CN111778218B - 噬菌体展示抗体库及基于其淘筛获得的针对新冠病毒SARS-CoV-2的单克隆抗体 - Google Patents
噬菌体展示抗体库及基于其淘筛获得的针对新冠病毒SARS-CoV-2的单克隆抗体 Download PDFInfo
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Abstract
本发明公开了一种噬菌体展示抗体库并筛选到了五株能够跟新冠病毒SARS‑CoV‑2的S蛋白结合的抗体,本发明基于合成生物学与噬菌体展示技术,将突变导入抗体可变区的超可变区,并将此基因转至大肠杆菌中,从而构建了含有108种抗体的合成抗体库;本发明的噬菌体展示抗体库能够筛选获得具有特异性和检测功能的抗体,扩大了生物研究和医学诊断的有力资源;本发明还筛选到了五株能够跟新冠病毒的S蛋白结合的抗体,可以用于病毒的检测,部分抗体可以阻断病毒与细胞结合,具有中和新冠病毒传染力的能力,可用于制备新冠病毒检测产品、制备抑制新冠病毒药物和制备预防或治疗由新冠病毒引起的疾病的药物制剂,具有广泛的应用前景。
Description
技术领域
本发明涉及生物医学和分子生物学技术领域,具体说是一种噬菌体展示抗体库及基于噬菌体展示抗体库淘筛获得的针对新冠病毒SARS-CoV-2的单克隆抗体。
背景技术
SARS-CoV-2相比原有的冠状病毒相比,不仅是重症患者具有传播性,症状轻微甚至潜伏期患者的传染性也很强,并且传播的途径很多,经飞沫、接触均能传播,给预防带来了很大的困难。
各国针对新冠肺炎的治疗提出了多种治疗方案,主要有使用小分子药物瑞德西韦、氯喹与羟氯喹联用、洛匹那韦与利托那韦联用及洛匹那韦、利托那韦与干扰素的三药联用,很遗憾的是都并没有明显的治疗效果且有些存在严重的毒副作用。另一方面,抗体药物在传染病、自身免疫疾病及肿瘤等的治疗上发挥了重要作用。在当前面临的SARS-CoV-2变异率高、新冠肺炎疫苗开发困难且时期漫长、传统药物副作用大甚至没有效果的情况下,使用康复患者的血清治疗重症患者为一个相对有效的治疗方案,在实际治疗中起了一定的作用,这表明新冠病毒的抗体能够有效的减弱SARS-CoV-2病毒的传染能力,在新冠肺炎患者治疗中起了一定的作用,给医药研发人员指出了一条新的道路。
SARS-CoV-2病毒直径为75-160纳米,其基因组为连续线性单链RNA,其基因组依次编码核蛋白(nucleoprotein)、包膜蛋白(envelope protein)、膜蛋白(membrane protein)及棘突蛋白(spike protein,又称S-protein或S蛋白),其中棘突蛋白是其表面最重要的蛋白,其主要功能为决定病毒的宿主范围和特异性,与宿主细胞细胞膜受体结合并融合,实现对细胞的感染。棘突蛋白具有两个亚基S1和S2,S1中的受体结合区(receptor bindingdomain,RBD),与人类SARS-CoV受体血管紧张素转化酶II(ACE2)分子互作,而S2则含有膜融合过程所需要的基本元件,实现病毒与细胞的融合。因此S蛋白的人源单克隆抗体理论上可以阻断病毒跟细胞的结合,具有减弱病毒传染的能力,可以作为抗体药物用于治疗新冠病毒肺炎患者。
抗体是哺乳动物免疫***中的一个重要糖蛋白分子。抗体分子由两条重链(Heavychain)和两条轻链(Light chain)组成,其中重链由分为可变区(Variable region ofheavy chain,VH)和三个恒定区(Constant region of heavy chain;CH1,CH2,CH3),轻链则由一个可变区(VL)和一个恒定区(CL)组成。可变区具有跟抗原结合的功能,因抗体个体不同而不同,而抗体的恒定区则因抗体的种属及亚型不同而不同。抗体的可变区又分为4个框架区(Framework;FR1,FR2,FR3,FR4)和三个互补决定区(Complementarity-determiningregions;CDR1,CDR2,CDR3),其中抗体重链可变区的结构为HFR1-CDRH1-HFR2-CDRH2-HFR3-CDRH3-HFR4,抗体轻链可变区的结构为LFR1-CDRL1-LFR2-CDRL2-LFR3-CDRL3-LFR4,其中互补决定区又称为高可变区,直接与抗原的表位结合。
发明内容
为解决上述问题,本发明的目的是提供一种噬菌体展示抗体库及基于噬菌体展示抗体库淘筛获得的针对新冠病毒SARS-CoV-2的单克隆抗体。
本发明为实现上述目的,通过以下技术方案实现:
一种噬菌体展示抗体库,包括利用NNK密码子,将抗体重链可变区的所有突变位置对应的氨基酸突变为任意氨基酸,对应得到的抗体重链可变区的DNA序列;及将在抗体轻链可变区的所有突变位置对应的氨基酸突变为任意氨基酸,对应得到的抗体轻链可变区的DNA序列;并将抗体重链可变区基因和抗体轻链可变区基因与噬菌体载体重组构建质粒后转化大肠杆菌,从而构建成噬菌体展示抗体库;
其中抗体重链可变区的突变位置为50,52a,52b,53,55,56,58,97,98,99,100,101,102,103,104,105;抗体轻链可变区的突变位置为50,51,52,53,89,90,91,92,93,94,95,96,97;
其中抗体库重链可变区的DNA序列为SEQ ID NO:53;
抗体库轻链可变区的DNA序列为SEQ ID NO:54。
本发明还包括噬菌体展示抗体库的构建方法,包括以下步骤:
①以抗体重链可变区DNA为模板,用引物SfiVHback和引物VHFR2For,通过聚合酶链式反应扩增抗体重链可变区VH的HFR1-CDHR1-HFR2基因片段VH1,并用简并引物CDRH2libback和简并引物CDRH3lib9for扩增CDRH2-HFR3-CDRH3-HFR4的基因即VH2,执行琼脂糖电泳,确认扩增的VH1和VH2;将两个基因片段切出纯化,再以两段基因序列为模板,用引物SfiVHback和XhoVHfor执行重叠PCR;执行琼脂糖电泳,确认扩增的DNA片段,纯化DNA片段,作为噬菌体展示抗体库的重链可变区基因;
其中抗体重链可变区模板DNA序列为SEQ ID NO:1;
引物SfiVHback的碱基序列为SEQ ID NO:2;
引物VHFR2For的碱基序列为SEQ ID NO:3;
简并引物CDRH2libback的碱基序列为SEQ ID NO:4;
简并引物CDRH3lib9for的碱基序列为SEQ ID NO:5;
XhoVHfor的碱基序列为SEQ ID NO:6;
抗体库重链可变区的DNA序列为SEQ ID NO:53;
②以抗体轻链可变区DNA为模板,用引物SalVLback和引物VLFR2For,通过聚合酶链式反应扩增抗体轻链VL的LFR1-CDRL1-LFR2基因片段VL1,用简并引物CDRL2libback和简并引物CDRL3libfor扩增CDRL2-LFR3-CDRL3-LFR4的基因片段VL2,执行琼脂糖电泳,确认扩增的VL1,VL2,将两个基因片段切出纯化,再以两段基因序列为模板,用引物SalVLback和NotVLfor执行重叠PCR;执行琼脂糖电泳,确认扩增的DNA片段,纯化DNA片段,作为噬菌体展示抗体库的轻链可变区基因;
其中抗体轻链可变区模板DNA序列为SEQ ID NO:7;
引物SalVLback的碱基序列为SEQ ID NO:8;
引物VLFR2For的碱基序列为SEQ ID NO:9;
简并引物CDRL2libback的碱基序列为SEQ ID NO:10;
简并引物CDRL3libfor的碱基序列为SEQ ID NO:11;
NotVLfor的碱基序列为SEQ ID NO:12;
抗体库轻链可变区的DNA序列为SEQ ID NO:54;
