVDLQMNSLKPEDTAVYYCKRELGPYASWGQGTQVTVSS(SEQ ID NO.44)；
11>iCA20-2-17-4-46:
QAQLVESGGGLVQPGGSLRLSCAASGFLFKMYAMGWYRQAPGKQRELVASIANDGSTGYGDSVKGRFIISRDNVKNTVDLQMNSLKPEDTAHYYCKRELGPFSSWGQGTQVTVST(SEQ ID NO.45)；
12>iCA20-2-17-4-49:
AVQLVESGGGLVQPGGSLRLSCAASGFNFNIYAMGWYRQAPGKQRELVATIAAGGSTGYADSVKGRFTISRDNVKNKVDLQMNSLKPEDTAVYYCKRELGPFASWGQGTQVTVSS(SEQ ID NO.46)；
13>iCA20-2-17-4-52:
AVQLVDSGGGLVQPGGSLRLSCAASGFNFNFYAMGWYRQAPGKQRELVATIAAGGSTNYADSVKGRFTISRHNEKNTVDLQMNSLKPEDTGVYYCKRELGPFASWGQGTQVTVSS(SEQ ID NO.47)；
14>iCA20-2-17-2-15:
QAQLVEPGGSLRLSCATSKSAFAIFAMSWYRQAPGKECEWVATITITGGNSNYADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAVYYCNADPGCPLGQGTQVTVSS(SEQ ID NO.48)；
15>iCA20-2-17-2-19:
MAQVQLVESGGGLVQPGGSLTLSCSASGFFFNGYNMGWYRQAPGKQRELVATISEAGTTGYADSVKGRFTISRDNVKNTVDLHMNSLKPEDTAVYYCKRELGPFSSWGQGTQVTVSS(SEQ ID NO.49)；
16>iCA20-2-17-2-32:
EVQLVESGGGLVQPGGSLTLSCAASGFTFSSYAMAWYRQAPGKECEWVATITITGGNSNYADSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCNADPSCPLGQGTQVTVSS(SEQ ID NO.50)；
17>iCA20-2-17-2-56:
QVQLVESGGGSVRAGESLRLSCAASGFTFSSYAMGWYRQAPGKECEWVATITITGGNSNYADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAVYYCNADPGCPLGQGTQVTVSS(SEQ ID NO.51)；
18>iCA20-2-17-2-69:
MAQVKLEESGGGLVQPGGSLRLSCGASGLTVSSGAFSWYRQTPGKERELVAAISSGGGTRSYGASVKGRFTISRDNAKNTVYLQMNSLQPEDTAVYLCYAARSWGGDYWGQGTQVTVSS(SEQ ID NO.52)。
实施例2
重组单域抗体表达纯化
根据Phage ELISA及Sanger测序的结果，将筛选到的单域抗体编码区核酸序列进行全基因合成，并亚克隆至真核表达载体pcDNA3.4-hIgG1-Fc中，获取的重组表达载体经Sanger测序验证无误后，使用Qiagen质粒大抽试剂盒进行质粒抽提，并采用PEI瞬时转染至293F细胞中，连续表达5天后，收集培养基上清，采用protein A纯化重组VHH单域抗体，用于后续的ELISA、FACS及功能试验。
实施例3
重组单域抗体ELISA、FACS验证
1)ELISA试验
i.用CBS稀释目标抗原S蛋白至5μg/ml，100μl/well，将孔板置于4℃包被过夜。
ii.将孔内的包被缓冲液去除，加入5％的PBSA进行室温封闭1小时。
iii.将上述制备的重组单域抗体使用PBSA进行稀释至终浓度1ug/mL；将稀释后的抗体加入到封闭后的孔板内，每孔加入100uL，室温孵育30分钟。
iv.使用PBST缓冲液洗涤3次，每次5分钟。
v.加入稀释后的HRP-Anti-Human IgG1Fc二抗，室温孵育30分钟。
vi.使用PBST缓冲液洗涤3次，每次5分钟。
vii.加入100μL TMB常温显色10min，50μL 0.2M盐酸终止显色反应，读取OD450的吸光值。
结果见图1，结果表明，从实施例1筛选到的各单域抗体均能够特异性的与刺突S蛋白结合。
2)流式细胞试验
i.取过表达S蛋白全长的重组HEK293T细胞，将细胞密度调整至1*10^6个/mL；
ii.向细胞悬液中加入重组单域抗体至终浓度为1ug/mL，室温孵育30分钟；
iii.500g室温离心5分钟，去除上清；使用PBS洗涤细胞3次；
iv.向细胞悬液中加入1:5000稀释的PE-Anti-human IgG二抗，室温避光孵育30分钟；
v.500g室温离心5分钟，去除上清；使用PBS洗涤细胞3次；最终用500uL PBS缓冲液重悬细胞沉淀；
vi.使用流式细胞仪分析单域抗体与细胞膜上S蛋白的结合情况。
结果见图2-4，结果表明，从实施例1筛选到的单域抗体均能够特异性的与细胞膜表达的刺突S蛋白结合。
实施例4
单域抗体中和活性实验
使用HEK293T细胞制备SARS-Cov-2假病毒，将HEK293T细胞接种至培养皿中，待细胞汇合度达到60％左右时，使用携带gag-pol基因、荧光素酶基因、S蛋白的质粒进行瞬时转染，48小时后，收集培养基上清，使用0.45微米的滤膜进行过滤，去掉细胞碎片，并将病毒小体积分装后置于-80度冰箱暂存。
复苏VeroE6细胞，并接种至24孔板中。将实施例1得到的单域抗体进行4倍梯度稀释后，加入相同体积假病毒室温孵育1小时，然后将抗体及假病毒的复合物加入VeroE6细胞中，置于37度、5％CO2培养箱中继续培养24小时；同时设置未加单域抗体的对照组；收集细胞，加入荧光素酶活性检测底物OneGlo试剂，使用Tecan M1000Pro读取荧光素酶活性数值。以未加单域抗体的组作为100％荧光素酶活性值，计算单域抗体对假病毒侵染靶细胞的影响。
结果见图5，实施例1筛选的单域抗体能够有效的抑制假病毒对于靶细胞的侵染，表现为随着抗体浓度的增加，假病毒对靶细胞的侵染效率逐步降低，表明筛选到的各单域抗体对于假病毒均具有抑制作用。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 宁波安诺柏德生物医药科技有限公司
<120> 针对新型冠状病毒的单域抗体、试剂盒和药物
