CN114560929B - 针对冠状病毒np蛋白的单克隆抗体及其应用 - Google Patents
针对冠状病毒np蛋白的单克隆抗体及其应用 Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了针对冠状病毒NP蛋白的单克隆抗体及其应用,上述单克隆抗体识别新型冠状病毒SARS‑CoV‑2的NP蛋白和/或严重急性呼吸综合征冠状病毒SARS‑CoV的NP蛋白。本发明还公开了编码所述单克隆抗体的核酸分子以及含有所述核酸分子的表达盒、重组载体、重组细胞系,以及上述单克隆抗体、核酸分子、重组载体、重组细胞系在制备检测或诊断试剂中的应用。本发明筛选获得的两株单克隆抗体可应用于多种检测方法中来检测SARS‑CoV‑2和SARS‑CoV的NP蛋白,呈现良好的广谱性和敏感性。
Description
技术领域
本发明涉及分子生物学及细胞免疫学的医药技术领域,尤其涉及针对冠状病毒NP蛋白 的单克隆抗体及其应用,具体地,上述单克隆抗体广谱识别新型冠状病毒(SARS-CoV-2) 的NP蛋白和严重急性呼吸综合征冠状病毒(SARS-CoV)的NP蛋白。
背景技术
新型冠状病毒肺炎(Corona Virus Disease 2019,COVID-19),是由新型冠状病毒(SARS-CoV-2)引起的一种严重的急性呼吸***传染病。该病感染性强,致死率高。根 据感染症状,分为无症状、轻症、重症等不同类型。常见症状有呼吸道症状、发热、咳嗽、 气促和呼吸困难等。在较严重病例中,感染可导致肺炎、严重急性呼吸综合征、肾衰竭, 甚至死亡。
SARS-CoV-2和已知的严重急性呼吸综合征冠状病毒(SARS-CoV)、中东呼吸综合征冠状病毒(MERS-CoV)同属于冠状病毒科的β冠状病毒属。SARS-CoV-2为单股正链RNA 病毒,大小约30kb。
早期诊断对于感染病人的及早发现和新冠疫情的防控都至关重要。针对新冠病毒,有 三种主要的检测方式:核酸检测、抗原检测和抗体检测。核酸检测、抗原检测和抗体检测 分别针对的是病毒核酸、病毒蛋白和人体的血清抗体,前两种是采集口鼻呼吸道的分泌物, 而抗体检测是要抽取患者的血液。病毒感染人体后,在靶细胞中有一个复制的过程,首先 能够检测到抗原和核酸。一定时间后,我们的人体会对病毒产生相应的抗体。核酸检测速 度快,可以大批量检测,但是受病程的影响较大,有无症状感染者在检测数十次为阴性后 才检测为阳性,而且检测专业要求高。目前全球大范围内新冠疫苗的接种,使得抗体检测 已经不太能作为病毒感染的指标。相比之下,抗原可以比较早进行检测,而且它对专业要 求不是很高,不需要特定的设备,非专业人士进行简单学习就可以操作,而且检测速度很 快,是核酸检测的一种有效补充手段,特别适合面对检测需求量变大情况下的初筛以及居 家自我检测。
SARS-CoV-2的核衣壳为螺旋对称结构,主要由衣壳蛋白(Nucleocapsid protein,NP) 组成,NP在宿主感染早期大量表达,含量最多,免疫原性较强,而且N蛋白在多数冠状病毒中属于相对保守的抗原,因此,NP可作为血清学诊断SARS-CoV-2感染的靶点。为提高 血清学诊断的效率,需要进一步寻找灵敏度更高、特异性更强的NP抗体。
发明内容
为了克服现有技术中存在的问题,解决目前亟需有效的针对早期广谱检测SARS-CoV-2 的问题,本发明筛选获得了两株针对新型冠状病毒NP蛋白的单克隆抗体,其具有高效、广 谱性、多用途的特点。本发明进一步提供了使用针对新型冠状病毒NP蛋白的单克隆抗体用 于多种方法检查新型冠状病毒,上述单克隆抗体对SARS-CoV-2NP蛋白具有强的特异性结 合能力,并可通过多种方法检测新型冠状病毒(SARS-CoV-2)的NP蛋白和严重急性呼吸 综合征冠状病毒(SARS-CoV)的NP蛋白,上述两个单克隆抗体有望成为血清学早期诊断 SARS-CoV-2和SARS-CoV的检测抗体,为检测新型冠状病毒、SARS-CoV及其突变株的 抗原检测提供了强有力的工具。
为实现上述目的,本发明采用如下技术方案:
本发明的第一个方面是提供两株单克隆抗体,其识别新型冠状病毒SARS-CoV-2的NP 蛋白和/或严重急性呼吸综合征冠状病毒SARS-CoV的NP蛋白。
进一步地,所述单克隆抗体识别SARS-CoV-2的NP蛋白和SARS-CoV的NP蛋白,其中,SARS-CoV-2包括SARS-CoV-2原始株及其突变株,所述突变株包括Alpha、Beta、Delta 和Omicron。
进一步地,所述单克隆抗体为广谱性人源单克隆抗体,其对SARS-CoV-2的NP蛋白和 SARS-CoV的NP蛋白具有反应性。
进一步地,所述单克隆抗体为第一种抗体(命名为P301-F7)、第二种抗体(命名为P301-H5)或两者的组合物;其中,所述第一种抗体的重链可变区VH的CDR序列(具体 为CDR1-H、CDR2-H、CDR3-H)如SEQ ID NO.1-SEQ ID NO.3所示,所述第一种抗体 的轻链可变区VL的CDR序列(具体为CDR1-L、CDR2-L、CDR3-L)如SEQ ID NO.4-SEQ ID NO.6所示;所述第二种抗体的重链可变区VH的CDR序列(具体为CDR1-H、CDR2-H、CDR3-H)如SEQ ID NO.17-SEQID NO.19所示,所述第二种抗体的轻链可变区VL的 CDR序列(具体为CDR1-L、CDR2-L、CDR3-L)如SEQ ID NO.20-SEQ ID NO.22所示。
进一步地,所述第一种抗体的重链可变区VH的氨基酸序列如SEQ ID No.13所示或与 其具有至少80%一致性的氨基酸序列,所述第一种抗体的轻链可变区VL的氨基酸序列如 SEQ ID No.14所示或与其具有至少80%一致性的氨基酸序列;所述第二种抗体的重链可变 区VH的氨基酸序列如SEQ ID No.29所示或与其具有至少80%一致性的氨基酸序列,所述 第二种抗体的轻链可变区VL的氨基酸序列如SEQ ID No.30所示或与其具有至少80%一致 性的氨基酸序列。
可理解的是的,上述单克隆抗体均由重链序列和与之对应的轻链序列组成,轻链和重 链的可变区都决定对抗原的结合识别和特异性,抗体的特异性取决于抗体结合位点和抗原 决定区间的结构互补,抗体结合位点由主要来自高度可变区或互补决定区(CDR)的残基 组成,而CDR间可通过***框架区(FR)进行连接,在获知CDR的氨基酸序列后,本领 域的技术人员可轻易确定框架区。因此,只要发现的重链序列和轻链序列的CDR序列不变, 都能够基本实现本发明P301-F7和P301-H5的功能和应用。因此,本发明的单克隆抗体, 其具体序列不仅限于以上具体的重链序列可变区和轻链序列可变区,以上具体序列的重链 序列可变区和轻链序列可变区只是本发明的一种实现方式中具体采用的单克隆抗体序列。