③使用限制酶SalI和NotI处理轻链可变区基因,纯化后将其克隆到噬菌体展示载体pDong1中,扩增后用限制酶SfiI和XhoI处理,将其与限制酶SfiI和XhoI处理后的重链可变区的基因使用T4 DNA连接酶连接,转化大肠杆菌TG-1;
④培养转化大肠杆菌TG-1,加入辅助噬菌体KM13感染,经后处理得到噬菌体展示抗体库。
本发明还包括噬菌体展示抗体库淘筛到的单克隆抗体A9,
A9抗体重链可变区氨基酸序列为SEQ ID NO:13,CDRH1氨基酸序列为SEQ ID NO:14;CDRH2氨基酸序列为SEQ ID NO:15;CDRH3氨基酸序列为SEQ ID NO:16;
A9抗体轻链可变区氨基酸序列为SEQ ID NO:17,CDRL1氨基酸序列为SEQ ID NO:18;CDRL2氨基酸序列为SEQ ID NO:19;CDRL3氨基酸序列为SEQ ID NO:20。
本发明还包括噬菌体展示抗体库淘筛到的单克隆抗体E11,
E11抗体重链可变区氨基酸序列为SEQ ID NO:21,CDRH1氨基酸序列为SEQ ID NO:22;CDRH2氨基酸序列为SEQ ID NO:23;CDRH3氨基酸序列为SEQ ID NO:24;
E11抗体轻链可变区氨基酸序列为SEQ ID NO:25,CDRL1氨基酸序列为SEQ ID NO:26;CDRL2氨基酸序列为SEQ ID NO:27;CDRL3氨基酸序列为SEQ ID NO:28。
本发明还包括噬菌体展示抗体库淘筛到的单克隆抗体F5,
F5抗体重链可变区氨基酸序列为SEQ ID NO:29,CDRH1氨基酸序列为SEQ ID NO:30;CDRH2氨基酸序列为SEQ ID NO:31;CDRH3氨基酸序列为SEQ ID NO:32;
F5抗体轻链可变区氨基酸序列为SEQ ID NO:33,CDRL1氨基酸序列为SEQ ID NO:34;CDRL2氨基酸序列为SEQ ID NO:35;CDRL3氨基酸序列为SEQ ID NO:36。
本发明还包括噬菌体展示抗体库淘筛到的单克隆抗体F10,
F10抗体重链可变区氨基酸序列为SEQ ID NO:37,CDRH1氨基酸序列为SEQ ID NO:38;CDRH2氨基酸序列为SEQ ID NO:39;CDRH3氨基酸序列为SEQ ID NO:40;
F10抗体轻链可变区氨基酸序列为SEQ ID NO:41,CDRL1氨基酸序列为SEQ ID NO:42;CDRL2氨基酸序列为SEQ ID NO:43;CDRL3氨基酸序列为SEQ ID NO:44。
本发明还包括噬菌体展示抗体库淘筛到的单克隆抗体H9,
H9抗体重链可变区氨基酸序列为SEQ ID NO:45,CDRH1氨基酸序列为SEQ ID NO:46;CDRH2氨基酸序列为SEQ ID NO:47;CDRH3氨基酸序列为SEQ ID NO:48;
H9抗体轻链可变区氨基酸序列为SEQ ID NO:49,CDRL1氨基酸序列为SEQ ID NO:50;CDRL2氨基酸序列为SEQ ID NO:51;CDRL3氨基酸序列为SEQ ID NO:52。
本发明还包括任一种噬菌体展示抗体库淘筛到的单克隆抗体(A9、E11、F5、F10、H9)在制备新冠病毒SARS-CoV-2检测产品中的应用;如可以制备成检测新冠病毒SARS-CoV-2的试剂盒,试剂盒中含有A9,E11,F5,F10,H9抗体中的任意一种或多种及其衍生物,如含有抗体可变区的全长抗体,scFv,或Fab片段或其他形式的含有可变区的融合蛋白及其他组装试剂盒的辅助材料。
本发明还包括任一种噬菌体展示抗体库淘筛到的单克隆抗体(A9、E11、F5、F10、H9)在制备抑制新冠病毒SARS-CoV-2药物中的应用。
本发明还包括任一种噬菌体展示抗体库淘筛到的单克隆抗体(A9、E11、F5、F10、H9)在制备预防或治疗由新冠病毒SARS-CoV-2引起的疾病的药物制剂中的应用。
本发明还包括任一改造的单克隆抗体,其抗原结合片段是选自任一单克隆抗体A9、E11、F5、F10、H9中的抗体片段,包括Fab、Fab’-SH、Fv、scFv或(Fab’)2片段。
抗体分可变区及恒定区,可变区决定抗体的抗原结合特性。本发明所涉及抗体可变区序列可用于制造具有相同抗原结合特异性及应用的Fab、Fab’-SH、Fv、scFv或(Fab’)2片段。
本发明还包括任一单克隆抗体,其重链及轻链的超可变区的氨基酸序列与任一单克隆抗体A9、E11、F5、F10或H9的相应序列具有80%以上的相同性。
将以上抗体的超可变区序列进行进一步的突变和进化,在保持80%的相同性的情况下,抗体的抗原结合特异性不变,抗原结合能力可显著提高。
本发明相比现有技术具有以下优点:
本发明基于合成生物学与噬菌体展示技术,将突变导入包括超可变区在内的抗体特定位置,并将此基因转至大肠杆菌中,从而构建了一个含有108种多样性的、能够在大肠杆菌中高效表达的合成抗体库;本发明的噬菌体展示抗体库能够用于筛选获得具有特异性和检测功能的抗体,扩大了生物研究和医学诊断的有力资源。
本发明还从该噬菌体展示抗体库中筛选到了五株能够跟新冠病毒SARS-CoV-2的S蛋白结合的抗体,抗体的超可变序列为新序列,是新型人源单克隆抗体;这些抗体可以阻断病毒与细胞结合,具有中和新冠病毒传染力的能力,可以用于制备新冠病毒SARS-CoV-2检测产品、制备抑制新冠病毒SARS-CoV-2药物和制备预防或治疗由新冠病毒SARS-CoV-2引起的疾病的药物制剂,具有广泛的应用前景。
附图说明
图1为PCR所得基因片段的琼脂糖电泳图;
图2为淘筛抗体库所得噬菌体的抗原结合能的酶联免疫分析结果;
图3为单克隆抗体抗原特异性的酶联免疫试验结果;
图4为利用淘筛的抗体检测新冠病毒原理示意图;
图5为利用A9抗体的Fab片段与F5,F10或H9组合检测病毒S蛋白的酶联免疫吸附试验结果;
图6为利用E11抗体的Fab片段与F5,F10或H9组合检测病毒S蛋白的酶联免疫吸附试验结果。
具体实施方式
本发明的目的是提供一种噬菌体展示抗体库及基于噬菌体展示抗体库淘筛获得的针对新冠病毒SARS-CoV-2的单克隆抗体,通过以下技术方案实现:
以下结合具体实施例来对本发明作进一步的描述。
实施例中使用的新冠病毒SARS-CoV-2的S蛋白和病毒S-RBD蛋白均采购于北京义翘神州生物科技有限公司。
噬菌体展示载体pDong1的合成方法参考文献为:Dong,et al.,AnalBiochem.2009,386(1):36-44。
实施例1
一、噬菌体展示抗体文库的构建
以合成的抗体重链可变区DNA(碱基序列为SEQ ID NO:1)为模板,用引物SfiVHback(碱基序列为SEQ ID NO:2)和VHFR2For(碱基序列为SEQ ID NO:3),通过聚合酶链式反应扩增抗体重链可变区VH的HFR1-CDRH1-HFR2基因片段VH1,并用简并引物CDRH2libback(碱基序列为SEQ ID NO:4)和简并引物CDRH3lib9for(碱基序列为SEQ IDNO:5)扩增CDRH2-HFR3-CDRH3-HFR4的基因即VH2,执行琼脂糖电泳,确认扩增的VH1和VH2,将两个基因片段切出纯化,再以两段基因序列为模板,用引物SfiVHback(碱基序列为SEQID NO:2)和XhoVHfor(碱基序列为SEQ ID NO:6)执行重叠PCR;
以上PCR反应均使用KOD-plus-neo(TOYOBO Bio),反应体系为50μL,DNA模板为50ng,引物的浓度为1μM,反应条件为94度变性2分钟,55度淬火30秒,DNA的伸长反应在68度下运行1分钟,共反应30个循环,执行琼脂糖电泳,确认扩增的DNA片段,切出并纯化DNA片段,作为噬菌体展示抗体库的重链可变区基因,其碱基序列为SEQ ID NO:53。
