CN111978395B - 抗新型冠状病毒rbd结构域抗原的单克隆抗体 - Google Patents

抗新型冠状病毒rbd结构域抗原的单克隆抗体 Download PDF

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CN111978395B
CN111978395B CN202010701787.XA CN202010701787A CN111978395B CN 111978395 B CN111978395 B CN 111978395B CN 202010701787 A CN202010701787 A CN 202010701787A CN 111978395 B CN111978395 B CN 111978395B
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唐麟
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Abstract

本发明公开了一种抗新型冠状病毒RBD结构域抗原的单克隆抗体，属于生物制品领域。本发明的单克隆抗体，其重链可变区氨基酸序列如SEQ ID NO.1～17任一所示，轻链可变区氨基酸序列如SEQ ID NO.18～34任一所示。本发明的单克隆抗体，具有良好的阻断新型冠状病毒RBD与其受体结合的能力，并经实验验证，可以有效阻止病毒感染细胞。

Description

抗新型冠状病毒RBD结构域抗原的单克隆抗体
技术领域
本发明属于生物制品领域。
背景技术
新型冠状病毒(SARS-CoV2)感染后引发的肺炎是当下全球共同面临的重大公共卫生问题。截止2020年6月24日，全球累计确诊病例超过920万例；虽然在全球范围内我国的疫情防控相对较好，但在全球疫情蔓延的大趋势背景下，关口前移工作与科研攻关迫在眉睫。然而截至目前，SARS-CoV2病毒引发的肺炎尚无特效药物，目前的研究已经证实，新型冠状病毒表面的刺突糖蛋白(Spike蛋白)在三维空间构象抗原性上与SARS抗原性非常接近；基于病毒的结构研究发现SARS-CoV2病毒上的受体结合域蛋白(receptor binding domain,RBD)能够特异性的识别人血管紧张素转化酶(ACE2)，这也是新冠病毒Spike蛋白介导细胞侵染的结构基础及分子机制。
基于该研究背景，研发能够高度特异性的识别该区域的治疗性抗体将有望阻止新型冠状病毒通过RBD与人ACE2蛋白进行结合，继而阻止病毒的侵入，因此，发展针对新型冠状病毒RBD结构域抗原的治疗性抗体，将有望为新型冠状病毒的诊断及治疗提供新的策略。
抗体(免疫球蛋白)含有4条多肽链，其中2条重链，2条轻链。重链和轻链中靠近N端氨基酸序列负责识别抗原，序列多样，被称为“可变区”，分别占重链和轻链的1/4和1/2；而靠近C端氨基酸序列则相对恒定，被称为“恒定区”。在治疗性抗体的研发过程中，重点需要寻找合适的可变区序列。
目前还没有针对新型冠状病毒RBD结构域抗原的治疗性抗体面世。
发明内容
本发明要解决的问题是：提供一种抗新型冠状病毒RBD结构域抗原的单克隆抗体。
本发明的技术方案如下：
一种抗新型冠状病毒的单克隆抗体，其重链可变区和轻链可变区序列为下表所列17种情形之一：
Figure BDA0002592905740000011
Figure BDA0002592905740000021
如前述的单克隆抗体，所述单克隆抗体是IgG1抗体。其恒定区同其他IgG1抗体。
编码前述新型冠状病毒抗体的核酸。
一种重组核酸载体，所述载体包括前述的核酸。
如前述的核酸载体，所述核酸载体是真核表达载体。
一种重组细胞，所述重组细胞内含有前述的载体。
前述抗体在制备抗新型冠状病毒的药物中的用途。
一种抗新型冠状病毒的药物，其特征在于：所述药物以前述抗体为活性成分。
本发明的有益效果：
本发明的单克隆抗体具有很强的抗新型冠状病毒的能力，其效果比目前已有的针对同为冠状病毒的中东呼吸综合征病毒(MERS)筛选得到的单克隆抗体的效果更强。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1：单克隆抗体纯化后电泳图。上图中1～9依次为DK1～9；左下图中1和2分别为DK10和DK11；右下图中1～6分别为DK12～17；M为marker。
具体实施方式
实施例1:抗RBD结构域抗原的单克隆抗体制备及检测
一、RBD抗原的制备
本实验针对小鼠使用的免疫抗原为SARS-CoV2病毒表面SPIKE蛋白的RBD结构域，采用昆虫表达系统，由于其为分泌蛋白，离心后收集培养基上清，使用镍柱进行分离纯化。纯化后的蛋白质使用12.5％的SDS-PAGE胶进行定量分析(见图1)。然后将纯化得到的蛋白质分装冷冻干燥-80C保存。(避免反复冻融导致蛋白质降解)。
二、RBD抗原免疫小鼠及多抗血清效价测定
选用6周龄的雌性小鼠。在未免疫前的1周，采用断尾取血的方法在小鼠的尾部静脉采集200-400ul的血液(作为后续实验的阴性对照)；在计划免疫小鼠前，首先将免疫抗原RBD溶于PBS缓冲液中，并与弗式佐剂按照1:1的方式在涡旋仪上持续震荡，充分乳化混合；乳化后在对小鼠进行免疫，免疫计划如表1所示：
Figure BDA0002592905740000031
注:将采集的小鼠尾静脉血液放置4℃过夜，待血块完全收缩，在(1000rpm，4℃，10min)离心，收集上清，-20℃下储存备用。
随后采用间接非竞争性ELISA法测定小鼠的多抗血清效价，用昆虫表达系统制备获得的免疫抗原RBD免疫小鼠，在经过4次免疫后，，小鼠多抗血清效价均符合要求，都可用于下一步细胞融合。
三、细胞融合及杂交瘤细胞的阳性筛选
按照实验标准流程对杂交瘤细胞SP2/0进行复苏及培养、同时制备饲养细胞，随后将上述符合要求(多抗血清效价在16000倍以上)的小鼠进行脾细胞制备；
1、细胞融合的实验步骤如下：(1)用20mL不完全1640培养基将3-5瓶长满培养瓶底且生长状态良好的SP2/0细胞吹打下来，并存于50ml无菌离心管中备用。(2)随后分别对脾细胞和SP2/0细胞进行计数，使得SP2/0细胞：脾细胞的数量之比为1：10。(3)分别将50ml脾细胞及SP2/0细胞吸入无菌的BD管中离心(20℃，1500rpm，5min)，弃上清。(4)用不完全1640培养基分别重悬两种细胞，并将两者进行混合，定容至20ml，随后在20℃，1500rpm，离心5min，弃上清，用灭过菌的试纸将无菌离心管中的溶液吸取干净，然后用手指连续敲击细胞使得细胞沉淀变成糊状，同时将其放置到40℃的无菌水浴中。(5)将1ml聚乙二醇PEG4500(需预热到37℃)在一分内先慢后快的加入到细胞沉淀中，轻轻摇晃离心管。(6)加入20ml不完全1640培养基(需预热到37℃)终止PEG4500的融合反应，同样采取先缓慢滴加再快速滴加的方式(需在4min内完成)。第1min加1ml，第2min加3ml，第3min加5ml，最后1min快速定容到20ml，随后静置10min，随后在20℃，1500rpm条件下离心5min，弃上清。(7)用100mlHAT培养基将细胞沉淀轻轻悬浮混匀，将细胞悬浮液定量的分配到制备好的饲养细胞的96孔细胞培养板中(每孔100ul)，随后置于37℃培养箱中培养，第二天查看96孔细胞培养板是否出现污染。
2、融合后杂交瘤细胞的培养
融合之后3天内的细胞尽可能少地移动，在4—7天内观察96孔细胞培养板内是否有杂交瘤细胞产生，并且观察细胞数量。如果看到有少量杂交瘤细胞长出，就可以更换一定体积的HAT培养基。在2周后可用HT培养基更换HAT培养基。当杂交瘤细胞集落面积占孔底面积约三分之一时，就开始对细胞集落进行计数，并吸取培养基上清用于ELISA检测,筛选阳性细胞。基于以上操作，一共筛选得到74个阳性克隆。
四、初步筛选和测序
利用biacore技术设计了74个阳性克隆产生单克隆抗体的阻断实验，筛选到了阻断率达到60％以上17个克隆。
将17个阳性克隆进行扩大培养，分别收集培养后的细胞进行总RNA的提取，经RT-PCR技术，获得抗RBD的17个单克隆抗体的轻链可变区(VL)、重链可变区(VH)基因序列，其对应的氨基酸序列如表2所示。