由与参照序列“至少80%一致性”的氨基酸序列组成的蛋白与参照序列相比可包含突 变如缺失、***和/或取代。在取代的情况中,由与参照序列至少80%、85%、90%、95%、 96%、97%、98%或99%相同的氨基酸序列组成的蛋白可对应源自不同于参照序列的物种 的同源序列。“氨基酸取代”可以是保守或非保守的。优选地,取代为保守取代,其中一个 氨基酸由具有相似结构和/或化学性质的另一氨基酸取代。具体地,重链或轻链可变区的序 列与参照序列区别仅在于保守氨基酸取代。
本发明的第二个方面提供与本发明第一个方面中任一所述的单克隆抗体相关的生物材 料,选自下述(A)~(B)中的一种:
(A)编码本发明第一个方面中任一所述的单克隆抗体的核酸分子;
(B)含有(A)中所述核酸分子的表达盒、重组载体、重组细胞系。
进一步地,所述表达盒、重组载体、重组细胞系可用于表达上述重链序列、轻链序列 或单克隆抗体。
进一步地,编码SEQ ID NO.1所示的第一种抗体的CDR1-H的核酸序列为SEQ IDNO. 7;编码SEQ ID NO.2所示的第一种抗体的CDR2-H的核酸序列为SEQ ID NO.8;编码SEQID NO.3所示的第一种抗体的CDR3-H的核酸序列为SEQ ID NO.9;编码SEQ ID NO.4所 示的第一种抗体的CDR1-L的核酸序列为SEQ ID NO.10;编码SEQ ID NO.5所示的第一 种抗体的CDR2-L的核酸序列为SEQ ID NO.11;编码SEQ ID NO.6所示的第一种抗体的 CDR3-L的核酸序列为SEQ ID NO.12;编码SEQ ID NO.17所示的第二种抗体的CDR1-H 的核酸序列为SEQID NO.23;编码SEQ ID NO.18所示的第二种抗体的CDR2-H的核酸序 列为SEQ ID NO.24;编码SEQ ID NO.19所示的第二种抗体的CDR3-H的核酸序列为SEQ ID NO.25;编码SEQ IDNO.20所示的第二种抗体的CDR1-L的核酸序列为SEQ ID NO.26; 编码SEQ ID NO.21所示的第二种抗体的CDR2-L的核酸序列为SEQ ID NO.27;编码SEQ ID NO.22所示的第二种抗体的CDR3-L的核酸序列为SEQ ID NO.28。
进一步地,编码SEQ ID NO.13所示的第一种抗体的VH的核酸序列为SEQ IDNO.15; 编码SEQ ID NO.14所示的第一种抗体的VL的核酸序列为SEQ ID NO.16;编码SEQID NO. 29所示的第二种抗体的VH的核酸序列为SEQ ID NO.31;编码SEQ ID NO.30所示的第二 种抗体的VL的核酸序列为SEQ ID NO.32。
可理解的是,以上具体的核酸序列只是本发明的一种实现方式种具体采用的核酸序列, 根据密码子的简并性,在保障编码序列不变的情况下,除了以上限定的核酸序列以外,可 以编码出相同重链序列或轻链序列的若干种核酸序列(例如保守核苷酸序列变体源于遗传 密码简并和沉默的变体,核苷酸的替代、缺失和增加也包含在内),都在本发明的保护范围 内。
上述生物材料中,含有编码抗体的核酸分子的表达盒,是指能够在宿主细胞中表达所 述抗体的DNA,该DNA不但可包括启动所述抗体基因转录的启动子,还可包括终止所述抗体基因转录的终止子。
上述生物材料中,载体是可将编码某蛋白的多聚核苷酸***其中并使蛋白获得表达的 一种核酸运载工具,载体可通过转化、转导或转染宿主细胞,使其携带的遗传物质元件在 宿主细胞内得以表达,载体可为可为质粒、黏粒、噬菌体或病毒载体。
上述生物材料中,宿主细胞指的是导入载体的细胞,包括原核细胞、真菌细胞、昆虫 细胞、动物细胞等,例如大肠杆菌、酵母细胞、S2果蝇细胞、BHK细胞、CHO细胞、HEK 293细胞。上述表达盒、重组载体、重组细胞系均可采用本领域常规方法制备。
上述P301-F7抗体、P301-H5抗体的重链、轻链的氨基酸序列及其编码核酸分子的核苷 酸序列如下表所示:
表1–序列信息表
本发明的第三个方面是提供本发明第一个方面中任一所述的单克隆抗体的制备方法, 其包括步骤:将含有本发明第一个方面中任一所述的单克隆抗体的核酸分子的重组载体转 染宿主细胞;培养转染后的宿主细胞并收集上清液,纯化获得所述单克隆抗体。
进一步地,所述重组载体为pCDNA3.4质粒,所述宿主细胞为293F细胞。
本发明的第四个方面是提供本发明第一个方面中任一所述的单克隆抗体、本发明第二 个方面中任一所述的生物材料或采用本发明第三个方面的方法制得的单克隆抗体在制备检 测或诊断SARS-CoV-2和/或SARS-CoV的产品中的应用。
本发明的第五个方面是提供一种用于检测或诊断SARS-CoV-2和/或SARS-CoV的产品, 所述产品包含本发明第一个方面中任一所述的单克隆抗体、或本发明第二个方面中任一所 述的生物材料或采用本发明第三个方面的方法制得的单克隆抗体。
进一步地,上述产品可为诊断试剂(诊断试剂盒)、检测试剂(检测试剂盒)等,其还可包括其他反应试剂。上述产品可应用于ELISA、Western blot、流式细胞术、IFA和免疫 斑点方法来检测SARS-CoV-2和SARS-CoV的NP蛋白。
本发明的第六个方面是提供本发明第五个方面中任一所述的产品的使用方法,或者提 供一种非诊断目的的检测SARS-CoV-2和/或SARS-CoV或其NP蛋白的方法,其包括采用所述单克隆抗体与待检样品混合以检测SARS-CoV-2和/或SARS-CoV的NP蛋白。
上述待检样品涵盖从受试者获得的多种样品类型且可在诊断或检测中使用,包括但不 限于血液和其他生物来源的液体样品、固体组织样品,涵盖临床样品、培养基中的细胞、 细胞上清、细胞裂解物、血清、血浆、生物液体和组织样品等。
进一步地,检测方法包括:ELISA、Western blot、流式细胞术、IFA和免疫斑点方法。
进一步地,所述单克隆抗体作为包被抗体和酶标二抗(检测抗体)使用。
可理解的是,在实际应用中,可分别单独使用第一种抗体(P301-F7抗体)或第二种抗 体(P301-H5抗体),也可联合使用两种抗体制备成的组合物,以检测SARS-CoV-2和/或SARS-CoV的NP蛋白。
与现有技术相比,本发明采用上述技术方案具有以下有益效果:
本发明筛选获得的针对新型冠状病毒NP蛋白单克隆抗体P301-F7和P301-H5,其与新 型冠状病毒NP蛋白和SARS-CoV的NP蛋白的结合能力强。本发明通过实施例验证了上述单克隆抗体在多种生物学方法中的应用,并且验证了其检测新型冠状病毒突变株的能力。上述单克隆抗体,具有广谱性,对多种新冠病毒突变株的NP蛋白均具有较好的结合能力,在SARS-CoV-2和SARS-CoV感染的早期诊断领域具广阔的应用前景。
附图说明
此处所说明的附图用来提供对本发明的进一步理解,构成本发明的一部分,本发明的 示意性实施例及其说明仅用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1为本发明一实施例中流式细胞仪分选新型冠状病毒NP特异性IgG+记忆B细胞的 结果图;
图2为本发明一实施例中分离到的单克隆抗体结合SARS-CoV-2和SARS-CoV的NP活性的ELISA结果示意图;
图3为本发明一实施例中通过竞争ELISA法检测P301-F7和P301-H5为非竞争单克隆 抗体的结果示意图;
图4为本发明一实施例中单克隆抗体P301-F7和P301-H5用于Western blot方法鉴定 SARS-CoV-2和SARS-CoV的NP蛋白的结果示意图;
图5为本发明一实施例中单克隆抗体P301-F7用于IFA检测SARS-CoV-2的NP蛋白的结果示意图;
图6为本发明一实施例中单克隆抗体P301-F7和P301-H5用于流式细胞术检测细胞内 的SARS-CoV-2和SARS-CoV的NP蛋白的结果示意图;其中,293T-NP(SARS-CoV-2)为 转染表达pCMV-SARS-CoV-2-N的293T细胞,293T-NP(SARS-CoV)为转染 pCMV-SARS-CoV-N的293T细胞;
图7为本发明一实施例中单克隆抗体P301-F7用于免疫斑点实验检测SARS-CoV-2及 其突变株的NP蛋白的结果示意图。
具体实施方式
下面将结合本发明实施例中的附图,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的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。下列实施例中未注明具体条件的实验方法,通常按照 国家标准测定。下述实施例中未注明出处的实验材料,均为市售原料。下述实施例中的各 步骤中采用的设备均为常规设备。若没有相应的国家标准,则按照通用的国际标准、常规 条件、或按照制造厂商所建议的条件进行。除非另外说明,否则所有的份数为重量份,所 有的百分比为质量百分比。除非另有定义或说明,本发明中所使用的所有专业与科学用语 与本领域技术熟练人员所熟悉的意义相同。此外任何与所记载内容相似或均等的方法及材 料皆可应用于本发明方法中。
需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组 合。下面结合附图和具体实施例对本发明作进一步说明,但不作为本发明的限定。
实施例1-抗体筛选和表达纯化
本实施例从新冠感染康复者体内分析、筛选并纯化单克隆抗体,其具体步骤如下:
(1)抗体的筛选分析
外周血单个核细胞(PBMC)样品采集自新冠感染康复者并保存于液氮中。
复苏冻存的PBMC,用10mL含有10%胎牛血清的1640培养基(72400047,Gibco) 洗涤2次。在50μL染色缓冲液(PBS(B540626,上海生工)+2%胎牛血清)中加入Live/Dead(L34964、Invitrogen),重悬外周血单个核细胞,于4℃染色30分钟。PBS洗涤2次后, 在50μL染色缓冲液中加入CD19-PE-Cy7、CD3-Pacific Blue、CD8-Pacific Blue、 CD14-PacificBlue、CD27-APC/Cy7、IgG-FITC(557835、558117、558207、558121、560222、 555786,BDPharmingen)和带有His标签的新型冠状病毒株NP(40588-V07E,Sino Biological)探针混合物,重悬外周血单个核细胞,于4℃染色30分钟。PBS洗涤2次后, 在50μL染色缓冲液中加入Anti-6×His-APC和Anti-6×His-PE(ab72579、ab72467,Abcam), 重悬外周血单个核细胞,于4℃染色30分钟。PBS洗涤2次后,通过BD FACS Aria II分 选型流式细胞仪分选新型冠状病毒株NP特异性IgG+记忆B细胞。
将单个B细胞分选到含有裂解缓冲液的96孔PCR板中,然后按照文献(Liao HX,Levesque MC,Nagel A,Dixon A,Zhang R,Walter E,et al.High-throughput isolationof immunoglobulin genes from single human B cells and expression asmonoclonal antibodies. Journal of virological methods.2009;158:171-9.)的方法进行RT-PCR和巢式PCR,分别扩增 重链和轻链可变区域。将PCR扩增产物送至生工生物工程(上海)股份有限公司进行测序。 将测序获得的抗体可变区序列送金斯瑞生物科技有限公司合成,并由该公司将抗体重链和 轻链的可变区域分别克隆到全长IgG1重链和轻链表达载体pCDNA3.4(金斯瑞生物科技有 限公司)中,并大量制备抗体重链和轻链的质粒。
(2)单克隆抗体的表达和纯化
利用PEI转染试剂将成对的重链和轻链表达质粒共转染293F细胞(以500mL为例)。具体步骤为:293F细胞培养于8%CO2,37℃培养箱中,将293F细胞浓度调整为1.2×106/mL,继续培养2小时;配制A液:向12.5mL的opti-MEM(31985070,Gibco)加入250μg抗 体重链质粒和250μg抗体轻链质粒;配制B液:向12.5mL的opti-MEM加入2.5mL 1mg/mL 的PEI转染试剂(24885-2,Polyscieces),静置5分钟;将A液和B液混合,静置20分钟, 获得AB混合液;将25mL的AB混合液逐滴加入到500mL的293F细胞中,边滴加边摇 匀;将细胞继续培养5天;然后离心293F细胞,3000g,离心20分钟,收集上清,使用 0.45μm滤膜过滤;打开Protein A重力柱中盖子,利用重力让柱子中20%的乙醇溶液完全 流出,用5倍柱体积的10mM的PBS溶液平衡Protein A重力柱;向Protein A重力柱内加 入过滤后的细胞上清液,依靠重力作用流出;用3倍柱体积的PBS溶液清洗Protein A重力 柱,再用5倍体积的0.1M的甘氨酸-盐酸溶液(pH=3.0)洗脱;将洗脱液置于30KD的超 滤浓缩管中,用PBS补满,4℃下,3500g离心40分钟,弃去收集管中废液,加入20mL 的PBS溶液,4℃下,3500g离心40分钟,吸取浓缩置换后的抗体溶液,测定抗体蛋白浓 度。