以合成的抗体轻链DNA(碱基序列为SEQ ID NO:7)为模板,用引物SalVLback(碱基序列为SEQ ID NO:8)和VLFR2For(碱基序列为SEQ ID NO:9),通过聚合酶链式反应扩增抗体轻链VL的LFR1-CDRL1-LFR2基因片段VL1,用简并引物CDRL2libback(碱基序列为SEQ IDNO:10)和简并引物CDRL3libfor(碱基序列为SEQ ID NO:11)扩增CDRL2-LFR3-CDRL3-LFR4的基因片段VL2,执行琼脂糖电泳,确认扩增的VL1,VL2,将两个基因片段切出纯化,再以两段基因序列为模板,用引物SalVLback(碱基序列为SEQ ID NO:8)和NotVLfor(碱基序列为SEQ ID NO:12)执行重叠PCR。
以上PCR反应均使用KOD-plus-neo(TOYOBO Bio),反应体系为50μL,DNA模板为50ng,引物的浓度为1μM,反应条件为94度变性2分钟,55度淬火30秒,DNA的伸长反应在68度下运行1分钟,共反应30个循环。执行琼脂糖电泳,纯化PCR扩增的DNA片段,作为噬菌体展示抗体库的轻链基因,其碱基序列为SEQ ID NO:54。
使用10单位的限制酶SalI和NotI在37度下处理轻链可变区基因3小时,纯化后将其克隆到噬菌体展示载体pDong1。将克隆成功的质粒(含有轻链可变区基因)扩增,并进一步使用10单位的SfiI和XhoI处理,将其与使用相同酶(SfiI和XhoI)处理的抗体重链可变区基因在16℃下使用T4 DNA连接酶连接,转化大肠杆菌TG-1;
37℃培养转化大肠杆菌25mL至OD600为0.5,加入辅助噬菌体KM13,37℃感染1小时后,3000g离心30分钟,弃上清,使用含有100μg/ml氨苄青霉素、50μg/mL卡那霉素及0.1%葡萄糖的2YT培养基50mL(1.6%Tryptone,1%Yeast Extract,0.5%NaCl)悬浮菌体,30℃,250rpm,摇菌16小时,次日5000g,30分钟离心培养液,分离回收上清40mL,在上清液中加入10mL的PEG/NaCl溶液,混合均匀后在冰上放置30分钟,5000g,30分钟离心,弃上清,加入2mL的灭菌PBS溶液将沉淀溶解,作为噬菌体展示抗体库溶液。
扩增的合成抗体库可变区基因琼脂糖电泳图如图1所示,VL1,VL2为合成抗体库轻链可变区基因的两个片段,VL为抗体库轻链可变区的基因;VH1,VH2为合成抗体库重链可变区基因的两个片段,VH为抗体库重链可变区基因;泳道M为DNA marker;抗体可变区基因成功扩增,其中重链可变区基因的长度约为400bp,轻链可变区基因的长度约为370bp,将以上基因片段克隆至噬菌体展示载体pDong1,利用大肠杆菌TG-1制作噬菌体,最后获得一个含有约108种抗体的噬菌体展示抗体库。
所得的噬菌体展示抗体库,包括利用NNK密码子,将抗体重链可变区的突变位置对应的氨基酸突变为任意氨基酸,对应得到的抗体重链可变区的DNA序列;及将在抗体轻链可变区的突变位置对应的氨基酸突变为任意氨基酸,对应得到的抗体轻链可变区的DNA序列;并将抗体重链可变区基因和抗体轻链可变区基因与噬菌体载体重组构建质粒后转化大肠杆菌,从而构建成噬菌体展示抗体库;
其中抗体重链可变区的突变位置为50,52a,52b,53,55,56,58,97,98,99,100,101,102,103,104,105;抗体轻链可变区的突变位置为50,51,52,53,89,90,91,92,93,94,95,96,97;
其中抗体重链可变区的DNA序列为SEQ ID NO:53;
抗体轻链可变区的DNA序列为SEQ ID NO:54。
二、噬菌体展示抗体库的淘筛
在96孔微孔板的10个孔内各加入100μL的含有10μg/ml SARS-CoV-2病毒S蛋白的PBS溶液,4℃过夜孵育,次日弃抗原溶液,每孔加入200μL含有2%脱脂奶粉的PBS溶液,25℃孵育2小时进行封闭,用PBST洗涤3次后,每孔加入100μL的噬菌体溶液(R0;每孔含有109cfu的噬菌体),室温下孵育2小时,用PBST洗涤后,每孔加入100μL的胰蛋白酶将结合到病毒S蛋白的噬菌体洗脱;
培养TG-1大肠杆菌至OD600为0.4,取4mL菌液,将500μL溶出的噬菌体溶液加入到菌液中,37℃感染30分钟,5000g离心20分钟,弃上清,用含有100μg/mL氨苄青霉素、50μg/mL卡那霉素及0.1%葡萄糖的2YT培养基悬浮菌体,30℃,250rpm摇菌16小时,次日5000g,30分钟离心培养液,分离回收上清,在上清溶液中加入1/5容积的PEG/NaCl溶液,混合均匀后在冰上放置30分钟,5000g,30分钟离心,弃上清,加入200μL的灭菌PBS溶液,作为第一次富集后的噬菌体溶液(R1);重复以上步骤,分别获取噬菌体溶液R2和R3;执行酶联免疫吸附试验,验证淘筛过程中获得的噬菌体展示抗体库跟新冠病毒SARS-CoV-2的结合特异性与结合性能。
酶联免疫吸附试验的操作如下:在96孔酶标板加入100μL含有新冠病毒S蛋白溶液(2μg/mL)或牛血清白蛋白BSA(2μg/mL)的PBS溶液,4℃过夜,次日弃抗原溶液,加入200μL含有2%脱脂奶粉的溶液,在25℃下孵育2小时,对酶标板进行封闭,用含有0.1%吐温20的PBS溶液洗涤酶标板3次,加入稀释后的R0、R1、R2和R3噬菌体溶液(109cfu/well)25℃孵育1小时,用PBST溶液洗涤酶标板,加入HRP标记的小鼠抗M13噬菌体抗体,孵育1小时后,用PBST洗板,加入HRP底物TMBZ(用pH6.0的乙酸钠溶液配制,含1/10000稀释的30%H2O2),显色后用酶标仪测在450nm处的吸光度,绘制柱状图,比较各步获得的噬菌体抗体与S蛋白及BSA的结合性能。
酶联免疫吸附试验的结果如图2所示,比较噬菌体淘筛过程中获得的噬菌体库R0、R1、R2及R3跟S蛋白的结合能力时发现,第三轮淘筛时所得噬菌体溶液R3与S蛋白的结合能力显著增加,而对BSA的结合性能非常微弱且没有变化,说明构建的噬菌体展示抗体库中抗新冠病毒S蛋白的抗体得以富集。
三、单克隆抗体筛选
培养TG-1大肠杆菌至OD600为0.4,取100μL淘筛R3噬菌体抗体库溶出的噬菌体溶液,用于感染200μL的大肠杆菌菌液,37℃孵育30分钟后,将菌液涂布到含有100μg/mL氨卞青霉素,50μg/mL卡那霉素及1%葡萄糖的2YT培养基平板上,37℃过夜培养,次日挑96个菌落接种到96孔的培养板上,37℃培养至OD600为0.4,每个孔内加入M13噬菌体,感染后5000g离心20分钟,去除上清,每孔加入200μL含有100μg/mL氨苄青霉素、50μg/mL卡那霉素及0.1%葡萄糖的2YT培养基,悬浮菌体,30℃,250rpm的速度培养16小时;次日5000g,30分钟离心培养液,分离回收上清,执行酶联免疫吸附试验,验证各单克隆抗体跟新冠病毒S蛋白的结合特异性及结合性能。
酶联免疫吸附试验的操作如下:在96孔酶标板内加入100μL含有病毒S蛋白(1μg/mL)的PBS溶液,4℃过夜,次日弃抗原溶液,加入200μL含有2%脱脂奶粉的溶液,在25℃下孵育2小时,对酶标板进行封闭。用含有0.1%吐温20的PBS溶液洗涤酶标板3次,加入噬菌体溶液,25℃孵育1小时,用PBST溶液洗涤酶标板,加入HRP标记的小鼠抗M13抗体,孵育1小时后,用PBST洗板,加入HRP底物TMBZ(用pH6.0的乙酸钠溶液配制,含1/10000稀释的30%H2O2),显色后用酶标仪测450nm的吸光度,比较利用各克隆制作的噬菌体抗体跟S蛋白及牛血清蛋白的结合性能。
根据试验结果,有5株抗体A9、E11、F5、F10、H9与S蛋白结合,抽取质粒进行基因测序,获得5株不同序列的抗体基因,通过与抗体基因库中登录抗体序列进行比对,没有发现与本发明所述抗体基因相同的序列,因此这些序列为新型抗体,抗体的氨基酸序列如下:
A9抗体重链可变区序列为SEQ ID NO:13,CDRH1序列为SEQ ID NO:14;CDRH2