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Gln Ala Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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115
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115
<210> 47
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Ala Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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115
<210> 48
<211> 107
<212> PRT
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Gln Ala Gln Leu Val Glu Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
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<210> 49
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Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
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115
<210> 50
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
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Ala Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Cys Glu Trp Val
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Asn Ala Asp Pro Ser Cys Pro Leu Gly Gln Gly Thr Gln Val Thr Val
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Ser Ser
<210> 51
<211> 114
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<400> 51
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Arg Ala Gly Glu
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
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Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Cys Glu Trp Val
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Ala Thr Ile Thr Ile Thr Gly Gly Asn Ser Asn Tyr Ala Asp Ser Val
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Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys
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Asn Ala Asp Pro Gly Cys Pro Leu Gly Gln Gly Thr Gln Val Thr Val
100 105 110
Ser Ser
Claims (6)
1.一种针对新型冠状病毒的单域抗体，其特征在于，其具有互补决定区CDR1、CDR2和CDR3；
其中，
所述单域抗体的各互补决定区的氨基酸如下表中（2）、（4）、（5）和（7）中的任意一项所示：
。
2.根据权利要求1所述的单域抗体，其特征在于，所述单域抗体的氨基酸序列如SEQ IDNO.36、SEQ ID NO.38、SEQ ID NO.39和SEQ ID NO.41中任意一者所示。
3.一种检测新型冠状病毒的试剂或试剂盒，其特征在于，其包括权利要求1-2任一项所述的单域抗体。
4.一种预防或治疗由新型冠状病毒引起的疾病的药物，其特征在于，其包括权利要求1-2任一项所述的单域抗体。
5.分离的核酸分子，其特征在于，其编码如权利要求1-2任一项所述的单域抗体。
6.含有权利要求5所述的核酸分子的载体或重组细胞。
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| CN111303279A (zh) * | 2020-03-17 | 2020-06-19 | 中国医学科学院病原生物学研究所 | 一种针对新型冠状病毒的单域抗体及其应用 |
| CN111303280A (zh) * | 2020-03-22 | 2020-06-19 | 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 |
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| CN111269313A (zh) * | 2020-03-07 | 2020-06-12 | 北京岳昊科技发展有限公司 | 一种用于检测新型冠状病毒的单克隆抗体及其制备试剂盒的用途 |
| CN111303279A (zh) * | 2020-03-17 | 2020-06-19 | 中国医学科学院病原生物学研究所 | 一种针对新型冠状病毒的单域抗体及其应用 |
| CN111303280A (zh) * | 2020-03-22 | 2020-06-19 | 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 |
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