通过流式细胞术从一个新冠感染康复者(P301)外周血中分离新型冠状病毒(SARS-CoV-2)NP特异性IgG+记忆B细胞。结果如图1所示,外周血淋巴细胞比例约 占51.4%,通过CD3、CD8、CD14等排除T淋巴细胞、单核细胞,通过CD19分子选出 B细胞,通过CD27分子选出记忆B细胞,并进一步选出IgG阳性的细胞,最后通过带 有His标签的新型冠状病毒株NP探针和Anti-6×His-APC和Anti-6×His-PE抗体选出新型 冠状病毒NP特异性记忆B细胞。康复者外周血中新型冠状病毒NP特异性记忆B细胞占 IgG阳性的记忆B细胞的比例分0.57%。
实施例2-酶联免疫吸附试验(ELISA)验证NP抗体的结合能力
从获得的抗体中最终选定5株抗体用于单克隆抗体的表达及相关功能验证,上述5株 抗体分别命名为P301-C12、P301-D9、P301-F6、P301-F7、P301-H5。
其中,P301-F7的序列信息如下:重链可变区VH的CDR1-H、CDR2-H、CDR3-H序 列分别如SEQ ID NO.1-SEQ ID NO.3所示,编码其的核酸分子的序列分别如SEQ ID NO. 7-SEQID NO.9所示;轻链可变区VL的CDR1-L、CDR2-L、CDR3-L的序列分别如SEQ ID NO.4-SEQ IDNO.6所示,编码其的核酸分子的序列分别如SEQ ID NO.10-SEQ ID NO.12所示;重链可变区VH的氨基酸序列如SEQ ID No.13所示,编码其的核酸分子的序 列如SEQ ID NO.15所示,重链可变区VL的氨基酸序列如SEQ ID No.14所示,编码其的 核酸分子的序列如SEQ IDNO.16所示。
P301-H5的序列信息如下:重链可变区VH的CDR1-H、CDR2-H、CDR3-H序列分别 如SEQ ID NO.17-SEQ ID NO.19所示,编码其的核酸分子的序列分别如SEQ ID NO.23- SEQID NO.25所示;轻链可变区VL的CDR1-L、CDR2-L、CDR3-L的序列分别如SEQ ID NO.20-SEQID NO.22所示,编码其的核酸分子的序列分别如SEQ ID NO.26-SEQ ID NO. 28所示;重链可变区VH的氨基酸序列如SEQ ID No.29所示,编码其的核酸分子的序列如 SEQ ID NO.31所示,重链可变区VL的氨基酸序列如SEQ ID No.30所示,编码其的核酸 分子的序列如SEQID NO.32所示。
上述5株单克隆抗体的制备方法与实施例1中-(2)单克隆抗体的表达和纯化的方法相 同。
通过ELISA实验检测所分选获得的NP单克隆抗体与SARS-CoV-2和SARS-CoV的NP的特异性结合能力。96孔酶标板中分别将SARS-CoV-2NP蛋白(40588-V07E,SinoBiological) 和SARS-CoV NP蛋白(40143-V08B,Sino Biological)按照2μg/mL在4℃包被过夜,每 孔100μL。PBST洗5次(含0.5%Tween-20的PBS溶液);用封闭液常温封闭1小时,每孔150μL,后用PBST洗5次,封闭液配方:5%脱脂奶+2%BSA(PBS配制),后面的所有 抗体稀释液配方和封闭液相同;将待检测的抗体从最高浓度10μg/mL开始连续5倍倍比稀 释,共7个稀释度,每孔100μL,37℃孵育1小时,后用PBST洗5次;按1:5000加入 HRP-山羊抗人IgG(ZB-2304,中杉金桥),37℃孵育1小时,后用PBST洗5次;显色A 液和B液(生工)按1:1混合,每孔100μL,避光常温作用20分钟;然后用50μL 2M H2SO4终止反应。用Varioskan LUX多模酶标仪(Thermo Scientific)在450nm(OD)下测量光密 度。
通过ELISA实验分析从康复者外周血中分离到的单克隆抗体对SARS-CoV-2和SARS-CoV的NP的结合活性。结果如图2所示,尽管强度不同,5个单克隆抗体均可特异 性结合SARS-CoV-2和SARS-CoV的NP,其中抗体P301-F7、P301-H5对SARS-CoV-2和 SARS-CoV的NP蛋白均具有更优的亲和力。
实施例3-竞争ELISA法检测P301-F7和P301-H5
本实施例采用竞争ELISA法检测P301-F7和P301-H5,确定两者为非竞争单克隆抗体, 其采用的实验步骤具体如下:
首先用HRP标记试剂盒(ab102890,Abcam)标记P301-F7。步骤如下:将100μg待 标记的抗体用PBS稀释至100μL,加入10μL Modifier试剂,轻柔吹打混匀。打开HRP偶 联混合物的瓶盖,用移液枪头吸取抗体样本(已加入Modifier试剂),直接加在冻干粉材料 上,轻柔重悬。盖上瓶盖,室温(20℃-25℃)下避光放置3小时。孵育3小时(或更久) 之后,在每10μL反应中的抗体内加入1μL Quencher试剂,轻柔混匀。30分钟后偶联抗体 即可使用,再加入100μL甘油,混匀后-20℃保存(约0.5μg/mL)。
将SARS-CoV-2的NP蛋白(2μg/mL)分别包被于96孔板中并4℃过夜,然后用封闭 液进行封闭,37℃封闭1小时。将10μg/mL P301-F7、P301-H5、IgG1(阴性对照)用1:60000 稀释的HRP酶标的P301-F7溶液做5倍倍比稀释,共7个稀释度。加入96孔板中,每孔 100μL,37℃孵育1小时。后用PBST洗5次;A液和B液(生工)按1:1混合,每孔100μL, 避光常温作用20分钟;然后用50μL 2M H2SO4终止反应。用Varioskan LUX多模酶标仪 (Thermo Scientific)在450nm(OD)下测量光密度。实验结果如图3所示。
由实验结果可得,OD450值不随着P301-H5单克隆抗体和IgG1(阴性对照)抗体浓度的变化而有显著变化,而P301-F7呈剂量依赖地降低OD450读值,表明P301-H5不影响 P301-F7与SARS-CoV-2NP蛋白的结合,这也表明P301-F7和P301-H5为非竞争单克隆抗 体。