序列为SEQ ID NO:15;CDRH3序列为SEQ ID NO:16;
A9抗体轻链可变区序列为SEQ ID NO:17,CDRL1序列为SEQ ID NO:18;CDRL2序列为SEQ ID NO:19;CDRL3序列为SEQ ID NO:20;
E11抗体重链可变区序列为SEQ ID NO:21,CDRH1序列为SEQ ID NO:22;CDRH2序列为SEQ ID NO:23;CDRH3序列为SEQ ID NO:24;
E11抗体轻链可变区序列为SEQ ID NO:25,CDRL1序列为SEQ ID NO:26;CDRL2序列为SEQ ID NO:27;CDRL3序列为SEQ ID NO:28;
F5抗体重链可变区序列为SEQ ID NO:29,CDRH1序列为SEQ ID NO:30;CDRH2序列为SEQ ID NO:31;CDRH3序列为SEQ ID NO:32;
F5抗体轻链可变区序列为SEQ ID NO:33,CDRL1序列为SEQ ID NO:34;CDRL2序列为SEQ ID NO:35;CDRL3序列为SEQ ID NO:36;
F10抗体重链可变区序列为SEQ ID NO:37,CDRH1序列为SEQ ID NO:38;CDRH2序列为SEQ ID NO:39;CDRH3序列为SEQ ID NO:40;
F10抗体轻链可变区序列为SEQ ID NO:41,CDRL1序列为SEQ ID NO:42;CDRL2序列为SEQ ID NO:43;CDRL3序列为SEQ ID NO:44;
H9抗体重链可变区序列为SEQ ID NO:45,CDRH1序列为SEQ ID NO:46;CDRH2序列为SEQ ID NO:47;CDRH3序列为SEQ ID NO:48;
H9抗体轻链可变区序列为SEQ ID NO:49,CDRL1序列为SEQ ID NO:50;CDRL2序列为SEQ ID NO:51;CDRL3序列为SEQ ID NO:52;
四、单克隆抗体的抗原特异性
在96孔酶标板内加入100μL,浓度各为1μg/mL的新冠病毒S蛋白,S-RBD蛋白,及BSA,4℃过夜,次日弃蛋白溶液,加入200μL 2%脱脂奶粉溶液,在25℃下孵育2小时,对酶标板进行封闭;用含有0.1%吐温20的PBS溶液洗涤酶标板3次后,每孔加入100μL稀释的噬菌体展示抗体溶液,25℃孵育1小时,用PBST溶液洗涤酶标板,加入HRP标记的小鼠抗M13抗体,孵育1小时后,用PBST洗板,加入HRP底物TMBZ(用pH6.0的乙酸钠溶液配制,含1/10000稀释的30%H2O2),显色后用酶标仪测450nm的吸光度,绘制柱状图,比较利用各克隆制作的噬菌体抗体跟包被蛋白的结合性能。
酶联免疫吸附试验的结果如图3所示,A9及E11抗体与S蛋白与S-RBD蛋白结合,为识别S蛋白RBD区的抗体,F5,F10与H9抗体与S蛋白结合但是与RBD结合能力弱,说明他们主要跟S蛋白的其他区域结合。并且上述抗体与包被的BSA均不结合,说明这些抗体不与BSA结合,因此可以断定抗体A9、E11、F5、F10、H9与S蛋白及S-RBD蛋白的结合具有特异性。
五、使用A9抗体Fab片段与噬菌体展示的F5、F10或H9抗体对新冠病毒S蛋白进行检测
检测原理如图4所示,利用A9蛋白及噬菌体展示的F5、F10及H9,对新冠病毒的S蛋白进行检测,在96孔微孔板的孔内包被A9抗体的Fab片段,封闭微孔板后,添加S蛋白或BSA蛋白,洗板后加入噬菌体展示F5抗体,之后加入标记辣根过氧化物酶的抗噬菌体抗体,洗板后加入底物显色,当样品中没有新冠病毒S蛋白或者为BSA蛋白时,反应体系不显色,有病毒S蛋白时体系显色,且样品中的病毒S蛋白越多,则通过病毒S1蛋白捕获到酶标板的F5噬菌体就越多,相应的捕获的抗噬菌体抗体也就越多,加入酶的底物显色后颜色越深,用此方法可以判断样品中是否有病毒S1蛋白,该方法也可用于检测样品中是否存在SARS-CoV-2病毒。
具体操作如下:在96孔酶标板的孔内加入A9抗体Fab片段,4℃过夜,次日弃孔内液体,加入200μL 2%脱脂奶粉溶液,室温放置两个小时,对酶标板进行封闭。洗板后在微孔内加入100μL含有新冠病毒S蛋白的溶液或牛血清蛋白(BSA)溶液,25℃孵育1小时,去除孔内溶液,用含有0.1%吐温的PBS溶液(PBST溶液)洗涤酶标板,加入噬菌体展示的F5抗体溶液(109cfu/mL),25℃孵育1小时,洗板后加入标记有辣根过氧化物酶(HRP)的抗噬菌体抗体溶液(1μg/mL),25℃孵育1小时,去除孔内溶液后用PBST洗板3次,最后加入HRP底物3,3,5,5-四甲基联苯胺盐酸盐(TMBZ)溶液显色,测定孔内溶液在450nm处的吸收度,制作柱状图,比较抗体跟新冠病毒S蛋白及牛血清蛋白的结合能力。
将噬菌体展示的F5抗体溶液换成F10或H9进行上述同样的操作制作柱状图,比较抗体跟新冠病毒S蛋白及牛血清蛋白的结合能力。
如图5所示,A9抗体分别与噬菌体展示的F5、F10或H9组合检测病毒S蛋白的结果,横轴为与A9组合的抗体名称,纵轴为对应酶标孔内溶液的吸光度。A9与F5组合的溶液中含有病毒S蛋白酶标板孔的吸光度为0.45,而添加BSA的孔的吸光度为0.03,说明A9与F5的组合可用于检测溶液中的新冠病毒S蛋白,A9抗体与F10及H9的组合得到了相似的结果,说明A9抗体与F5,F10或H9抗体的组合能够检测样品中是否有新冠病毒S蛋白及新冠病毒。
六、使用E11抗体Fab片段与噬菌体展示的F5、F10或H9抗体对新冠病毒S蛋白进行检测
检测原理如图4所示,利用E11抗体的Fab片段及噬菌体展示的F5、F10及H9对新冠病毒S蛋白进行检测。在96孔微孔板的孔内包被E11抗体的Fab片段,封闭微孔板后,添加S蛋白,洗板后加入噬菌体展示F5抗体,之后加入标记辣根过氧化物酶的抗噬菌体抗体,洗板后加入底物显色。当样品中没有新冠病毒S蛋白或为BSA时,反应体系不显色;有病毒S蛋白时体系显色且样品中的病毒S蛋白越多,则通过病毒S1蛋白捕捉到酶标板的F5噬菌体就越多,相应的捕获的抗噬菌体抗体也就越多,加入酶的底物显色后颜色越深。应用此方法可以判断出样品是否有病毒的S1蛋白,该方法也可以用于检测样品中是否含有SARS-CoV-2病毒。
具体操作如下:
在96孔酶标板的孔内加入E11抗体片段,4℃过夜,次日弃孔内液体,加入200μL2%脱脂奶粉溶液,室温放置两个小时,对酶标板进行封闭。洗板后在微孔内加入100μL含有新冠病毒S蛋白的溶液或牛血清蛋白(BSA)溶液,25℃孵育1小时,去除孔内溶液,用含有0.1%吐温的PBS溶液(PBST溶液)洗涤酶标板,加入噬菌体展示的F5抗体溶液(109cfu/mL),25℃孵育1小时,洗板后加入标记有辣根过氧化酶(HRP)的抗噬菌体抗体溶液(1μg/mL),25℃孵育1小时,去除孔内溶液后用PBST洗板3次,最后加入HRP底物3,3,5,5-四甲基联苯胺盐酸盐(TMBZ)溶液显色,测定孔内溶液在450nm处的吸收度,制作柱状图,比较抗体跟新冠病毒S蛋白及牛血清蛋白的结合能力。将噬菌体展示的F5抗体溶液换成F10或H9进行上述同样的操作,制作柱状图,比较抗体跟新冠病毒S蛋白及牛血清蛋白的结合能力。
图6为E11抗体Fab片段分别与噬菌体展示的F5、F10及H9组合检测病毒S蛋白的结果。横轴表示与E11组合的抗体名称,纵轴为对于酶标孔内溶液的吸光度。E11与F5组合的溶液中含有病毒S蛋白酶标板孔的吸光度为0.36,而添加BSA的孔的吸光度为0.02,说明E11与F5的组合可以用于检测溶液中的新冠病毒S蛋白。同样的,E11抗体片段与F10或H9的组合也获得了相似的结果,说明E11抗体与F5、F10或H9抗体的组合均可用于检测样品中是否含有新冠病毒S蛋白或新冠病毒。
序列信息如下:
抗体重链可变区模板DNA为SEQ ID NO:1
gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaatattcatgcgagtggtatgcgtacatcgtacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgagaaaagtagtggtacgtttgacttctggggccagggaaccctggtcaccgtctcgagc;
SfiVHback为SEQ ID NO:2:
tcgcggcccagccggccatggccgaggtgcagctgttgga;
VHFR2for为SEQ ID NO:3:
tgagacccactccagccccttccct;
CDRH2libback为SEQ ID NO:4:
tggagtgggtctcannkattnnknnknnkggtnnknnkacannktacgctgactccgt;
CDRH3lib9for为SEQ ID NO:5:
ttccctggccccamnnmnnmnnmnnmnnmnnmnnmnnmnnacagtaatatacggccgt;
XhoVHfor为SEQ ID NO:6:
tgagctcgagacggtgaccagggttccctggcccca;
抗体轻链可变区模板DNA为SEQ ID NO:7:
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatcttggatgccatttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtggacaggctggaggacggggattgagcttcggccaagggaccaaggtggaaatcaaacgg;
SalIVLback为SEQ ID NO:8:
ctcagtcgacggacatccagatgacccagt;
VLFR2for为SEQ ID NO:9:
aggagcttaggggctttccctggttt;
CDRL2libback为SEQ ID NO:10:
aaagcccctaagctcctgatctatnnknnknnknnkttgcaaagtggggt;
CDRL3libfor为SEQ ID NO:11:
ttggtcccttggccgaamnnmnnmnnmnnmnnmnnmnnmnnmnnacagtagtaagtt;
NotVLfor为SEQ ID NO:12:
gcctgcggccgcccgtttgatttccaccttggtcccttggccga;
A9抗体重链可变区序列为SEQ ID NO:13:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITNSGSSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTDAFDYWGQGTLVTVSS;
CDRH1序列为SEQ ID NO:14:GFTFSSYA;
CDRH2序列为SEQ ID NO:15:ITNSGSST;
CDRH3序列为SEQ ID NO:16:AKGTDAFDY;
A9抗体轻链可变区序列为SEQ ID NO:17:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNCSPATFGQGTKVEIKR;
A9抗体CDRL1序列为SEQ ID NO:18:QSISSY;
A9抗体CDRL2序列为SEQ ID NO:19:AAS;
A9抗体CDRL3序列为SEQ ID NO:20:QQSNCSPAT;
E11抗体重链可变区序列为SEQ ID NO:21:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDSSGYYTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNSDSFDYWGQGTLVTVSS;
E11抗体CDRH1序列为SEQ ID NO:22:GFTFSSYA;
E11抗体CDRH2序列为SEQ ID NO:23:IDSSGYYT;
E11抗体CDRH3序列为SEQ ID NO:24:AKNSDSFDY;
E11抗体轻链可变区序列为SEQ ID NO:25:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDDGPNTFGQGTKVEIKR;
E11抗体CDRL1序列为SEQ ID NO:26:QSISSY;
E11抗体CDRL2序列为SEQ ID NO:27:SAS;
E11抗体CDRL3序列为SEQ ID NO:28:QQYDDGPNT;
F5抗体重链可变区序列为SEQ ID NO:29:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIDSAGNSTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNDSSFDYWGQGTLVTVSS;
F5抗体CDRH1序列为SEQ ID NO:30:GFTFSSYA;
F5抗体CDRH2序列为SEQ ID NO:31:IDSAGNST;
F5抗体CDRH3序列为SEQ ID NO:32:AKNDSSFDY;
F5抗体轻链可变区序列为SEQ ID NO:33:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASWLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSDGPDTFGQGTKVEIKR;
F5抗体CDRL1序列为SEQ ID NO:34:QSISSY;
F5抗体CDRL2序列为SEQ ID NO:35:SAS;
F5抗体CDRL3序列为SEQ ID NO:36:QQYSDGPDT;
F10抗体重链可变区序列为SEQ ID NO:37:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIDSAGYYTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSDTFDYWGQGTLVTVSS;
F10抗体CDRH1序列为SEQ ID NO:38:GFTFSSYA;
F10抗体CDRH2序列为SEQ ID NO:39:IDSAGYYT;
F10抗体CDRH3序列为SEQ ID NO:40:AKDSDTFDY;
F10抗体轻链可变区序列为SEQ ID NO:41:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSTPATFGQGTKVEIKR;,
F10抗体CDRL1序列为SEQ ID NO:42:QSISSY;
F10抗体CDRL2序列为SEQ ID NO:43:AAS;
F10抗体CDRL3序列为SEQ ID NO:44:QQAYSTPAT;
H9抗体重链可变区序列为SEQ ID NO:45:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDITDNGASTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTNTFDYWGQGTLVTVSS;
H9抗体CDRH1序列为SEQ ID NO:46:GFTFSSYA;
H9抗体CDRH2序列为SEQ ID NO:47:ITDNGAST;
H9抗体CDRH3序列为SEQ ID NO:48:AKSTNTFDY;
H9抗体轻链可变区序列为SEQ ID NO:49:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYINWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNSNSPSTFGQGTKVEIKR;
H9抗体CDRL1序列为SEQ ID NO:50:QSISSY;
H9抗体CDRL2序列为SEQ ID NO:51:DAS;
H9抗体CDRL3序列为SEQ ID NO:52:QQNSNSPST;
抗体库重链可变区的DNA序列为SEQ ID NO:53:
gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcannkattnnknnknnkggtnnknnkacannktacgctgactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtnnknnknnknnknnknnknnknnknnktggggccagggaaccctggtcaccgtctcgagc;
抗体库轻链可变区的DNA序列为SEQ ID NO:54:
Gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatnnknnknnknnkttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtnnknnknnknnknnknnknnknnknnkttcggccaagggaccaaggtggaaatcaaacgg。
序列表
<110> 山东宽和正生物医药有限公司
<120> 噬菌体展示抗体库及基于其淘筛获得的针对新冠病毒SARS-CoV-2 的单克隆抗体
<130> 20200604A-1
<141> 2020-06-04
<160> 54
<170> SIPOSequenceListing 1.0
<210> 1
<211> 348
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaaat attcatgcga gtggtatgcg tacatcgtac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtga gaaaagtagt 300
ggtacgtttg acttctgggg ccagggaacc ctggtcaccg tctcgagc 348
<210> 2
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tcgcggccca gccggccatg gccgaggtgc agctgttgga 40
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgagacccac tccagcccct tccct 25
<210> 4
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tggagtgggt ctcannkatt nnknnknnkg gtnnknnkac annktacgct gactccgt 58
<210> 5
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttccctggcc ccamnnmnnm nnmnnmnnmn nmnnmnnmnn acagtaatat acggccgt 58
<210> 6
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgagctcgag acggtgacca gggttccctg gcccca 36
<210> 7
<211> 324
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatctt ggatgccatt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtggacag gctggaggac ggggattgag cttcggccaa 300
gggaccaagg tggaaatcaa acgg 324
<210> 8
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctcagtcgac ggacatccag atgacccagt 30
<210> 9
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aggagcttag gggctttccc tggttt 26
<210> 10
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aaagccccta agctcctgat ctatnnknnk nnknnkttgc aaagtggggt 50
<210> 11
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ttggtccctt ggccgaamnn mnnmnnmnnm nnmnnmnnmn nmnnacagta gtaagtt 57
<210> 12
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gcctgcggcc gcccgtttga tttccacctt ggtcccttgg ccga 44
<210> 13
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Glu Val Gln Leu Leu Glu 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 Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Thr Asn Ser Gly Ser Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Gly Thr Asp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 14
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 15
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Ile Thr Asn Ser Gly Ser Ser Thr
1 5
<210> 16
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Ala Lys Gly Thr Asp Ala Phe Asp Tyr
1 5
<210> 17
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
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 Asn 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 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 Asn Cys Ser Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 18
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Gln Ser Ile Ser Ser Tyr
1 5
<210> 19
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Ala Ala Ser