Figure BDA0002592905740000051
Figure BDA0002592905740000061
Figure BDA0002592905740000071
Figure BDA0002592905740000081
对17个单抗的轻重链可变区进行PCR扩增，结果显示抗体的重链可变区和轻链可变区大小与预期相符合。
五、17个阳性单克隆抗体的真核表达与纯化
将获得的上述17个单克隆抗体基因片段的全长(包括可变区和恒定区)克隆到真核表达载体(pCDNA3.1)，构建无肉毒素的重组质粒，利用PEI转染试剂将重组质粒转染到293T细胞进行表达纯化，由于该系列抗体以分泌蛋白的方式表达，所以离心细胞后收集上清进行纯化即可。
转染的实验流程如下：
(1)转染前一天(24小时左右)用胰蛋白酶消化细胞并计数，细胞铺板后使其在转染时的汇合度达到70-90％之间。
(2)转染前1小时，将细胞培养基更换为DMEM基础培养基；
(3)用一定体积的DMEM培养基稀释重组质粒，小心混匀，记为A液；用相同体积的DMEM稀释PEI，小心混匀，记为B液；同时使质粒：PEI＝1：2(W/W),室温静止5min；
(4)使用旋涡振荡器，将B液缓慢加入A液，边加入边震荡，室温静置20min；
(5)将DNA-PEI混合液缓慢加入到细胞培养基中，轻轻混匀
(6)转染四小时后更换培养基。
纯化结果如图1所示。
六、DK1-DK17与RBD结合能力的检测
本部分研究利用biacore技术验证重组表达的DK1-DK17等17个单克隆抗体分别与RBD的结合能力。
解离常数越小，结合能力越强。如表3所示，DK1～3、5、6的解离常数都在1nM以下，为17个单克隆抗体中与RBD结合能力较强的。
表3 DK1-DK17与RBD的结合的解离常数Kd
Figure BDA0002592905740000082
Figure BDA0002592905740000091
七、利用假病毒系统检测上述DK1-DK17对SARS-CoV2感染的阻断活性
SARS-CoV2冠状病毒的假病毒通过将SARS-CoV2病毒的囊膜蛋白SPIKE参与到带有报告基因的缺损型病毒基因组的包装中从而得到该病毒的假病毒，其中所述的报告基因有GFP,EGFP,RFP,YFP,BFP,LacZ等；
实验步骤如下：
(1)将真核表达的RBD-单克隆抗体(包括DK1-DK17)样品按比例稀释
(2)将SARS-CoV2的假病毒的浓度配制为10IU/ul(指病毒感染单位)
(3)将20ul的假病毒与按1：20稀释后的100ul血清共同孵育(37℃，30min)，加入24孔板中的293T细胞培养体系中(细胞培养体系为：500ulDMEM+10％FCS)
(4)增加不同稀释浓度的RBD-单克隆抗体，检测其阻断假病毒感染的能力。
(5)14小时后计算GFP阳性细胞的数量
实验结果如表5所示。可见DK1～7、DK9～11、DK14～15的半最大效应浓度均很低，最低为0.012μg/mL。相比之下，前人在针对中东呼吸综合征病毒(MERS)的RBD开发特异性单克隆抗体时，其抑制MERS假病毒的最低IC50(半最大抑制浓度，相当于这里的EC50)为0.23μg/mL(宛玉花，MERS-CoV中和抗体的筛选及人源化抗体的设计，安徽大学，2015)。说明本发明的抗体抗病毒效果优良。
表5单克隆抗体对假病毒的半最大效应浓度(EC50)
单克隆抗体名称 EC50(nM) EC50(μg/mL)
DK1 0.091 0.014
DK2 0.083 0.012
DK3 0.41 0.062
DK4 1.84 0.276
DK5 0.10 0.015
DK6 0.47 0.071
DK7 0.47 0.071
DK8 n.d. n.d.
DK9 0.11 0.017
DK10 1.63 0.245
DK11 0.34 0.051
DK12 n.d. n.d.
DK13 n.d. n.d.
DK14 0.41 0.062
DK15 1.27 0.191
DK16 n.d. n.d.
DK17 n.d n.d.
注：n.d.，未检测。
综上，本发明的抗新型冠状病毒RBD结构域抗原的单克隆抗体，抗病毒效果优良，十分有利于控制新型冠状病毒疫情。
SEQUENCE LISTING
<110> 四川大学
<120> 抗新型冠状病毒RBD结构域抗原的单克隆抗体
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85 90 95
Ala Arg Asp Gly Tyr Asp Val Gly Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 4
<211> 119
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 4
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Tyr Asp Val Gly Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 5
<211> 122
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 5
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ser Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Tyr His Tyr Asp Gly Tyr Tyr Val Thr Leu Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 6
<211> 122
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 6
Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ser Ile His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Tyr His Tyr Asp Gly Tyr Tyr Val Thr Leu Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 7
<211> 122
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 7
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ser Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Tyr His Tyr Asp Gly Tyr Tyr Val Thr Leu Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 8
<211> 121
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 8
Glu Val Met Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg His Asp Gly Asn Tyr Val Asp Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 9
<211> 118
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 9
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Cys Cys
85 90 95
Ala Arg Glu Asp Tyr Gly Ser Ser Tyr Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 10
<211> 122
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 10