实施例4-利用Western blot实验验证P301-F7和P301-H5单克隆抗体的功能
本实施例将单克隆抗体P301-F7和P301-H5应用于Western blot方法以鉴定SARS-CoV-2和SARS-CoV的NP蛋白,其实验步骤具体包括:
将1μg SARS-CoV-2、SARS-CoV、MERS-CoV的重组NP蛋白(40588-V07E,40143-V08B,40068-V08B,Sino Biological)经SDS-PAGE电泳后,转印至NC膜中,5%脱脂乳封闭1 小时后,PBST洗3次,然后分别用P301-F7和P301-H5(10μg/mL)和抗His兔单克隆抗 体(ab245114,Abcam)孵育1小时后,PBST洗涤3次,再分别用1:5000稀释的山羊抗 人IgG(ZB-2304,中杉金桥)和山羊抗兔IgG(ab6721,Abcam)孵育1小时,PBST洗涤 3次后进行显色。实验结果如图4所示。
Western blot实验结果显示所述单克隆抗体P301-F7和P301-H5均能特异性结合重组表 达的SARS-CoV-2和SARS-CoV的NP蛋白,不能结合MERS-CoV-2NP蛋白。
实施例5-利用免疫荧光实验验证P301-F7单克隆抗体的功能
本实施例以单克隆抗体P301-F7为例将其用于IFA检测SARS-CoV-2的NP蛋白,其实验步骤具体包括:
本实验在认证的生物安全三级实验室中进行。96孔板中接种Vero E6细胞悬液(4×104个/孔),将培养板放在培养箱中预培养12小时,细胞贴壁生长;分别将(0、5、20μM) 浓度的Remdesivir(HY-104077,MCE)与SARS-CoV-2等体积混合,置于96孔底板,37℃ 孵育1小时;然后将混合物转移到接种有Vero E6细胞的96孔板上,保证每孔含有200PFU 的活病毒,在37℃下孵育1小时后换新鲜的完全培养基,37℃孵育24小时;然后用4%多 聚甲醛溶液固定细胞;弃液,用含0.1%Triton X-100的缓冲液对细胞进行渗透10分钟;加 入新鲜配制的含5%BSA的PBS溶液,室温作用1小时,PBS洗两次,每次5分钟;加入2 μg/ml P301-F7,常温孵育1小时;用PBS洗3次后再按1:1000加入Alexa488-Goat anti-Human IgG(H+L)Secondary Antibody(A-11013,Thermo Fisher)并常温孵育1小时; 加入Heochst染色液(33342,Heochst AG)作用3分钟,PBST清洗2次后利用荧光显微镜 进行观察。实验结果如图5所示。
结果显示,荧光强度随着Remdesivir浓度的降低而增加,视野下观察到足够的荧光强 度和良好的剂量反应模式,提示P301-F7可作为一种特异性检测SARS-CoV-2NP的抗体应 用于各种免疫荧光检测。
实施例6-利用流式细胞术验证P301-F7和P301-H5单克隆抗体的功能
本实施例将单克隆抗体P301-F7和P301-H5用于流式细胞术检测细胞内表达的SARS-CoV-2和SARS-CoV的NP蛋白,其实验步骤具体包括:
首先分别将SARS-CoV-2和SARS-CoV的N蛋白表达质粒pCMV-SARS-CoV-2-N和pCMV-SARS-CoV-N(VG40588-NH,VG40143-G-N,Sino Biological)分别转染293T细胞, 36小时后,用胰蛋白酶将细胞消化后加入完全培养基(10%FBS,DMEM)终止。用移液 枪将消化下来的细胞吹打混匀,吹打20下后在倒置显微镜下观察,保证90%以上的细胞处 于单细胞状态。将单细胞悬液吸取到1.5mL的EP管中,350g离心5分钟,弃去上清。使 用固定/渗透溶液试剂盒(BD Biosciences)对细胞进行固定和渗透,350g离心5分钟,弃 去上清。在细胞沉淀中加入1mL的Cell Staining Buffer,重悬细胞后,350g离心5分钟, 弃掉上清。用CellStaining Buffer重悬细胞,计数活细胞数,按照细胞个数为5×106cells/ 管。将细胞悬液以每管100μL分装至新的1.5mL EP管中,每个细胞样品3管。然后每个 EP管中加入5μL的Human TruStain FxXTM,室温封闭5-10分钟;分别加入10μg/mL P301-F7,P301-H5和pAb(40588-MM123,Sino Biological),室温作用1小时,然后用APC 偶联的二抗(LifeTechnologies)进行染色。洗涤后,细胞重悬,用FACSCalibur流式细胞 仪(BDBiososciences)采集,数据用FlowJo软件V10.6(BD Biososciences),实验结果如 图6所示。
结果显示,与pAb结果相似,利用P301-F7和P301-H5可以很好的检测细胞内表达的SARS-CoV-2和SARS-CoV的N蛋白。
实施例7-利用活病毒免疫斑点实验验证P301-F7单克隆抗体检测SARS-CoV-2及其变异株
本实施例以单克隆抗体P301-F7为例将其用于免疫斑点实验检测SARS-CoV-2的NP蛋 白,具体实验步骤包括:
本实验在认证的生物安全三级实验室中进行。将SARS-CoV-2及变异株(Alpha,Beta, Delta和Omicron)置于96孔板,37℃孵育1小时;然后将病毒液(PFU=200)转移到接种 有Vero E6细胞的96孔板上,在37℃孵育1小时后取出;洗涤后,加入覆盖培养基(含1.6羧甲基纤维、2%胎牛血清的MEM),37℃孵育24小时;去除覆盖介质后,将细胞用4%多 聚甲醛固定;用含1%Triton X-100的Perm/Wash缓冲液(BD Biosciences)渗透;加入新鲜 配制的含5%BSA的PBS溶液,室温作用1小时,PBS洗两次,每次5分钟;加入1:2000 稀释的HRP标记的P301-F7,常温孵育1小时;该反应用KPL TrueBlue过氧化物酶底物 (Seracare LifeSciences)进行反应。使用EliSpot阅读器(Cellular Technology Ltd)计算 SARS-CoV-2的病灶数量。实验结果如图7所示。
野生型SARS-CoV-2及其变异株感染Vero E6细胞后进行免疫斑点实验,并利用P301-F7 检测不同变异株的NP,结果显示,P301-F7可以识别WT和变异株的NP,表明P301-F7可 以用于免疫斑点实验检测WT,Alpha,Beta,Delta和Omicron的NP。