1
<210> 20
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Gln Gln Ser Asn Cys Ser Pro Ala Thr
1 5
<210> 21
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Glu Val Gln Leu Leu Glu 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 Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asp Ser Ser Gly Tyr Tyr Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asn Ser Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 22
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 23
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Ile Asp Ser Ser Gly Tyr Tyr Thr
1 5
<210> 24
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Ala Lys Asn Ser Asp Ser Phe Asp Tyr
1 5
<210> 25
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
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 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr 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 Tyr Asp Asp Gly Pro Asn
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 26
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Gln Ser Ile Ser Ser Tyr
1 5
<210> 27
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Ser Ala Ser
1
<210> 28
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Gln Gln Tyr Asp Asp Gly Pro Asn Thr
1 5
<210> 29
<211> 116
<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 Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Asp Ser Ala Gly Asn Ser Thr Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asn Asp Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 30
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 31
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Ile Asp Ser Ala Gly Asn Ser Thr
1 5
<210> 32
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Ala Lys Asn Asp Ser Ser Phe Asp Tyr
1 5
<210> 33
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
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 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Trp 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 Tyr Ser Asp Gly Pro Asp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 34
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Gln Ser Ile Ser Ser Tyr
1 5
<210> 35
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Ser Ala Ser
1
<210> 36
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Gln Gln Tyr Ser Asp Gly Pro Asp Thr
1 5
<210> 37
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Glu Val Gln Leu Leu Glu 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 Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Asp Ser Ala Gly Tyr Tyr Thr Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Ser Asp Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 38
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 39
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Ile Asp Ser Ala Gly Tyr Tyr Thr
1 5
<210> 40
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Ala Lys Asp Ser Asp Thr Phe Asp Tyr
1 5
<210> 41
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
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 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Tyr 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 Ala Tyr Ser Thr Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 42
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Gln Ser Ile Ser Ser Tyr
1 5
<210> 43