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ser Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Thr Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Tyr His Tyr Asp Gly Tyr Tyr Val Thr Leu Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 11
<211> 114
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 11
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala His
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Gly Arg Gly Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
100 105 110
Ser Ser
<210> 12
<211> 121
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 12
Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Met Phe Ser Arg Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Asn Cys
85 90 95
Ala Arg His Asp Gly Asn Tyr Val Asp Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 13
<211> 117
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 13
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Asn Arg Tyr Gly Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 14
<211> 117
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 14
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Ser Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Glu Leu Trp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 15
<211> 121
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 15
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Phe Val Met Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Asp Thr Ser Asp Thr Tyr Thr Arg Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Leu Tyr Tyr Tyr Gly Asn Pro Thr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 16
<211> 125
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 16
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Ser Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Gly Gly Ser Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Lys His Pro Leu Tyr Tyr Gly Ser Asn Ser Asp Gly Tyr Tyr Ala Met
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120 125
<210> 17
<211> 117
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 17
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr
20 25 30
Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asp Pro Ser Asp Ser Tyr Ser Asn Tyr Asn Gln Asn Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Glu Leu Trp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 18
<211> 113
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 18
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Val Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 19
<211> 107
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 19
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 20
<211> 119
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 20
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Tyr Asp Val Gly Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 21
<211> 113
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 21
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Arg Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 22
<211> 107
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr
20 25 30
Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 23
<211> 107
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 23
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr
20 25 30
Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 24
<211> 107
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 24
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr
20 25 30
Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 25
<211> 104
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 25
Gln Leu Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly
1 5 10 15
Glu Glu Ile Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr
35 40 45
Thr Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Asp Tyr Tyr Cys His Gln Trp Ser Cys Trp Thr Phe Gly
85 90 95
Gly Gly Thr Lys Leu Glu Ile Lys
100
<210> 26
<211> 113
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 26
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Asn
20 25 30
Ser Asn Gln Lys Lys Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 27
<211> 107
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 27
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr
20 25 30
Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 28
<211> 107
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 28
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Phe Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Glu His Tyr Ser Asn Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 29
<211> 104
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 29
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly
1 5 10 15
Glu Glu Ile Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser His Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr
35 40 45
Ser Thr Phe Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Asp Tyr Tyr Cys His Gln Trp Ser Ser Trp Thr Phe Gly
85 90 95
Gly Gly Thr Lys Leu Glu Ile Lys
100
<210> 30
<211> 112
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 30
Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Ile Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Lys Ser Thr Lys Arg Leu Leu Asn Ser
20 25 30
Asp Gly Phe Thr Phe Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn Arg Phe 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 Leu Gly Val Tyr Tyr Cys Phe Gln Ser
85 90 95
His Tyr Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210> 31
<211> 112
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 31
Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly
1 5 10 15
Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
His Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn
85 90 95
Leu Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg
100 105 110
<210> 32
<211> 107
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 32
Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly
1 5 10 15
Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 33
<211> 107
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 33
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ile Val
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 34
<211> 111
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 34
Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly
1 5 10 15
Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
His Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn
85 90 95
Leu Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110

Claims (8)

1. 一种抗新型冠状病毒的单克隆抗体，其特征在于：其重链可变区序列如SEQ IDNO.1所示，其轻链可变区序列如SEQ ID NO.18所示。
2.如权利要求1所述的单克隆抗体，其特征在于：所述单克隆抗体是IgGl抗体。
3.编码权利要求1或2所述的单克隆抗体的核酸。
4.一种重组核酸载体，其特征在于：所述重组核酸载体包括权利要求3所述的核酸。
5.如权利要求4所述的重组核酸载体，其特征在于：所述核酸载体是真核表达载体。
6.一种重组细胞，其特征在于：所述重组细胞内含有权利要求4或5所述的重组核酸载体。
7.权利要求1或2所述的单克隆抗体在制备抗新型冠状病毒的药物中的用途。
8.一种抗新型冠状病毒的药物，其特征在于：所述药物以权利要求1或2所述的单克隆抗体为活性成分。
CN202010701787.XA 2020-07-20 2020-07-20 抗新型冠状病毒rbd结构域抗原的单克隆抗体 Active CN111978395B (zh)

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