由上述实施例可知,本发明筛选获得的2株单克隆抗体与新型冠状病毒NP蛋白和SARS-CoV的NP蛋白均具有较强的结合能力,且采用多种检测方式来检测新型冠状病毒和SARS-CoV的NP蛋白,呈现良好的广谱性和敏感性。
以上所述仅为本发明较佳的实施例,并非因此限制本发明的实施方式及保护范围,对 于本领域技术人员而言,应当能够意识到凡运用本发明说明书及图示内容所作出的等同替 换和显而易见的变化所得到的方案,均应当包含在本发明的保护范围。
序列表
<110> 深圳国家感染性疾病临床医学研究中心
深圳市第三人民医院
<120> 针对冠状病毒NP蛋白的单克隆抗体及其应用
<160> 32
<170> SIPOSequenceListing 1.0
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Gly Gly Ser Ile Ser Ser Thr Ser Tyr Tyr
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Ile Tyr Tyr Ser Gly Ser Thr
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<212> PRT
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Ser Ser Asn Ile Gly Asn Asn Tyr
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Asp Asn Asn
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<212> PRT
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Gly Thr Trp Asp Ser Ser Leu Ser Ala Gly Gln Val Val
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<212> DNA
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atctattata gtgggagcac c 21
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gcgagatttt cgctgtattg tagtagtacc agctgctatg aaaactgg 48
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<212> DNA
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agctccaaca ttgggaataa ttat 24
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gacaataat 9
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ggaacatggg atagcagcct gagtgctggc caagtggta 39
<210> 13
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<212> PRT
<213> P301-F7 VH(Artificial Sequence)
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Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
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Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Thr
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Cys Ala Arg Phe Ser Leu Tyr Cys Ser Ser Thr Ser Cys Tyr Glu Asn
100 105 110
Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 14
<211> 112
<212> PRT
<213> P301-F7 VL(Artificial Sequence)
<400> 14
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln
1 5 10 15
Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn
20 25 30
Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln
65 70 75 80
Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu
85 90 95
Ser Ala Gly Gln Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 15
<211> 381
<212> DNA
<213> P301-F7 VH_DNA(Artificial Sequence)
<400> 15
cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60
acctgcactg tctctggtgg ctccatcagc agtactagtt actactgggg ctggatccgc 120
cagcccccag ggaaggggct ggagtggatt gggagtatct attatagtgg gagcacctac 180
tacaacccgt ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 240
tccctgaagc tgagctctgt gaccgccgcg gacacggccg tgtattactg tgcgagattt 300