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Ala Ala Ser
1
<210> 44
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
Gln Gln Ala Tyr Ser Thr Pro Ala Thr
1 5
<210> 45
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
Glu Val Gln Leu Leu Glu 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 Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Asp Ile Thr Asp Asn Gly Ala Ser Thr Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Thr Asn Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 46
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 46
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 47
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 47
Ile Thr Asp Asn Gly Ala Ser Thr
1 5
<210> 48
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 48
Ala Lys Ser Thr Asn Thr Phe Asp Tyr
1 5
<210> 49
<211> 108
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 49
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
Ile Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp 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 Asn Ser Asn Ser Pro Ser
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 50
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 50
Gln Ser Ile Ser Ser Tyr
1 5
<210> 51
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 51
Asp Ala Ser
1
<210> 52
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 52
Gln Gln Asn Ser Asn Ser Pro Ser Thr
1 5
<210> 53
<211> 348
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcannk attnnknnkn nkggtnnknn kacannktac 180
gctgactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtnn knnknnknnk 300
nnknnknnkn nknnktgggg ccagggaacc ctggtcaccg tctcgagc 348
<210> 54
<211> 324
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatnnk nnknnknnkt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtnnknnk nnknnknnkn nknnknnknn kttcggccaa 300
gggaccaagg tggaaatcaa acgg 324
Claims (4)
1.一种单克隆抗体A9,其特征在于:所述单克隆抗体A9能够与新冠病毒SARS-CoV-2S蛋白特异性结合;
A9抗体重链可变区氨基酸序列为SEQ ID NO:13,CDRH1氨基酸序列为SEQ ID NO:14;CDRH2氨基酸序列为SEQ ID NO:15;CDRH3氨基酸序列为SEQ ID NO:16;
A9抗体轻链可变区氨基酸序列为SEQ ID NO:17,CDRL1氨基酸序列为SEQ ID NO:18;CDRL2氨基酸序列为SEQ ID NO:19;CDRL3氨基酸序列为SEQ ID NO:20。
2.权利要求1所述的单克隆抗体A9,其特征在于:在制备新冠病毒SARS-CoV-2检测产品中的应用。
3.权利要求1所述的单克隆抗体A9,其特征在于:在制备抑制新冠病毒SARS-CoV-2药物及在制备治疗由新冠病毒SARS-CoV-2引起的疾病的药物制剂中的应用。
4.任一改造的单克隆抗体,其特征在于,其抗原结合片段是选自权利要求1所述的单克隆抗体A9的抗体片段,包括Fab、Fab’-SH、Fv、scFv或(Fab’)2片段。
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CN202110744069.5A CN113336846B (zh) | 2020-06-04 | 2020-06-04 | 针对新冠病毒SARS-CoV-2的单克隆抗体E11 |
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CN105131116B (zh) * | 2015-09-25 | 2018-10-09 | 中国人民解放军第四军医大学 | 靶向HER2的人源化改造的可内化单链抗体P1h3及制备方法和应用 |
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CN108191976A (zh) * | 2017-12-18 | 2018-06-22 | 青岛珅奥基生物工程有限公司 | 一种抗ER-α36单克隆抗体 |
CN109738645B (zh) * | 2018-12-26 | 2021-10-12 | 山东宽和正生物医药有限公司 | 一种检测盐酸克伦特罗含量的非竞争法酶联免疫分析方法 |
CN111217918B (zh) * | 2020-03-04 | 2020-11-10 | 中山大学 | 一种基于2,4-二氧四氢喋啶合酶的新型冠状病毒s蛋白双区域亚单位纳米疫苗 |
CN111217919B (zh) * | 2020-03-04 | 2020-12-01 | 中山大学 | 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗 |
CN111217920B (zh) * | 2020-03-10 | 2020-11-17 | 河北精硕生物科技有限公司 | 新冠病毒n-s优势表位融合蛋白、制备方法、应用,及表达蛋白、微生物、应用,试剂盒 |
CN111088283B (zh) * | 2020-03-20 | 2020-06-23 | 苏州奥特铭医药科技有限公司 | mVSV病毒载体及其病毒载体疫苗、一种基于mVSV介导的新冠肺炎疫苗 |
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