tcgctgtatt gtagtagtac cagctgctat gaaaactggt tcgacccctg gggccaggga 360
accctggtca ccgtctcctc a 381
<210> 16
<211> 336
<212> DNA
<213> P301-F7 VL_DNA(Artificial Sequence)
<400> 16
cagtctgtgt tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60
tcctgctctg gaagcagctc caacattggg aataattatg tatcctggta ccagcagctc 120
ccaggaacag cccccaaact cctcatttat gacaataata agcgaccctc agggattcct 180
gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240
actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tgctggccaa 300
gtggtattcg gcggagggac caagctgacc gtccta 336
<210> 17
<211> 8
<212> PRT
<213> P301-H5 CDR1-H(Artificial Sequence)
<400> 17
Gly Phe Thr Phe Ser Gly Ser Ala
1 5
<210> 18
<211> 10
<212> PRT
<213> P301-H5 CDR2-H(Artificial Sequence)
<400> 18
Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr
1 5 10
<210> 19
<211> 8
<212> PRT
<213> P301-H5 CDR3-H(Artificial Sequence)
<400> 19
Asn Phe Arg Gly Ala Phe Asp Tyr
1 5
<210> 20
<211> 6
<212> PRT
<213> P301-H5 CDR1-L(Artificial Sequence)
<400> 20
Ala Leu Pro Lys Gln Tyr
1 5
<210> 21
<211> 3
<212> PRT
<213> P301-H5 CDR2-L(Artificial Sequence)
<400> 21
Lys Asp Ser
1
<210> 22
<211> 11
<212> PRT
<213> P301-H5 CDR3-L(Artificial Sequence)
<400> 22
Gln Ser Ala Asp Ser Ser Gly Thr Tyr Val Val
1 5 10
<210> 23
<211> 24
<212> DNA
<213> P301-H5 CDR1-H_DNA(Artificial Sequence)
<400> 23
gggttcacct tcagtggctc tgct 24
<210> 24
<211> 30
<212> DNA
<213> P301-H5 CDR2-H_DNA(Artificial Sequence)
<400> 24
attagaagca aagctaacag ttacgcgaca 30
<210> 25
<211> 24
<212> DNA
<213> P301-H5 CDR3-H_DNA(Artificial Sequence)
<400> 25
aatttcaggg gggcttttga ctac 24
<210> 26
<211> 18
<212> DNA
<213> P301-H5 CDR1-L_DNA(Artificial Sequence)
<400> 26
gcattgccaa agcaatat 18
<210> 27
<211> 9
<212> DNA
<213> P301-H5 CDR2-L_DNA(Artificial Sequence)
<400> 27
aaagacagt 9
<210> 28
<211> 33
<212> DNA
<213> P301-H5 CDR3-L_DNA(Artificial Sequence)
<400> 28
caatcagcag acagcagtgg tacttatgtg gta 33
<210> 29
<211> 117
<212> PRT
<213> P301-H5 VH(Artificial Sequence)
<400> 29
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser
20 25 30
Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr Ala Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Asn Phe Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 30
<211> 108
<212> PRT
<213> P301-H5 VL(Artificial Sequence)
<400> 30
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly Thr Tyr
85 90 95
Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 31
<211> 351
<212> DNA
<213> P301-H5 VH_DNA(Artificial Sequence)
<400> 31
gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgaaactc 60
tcctgtgcag cctctgggtt caccttcagt ggctctgcta tgcactgggt ccgccaggct 120
tccgggaaag ggctggagtg ggttggccgt attagaagca aagctaacag ttacgcgaca 180
gcatatgctg cgtcggtgaa aggcaggttc accatctcca gagatgattc aaagaacacg 240
gcgtatctgc aaatgaacag cctgaaaacc gaggacacgg ccgtgtatta ctgtaatttc 300
aggggggctt ttgactactg gggccaggga accctggtca ccgtctcctc a 351
<210> 32
<211> 324
<212> DNA
<213> P301-H5 VL_DNA(Artificial Sequence)
<400> 32
tcctatgagc tgacacagcc accctcggtg tcagtgtccc caggacagac ggccaggatc 60
acctgctctg gagatgcatt gccaaagcaa tatgcttatt ggtaccagca gaagccaggc 120
caggcccctg tgctggtgat atataaagac agtgagaggc cctcagggat ccctgagcga 180
ttctctggct ccagctcagg gacaacagtc acgttgacca tcagtggagt ccaggcagaa 240
gacgaggctg actattactg tcaatcagca gacagcagtg gtacttatgt ggtattcggc 300
ggagggacca aactgaccgt ccta 324
Claims (8)
1.一种单克隆抗体,其特征在于,所述单克隆抗体识别新型冠状病毒SARS-CoV-2的NP蛋白和/或严重急性呼吸综合征冠状病毒SARS-CoV的NP蛋白;其中,所述单克隆抗体为第一种抗体、第二种抗体或两者的组合物;
其中,所述第一种抗体的重链可变区VH的CDR1-H序列如SEQ ID NO. 1所示,所述第一种抗体的重链可变区VH的CDR2-H序列如SEQ ID NO. 2所示,所述第一种抗体的重链可变区VH的CDR3-H序列如SEQ ID NO. 3所示;
所述第一种抗体的轻链可变区VL的CDR1-L序列如SEQ ID NO. 4所示,所述第一种抗体的轻链可变区VL的CDR2-L序列如 SEQ ID NO. 5所示,所述第一种抗体的轻链可变区VL的CDR3-L序列如 SEQ ID NO. 6所示;
所述第二种抗体的重链可变区VH的CDR1-H序列如SEQ ID NO. 17所示,所述第二种抗体的重链可变区VH的CDR2-H序列如SEQ ID NO. 18所示,所述第二种抗体的重链可变区VH的CDR3-H序列如SEQ ID NO. 19所示;
所述第二种抗体的轻链可变区VL的CDR1-L序列如SEQ ID NO. 20所示,所述第二种抗体的轻链可变区VL的CDR2-L序列如 SEQ ID NO. 21所示,所述第二种抗体的轻链可变区VL的CDR3-L序列如 SEQ ID NO. 22所示。
2.根据权利要求1所述的一种单克隆抗体,其特征在于,所述第一种抗体的重链可变区VH的氨基酸序列如SEQ ID No. 13所示或与其具有至少80%一致性的氨基酸序列,所述第一种抗体的轻链可变区VL的氨基酸序列如SEQ ID No. 14所示或与其具有至少80%一致性的氨基酸序列;所述第二种抗体的重链可变区VH的氨基酸序列如SEQ ID No. 29所示或与其具有至少80%一致性的氨基酸序列,所述第二种抗体的轻链可变区VL的氨基酸序列如SEQID No. 30所示或与其具有至少80%一致性的氨基酸序列。
3.一种与权利要求1~2中任一所述的单克隆抗体相关的生物材料,其特征在于,所述生物材料选自下述(A)~(B)中的一种:
(A)编码权利要求1~2中任一所述单克隆抗体的核酸分子;
(B)含有(A)中所述核酸分子的表达盒、重组载体、重组细胞系。
4.根据权利要求3所述的生物材料,其特征在于,
编码SEQ ID NO. 1所示的第一种抗体的CDR1-H的核酸序列为SEQ ID NO. 7;
编码SEQ ID NO. 2所示的第一种抗体的CDR2-H的核酸序列为SEQ ID NO. 8;
编码SEQ ID NO. 3所示的第一种抗体的CDR3-H的核酸序列为SEQ ID NO. 9;
编码SEQ ID NO. 4所示的第一种抗体的CDR1-L的核酸序列为SEQ ID NO. 10;
编码SEQ ID NO. 5所示的第一种抗体的CDR2-L的核酸序列为SEQ ID NO. 11;
编码SEQ ID NO. 6所示的第一种抗体的CDR3-L的核酸序列为SEQ ID NO. 12;
编码SEQ ID NO. 17所示的第二种抗体的CDR1-H的核酸序列为SEQ ID NO. 23;
编码SEQ ID NO. 18所示的第二种抗体的CDR2-H的核酸序列为SEQ ID NO. 24;
编码SEQ ID NO. 19所示的第二种抗体的CDR3-H的核酸序列为SEQ ID NO. 25;
编码SEQ ID NO. 20所示的第二种抗体的CDR1-L的核酸序列为SEQ ID NO. 26;
编码SEQ ID NO. 21所示的第二种抗体的CDR2-L的核酸序列为SEQ ID NO. 27;
编码SEQ ID NO. 22所示的第二种抗体的CDR3-L的核酸序列为SEQ ID NO. 28。
5.根据权利要求3所述的生物材料,其特征在于,
编码SEQ ID NO. 13所示的第一种抗体的VH的核酸序列为SEQ ID NO. 15;
编码SEQ ID NO. 14所示的第一种抗体的VL的核酸序列为SEQ ID NO. 16;
编码SEQ ID NO. 29所示的第二种抗体的VH的核酸序列为SEQ ID NO. 31;
编码SEQ ID NO. 30所示的第二种抗体的VL的核酸序列为SEQ ID NO. 32。
6.一种如权利要求1~2中任一所述的单克隆抗体的制备方法,其特征在于,包括步骤:将含有编码所述单克隆抗体的核酸分子的重组载体转染宿主细胞;培养转染后的宿主细胞并收集上清液,纯化获得所述单克隆抗体。
7.一种如权利要求1~2中任一所述的单克隆抗体、如权利要求3~5中任一所述的生物材料或由权利要求6所述的方法制得的单克隆抗体在制备用于检测或诊断SARS-CoV-2和/或SARS-CoV的产品中的应用。
8.一种用于检测或诊断SARS-CoV-2和/或SARS-CoV的产品,其特征在于,所述产品包含如权利要求1~2中任一所述的单克隆抗体、如权利要求3~5中任一所述的生物材料或由权利要求6所述的方法制得的单克隆抗体。
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